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PALACKY UNIVERSITY OLOMOUC
Faculty of Natural Sciences
Department of Biochemistry
Target identification of potential antitumor drugs
inducing changes in the cell cycle
MASTER THESIS
Author: Alžběta Kameníčková
Study program: B1406 Biochemistry
Study branch: Biochemistry
Study mode: Full – time
Supervisor: MUDr. Petr Džubák, Ph.D.
Submitted: 26.4.2010
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I declare that this master thesis has been written solely and all the sources used in this
thesis are cited and included in the reference part.
In Olomouc 2. 5. 2010
…………………………….
Acknowledgments
First of all I would like to express thanks to my supervisor MUDr. Petr Džubák, Ph.D.
for special leading, valuable reminder, advices and gained experiences during my
work. Other thanks are regarded to all employees of Laboratory of Experimental
Medicine, Department of Pediatrics, Faculty of Medicine, Palacký University and
Faculty Hospital in Olomouc for their help. Last but not least, I would like to thank my
parents and roommates for friendly atmosphere in which this work could originate.
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Bibliografická identifikace:
Jméno a příjmení Alžběta Kameníčková
Název práce
Typ práce:
Identifikace cílů potenciálních protinádorových léčiv indukující změny v buněčném cyklu
Diplomová
Pracoviště Laboratoř experimentální medicíny při Dětské klinice LF UP a FN Olomouc
Vedoucí práce MUDr. Petr Džubák, Ph.D.
Rok obhajoby práce 2010
Abstrakt Počet případů onemocnění rakovinou se neustále zvyšuje, proto je vyvíjeno úsilí identifikovat nová léčiva vykazující protinádorovou aktivitu. Předmětem této práce je identifikovat cíle potenciálních protinádorových derivátů kyseliny betulinové u nichž byly pozorovány zajímavé změny v buněčném cyklu. Kyselina betulinová se řadí mezi přírodní triterpenoidní sloučeniny a je prokázána její vysoká cytotoxická účinnost vůči nádorovým buňkám. Vzhledem k jejím farmakologickým vlastnostem, které nejsou příliš výhodné, se laboratoře snaží o syntézu lépe biologicky dostupných a účinějších derivátů. V naší práci jsme identifikovali geny, které byly ovlivněny účinkem derivátu kyseliny betulinové JS8. V předchozí práci byly identifikovány deriváty kyseliny betulinové, které indukovaly bloky v S a G2/M fázi buněčného cyklu, včetně korespondující fosforylace serinu 10 na histonu H3. Také byl pozorován vliv na syntézu DNA a RNA ve smyslu její aktivace/inhibice. V předkládané práci se zabýváme genovou expresí na základě působení derivátu JS8 na buněčnou nádorovou lini lymfoblastické leukemie CEM pomocí Affymetrix technologie a tím zjistit potenciální cíle účinku tohoto derivátu.
Klíčová slova Kyselina betulinová, rakovina, exprese genů
Počet stran 69
Počet příloh 0
Jazyk
Anglický
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Bibliographical identification:
Author’s first name and surname Alžběta Kameníčková
Title Target identification of antitumor drugs inducing changes in the cell cyle
Type of thesis
Master
Department Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacký University and Faculty Hospital in Olomouc
Supervisor MUDr. Petr Džubák, Ph.D.
The year of presentation 2008
Abstract The number of cancer disease is still increasing and that’s why new potentially anti-tumor derivates should be tested. The aim of this work is to identify the targets of potential antitumor derivatives of betulinic acid which were observed to induce interesting changes in the cell cycle. Betulinic acid is natural triterpenoid compound and exhibits high cytotoxic activity against several tumor cells. But it’s pharmacological properties aren’t very good and that’s why there’s an effort to synthesize new derivates, which could be more bioavailable and effective. In our study we have identified number of genes that were affected by the action of betulinic acid derivative JS8. In previous work were identified derivatives inducing block in the S and G2/M phase of cell cycle instead of corresponding phosphorylation of the serine 10 on the histone H3. Affected, activated/inhibited DNA and RNA synthesis was observed. In the present work we deal with gene expression by exposure the lymphoblastic leukemia cell line CEM to JS8 derivative using Affymetrix technology, and thereby identify potential targets of action of JS8.
Keywords Betulinic acid, cancer, gene expression
Number of pages 69
Number of appendices 0
Language English
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Table of content
1. THEORETICAL PART .............................................................................................. 5
1.1 Active triterpenoids as antitumor agents ........................................................... - 9 -
1.1.2 Betulinic acid ................................................................................................ - 9 -
1.1.4 Ursolic acid ................................................................................................. - 14 -
1.1.5 Oleanic acid ............................................................................................... - 16 -
1.1.5.1 CDDO, CDDO-Me, CDDO-Im .............................................................. - 16 -
1.1.6 Glycyrrhetinic acid ...................................................................................... - 17 -
1.1.7 Boswellic acid ............................................................................................. - 18 -
1.1.8 Ginsenosides ............................................................................................. - 19 -
1.1.9 Ganoderic acid ........................................................................................... - 19 -
1.1.10 Cucurbitacins ........................................................................................... - 20 -
1.1.11 Avicins ...................................................................................................... - 20 -
1.1.12 Beta-aescin .............................................................................................. - 21 -
1.1.13 Ardisiacrispin (A+B) .................................................................................. - 22 -
1.2 Methods .......................................................................................................... - 23 -
1.2.2 UV-VIS spectroscopic analysis ................................................................... - 23 -
1.2.3 DNA microarray .......................................................................................... - 23 -
1.2.3.1 Affymetrix technology .......................................................................... - 24 -
2. PRACTICAL PART ............................................................................................. - 26 -
2.1 Chemicals, reagents, instruments .................................................................. - 27 -
2.2 Methods ......................................................................................................... - 28 -
2.2.1 Passage of the suspension cells ................................................................ - 28 -
2.2.2 Isolation of RNA ......................................................................................... - 29 -
2.2.3 Measuring of RNA concentration ................................................................ - 30 -
2.2.4 Whole transcript sense target labeling assay .............................................. - 30 -
2.2.5 UV-VIS spectroscopic analysis .................................................................. - 40 -
2.3 Results ........................................................................................................... - 41 -
2.3.1 Determination of RNA, cRNA concentration ............................................... - 41 -
2.3.2 Determination of cDNA concentration ......................................................... - 42 -
2.3.3 cDNA electrophoresis ................................................................................. - 43 -
2.3.4 Statistical evaluation ................................................................................... - 45 -
2.3.5 Identification and annotation of expressed genes ....................................... - 48 -
2.3.6 UV-VIS spectroscopy analysis ................................................................... - 50 -
2.3 Discussion ...................................................................................................... - 54 -
3. Conclusion .......................................................................................................... - 57 -
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References ............................................................................................................. - 59 -
Abbreviations .......................................................................................................... - 67 -
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Aims of the work
1. Try to find target identification of derivates of betulinic acid inducing changes in
the cell cycle.
2. Reveal genes and their changes in expression involved in cancer cell line CEM
by treatment with derivatives of betulinic acid.
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1. THEORETICAL PART
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1.1 Active triterpenoids as antitumor agents
Natural products are great reservoir of biologically active compounds. Extracts
from natural products have been a main source of folk medicines for thousands of
years, and even today, many cultures still use them for medicinal purposes. Among the
classes of identified natural products, triterpenoids, one of the largest families, have
been studied intensively for their diverse structures and variety of biological activities.
This work is aimed on triterpenoids, which antitumor activity was observed in the last
two years (2008-2009). Practical part is focused mainly on the betulinic acid and its
derivative.
1.1.2 Betulinic acid
Betulinic acid is naturally occurring pentacyclic triterpene belonging to lupane
family. It is widely distributed in many terrestrial plant species. First, it was assumed,
that betulinic acid is a selective cytotoxic compound against melanoma cells (Pisha et
al., 1995) but the antitumor cytotoxicity of BetA has been extensively studied over the
last years in a large variety of cancer cell lines, primary tumor samples and xenograft
mouse models. Recent studies showed antiproliferative activity against breast
adenocarcinoma (MCF-7 cells), as well as neuroblastoma (SKNAS),
rhabdomyosarcoma-medulloblastoma (TE671), lung carcinoma (A549), colon
adenocarcinoma (HT-29), multiple myeloma (RPMI8226), cervical carcinoma (HPCC).
(Fulda S., 2009). BetA also showed broad-spectrum cytotoxicity towards lung,
colorectal, breast, prostate and cervical cancer cell lines as well as drug-resistant colon
adenocarcinoma cell lines (Jung et al., 2007). Recent data showed potential antitumor
effect of BetA against AGS cells (gastric adenocarcinoma), where was detected the cell
cycle arrest and induction of apoptosis (Yang et al., 2010). Derivates of betulinic acid
and betulin were also tested against HepG2 (human hepatocellular carcinoma), Jukart
(human leukemia) and HeLa (human cervical adenocarcinoma), where the derivates
markedly inhibited the proliferation of these cell lines (Santos at al., 2009). Betulin, the
reduced congener of BetA, showed apoptotic effect on human lung cancer cells related
characteristic proteins. Recent study revealed some proteins that were differentially
expressed by using nano-HPLC/MS method. There is a list of proteins identify to be
down or up regulated by treating with betulin. Downregualted proteins were heat shock
protein 90 – alpha 2, poly (rC) – binding protein 1, enoyl CoA hydratase, isoform 1 of 3
– hydroxyacyl – CoA dehydrogenate type 2. Proteins that were analyzed to be
upregulated were aconitase hydratase, malate dehydrogenase and splicing factor
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arginine/serine – rich 1 (Pyo 2009). New observation of betulin studies investigate that
this compound has potent anti-tumor activity especially in combination with cholesterol
that sensitized cells to betulin-induced apoptosis. Comparing to betulin acid, there was
no effect of cholesterol on it (Mullauer et al., 2009). There is the effort to synthesise
new derivates of betulinic acid because of their better pharmacological and physico-
chemical properties. The derivate NVX-207 was described to be well tolerated with
significant anti-cancer activity in vivo and in vitro (Willmann et al., 2009).
I II
Figure 1: Structures of betulinic acid (I) and betulin (II)
It was revealed that BetA induces apoptosis via direct mitochondrial
perturbations (Fulda et al., 1997) This pathway is normally regulated by carefully
balanced interplay between pro and anti-apoptotic members of Bcl-2 family. Over
expression of pro-survival molecules, such as Bcl-2, Bcl-xL or Mcl-1 or deletion of pro-
apoptotic members, such as Bak and Bax, or alternatively deregulation of BH-3 only
molecules like Puma or Bim, is often in tumors and causes resistance of these cells to
intrinsic death stimuli (Adams & Cori, 2007). A typical decrease in Bcl-2 and cyclin D1
gene expression and increase on bax gene expression was observed in several cancer
lines treated with BetA (Rzeski et al., 2006). Subsequent studies on anticancer
mechanism of BetA revealed that it is a potent activator of the chymotrypsin like activity
of the proteasome (Huang et al., 2007). In addition, BetA decreases expression or
vascular endothelial growth factor (VEGF) and the antiapoptotic protein survivin in
prostate cancer cells (LNCaP) by activating selective proteasome-dependent
degradation of the transcription factor´s specifity proteins (Chintharlapalli et al., 2007).
