NORDAMNACANTHAL INDUCED APOPTOSIS AND MITOTIC … · nordamnacanthal (0.03, 0.01, 0.3, 1, 3, 10 and...

Open Access Research Journal, www.pieb.cz Medical and Health Science Journal, MHSJ

ISSN: 1804-1884 (Print) Volume 2, 2010, pp. 27-38

- 27 - © 2010 Prague Development Center

NORDAMNACANTHAL INDUCED APOPTOSIS AND MITOTIC-G2/M ARREST WITH DOWNREGULATION OF BCL-2 IN THE HUMAN BREAST CANCER CELL LINE (MCF-7)

Nordamnacanthal, an anthraquinone extracted from the root of Morinda elliptica from Rubiaceae family has cytotoxic properties towards cancer cell lines and antitumor promoting activities. This study was conducted to determine the cytotoxic effect of nordamnacanthal towards MOLT-4 and MCF-7 cell lines. Nordamnacanthal was found to be more cytotoxic towards MOLT-4 than MCF-7 with the IC50 of 3.8 µg/ml and 54 µg/ml, respectively, as detected by using the trypan blue dye exclusion test. The nordamnacanthal-treated cells showed characteristics of apoptosis such as membrane blebbing, chromatin condensation and formation of apoptotic bodies as observed under an inverted light microscope. Fluorescence analysis of cell death using acridine orange and propidium iodide staining showed that the population of MOLT-4 and MCF-7 cells underwent apoptosis at the IC50 value was 32% and 30.4%, respectively. Cell cycle analysis by flow cytometry indicated that nordamnacanthal did arrest MCF-7 cells at the G2/M phase. For MOLT-4, no cell cycle arrest was observed. Bcl-2 and Bax were downregulated in nordamnacanthal-treated MCF-7 cells. On the other hand, expression of the proteins in MOLT-4 was not significantly different from the control. In conclusion, nordamnacanthal was more cytotoxic towards MOLT-4 than MCF-7 cell line. The compound induced apoptosis in both cell lines, but with G2/M arrest and the involvement of Bcl-2 and Bax only in MCF-7.

1NORSYAFINI ISHAK, 1,2 LATIFAH SAIFUL YAZAN*, 3NORDIN HAJI LAJIS

1 Laboratory of Molecular Biomedicine, Institute of Bioscience, 2 Department of Biomedical Science, Faculty of Medicine and Health Sciences, 3 Natural Product Laboratory, Institute of Bioscience, Universiti Putra Malaysia

Keywords: Nordamnacanthal, cytotoxicity, apoptosis, G2/M arrest, Bcl-2, Bax.

UDC: 577

Introduction†

Nordamnacanthal or 2-formyl-1,3-dihydroxyanthraquinone (C15H8O5) is one of the 11 anthraquinones (AQs) which has been extracted from roots Morinda elliptica (Nor Hadiani et al., 1997), a small plant from the Rubiaceae family also known as “mengkudu kecil”. It is a native plant of Asia and Polynesia used in traditional folk medicine such as cholera, diarrhea, piles, headache and to increase appetite (Ali et al., 2000). Roots of the plant are usually rich in AQs, which most often occur as glycones and glycosides. There are about 90% of AQs from the Rubiacae family that occur as derivatives of 9,10-anthracenedione with several hydroxyl and other functional groups, such as methyl, hydroxymethyl and carboxyl (Wijnsma and Verpoorte, 1986). Hydroxyanthraquinones are the active principles of many phyto-therapeutic drugs (Westendorf et al., 1990).

Some studies have indicated that nordamnacanthal has a number of biological properties including antioxidant activities and antitumor effects on human B-lymphoblastoid cell line (Jasril et al., 2003). The compound also exhibits antiviral, antimicrobial and cytotoxic properties (Ali et al., 2000). A study has successfully shown that nordamnacanthal inhibits the activities of mammalian DNA polymerases families in colon cancer cell line (HCT116)

* Corresponding author: Latifah Saiful Yazan, [email protected]

† This work was supported by Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education (MOHE) of Malaysia (vote no: 5523104).

Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print)


due to the aldehyde group at the C-2 position that ultimately suppress growth of the cells (Kohei et al., 2010).

In this study, the cytotoxic effect and mode of cell death induced by nordamnacanthal in breast adenocarcinoma (MCF-7) and acute T-lymphoblastic leukaemia (MOLT-4) cell lines were determined.

Materials and methods

Cell culture

The human breast adenocarcinoma (MCF-7) and acute T-lymphoblastic leukaemia (MOLT-4) cell lines were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland, USA. The cell lines were cultured in RPMI-1640 medium (PAA, Germany) containing 10% fetal bovine serum (PAA, Germany) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) (PAA, Germany). The cells were incubated at 37 0C under 5% CO2 in a humidified atmosphere. Cell number and viability were determined by staining the cells with trypan blue dye (Sigma, Germany) and counted using a hemocytometer viewed under an inverted light microscope. The cells were subcultured when reach confluent at 99%.

Compound

Nordamnacanthal was kindly supplied by the Natural Product Laboratory, Institute of Bioscience, Universiti Putra Malaysia. The compound was dissolved in dimethyl sulphoxide (DMSO) to give the stock concentration of 10 mg/ml.

Treatment

The cell density of 1 x 105 cells/ml was treated with various concentrations of nordamnacanthal (0.03, 0.01, 0.3, 1, 3, 10 and 30 µg/ml for MOLT-4 and 30, 50, 70, 90 and 110 µg/ml for MCF-7) for 72 hours at 37 oC in 5% CO2. Control which contains cell without compound was also included. Following the treatment, the cells were harvested by centrifugation (450 x g, 10 minutes), washed with phosphate-buffered saline (PBS) twice before analysis.

Cytotoxicity of nordamnacanthal on MOLT-4 and MCF-7 cell lines

a. 3-[4.5-dimethylthizol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) Assay. Following the treatment, 20 µl of MTT solution was added into the cell suspension and left for 4 hours at 37oC in dark condition. The plate was spun at 450 x g for 10 minutes and 50 µl of media was sucked out. Subsequently, the formazan salt was dissolved by 100 µl of DMSO and left for 10 minutes at room temperature. The optical density (OD) was measured by using an ELISA reader (Sunrise, Tecan) at 570 nm test wavelength and 630 nm reference wavelength. Concentration that inhibits 50% of the cell growth compared to the untreated sample (IC50) was determined by the absorbance (OD) versus concentration curve.

b. Trypan Blue Dye Exclusion Method. Briefly, the treated cells were stained with 0.25% trypan blue (Sigma, USA) at a dilution of 1:1, and counted using a Neubauer hemocytometer under an inverted light microscope. The counting was done in triplicate. Concentration that inhibits 50% of the cell growth (IC50) compared to the untreated sample was determined by plotting the graph of percentage of cell viability versus concentration of the compound.



Morphological changes of MOLT-4 and MCF-7 cells treated with nordamnacanthal

The morphological changes were observed under an inverted light microscope at 400X magnification.

Determination of mode of cell death

The cells were stained with 100 µg/ml of acridine orange (Sigma, USA) and 100 µg/ml of propidium iodide (Sigma, USA) at 1:1 mixture after treatment with nordamnacanthal (Gorman et al., 1996). The suspension was placed onto a clean microscopic slide and viewed under a fluorescence microscope at 200X magnification. A minimum of 200 cells was counted in every sample. The number of viable, apoptotic and necrotic cells was counted and expressed as a proportion of the total cell number (%).

Cell cycle analysis

The cells were harvested by centrifugation at 644 x g for 10 minutes and washed twice with PBS. The fixation of the cells was done according to Klucar and al-Rubeai (1997) with some modifications