Other studies show the relationship between NF-ĸB expression and BetA (Kasperczyk
et al., 2005). The transcription factor, NF-kB is a key mediator of the cellular stress
response and in the cells exposed to an anticancer therapy NF-kB typically activates
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survival pathways (Aggraval et al., 2006).
It was revealed that BetA cooperated with TRAIL to induce apoptosis in tumor
cells (Fulda et al., 2004). Though functional complementation, simultaneous stimulation
of mitochondrial pathway by TRIAL resulted in complete activation of effectors’
caspases, apoptosis and inhibition of clonogenic survival (figure 1). BetA and TRAIL
cooperation leads loss of mitochondrial membrane potential and release cytochrome c
and Smac from mitochondria. These reports suggest that using BetA as sensitizer in
chemotherapy, radiotherapy or TRAIL-based combination regiments may be a novel
strategy to enhance the efficacy of anticancer therapy (Fulda S. 2009). Betulinic acid
also inhibits in vitro an enzyme aminopeptidase N that is involved in the regulation of
angiogenesis, overexpressed in several cancer (Fulda S. 2009).
Figure 2: Activation of effectors’ caspases by TRAIL (Fulda S., 2009).
Recent findings also suggest an inhibitory role of NF-kB in carcinogenesis and
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tumorigenesis (Chen et al., 2007). It has been demonstrated that inhibition of NF-kB
activity in cancer cell lines could reduce cell proliferation and metastatic capabilities in
vivo (Haung et al., 2001). Treatment of cells with BetA resulted in significant inhibition
of NF-kB activation and that this effect was mediated via the IkBa pathway. BetA shows
potential for use as an agent targeting NF-kB blockage in androgen-refractory human
prostate cancer cells (Rabi et al., 2008). BetA is also reported as an inhibitor of human
topoisomerases I and II, but it does not show synergistic effects with other topo-I
inhibitors. In contrast, BetA inhibited the formation of topo-I DNA cleavable complexes
induced by camptothecin, stautosporine and etoposide in prostate cancer cells. This
study also indicated that the tumor death mediated by BetA can be counteracted by the
mitogen-activated protein kinase 1 (MAPK1) inhibitor U0126 in melanoma cells
(Ganguly et al., 2007).
1.1.3 Lupeol
Lupeol is very common lupane-triterpene widely distributed in the plant
kingdom, occurs across a multitude of taxonomically diverse genera and geographical
areas. In earlier studies was investigated induction of apoptosis and cell arrest in
various cancer cells, further molecular-phenomena signaling pathway were also
revealed.
Figure 3: structure of lupeol
The following in vitro and in vivo studies show similar results that lupeol
inhibited the growth of CaP cells and significantly reduced testosterone-induced
prostate changes in mice (Prasad et al., 2008). Lupeol also significantly reduces the
proliferative and clonogenic potential of androgen-sensitive as well as androgen-
intensive CaP cells by modulating ß-catenin-signaling pathway in which lupeol
decreased the level of ß-catenin in nuclei of CaP cells thus inhibiting the transcription
of proliferation-associated genes in CaP cells (Saleem et al., 2009). One way of
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apoptosis induced by lupeol is characteristic by targeting Fas receptor and
consequently activation the extrinsic apoptotic pathway via caspase 8. Lupeol at 20 μM
concentration increased the expression of the FADD protein and Fas receptor on
androgen sensitive prostate cancer cells (Saleem et al., 2005). Apoptosis resistance is
reported to be overcome by activation of tumor suppressor genes and oncogene
inhibition when lupeol is added. When is lupeol at a concentration of 30 μM there is
also significant reduction of Ras protein that is commonly overexpressed and it leads to
subsequent inhibition of the PI3/Akt pathway that is known for promoting cell growth.
Coincidentally, observation of decreased levels of NF-κB and expression of phospho-
p38 MAPK, which triggers an antiapoptotic response, were observed (Saleem et al,
2005). Lupeol is also reported to inhibit the growth and proliferation of highly
aggressive pancreatic cancer and melanoma cells and it also inhibits the skin
tumorigenesis in mouse model through the modulation of signaling pathways such as
PI3K/AKt, nuclear factor kappa B1 and Ras/PKCα (Saleem et al., 2009). Another study
investigated that lupeol causes apoptosis in chemoresistant pancreatic cells by
recombinant TRAIL via suppression on cFLIP (Murtaza et al., 2009).
Figure 4: Multiple signaling pathways induced by lupeol (Chaturvedi et al., 2008).
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1.1.4 Ursolic acid
Ursolic acid is a prevalent pentacyclic triterpenoid carboxylic acid (figure 5).
Figure 5: Ursolic acid
Recent results of pro-apoptotic activity of ursane acid indicated a modulation of the Bcl-
2 protein family due to a suppression NF-kB, CREB, ATF-2, c-Fos and pro-
inflammatory cytokines by ursolic acid (50 μM) in B16 - F10 mouse melanoma cells.
Induction of apoptosis was accompanied by activation of p53 and caspase-3 gene
expression (figure 2) (Manu et al., 2008). Inhibitory effects were also observed on both
the PI3-Akt and MAPK P44/42 pathways, which are associated with cell apoptosis in
endometrial cancer cell lines (SNG-II and HEC108) (Achiwa et al., 2007). Ursolic acid
also inhibited endogenous reverse transcriptase (RT) in melanoma (A375) and
anaplastic carcinoma (ARO) cell lines, in which was shown down-regulation of c-myc
and cyclin D-1 by ursolic acid (Bonaccorsi et al., 2008). Another study investigated that
ursolic acid directly inhibits the interaction between ZIP/p62 and PKC-z and further
reduced matrix metalloproteinase-9 (MMP-9) activity and expression via blockade of
the NF-kB-dependent pathway induced by IL-1β or TNF-α in C6 glioma cells.
Therefore, it is suggested that inhibition of matrix metalloproteinase-9 expression by
ursolic acid is responsible for dysfunctions in tumor invasion. On the other hand, MMP-
9 does not only influence tumor invasion, but it also degrades the extracellular matrix,
including collagens, laminins, and the blood–brain barrier of angiogenesis in glial
tumors (Huang et al., 2009). Furthermore, it was demonstrated that ursolic acid
effectively induces apoptosis, probably via JNK-mediated Bcl-2 phosphorylation and
degradation in the androgen-independent human prostate cancer cell line DU145.
These findings suggest that UA-induced apoptosis could be a potential therapeutic
approach in advanced prostate cancer (Zhang et al, 2009).
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In addition, astilbortriterpenic acid, new triterpenoid compound structurally very
similar to ursolic acid was identified. This compound was tested against human breast
carcinoma Bcap37 cells, human cervical cancer HeLa cells, human hepatoma HepG2
cells, human ovarian carcinoma HO-8910 cells, human leukemia K562 cells, human
lung adenocarcinoma PAA cells, human gastric carcinoma SGC7901 cells, and murine
leukemic P388 cells where has been shown cytotoxic effect and induction of apoptosis
(Zhang et al., 2007). Subsequently, another study revealed mechanism of action of this
compound and investigate the involvement of mitochondria caspase activation and
reactive oxygen species, loss of mitochondrial transmembrane potential,
downregulation of Bcl-2 and up-regulation of Bax during induction of apoptosis by
astilbortriterpenic acid (Zhang et al., 2009).
Figure 6: Effect of ursolic acid of pro- and anti-apoptotic pathway (Manu et al., 2008).
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1.1.5 Oleanic acid
Oleanic acid (80 μM) showed apoptosis induction in leukemia cells (HL60) via
activation of caspase-9 and caspase-3 accompanied by the cleavage of poly (ADP-
ribose) polymerase (Zhang et al., 2007).
Results of another study revealed that ursolic acid, oleanic acid, and a mixture of
ursolic acid and oleanic acid (figure 7) significantly reduced 1,2 dimethylhydrazine-
induced aberrant crypt foci (ACF), which are considered to be an important early step
in colorectal carcinogenesis and tumors of the rat colon (Furtado et al., 2008).
Figure 7: oleanic acid
1.1.5.1 CDDO, CDDO-Me, CDDO-Im
Synthetic A-ring modified oleanic acid analogues with improved cytotoxicity were
pursued and 2-cyano-3, 12-dioxoolean-1,9-dien-28-oic acid (CDDO) as well as its C-28
methyl ester, CDDO-Me were designed and synthesized. Further derivatization of
CDDO was designed (CDDO-Im). CDDO and CDDO-Me are currently in phase I/II
clinical trials for cancer treatment. CDDO-Me and other derivatives with modifications
at the C-28 position induce apoptosis of human myeloid leukemia (Ito et al., 2000),
multiple myeloma (Ikeda et al., 2004) osteosarcoma (Ito et al., 2001), lung cancer (Kim
et al., 2002), breast cancer (Lapillonne et al., 2003) and pancreatic cancer (Samudio et
al., 2005).
CDDO-Me and related derivatives induce apoptosis in vitro by increasing reactive
oxygen species and decreasing intracellular glutathione. CDDO-Me depletes
intracellular GSH, resulting in ER stress. Subsequently, it activates JNK, leading to
CHOP-dependent DR5 upregulation and apoptosis (Zou et al., 2009). Recent results
also showed that CDDO-Me directly inhibits STAT3. These findings thus indicate that
CDDO-Me blocks the JAK1 STAT3 pathway by directly inhibiting both JAK1 and STAT3
(Rahmad et al., 2008). Furthermore, CDDO-Me at nanomolar or low micromolar
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concentrations can induce tumor cell death by a GSK3-mediated pathway indicates
that CDDO-Me could overcome cell death resistance in tumors unresponsive to
conventional chemotherapy (Vené et al., 2008). Another study showed that CDDO-Im
exerted a marked cytotoxic activity against primary ovarian cancer cells and a panel of
ovarian cancer cell lines, including chemoresistant A2780/ADR cells. It is proposed that
the inhibition of STAT3 activation induced by CDDO-Im determines a decrease of c-
FLIP levels, thus making the tumor cells sensitive to caspase-8 mediated cell death
(Petronelli et al., 2009).
1.1.6 Glycyrrhetinic acid
Glycyrrhetinic acid (GA) is a pentacyclic triterpenoid acid that is found as a conjugate
(glycyrrhizin) in licorice extracts. GA (figure 8) is one of the medicinally active
compounds of licorice and exhibits multiple activities which include the enhancement of
corticosterone levels which contributes to decreased body fat index in human studies
with GA (Armanini et al., 2003).
Figure 8: glycyrrhetinic acid
Although several studies have found that glycyrrhetinic acid give rise to cytotoxic or
apoptotic activities, most of the results have shown only moderate or low potency.
Thus, further investigation has focused more on the preparation of active derivatives
and chemosensitizing activity like 2-cyano-3, 11-dioxo-18b-olean-1, 12-dien-30-oic acid
(CDODA) and its methyl ester (CDODA-Me) from glycyrrhetinic acid and it was
demonstrated that these compounds are highly cytotoxic in colon, prostate (Papineni et
al., 2008) bladder, and pancreatic cancer cells (Chadalapaka et al., 2008). The most
active member of these glycyrrhetinic derivatives is CDODA-Me (18b isomer) which
activates peroxisome proliferator-activator receptor g (PPARg) and induces both
receptor dependent and independent responses in colon and prostate cancer cells
(Papineni et al., 2008). In subsequent study was shown that CDODA-Me inhibits
growth and induces apoptosis in pancreatic cancer cells. Although CDODA-Me
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activates PPARg in Panc28 and Panc1 cells where the induction of growth inhibitory
and proapoptotic proteins and activation of multiple kinase activities is receptor-
independent (Jutooru et al., 2009).
1.1.7 Boswellic acid
The most explored boswellic acid (figure 9) is compound commonly known as AKBA (3-
O-acetyl-11-keto-β-boswellic acid).
Figure 9: 3-O-acetyl-11-keto-β-boswellic acid
A recent discovery revealed that AKBA showed moderate to low toxicity against human
skin-derived normal cell lines (Burlado et al., 2008). However, AKBA induced apoptosis
also in PC-3 and LNCaP cells through a death receptor (DR-5)-mediated pathway,
which is a signal transduction, cascade involving the activation of caspase-8 and
caspase-3 in apoptosis (Lu et al., 2008). Another study investigated that AKBA inhibited
STAT 3 activation in human multiple myeloma cancer cells that in turn leads to the
suppression of gene products involved in proliferation (cyclin D1), survival (Bcl-xL, Bcl-
2 and Mcl-1) and angiogenesis (VEGF). According to these results, AKBA is suggested
to be a novel inhibitor of STAT 3 activation and has potential in cancer treatment
(Kunnumakkara et al., 2009). Finally, it was discovered that AKBA inhibits human
prostate tumor growth through inhibition of angiogenesis induced by VEGFR2 signaling
pathways (Pang et al., 2009).
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1.1.8 Ginsenosides
Ginsenosides are a series of compounds comprised of more than 60 triterpenes and
related glycosides isolated from the leaves, stems, berries and roots of different Panax
species. Recently ginsenosides from natural resources were evaluated for their
antitumor activity. But comparing to another active triterpenoids, most ginsenosides
showed very low cytotoxicity against various cancer cells. The anti-angiogenic
properties of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol were evaluated in vivo
angiogenesis assay using HUVECs, in which the compounds showed strong anti-
proliferative activity (Usami et al., 2008). Another type, ginsenosides R3 (GS-R3), were
also tested for their inhibitory effect on the process of tumor angiogenesis and it was
revealed that GS-R3 down-regulate the expression of VEGF and its kinase insert
domain receptor KDR in human lung cancer SK-MES-1 cell line (Wang et al., 2009).
Furthermore, 20(S)-25-methoxyprotopanaxadiol induces apoptosis and cell cycle arrest
in the G1 phase, inhibited proliferation of T98G, HPAC, A549 and PC-3 cell lines and
was 5- to 15-fold potent than 20(S)-protopanaxatriol (Wang et al., 2008). In addition,
three semi-synthetic derivates with a C-20 sugar moiety showed significant cytotoxicity
against MCF, SK-MEL-2 and B16 cancer cell lines (Lei et al., 2007).
1.1.9 Ganoderic acid
Ganoderic acid (figure 9) was assed for anti-proliferation activity and showed an IC50
value of 17,3 μM against human cervical carcinoma cells (HeLa), which were due to
treatment with this compound arrested at the G2/M phase of the cell cycle with
apoptosis.
Figure 9: ganoderic acid
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A simultaneous proteomic study led to the identification of 21 genes regulated by
ganoderic acid (Yue et al., 2008). Structurally related ganoderic acid A, F and H (GA-A,
GA-F, GA-H) were identified and tested on human breast cancer cells where results
suggested that GA-A and GA-H act through the inhibition of transcription factors AP-1
and nuclear factor kappa B, leading to down-regulation of Cdk4 expression and
suppression of secretion of urokinase-type plasminogen activator (Jiang et al., 2008).
The results of one study demonstrated that another ganoderic acid-Me effectively
inhibited tumor invasion, and it might act as a new matrix metalloproteinase 2/9
inhibitor for anti-metastasis treatment of carcinoma cells (Chen et al., 2008).
1.1.10 Cucurbitacins
Cucurbitane-type triterpenes, isomers of lanostanes, constitute a group of diverse
substances, are known for their cytotoxicity and anticancer activity. Cucurbitacins have
been reported to inhibit several types of cancers including those originating in the
prostate, lung and breast as well as choriocarcinoma, glioblastoma multiform, and
myeloid leukemia cells. Among the various cucurbitacins, the most abundant is
cucurbitacin B (CuB).
Recent studies showed, that the antiproliferative activity of CuB against breast cancer
cells in vitro and in vivo, irrespective of their ER, Her-2/neu or p53 status, is intriguing
and is worthy of further investigation (Wakimoto et al., 2008). It was also showed that
cucurbitacin B possessed inhibitory effects on laryngeal squamous cell carcinoma
(Hep-2), which can be due to the inhibition of STAT-3, a transcription activator in cell
growth (Liu et al., 2008). Another recent study investigated that cucurbitacin B has
profound antipancreatic cancer activity in vitro and in vivo. The drug induces cell cycle
arrest and apoptosis in pancreatic cancer cell lines by inhibiting the JAK/STAT pathway,
increasing the expression of p21/WAF1 independently of p53 activity, inducing the
caspase cascade and the drug also supports the proliferative activity of the nucleoside
analogue gemcitabine (Thoennissen et al., 2009).
1.1.11 Avicins
Avicins are a family of plant triterpene electrophiles with ability to trigger apoptosis-
associated tumor cell death, and suppress chemical induced carcinogenesis by its anti-
inflammatory, anti-mutagenic, and antioxidant properties (Xu et al., 2009). In vitro,
avicins inhibit cell growth and induce apoptosis in leukemia and epithelial cancer cell
lines. In a mouse skin carcinogenesis model, avicins have been shown to suppress
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both initiation and promotion phases of chemical carcinogenesis, as well as UV light B
damage with resultant suppression of oxidative DNA and lipid damage (Haridas et al.,
2004).
Avicins induce apoptosis by affecting mitochondrial function and activating the
intrinsic caspase pathway and close the voltage-dependent anion channel,
subsequently leading to lower cell energy metabolism and triggering cell apoptosis by
permeabilization of the outer mitochondrial membrane and release of cytochrome c
(Haridas et al., 2007). Avicins also suppress multiple pro-inflammatory components of
the innate immune system, including the transcriptional factor NF-kB (Haridas et al.,
2001), the phosphoinositide-3 kinase/AKT signaling pathway (Mujoo et al., 2001), as
well as heat shock proteins (Gaikwad et al., 2005). Recent study evaluated the anti-
tumor effects of avicin D on induction of apoptosis and modulation of signal transducer
and activator of transcription-3 (STAT-3) and apoptosis-related proteins in CTCL cell
lines and patients’ Sézary cells. These data suggest that selective induction of tumor T-
cell apoptosis, inhibition of STAT-3 activation and downregulation of bcl-2 and survivin
underline results demonstrate that beta-aescin is a potent natural inhibitor of cell
proliferation and inducer of apoptosis in HL-60 acute myeloid leukemia cells and the
therapeutic potential of avicin D in patients with Sézary syndrome (Zhang et al., 2008).
Another recent study investigated that avicin D might lead to use of raft-dependent and
intracellular activated Fas-mediated killing in cancer chemotherapy, representing a new
way to target tumor cells (Xu et al., 2009). It was also revealed that avicins can induce
not only apoptotic cell death, but also autophagic programmed cell death by depletion
of cell energy supply. These results showed that even when proapoptotic genes are
deleted or caspases are inhibited, avicins are able to continuously trigger cell death,
implicating the potentional therapeutic application of avicins in apoptosis – resistant
cancers (Xu et al., 2007).
1.1.12 Beta-aescin
Beta-aescin, a natural triterpenoid saponin isolated from the seed of Chinese horse
chestnut (Aesculus chinensis), is known to generate a wide variety of biochemical and
pharmacological effects. In recent study was investigating the antiproliferative and
apoptotic effects of beta-aescin in human chronic myeloid leukaemia K562 cell line in
vitro. The results showed that beta-aescin exhibited potent dose- and time-dependent
anti-proliferative effects in K562 cells. Morphological evidence of apoptosis, a
significant increase of annexin Vţ and PI7 cells (early apoptotic) and apoptotic DNA
fragmentation were observed in cells treated with beta-aescin. It was also observed,
- 22 -
that beta-aescin could lead to an accumulation of sub G1 population in K562 cells, and
suggesting a potential G1 phase accumulation in cell cycle profile of K562 cells. These
findings revealed that beta-aescin is a potent natural inhibitor of proliferation and
inducer of apoptosis in K562 cells, and beta-aescin may be a candidate lead
compound to explore potential antileukemia drugs (Niu et al., 2008). Another study
revealed the effect of mechanism of beta-aescin and 5-fluorouracil on human
hepatocellular carcinoma SMMC-7721 cells. This observation led to conclusion that the
mechanism of action could be through the synergistic arrest of the cell cycle, induction
of apoptosis, caspase 3,8 and 9 activation and down-regulation of Bcl-2 expression
(Ming et al., 2010).
1.1.13 Ardisiacrispin (A+B)
Ardisiacrispin (A + B) is a mixture of ardisiacrispins A and B, derived from Ardisia
crenata with a fixed proportion (2:1). The recent study showed that this mixture has
effect on proliferation of several human cancer cells. The (IC50) of ardisiacrispins (A +
B) were in the range of 0.9–6.5mg/ml by sulphorhodamine B-based colorimetric assay,
in which Bel-7402 was the most sensitive cell line. Ardisiacrispin (A + B) could inhibit
the proliferation of human hepatoma cells (Bel-7402) via inducing apoptosis and
disassembling microtubule. The finding that ardisiacrispins (A + B) has a remarked
anticancer activity on Bel-7402 cells also opens interesting perspectives for further
exploration of the triterpenoid saponins and compounds of as potential anticancer
agents (Li et al., 2008).
- 23 -
1.2 Methods
1.2.2 UV-VIS spectroscopic analysis of interactions between cytochrome c
and betulinic acid derivates
This method was used for monitoring of interactions between cytochrome c and
another derivates of betulinic acid. Triterpenoid compounds, especially betulin and
betulinic acid are well known for their property to decrease mitochondrial potential and
release cytochrome c from mitochondria. Previous study revealed that 3β,28-diacetoxy-
18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid (code JS8) compound form non-
covalent complex with cytochrome c (Dzubak et al., 2007) and other several derivates
were analysed. The changes in cytochrome c absorption spectra caused by the
respective interactions with derivates were visualized as the difference spectra of
betulinic acid derivatives (code JS3x) treated samples of cytochrome c. Differences in
the maximum absorption were used to quantify the effects on the formation/stability of
cytochrome c/JS3 complexes. The range of wave length where the maximal absorption
of cytochrome c occurred was from 350 to 575 nm. The figure 10 shows a typical
absorption spectrum of cytochrome c.
Figure 10: absorption spectrum of cytochrome c
1.2.3 DNA microarray
DNA microarrays is said to be modern platform enabling high sophisticated analysis of
the gene expression, expression of specific exons, microRNA, DNA methylation,
0
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0,15
0,2
0,25
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35
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37
0
38
0
39
0
40
0
41
0
42
0
43
0
44
0
45
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0
47
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- 24 -
analysis of nucleotide polymorphism and others in the range of the whole genome or
transcriptome. This method is very important for understanding in the complex access
the complicated mechanism and signal pathways that play role in biological processes.
A lot of data can be obtained during this analysis to provide overview on the
complicated processes such as cancerogenesis, metastasis or drug resistant of the
patient on the treatment. One of the most significant contributions of this method is fact
that there are new staging parameters that are able to identify the patients with high or
low risk on the basis of molecular description of the tumor. As for this work, DNA
microarray was used for the potential target identification and clarification the
mechanism of action of new potential antitumor drugs by comparing the gene
expression of tested tumor cell lines in the presence of the drug.
1.2.3.1 Affymetrix technology
Expression microarrays use probes targeting specific genes based on nucleotide
sequence complementarity to quantitatively measure mRNA levels for tens of
thousands of genes. Each set of the probe contains 22 oligonucleotides representing
one gene where 11 of them are perfect matched and precisely copy of gene sequence
is provided. On the other hand, next eleven oligonucleotides are mismatched and they
differ from each other by one nucleotide in central position (Li & Wong, 2001).
Genetic information from the human is transferred into the gene chip probe arrays by
using process called photolithography. This manufacturing process uses a light to
selectively activate the chip surface by the photochemical reaction with the DNA
building blocks. Repeated cycles of selectively activation of the surface in three
determined patterns allow many different DNA probes to be built up in a small number
of steps. As the size of the feature (the square locations on the array) is reduced, the
amount of information contained on the single gene chip array increases exponentially.
As first, high quality of RNA from sample is extracted and then is reverse transcribe
into more stable complementary DNA (cDNA) with subsequent labeling and
fragmentation. Using the gene chip fluidic station, a test sample is introduced into the
DNA probe array where hybridization occurs. Each probe on the chip is a single
stranded DNA. Hybridization occurs when two complementary strands of DNA come
together to form a complex. When a single stranded sample is washed over the
surface, the sample will bind to its complementary strand. After this hybridization step,
the chips are put into the laser scanner to electronically capture the data. The chip is
scanned with the laser activating fluorescence dyes on the sample´s complementary
DNA. The computer captures this information and calculates the ratio of each spot.
- 25 -
Since the sequence of each location on the chip is known, it is possible to determine
the sample DNA sequence and to see if a target gene is present and whether it
contains mutation (http//media.affymetrix.com).
Figure 10: Steps involved in the Affymetrix technology
(www.umassmed.edu/gcf/indes.aspx)
- 26 -
2. PRACTICAL PART
- 27 -
2.1 Chemicals, reagents, instruments
Reagents:
GeneChip® WT sense target labeling and control reagents contains one of each of the
following kits - GeneChip® eukaryotic Poly-A RNA control kit , GeneChip® WT cDNA
synthesis and amplification kit, GeneChip® WT terminal labeling kit, GeneChip®
sample cleanup module, GeneChip® IVT cRNA Cleanup Kit, GeneChip® hybridization
Control Kit (Affymetrix 900652), GeneChip® eukaryotic Poly-A RNA control kit contains
Poly-A control stock and Poly-A control dilution buffer (Affymetrix 900433), GeneChip®
WT cDNA synthesis and amplification kit Sub-kit 1: GeneChip® WT cDNA synthesis
kit contains T7-(N)6 primers, 2.5 μg/μL, 5X 1st strand buffer, DTT 0.1M, dNTP, 10 mM,
RNase Inhibitor, SuperScript™ II, MgCl2 1M, DNA polymerase I, RNase H, random
primers, 3 μg/μL, dNTP+dUTP, 10 mM, RNase-free water, Sub-kit 2: GeneChip® WT
cDNA amplification kit contains 10X IVT Buffer, IVT NTP Mix, IVT enzyme mix, IVT
control (Affymetrix 900673), GeneChip® WT terminal labeling kit contains 10X cDNA
fragmentation buffer, UDG 10 U/μL, APE1 1,000 U/μL, 5X TdT buffer, TdT, 30 U/μL,
DNA labeling reagent 5 mM, RNase-free water (Affymetrix 900671), GeneChip® IVT
cRNA cleanup kit contains IVT cRNA cleanup spin columns, IVT cRNA binding buffer,
IVT cRNA wash buffer 5 mL concentrate, RNase-free water, 1,5 ml collection tubes (for
elution), 2 ml collection tubes (Affymetrix 900547), GeneChip® sample cleanup module
contains cDNA Cleanup Spin Columns, cDNA Binding Buffer, cDNA Wash Buffer 6ml
concentrate, cDNA elution buffer, IVT cRNA cleanup spin columns, IVT cRNA binding
buffer, IVT cRNA wash buffer 5 ml concentrate, RNase-free water, 1,5 ml collection
tubes (for elution), 2 ml collection tubes, 5X fragmentation buffer (Affymetrix 900371),
GeneChip® hybridization control kit contains 20X hybridization controls 3 nM control
oligo B2 (Affymetrix 900454), GeneChip® hybridization, wash and stain kit containing
pre-hybridization mix, 2x hybridization mix, DMSO, nuclease-free water, stain cocktail
1, stain cocktail 2, array holding buffer (Affymetrix 900720), wash buffer A (Affymetrix
900721), wash buffer B (Affymetrix 900722), absolute ethanol (Serva), RNA 6000 nano
kit (Agilent), TRI reagent (Sigma T9242), pyridine (Sigma P3776), cytochrome c from
horse heart (Sigma 105201), Medium RPMI-1640 (Sigma), fetal bovine serum (Biocom,
CZ), streptomycine 100 µg/ml (Sigma S 9137), peniciline 100 µg/ml (Biotika, SK),
propidium iodide (Sigma), ribonuclease A (Sigma).
Instruments:
NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), GeneChip®
Hybridization Oven 645 (Affymetrix), Centrifuge (Eppendorf 5810R), Picofuge
- 28 -
(Eppendorf), GeneChip® Fluidics Station 450 (Affymetrix), GeneChip® Scanner 3000
(Affymetrix), GeneChip® AutoLoader with external barcode reader (Affymetrix),
thermocycler (Biotech), Bioanalyzer 2100 (Agilent), heating blocks (Eppendorf), vortex
(Genie), biohazard box (Forma Scientific), PowerWave spectrophotometer (BIO-TEK),
pH meter (Metler Toledo), light microscope, CO2 incubator (Jouan)
Solutions:
10 x concentrated PBS
80 g NaCl, 32,1 g Na2HPO4 . 12 H2O, 2 g KH2PO4 dissolved in 700 ml of distilled water,
pH adjusted at 7,4 by HCl, fill with distilled water to reach 1 litre.
1 x PBS
Prepare by dilution of 10 x concentrated PBS with distilled water.
5 mM Pyridine/HCl, pH 6,0 buffer
Concentration of the stock solution is 0,01M. For preparation of 5 mM buffer take 50 ml
of the stock solution and fill with distilled water into the final volume 100 ml. Adjust pH
on 6,0 with HCl.
4 μM cytochrome c
For 50 ml of buffer 2,46 mg of cytochrome c was needed
2.2 Methods
2.2.1 Passage of the suspension cells
Standardise cell line CEM was used for flow cytometry analysis and for DNA
microarray. This line is cancer line of human lymphoblastic leukemia. The cells were
regularly passaged for their later using in experiments.
Protocol:
1. The cells were observed under microscope before passaging to see if the
contamination occurred.
2. The cultivation flask was shaked.
3. The cover of cultivation flask was disinfected.
4. The cells were put into 50 ml tube.
5. 2 ml of the cell suspension was inoculated on another 175 ml flask and 30 ml of
the culturing medium were added.
6. The flask was disinfected and put back into the incubator for other cultivation.
7. The removed cells were counted and used for experiments.
- 29 -
2.2.2 Isolation of RNA
Protocol:
1) Cells were counted and to reach the final concentration of 1 x 106/1 ml,
according amount of cultural medium RPMI was added.
2) 5,6 ml of the cell suspension was pipette into 25 cm2 culture flask.
3) Cells were incubated for 24 hours.
4) After cultivation time, the derivate of betulinic acid JS8 was added.
5) The incubation with derivate last 90 minutes.
6) After incubation, cells were centrifugated at 4oC for 10 minutes at 2000 rpm,
supernatant was aspirated.
7) Cells were washed with 20 ml of 1x PBS at the same speed, time and
temperature, supernatant was aspirated.
8) Cells were suspended in 1 ml of 1x PBS and transferred into 1,5 ml tube.
9) Samples were centrifugated at 2000 rpm for 4 minutes.
10) 1 ml of TRI reagent was added to each sample, mixed carefully and the mixture
was stored for 5 minutes at room temperature.
11) 200 µl of chloroform were added and then vortex was used until homogenous
mixture results.
12) Samples were incubated for 10 minutes and then centrifugated at 12000 rpm
for 15 minutes at 4oC.
13) The mixture was separated into three phases: 1. lower red (containing
proteins) 2. interphase (containing DNA) 3. upper aqueous phase (containing
RNA).
14) The upper phase was transferred into fresh tubes and 500 µl of isopropanol
was added to 500 µl of aqueous phase. Tubes were mixed by turning up and
down.
15) Samples were incubated at room temperature for 5 minutes and centrifugated
at 12000 rpm for 10 minutes at 4oC.
16) The supernatant was removed and 1,5 ml of 75% ethanol was added and the
pellet was washed by gentle shaking without degradation.
17) Samples were centrifugated at 12000 rpm for 5 minutes at 4oC.
18) The ethanol was removed and the pellet was left to dry.
19) Next step involved dissolving the pellet in water. Water addition was according
to the size of the pellet.
20) Samples were incubated at 60oC in dry bath incubator for 5 minutes.
- 30 -
2.2.3 Measuring of RNA concentration
Concentration of RNA was measured by nanodrop method. 1,5 µl of RNA solution was
used for measuring and water was used as a blank. Final concentration of RNA was
given in ng/µl.
Nanodrop spectrometer operation
1) The adapter power was switch on and the programme was opened by clicking
ND on the desktop.
2) The programme RNA-40 was selected.
3) 1,5 µl of DEPC water was loaded on nanodrop.
4) The blank was measured.
5) Water was removed by pipette and the surface of loading area was cleaned
with tissue paper.
6) 1,5 µl of sample was loaded, the name was given and clicked measure.
7) The value of RNA concentration was counted.
8) The sample of RNA was removed with pipette and cleaned with tissue paper.
9) Water was loaded and measured again.
10) The last step involved pressing exit, escape and save.
2.2.4 Whole transcript sense target labeling assay
This protocol was provided by Affymetrix.
1st day
Preparation of dilutions of poly-A RNA controls
Total RNA labeling protocol
First cycle, first-strand cDNA synthesis
First cycle, second-strand cDNA synthesis
First cycle, cRNA synthesis and cleanup
2nd day
Second cycle, first-strand cDNA synthesis
Hydrolysis of cRNA and cleanup of single-stranded DNA
Fragmentation of single-stranded DNA
Labeling of fragmented single-stranded DNA
Hybridization
3rd day
Washing, staining and scanning
1st day
- 31 -
A) Preparation of dilution of poly-A RNA controls
For this procedure, GeneChip® Eukaryotic Poly-A RNA Control Kit
1) 2 µl of poly-A RNA control stock were added to 38 µl of poly-A control dilution
buffer to make the first dilution (1:20)
2) The solution was mixed and spined to collect it at the bottom of the tube.
3) 2 µl of the first dilution were added to 98 µl of poly-A dilution buffer to make the
second dilution (1:50)
4) The solution was mixed and spined to collect it at the bottom of the tube.
5) 1 µg of total RNA was used as a starting material. 2 µl of the second dilution
were added to 98 µl of poly-A dilution buffer to make third dilution (1:50).
B) Preparation of T7-(N)6 primers/poly-A RNA controls
1) A fresh 250 ng/µl T7-(N)6 primers dilution was prepared (from a 2,5 µg/µl stock)
by adding the concentrated T7-(N)6 primers to the diluted poly-A RNA controls
using non-stick RNase-free microfuge tube as follows:
Table 1: first-cycle, primer/poly-A RNA controls
component
T7-(N)6 primers, 2,5 µg/µl 2 µl
diluted poly-A RNA controls 6 µl
RNase free water 12 µl
total volume 20 µl
2) The solution was flick-mixed, spined down and placed on ice.
C) Preparation of total RNA/T7-(N)6 primers/poly-A RNA controls
1) Total RNA and T7-(N)6 primers/poly-A RNA controls solution was mixed as listed
in table 2.
Table 2: first-cycle, total RNA/primer/poly-A RNA controls
component volume in 1 Rxn
Total RNA, 300 ng 3 µl
T7-(N) primers/poly-A RNA controls
solution 2 µl
Total volume 5 µl
2) The solution was flick-mixed, spined down the tube and incubated for 5 minutes
at 70oC. Then, the sample was cooled for 2 minutes at 4oC and spined down
and placed on ice.
D) First cycle, first strand cDNA synthesis
- 32 -
This procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.
1) First-cycle, first strand master mix was prepared as shown in table3. The
enzyme SuperScript II was added last to the master mix and proceeded
immediately to aliquot into the tubes from procedure C.
Table 3: first-cycle, first-strand master mix
component volume in 6 Rxn
5x 1st strand buffer 12 µl
0,1 M DTT 6 µl
10 mM dNTP mix 3 µl
Rnase inhibitor 3 µl
SuperScript II 6 µl
Total volume 30 µl
2) 5 µl of the first-cycle, first-strand master mix were added to the tube containing
the concentrated rRNA reduced total RNA/poly-A controls/T7-(N)6 primers mix
from procedure C, flick mixed and spined down.
3) The reaction was incubated at:
25 oC for 10 minutes
42 oC for 60 minutes
70 oC for 10 minutes
4) The reaction was cooled to 4 oC at least 2 minutes before immediately
continuing to the first-cycle, second-strand cDNA synthesis.
E) First cycle, second strand cDNA synthesis
This procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.
1) Fresh dilution of 17,5 mM MgCl2 was made by mixing 2 µl of 1 M MgCl2 with 112
µl of RNase-free water.
2) The first-cycle, second-strand master mix was prepared as described in table 4.
The RNase H and DNA polymerase I enzymes were added to the master mix
last and proceeded immediately to aliquot into the tubes from procedure D.
Table 4: first-cycle, second-strand master mix
component volume in 6 Rxn
Rnase-free water 28,8 µl
17,5 mM MgCl2 24 µl
10 mM dNTP Mix 2,4 µl
DNA polymerase I 3,6 µl
Rnase H 1,2 µl
Total volume 10 µl
- 33 -
3) 10 µl of the first-cycle, second-strand master mix were added to the reaction
tube from the first-strand cDNA synthesis reaction in procedure B for a total
reaction volume of 20 µl. The tubes were gently vortexed and spined down.
4) The reaction was incubated in a thermal cycler at:
16 oC for 120 minutes without heated lid
75 oC for 10 minutes with heated lid
5) The sample was cooled at least 2 minutes at 4oC before immediately proceeding
to the next procedure.
F) First-cycle, cRNA synthesis and cleanup
This procedure requires the use of the GeneChip® WT cDNA Amplification Kit and
the GeneChip® Sample Cleanup Module.
1) The IVT master mix was assembled in a separate tube at room temperature as
listed in table 5. The IVT enzyme mix was added to the master mix last and
proceeds immediately to aliquot into the tubes from procedure E.
There can be problem if a white precipitate is still present in the 10x IVT buffer
after thawing. It is recommended to incubate the tube at 37 oC until the
precipitate gets dissolved.
Table 5: First-cycle, IVT master mix
component volume in 6 Rxn
10x IVT Buffer 30 µl
IVT NTP Mix 120 µl
IVT enzyme mix 30 µl
Total volume 180 µl
2) 30 µl of the IVT master mix were added to each first-cycle cDNA synthesis
reaction sample from procedure E to final volume of 50 µl. The solution was
flick-mixed and briefly spined in a microfuge.
3) The reaction was incubated for 16 hours at 37 oC in a thermal cycler.
2nd day
1) The cleanup procedure for cRNA was proceeded using the cRNA cleanup spin
columns from the GeneChio Sample Cleanup Module.
2) 20 ml of ethanol (100%) were added to the cRNA wash buffer supplied in the
GeneChip Sample Cleanup Module.
3) 50 µl of RNase-free water were added to each IVT reaction to a final volume of
100 µl.
4) 350 µl of cRNA binding buffer were added to each sample and vortexed for 3
seconds.
5) 250 µl of 100% ethanol were added to each reaction and flick-mixed.
- 34 -
6) The sample was applied to the IVT cRNA cleanup spin column sitting in a 2 ml
collection tube.
7) The sample was centrifugated for 15 seconds at more than 8000 x g. The flow-
through was discarded.
8) The IVT cRNA cleanup spin column was transferred to a new 2 ml collection
tube. 500 µl of cRNA wash buffer were added to celumn and centrifugated for
15 seconds at more than 8000 x g. The flow-through was discarded.
9) The next washing was performed with 500 µl of 80% (v/v) ethanol. Samples
were centrifugated for 15 seconds at more than 8000 x g and the flow-through
was discarded.
10) The column cap was opened and spined at maximum speed for 5 minutes with
the caps open.
11) The IVT cRNA cleanup spin column was transferred to a new 1,5 ml collection
tube and 15 µl of RNase-free water were added directly to the membrane.
Incubation proceeded at room temperature for 5 minutes and then spined at
maximum speed for 1 minute.
12) The flow-through in the collection tube (something about 13,5 µl) was eluted a
second time by pipetting back onto the spin column membrane. The spin
column was placed back into the collection tube and incubated at room
temperature for 5 minutes and then spined at maximum speed for 1 minute.
13) The volume of eluted cRNA was approximately 13,5 µl and the concentration
was determined by the NanoDrop.
G) Second-cycle, first-strand cDNA synthesis
1) The volume of cRNA to 10 µg was determined.
2) cRNA samples were mixed with the random primers in a strip tubes as listed in
table below.
Table 6: Second-cycle, cRNA/random primers mix
component volume in 1 Rxn
10 µg of cRNA variable
3 µg/µl Random pirmers 1,5 µl
Rnase free water up to 8 µl
Total volume 8 µl
3) The mixture was flick-mixed and spined down the tubes.
4) The second-cycle, cRNA/random primers mix was incubated at:
70 oC for 5 minutes
25 oC for 5 minutes
5) The samples were cooled at 4 oC at least 2 minutes.
- 35 -
6) The second-cycle, reverse transcription master mix was prepared in a separate
tube and the components are described in the table 7.
Table 7: second-cycle, first-strand cDNA synthesis master mix
component volume in 6 Rxn
5x 1st strand buffer 24 µl
0,1 M DTT 12 µl
10 mM dNTP+dUTP 7,5 µl
SuperScript II 28,5 µl
Total volume 72 µl
7) 12 µl of the second-cycle, first-strand cDNA synthesis master mix were
transferred to the second.cycle, cRNA/random primers mix from previous
procedure for a total reaction volume of 20 µl and briefly centrifugated.
8) The reaction was incubated at:
25 oC for 10 minutes
42 oC for 90 minutes
70 oC for 10 minutes
4 oC for at least 2 minutes
H) Hydrolysis of cRNA and cleanup of single.stranded DNA
This procedure requires the use of GeneChip® WT cDNA Synthesis Kit and the
GeneChip® Sample Cleanup Module
1) 1 µl of RNase H was added to each of samples and incubated at:
37 oC for 45 minutes
95 oC for 5 minutes
4 oC for 2 minutes
2) The cleanup step was proceeding by using the cDNA cleanup spin columns from
the GeneChip Sample cleanup module.
3) 80 µl of RNase-free water were added to each sample, followed by 370 µl of
cDNA binding buffer and vortexed for 3 seconds.
4) The entire sample was applied to a cDNA spon column sitting in a 2 ml
collection tube.
5) The samples were spined at more than 8000 x g for 1 minute and the through
flow was discarded.
6) The cap of the cDNA clean up spin column was left to be opened and then the
samples were spined at the maximal speed for 5 minutes. The flow through was
discarded and the column was placed in a 1,5 ml collection tube.
7) 15 µl of the cDNA elution buffer were pipetted directly to the column membrane
and incubated at room temperature for 1 minute and subsequently spined at
maximum speed for 1 minute.
- 36 -
8) The elution step was repeated by pipetting another 15 µl of the cDNA elution
buffer directly to the column membrane and incubated at room temperature for 1
minute, then spined at maximum speed for 1 minute.
9) The total volume of the eluted single stranded DNA was approximately 28 µl. 2 µ
l were taken from each sample to determine the yield by using the NanoDrop to
measure the concentration.
I) Fragmentation of single-stranded DNA
This procedure requires the use of the GeneChip® WT Terminal Labeling Kit.
1) The fragmentation reaction was set up in 0,2 ml tube.
2) The fragmentation master mix was prepared according to table 8.
Table 8: Fragmentation master mix
component volume in 6 Rxn
RNase-free water 60 µl
10x cDNA fragmentation buffer 28,8 µl
10 U/µl UDG 6 µl
1000 U/µl APE 6 µl
Total volume 100,8 µl
3) 16,8 µl of the above fragmentation master mix were added to the samples
prepared on step 1. The tubes were gently vortexed and spined down.
4) The reaction was incubated at:
37 oC for 60 minutes
93 oC for 2 minutes
4 oC for at least 2 minutes
5) The samples were flick-mixed, spined down the tubes and 45 µl of the sample
were transferred to a new 0,2 ml strip tube. The remainder of the sample was
used for size analysis using a RNA 6000 Nano assay kit supplied with Agilent
2100 bioanalyzer. The range of peak size of fragmented samples should be
approximately 50 to 100 nt.
Procedure:
One of the wells of electrode was fill with 350 µl of RNAseZAP and put in
the Agilent 2100 bioanalyzer, the lid was left to be closed for 1 minute.
Another well of electrode cleaner was filled with 350 µl of RNase-free
water, placed in the bioanalyzer, the lid was left to be closed for 10
- 37 -
seconds, then opened, the electrode cleaner was removed and then left
for 10 second to evaporated.
For the gel preparation, 550 µl of RNA Nano gel matrix were pipetted
directly on the filtr and spined at 1500 g for 10 minutes at room
temperature.
65 µl of the gel were transferred into an RNase-free 1,5 microfuge tubes
and 1 µl of RNA dye concentrate was added. To reach the mixture to be
homogenous, it was centrifugated at 13 000 g for 10 minutes at room
temperature.
The samples for denaturation were prepared, 1,2 µl of the sample and
ladder was pipetted into 0,2 ml tubes, then denaturated in cycler for 2
minutes at 70 oC and subsequently cooled on the ice.
A new RNA chip was taken and placed in the Chip Priming Station.
9 µl of the gel-dye mix were drawn up with a pipette and placed at the
bottom of the marked well. The gel-dye mix was dispensed. After
ensurance the plunger is at 1 ml, the priming station was closed.
After 30 seconds, the plunger was released with the clip release
mechanism.
The plunger was pulled back to the 1 ml position, the priming station was
opened and 9 µl of the gel-dye mix were pipetted in each of the wells
marked.
5 µl of marker were dispensed into each of the 12 sample wells.
1 µl of denaturated samples was pipetted into the wells 1-12 and 1 µl of
denaturated ladder was pipetted into the ladder-marked well.
The chip was vortexed for 1 minute at 2000 rpm and then placed in the
analyzer where the analysis was immediately started.
J) Labeling of single-stranded DNA
This procedure requires the use of the GeneChip® WT Terminal Labeling Kit.
1) The labeling reactions were prepared as listed in table 9. A master mix using 5x
TdT buffer, TdT and DNA labeling reagent were prepared just before aliquoting
15 µl into the 0,2 ml strip tubes containing the 45 µl of fragmented single-
stranded DNA.
- 38 -
Table 9: Labeling reaction
component volume in 1 Rxn
Fragmented single-stranded DNA 45 µl
5x TdT Buffer 12 µl
TdT 2 µl
5 mM DNA labeling reagent 1 µl
Total volume 60 µl
2) After the labeling reagents were adding to the fragmented DNA, the samples
flick-mixed and spined down.
3) The reaction was incubated at:
37 oC for 60 minutes
70 oC for 10 minutes
4 oC for at least 2 minutes
K) Hybridization
This procedure requires the GeneChip® Hybridization, Wash and Stain Kit.
1) The hybridization coctail was prepared in a 1,5 ml RNase-free microtube as
shown in table 10.
Table 10: Hybridization coctail
component
volume for one 169 format
array
fragmented and labeled DNA target 27 µl
3 nM control oligonucleotide B2 1,7 µl
20x eucaryotic hybridization
controls 5 µl
2x hybridization mix 50 µl
DMSO 7 µl
Nuclease-free water 9,3 µl
Total volume 100 µl
It is necessary to heat the frozen stock of 20x eukaryotic hybridization control to
65 oC for 5 minutes to completely resuspend the cRNA before aliquoting.
2) The tubes were gently vortexed and spined down.
3) The hybridization coctail was heated at 99 oC for 5 minutes and subsequently
cooled to 45 oC for 5 minutes and centrifugated at maximum speed for 1 minute.
4) The GeneChip ST array was equilibrated at room temperature immediately
before use. The array was labeled with the name of the sample.
5) 80 µl of the sample were injected into the array through one of the septa.
- 39 -
Two pipette tips are necessary to use when filling the probe array cartridge. The
bubble is important to get contact with hybridization coctail and all portions of the
array.
6) The array was placed in 45 oC hybridization oven at 60 rpm and incubated for 17
hours.
3rd day
Before following procedures washing, staining and scanning, the samples must be
registered in Affymetrix GeneChip Command Console (AGCC).
A) Priming the fluidics station
1) The intake buffer reservoirs were filled with the appropriate Wash A and Wash
B solutions.
2) The water reservoir was filled with deionized water.
3) 3 empty 1.5 ml microfuge tubes were placed into the stain holder positions 1, 2
and 3.
4) The wash block lever was placed into the engaged/closed position and the
needle lever was pushed into the down position.
5) The Prime_450 maintenance protocol was set and run.
B) Wash and stain
1) The array was removed after 16 hours of hybridization from the hybridization
oven.
2) The hybridization coctail was extracted and the probe array was completely
refilled with 100 µl of Wash Buffer A.
C) The stain reagents preparation
1) The reagents were aliquoted as described in table 11.
Table 11: volume of stain reagents
compound volume ( µl)
Stain coctail 1 600
Stain coctail 2 600
Array holding buffer 800
2) All the vials were spined down to remove the presence of any air bubble.
D) Using the fluidics station
1) The barcode of the chip was scanned with an external barcode reader.
2) The fluidics protocol was selected.
3) The appropriate probe array was inserted into the designated module of the
fluidics station.
- 40 -
4) The vial containing 600 µl of stain coctail 1 was placed in sample holder 1, the
vial containing 600 µl of stain coctail 2 was placed in sample holder 2 and the
last vial containing 800 µl of array holding buffer was placed in sample holder 3.
5) The needle was pressed down and the run was begun.
6) After complete protocol, the probe arrays were removed from the fluidics
station.
E) Scanning
1) One tough-spots was applied to each of two septa on the back of the probe
array cartridge.
2) The cartridge was inserted into the scanner.
3) The scanning protocol was set and start of analyse begun.
2.2.5 UV-VIS spectroscopic analysis of interactions between cytochrome c
and JS3 triterpenoid derivates
Protocol:
1) 6 µl of certain derivates were taken.
2) 2,46 g of cytochrome c were dissolved in the 5mM pyridine/HCl buffer.
3) Derivatives were mixed with cytochrome c in the buffer, 200 µl were prepared
for each derivative.
4) The mixture was pipetted in the final volume of 50 μl into 384 well plate.
5) Samples were measured by using the spectrophotometer, where the wave
length was set in the range from 200 to 750 nm.
6) The data were analyzed into charts.
- 41 -
2.3 Results
2.3.1 Determination of RNA, cRNA concentration
For further whole transcript sense target labeling assay, 300 ng of RNA were required.
The final concentration of RNA is listed in the table 12. The volume of RNA and H2O to
reach 300 ng is listed in table 13. As the concentration of control 3 was under limit, the
sample was not analyzed. Another measurement of cRNA was done to ensure if
appropriate amount of cRNA was in each sample. Subsequently the volume of cRNA
was determined to reach 10 µg per sample (table 14). cRNA was diluted with water to
the final volume of 6,5 µl (table 15).
Table 12: The final concentration of RNA
the sample RNA concentration (ng/ µl)
control 1 666,7
control 2 588,23
control 3 12,6 (under limit)
JS8 a 1200
JS8 b 652,17
JS8 c 461,54
Table 13: The volume of RNA and H2O to reach 300 ng of RNA
the sample volume of RNA (µl) Volume of H2O (µl)
control 1 0,45 2,55
control 2 0,51 2,5
JS8 a 0,25 2,75
JS8 b 0,46 2,54
JS8 c 0,65 2,35
Table 14: The final concentration of cRNA
the sample concentration of cRNA µg/µl
control 1 2111,57
control 2 4162,92
JS8 a 4350,15
JS8 b 4358,98
JS8 c 4294,7
- 42 -
Table 15: The volume of cRNA and H2O
2.3.2 Determination of cDNA concentration
cDNA concentration had to be measured to see if there was enough cDNA in the
sample and if was able to continue with experiment. The right concentration of cDNA
was needed for the fragmentation procedure (tab). The table shows volumes of cDNA
of each sample and RNase free water addition for the final volume of 30 µl needed for
fragmentation reaction.
Table 16: cDNA concentration
the sample cDNA concentration (ng/µl)
control 1 460,29
control 3 282,54
JS8 a 232,24
JS8 b 419,44
JS8 c 316,43
Table 17: Fragmentation reaction
the sample
volume of c DNA
(µl)
volume of Rnase free
water(µl)
control 1 23,68 7,52
control 3 19,43 11,73
JS8 a 11,95 19,25
JS8 b 13,11 18,09
JS8 c 17,3 13,82
the sample
µl of cRNA needed for 10
µg
µl of H2O needed up to 6,5
µl
control 1 4,74 1,76
control 2 2,4 4,1
JS8 a 2,3 4,2
JS8 b 2,3 4,2
JS8 c 2,33 4,17
- 43 -
2.3.3 cDNA electrophoresis
Fragmented single-stranded DNA samples were used for size analysis using a
Bioanalyzer to see the peak size of fragmented samples. The Agilent Rna chip was
used for successfully control of the fragmentation of single stranded DNA for Affymetrix
GeneChip which is very important for further analysis. After fragmentation of DNA, then
labeling is performed by deoxynucleotidyl transferase that is covalently linked to biotin
anticipating the sufficient target to be generated for hybridization to a single array.
Agilent RNA chip involves appropriate dye and the setting of electrical parameters of
the electrophoresis that is convenient for a single stranded DNA. The area of the peaks
should be in the range of 50-100 nucleotides (nt) thus ensuring the fact of the
successful fragmentation. The figures (11-16) show the right peak size, although there
is a sample JS8a showing another strange peak, but we decided to use all samples
for loading them on the Affymetrix GeneChip.
Figure 11: Electrophoresis run summary
- 44 -
Figure12: Bioanalyzer profile of single-stranded DNA control 1.
Figure 13: Bioanalyzer profile of single-stranded DNA of control 3.
Figure 14: Bioanalyzer profile of fragmented single-stranded DNA of JS8 a.
- 45 -
Figure 15: Bioanalyzer profile of fragmented single-stranded DNA of sJS8 b.
Figure 16: Bioanalyzer profile of fragmented single-stranded DNA of JS8 c.
2.3.4 Statistical evaluation
Array-array correlation method was performed to see the correlation of all arrays from
the experiment with each other. As was predicted, our results show high correlation
among the samples, because all the samples were from the same source, cancer cell
line CEM and no significant changes occurred. Another statistical evaluation, RLE
(relatively long expression) and NUSE (normalized unscaled standard error) was
accomplished. RLE confirmed the fact that only relatively few genes were differentially
expressed that we can see in boxes which are similar in range and are centred close to
value 0. The graphical representation NUSE represents normalized standard error
which estimates are normalized for each probe set and the median standard error
across all arrays is equal to 1. The cluster dendogram provides the information
concerning which observations are grouped together at various level of similarity.
- 46 -
Histogram of p-value was also established. As for p-values characteristic, minimal
value was 2,099 x 10-6, maximum was equal to 1, median reached 0,453 and mean
0,743. According to p-values, 314 genes were detected with p < 0,01 and 38 genes
with p < 0,001.
Figure 17: Array – array intensity correlation
- 47 -
Figure 18: RLE and NUSE statistical evaluation
Figure 19: Cluster dendogram
- 48 -
Figure20: Histogram of p-values
2.3.5 Identification and annotation of expressed genes
The genes of interest were chosen according to the treated/control samples ratio to see
which ones were over/underexpressed. As for underexpressed genes, the ratio <0,7
was established and for overespressed genes was the ratio >1,3. All the genes were
selected on the basis of their p-value = < 0,05. Searching for the genes annotation was
performed by Affymetrix and Ensembl databases. The tables show the genes whose
expression was changed by action of JS8 derivate.
- 49 -
Table 18: Identification of the overexpressed genes
gene ID ratio p-value chromosome location
official
full name
8021584 1,312348 0,021597 18 18q21.3
serpin peptidase
inhibitor, clade B
(ovalbumin), member 5
7905691 1,316503 0,027153 1 1q21 ribosomal
protein S27
8015230 1,319223 0,036293 17 17q12-
q21
keratin
associated
protein 4-11
7893796 1,331488 0,012632 6 6p21.33 ATP-binding cassette
sub-family F member 1
8178841 1,468844 0,032302 6 6p21.3
Transporter 2, ATP-binding
cassette, sub-family B
(MDR/TAP)
8125042 1,575965 0,04101 6 6p21.33
lymphocyte
antigen 6
complex, locus
G6C
8171087 1,619679 0,038681 X,Y Xp22.33;
Yp11.3
protein phosphatase 2
(formerly 2A), regulatory
subunit B'', beta
7895347 1,668704 0,011198 12 12q24.31
transmembrane emp24
domain trafficking
protein 2
8124855 2,490548 0,01321 6 6p21.33 surfactant
associated 2
8095364 3,080356 0,042023 4 4q13.2
transmembrane
protease, serine
11E
- 50 -
Table 19: Identification of the underexpressed genes
gene ID ratio p-value chromosome location
official full
name
8014633 0,570255 0,013925 17 17q12 TBC1 domain family,
member 3C
7894743 0,611395 0,024558 1 1q21.3
interleukin
enhancer binding
factor 2, 45kDa
7893795 0,624477 0,015187 2 2p13-p12
C1D nuclear
receptor co-
repressor
7892639 0,631789 0,029428 X Xq13.1
non-POU domain
containing, octamer-
binding
7892501 0,648596 0,009867 18 18q21 small nucleolar
RNA, C/D box 58B
7894401 0,679641 0,038444 22 22q13.1
eukaryotic translation
initiation factor 3,
subunit D
8020349 0,680136 0,002009 2 2q11.1 ankyrin repeat
domain 20B
2.3.6 UV-VIS spectroscopy analysis of interactions between cytochrome c
and derivates of betulinic acid
This method was performed in connection with the fact that betulinic acid and its
derivates are known to interact with cytochrome c. Absorption spectra of resulting
mixture consists of cytochrome c and derivates were recorded by this analysis. The
measurement was done in triplicate and 63 derivates were analyzed. The changes in
cytochrome c absorption spectra caused by their respective interactions with the
derivates were visualized as the difference spectra (∆A) of derivates treated samples of
cytochrome c. The absorption was measured three times and the average differences
in the maximum absorption (∆Amax) were used to quantify the effects of derivates on
cytochrome c. The final results are listed in the tables 20 and 21. The red labeled
values represent a similar or higher interaction of derivatives with cytochrome c,
comparing to the positive control, JS8. Example graphs (figure 21) show the typical
absorption spectrum of cytochrome c, where the range of wavelength was from 350-
- 51 -
750 nm. The blue line represents cytochrome c and the red one belongs to the certain
derivate.
Table 20: Final result of cytochrome c/derivative interaction
derivate max absorbance (A max)
wave
lenght ∆A max SD IC50
cyt c 0,329 0,339 0,327 405,5
JS8 0,248 0,257 0,231 405,5 0,08633 0,00839
BetA 0,303 0,305 0,297 405,5 0,03 0,004
1382 0,281 0,306 0,271 405,5 0,04567 0,01168 20,796
1384 0,293 0,295 0,284 405,5 0,041 0,00436 198,56
1385 0,274 0,294 0,276 405,5 0,05033 0,00503 10,7
1386 0,276 0,301 0,283 405,5 0,045 0,00755 12,386
1387 0,272 0,285 0,268 405,5 0,05667 0,00252 11,65
1411 0,319 0,332 0,331 405,5 0,00433 0,00737 27,96
1413 0,274 0,301 0,267 406 0,051 0,01153 14,63
1414 0,29 0,311 0,297 405,5 0,03233 0,00586 118,56
1415 0,264 0,282 0,29 405 0,053 0,01442 21,92
1416 0,274 0,289 0,282 405,5 0,05 0,005 24,37
1417 0,273 0,29 0,28 405,5 0,05067 0,00473 15,027
1418 0,277 0,302 0,279 405,5 0,04567 0,00777 37,94
1419 0,269 0,299 0,288 405 0,04633 0,01185 11,67
1420 0,276 0,3 0,284 405,5 0,045 0,00721 3,16
1421 0,284 0,3 0,272 405,5 0,04633 0,00808 249,4
1422 0,278 0,296 0,262 405,5 0,053 0,01114 15,6
1423 0,283 0,304 0,287 405,5 0,04033 0,00551 222,66
1424 0,295 0,304 0,301 405 0,03167 0,00493 39,85
1425 0,298 0,306 0,299 405,5 0,03067 0,00252 79,13
1426 0,288 0,312 0,282 405 0,03767 0,00945 18,92
1427 0,295 0,323 0,286 405,5 0,03033 0,0129 135,11
1428 0,284 0,303 0,289 405 0,03967 0,00473 14,71
1429 0,292 0,293 0,288 405 0,04067 0,00473 69,08
1431 0,314 0,323 0,309 405 0,01633 0,00153 29
1432 0,29 0,309 0,294 405 0,034 0,00458 27,47
1433 0,283 0,308 0,289 405,5 0,03833 0,00751 34,8
1434 0,285 0,302 0,284 405 0,04133 0,00379 9,64
1443 0,29 0,296 0,296 405,5 0,03767 0,00611 16,26
1444 0,217 0,235 0,238 406,5 0,10167 0,01168 24,58
- 52 -
Table 21: Final results of cytochrome c/derivate interaction
derivate max absorbance (A max)
wave
lenght ∆A max SD IC50
1445 0,287 0,299 0,288 405,5 0,04033 0,00153 14,96
1446 0,282 0,302 0,292 405,5 0,03967 0,00643 16,5
1447 0,288 0,303 0,29 405,5 0,038 0,00265 20,661
1448 0,284 0,293 0,294 405,5 0,04133 0,00723 5,73
1449 0,267 0,291 0,276 405,5 0,05367 0,00737 5,99
1450 0,284 0,302 0,287 405,5 0,04067 0,00404 23,07
1451 0,296 0,296 0,295 405,5 0,052 0,02571 2,62
1452 0,294 0,305 0,298 405,5 0,03267 0,00321 5,89
1453 0,308 0,322 0,299 405,5 0,022 0,00557 38,98
1454 0,289 0,295 0,289 405 0,04067 0,00306 37
1455 0,215 0,229 0,174 406,5 0,12567 0,02376 4,95
1456 0,282 0,298 0,27 405,5 0,04833 0,00808 15,11
1457 0,284 0,297 0,279 405,5 0,045 0,003 24,95
1458 0,287 0,3 0,283 405,5 0,04167 0,00252 9,22
1459 0,279 0,29 0,284 405,5 0,04733 0,00379 20,33
1460 0,306 0,326 0,308 405 0,01833 0,00503 14,096
1461 0,286 0,307 0,298 405,5 0,03467 0,00737 3,98
1462 0,278 0,303 0,27 405,5 0,048 0,01082 11,3
1463 0,289 0,296 0,288 405 0,04067 0,00208 15,17
1464 0,943 0,921 0,894 413 -0,5877 0,02401 15,57
1465 0,28 0,288 0,29 405,5 0,04567 0,00757 4,055
1466 0,28 0,302 0,288 405,5 0,04167 0,00643 28,44
1467 0,279 0,3 0,29 405,5 0,042 0,007 17,31
1468 0,29 0,304 0,302 405,5 0,033 0,00721 67,53
1469 0,282 0,298 0,29 405,5 0,04167 0,00503 22,27
1470 0,29 0,304 0,299 405,5 0,034 0,00557 60,147
1471 0,293 0,29 0,292 405,5 0,04 0,00781 53,1
1472 0,278 0,289 0,277 405,5 0,05033 0,00058 3,699
1473 0,283 0,3 0,283 405,5 0,043 0,00361 14,43
1474 0,267 0,303 0,261 405,5 0,05467 0,01629 3,93
1475 0,309 0,332 0,306 405 0,016 0,00781 51,099
1476 0,285 0,299 0,281 405,5 0,04333 0,00306 8,28
1477 0,284 0,276 0,29 405 0,04833 0,01332 3,88
- 53 -
Figure 21: Graphs showing different absorption maximum of cytochrome c and
derivates.
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
35
0
35
9
36
8
37
7
38
6
39
5
40
4
41
3
42
2
43
1
44
0
44
9
45
8
46
7
47
6
48
5
49
4
50
3
51
2
52
1
53
0
53
9
54
8
55
7
56
6
57
5
cyt c vs JS8
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
35
0
35
9
36
8
37
7
38
6
39
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40
4
41
3
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2
43
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44
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45
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46
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47
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48
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49
4
50
3
51
2
52
1
53
0
53
9
54
8
55
7
56
6
57
5
cyt c vs BetA
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
35
0
35
9
36
8
37
7
38
6
39
5
40
4
41
3
42
2
43
1
44
0
44
9
45
8
46
7
47
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48
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49
4
50
3
51
2
52
1
53
0
53
9
54
8
55
7
56
6
57
5
cyt c vs 1422
- 54 -
2.3 Discussion
The received results given more knowledge about the mechanism of action of
3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid (code JS8) on the
lymphoblastic leukemia cell line CEM. In our study we tried to identify target genes
whose expression was changed by JS8 action. Previous study suggested that primary
target of JS8 lays in mitochondria and that direct incubation of JS8 with cytochrome c
form non-covalent complex thus indicating cytochrome c to be a primary target for JS8
(Dzubak et al., 2007).
Our results from spectroscopic analysis confirm the fact that JS8 interacts with
cytochrome c. Other derivatives were also tested and the obtained results lead to
conclusion that they may have a similar effect of action like JS8, thus suggesting their
potential interaction with cytochrome c.
On the other hand, Affymetrix microarray analysis revealed several genes being
under/overexpressed in the base of JS8 action. One of the underexpressed gene was
C1D encodes nuclear-matrix associated protein C1D. C1D is nuclear matrix non-
histone protein which sequence level was characterized (Nehls et al., 1997). C1D was
also found to play role as a component of the complex involved in transcriptional
repression because of its association with the trancsriptional repressor RevErb and the
nuclear corepressors N-cor and SMRT (Zamir et al., 1997). C1D is also appears to
serve as a DNA-dependent protein kinase activator (Yavuzer et al., 1998), which plays
an important role in DNA double strand break (Jackson, 1997). Overexpression of C1D
leads to induction of apoptosis by increasing levels of p21Cip1 (WAF1), correlate with
specific interaction of C1D with DNA-PK that phosphorylates p53 protein on Ser 15
and Ser 37, resulting in p53 activation (Shieh et al., 1997). It was also revealed that
trancriptional and postranscriptional levels of C1D are tightly regulated. The
transcriptional activity of the basal C1D promoter is repressed by cis-acting sequences
comprised in a LINE-1 upstream element (Rothbarth et al., 2001) and proteasome-
mediated degradation appears to avoid random protein levels (Rothbath et al., 2002).
Taking into account all these information and comparing them with our result, where
the level of this gene was downregulated it can be said, that our derivative may play
role as C1D promoter repressor or protein degradation by ubiquitin and subseqeunt
caspase activation can occur. Another detected uderexpressed gene was interleukin
enhancer binding factor 2 ILF2, also known as NF 45. NF 45 together with NF 90 is
associated with the regulation of RNA gene at the levels of transcription, translation,
splicing and export (Zhao et al., 2004). This complex have been shown to stabilize the
association between Ku 70, Ku 80 and the DNA-dependent protein kinase catalytic
- 55 -
subunit DNA-PKcs, suggesting an involvement in DNA repair and chromosome stability
(Ting et al., 1998). It was revealed that increased expression of these proteins occurs
in transformed lymphoma, leukemia and hepatocellular carcinoma cells, suggesting
that aberrant upregulation of NF45 may contribute to the malignant phenotype,
especially in leukemias and lymphomas (Zhao et al., 2004). Taking into account this
information, it can be said that JS8 may play role in association with NF 45 in cancer
cell where DNA repairs fail or it can regulate protein at the transcriptional level with its
subsequent degradation. Another uderexpressed gene involved in DNA repair is
NONO non-POU domain containing, octamer-binding, also known as p54. p54 was
found to form complex with PSF that bind directly to DNA substrates of the end joining
reaction and subsequent cooperate with Ku protein, leading to functional complex
establishment. PSP and p54 are both phosphoproteins. Phosphorylation may be
involved in the relocalization of PSF during apoptosis and in regulating the binding
properties of p54 during mitosis. As this gene was uderexpressed, it can be suggested
that JS8 could again play role in DNA repair.
Next gene found to be underexpressed was TBC1 domain family, member 3C
(TBC1D3C) that includes TBC domain and it has been reported to posses GAP activity
towards RAB5A (Pei et al., 2002). Some in vitro studies and human tumor tissues
analysis revealed that TBC1D3C plays a role as an oncogene. In one study with
prostate cancer cells was this oncogene highly overexpressed and its oncogenic
activity is supposed to influence a tumorigenic phenotype of prostate cancer. The
mechanism of action of these oncogenity is said to be because of its overexpression
that leads to direct interaction of RAB5 due to the stimulation of its GTP hydrolysis (Pei
et al., 2002). In our study, the TBC1D3C gene was underexpressed comparing ratio
treated/control (p=0,014). Hypothetically, our derivative can play role in the mechanism
of TBC1D3C and its interaction with RAB5. Another explanation of the underexpressed
genes is the fact that most of them are regulated by transcription pathways which
should be affected by ubiquitin and caspases.
As for overexpressed genes, metallopanstimulin (MPS-1) was detected. MPS-1
is an ubiquitous multifunctional ribosomal S27/nuclear "zinc finger" protein which high
expression was revealed in a wide variety of cultured proliferating cells and tumor
tissues, including melanoma. MPS-1 is also thought to be involved in DNA repair and
recognition of altered mRNA. It has been investigated that the MPS-1 mRNA was
ubiquitously expressed in normal tissue and the increase message of the MPS-1 gene
was found to be increased in cancer cell lines (Fernandez et al., 1996). It was reported
that MSP-1 protiein contains ine zinc finger domain allowing to bind specifically the
DNA oligomer containing cAMP responsive elements sequence, considering MPS-1
- 56 -
protein might be involve in regulating the transcription of specific genes associated with
growth control and apoptosis (Fernandez, 1994). Another gene found to be
overexpressed is ATP-binding cassette sub family F, member 1 (ATP50) belongs to
ABC family proteins which have been characterised to lack recognizable
transmembrane domains. ATP50 was reported to increased its expression by
treatment of synoviocytes with tumor necrosis factor (Richard et al., 1998) and
subsequent study reveal that ATP50 plays an essential role in iniciation of translation
by binding to eIF2 and to both initiating and elongating ribosomes (Paytubi et al.,
2009). Another gene found to be overexpressed is ATP-binding cassette sub family F,
member 1 (ATP50) belongs to ABC family proteins which have been characterised to
lack recognizable transmembrane domains. ATP50 was reported to increased its
expression by treatment of synoviocytes with tumor necrosis factor (Richard et al.,
1998) and subsequent study reveal that ATP50 plays an essential role in iniciation of
translation by binding to eIF2 and to both initiating and elongating ribosomes (Paytubi
et al., 2009). Serpin B5 also known as Maspin was detected to be overexpressed.
Maspin belongs to serine protease inhibitor family and is known as class II tumor
suppressor, because although it is downregulated in many metastatic carcinoma cells,
no mutation occurs (Sager at al., 1996). Maspin also induce production of angiostatin
and inhibits vascular endothelial cell migration and tube formation to keep
angiogenesis in check (Narayan and Twining, 2009). There were revealed two
mechanisms of altered function of maspin in cancer. As first, the gene encodes maspin
is silenced in tumor by hypermethylation at Cp6 islands. In the second mechanism,
maspin was shown to be regulated by p53, in the case of abnormal function of p53,
maspin is not expressed (Zou et al., 2000). One study also revealed that maspin can
modulate tumor cell apoptosis through regulation of Bcl-2 family protein, where
reduced level of antiapoptotic protein but an increased proapoptotic protein Bax was
observed. This change in Bcl-2 family protein resulted in an increase release of
cytochrome c from mitochondria and subsequently the apoptosis is increased in
maspin-expressing tumor cells (Zhang et al., 2005). In our study was obviously
overexpressed level of maspin gene. This may be due to the cellular compensatory
action in the reaction on the described massive activation of the intracellular proteases.
Another overespressed gene was PPP2R3B protein phosphatase 2 (formerly 2A),
regulatory subunit B'', beta, PR48. It was revealed, that P48 mediated interaction
between Cdc6 and PP2A (serin/threonin phosphatase). This interaction is important for
Cdc6 dephosphorylation, ensuring the right cell cycle progression and DNA replication
control. Overexpression of PR48 causes the cell cycle arrest and has negative impact
on DNA replication (Yen et al., 1999). Comparing with our results, where gene encodes
- 57 -
this protein was overexpressed, it can be suggested that phosphatases compensatory
action may play role in the reaction with JS8. Another gene found to be overexpressed
is TAP-2, belonging to TAP family protein, which is involved in the transport of cytosolic
endogenous peptides to the endoplasmic reticulum mediating a link between antigen
processing and presentation. It was also observed that the transcription of this gene is
responsive to interferon gamma (IFN-gamma), indicating a common regulation and
concerted function of this gene in antigen processing. IFN gamma is produced by
mucosal lymphoid tissue exposed to a number of xenobiotic agents which function as
IFN gamma inducers and IFN-gamma encodes MHC proteins, the two specialized
proteasome subunits, leading to signal cell recognition by cytotoxic T-cell (Bocci et al.,
1981). Our result revealed upregulated TAP 2 via JS8 and it can be suggested that
JS8 enhances IFN-gamma to activate TAP 2.
In our study several potential targets of JS8 were revealed. JS8 is supposed to
be involved in DNA repair mechanism, GTP signaling pathway, cell cycle and in
apoptosis. The changes in the cell cycle were difficult to identify because of another
apoptosis mechanisms, which are much faster. For a specific interaction cytochrome c
is considerated, but other experiments are needed to be performed.
- 58 -
3. Conclusion
In this work, the effect of the JS8 on the expressional profile of CEM was analyzed.
JS8 is derivate of betulinic acid that is well known for its antitumor properties and one
of the modes of action is cytochrome c release and subsequent caspase activation
leading to programmed cell death. JS8 in comparison with betulinic acid showed
stronger activity against the tumor cell lines and the new observation were needed to
be revealed. That is why it was chosen for our experiments and by the method of
Affymetrix microarray assay the new potential targets and explanations of previous
experiments were identified.
- 59 -
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Links:
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- 67 -
Abbreviations
A549 lung carcinoma cell line
A2780/ADR ovarian cancer cell line
A375 melanoma cell line
ACF aberrant crypt foci
AGS gastric adenocarcinoma cells
AKBA 3-O-acetyl-11-keto-β-boswellic acid
ARO anaplastic carcinoma cell line
ATF-2 Activating transcription factor 2
B16 - F10 mouse melanoma cell line
Bak Bcl-2 homologous antagonist/killer
Bax Bcl-2–associated X protein
Bcap37 human breast carcinoma cell line
Bcl-2 B-cell CLL/lymphoma 2
Bcl-xL Bcl-2 family member
Bel-7402 hepatoma cell line
Bim Bcl-2 family member
CaP androgen-intensive cells
CDDO 2-cyano-3, 12-dioxoolean-1,9-dien-28-oic acid
Cdk cyclin dependent kinase
CDODA 2-cyano-3, 11-dioxo-18b-olean-1, 12-dien-30-oic acid
cFos proto-oncogene
CHOP ccaat-enhancer-binding protein
CTCL Cutaneous T cell lymphoma cell line
CREB cAMP response element-binding
CuB cucurbitacin B
DNA deoxyribonucleid acid
DU145 androgen-independent human prostate cancer cell line
ER endoplasmatic reticulum
FADD Fas-associated protein with death domain
Fas death receptor
GSH gglutathione synthetase
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GSK3 glycogen synthase kinase 3 alpha
GS-R3 ginsenosides R3
HEC108 endometrial cancer cell line
HeLa human cervical adenocarcinoma
HepG2 human hepatoma cells
HL-60 myeolid leukemia cell line
HO-8910 human ovarian carcinoma cells
HPAC human pancreatic cancer cell line
HPCC cervical carcinoma
HPLC/MS high pressure liquid chromatography/mass spectrometry
HT-29 colon adenocarcinoma cells
HUVECs human umbilical vein endothelial cells
IL-1β interleukin 1, beta
IkBa nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha
JAK 1 Janus kinase 1
JNK mitogen-activated protein kinase 8
K562 human leukemia cells
KDR kinase insert domain receptor
LNCaP prostate cancer cell line
MAPK1 mitogen activated protein kinase 1
MCF-7 breast adenocarcinoma cells
Mcl-1 induced myeloid leukemia cell differentiation protein
MMP-9 matrix metalloproteinase-9
NF-ĸB nuclear factor kappa B
p21/WAF1 cyclin-dependent kinase inhibitor 1
p53 tumor suppressor protein
p62 ubiquitin binding protein
P388 murine leukemic cell line
PAA human lung adenocarcinoma cells
PC-3 prostate cancer cell lines
PI3 phosphoinositide 3
PKCα protein kinase C alpha
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PPARg proliferator-activator receptor g
Ras family of genes encoding small GTPases
RPMI8226 multiple myeloma cell line
SGC7901 human gastric carcinoma cells
SK-MES-1 lung cancer cell line
SK-MEL-2 human melanoma cell line
SKNAS neuroblastoma cells
SMMC-7721 hepatocellular carcinoma cell line
SNG-II endometrial cancer cell line
STAT3 signal transducer and activator of
transcription 3
T98G glioblastoma cell line
TE671 rhabdomyosarcoma-medulloblastoma
cells
TNF-α tumor necrosis factor alpha
TRAIL TNF-related apoptosis-inducing ligand
VEGF vascular endothelial growth factor A
VEGFR 2 vascular endothelial growth factor
receptor 2
ZIP death-associated protein kinase 3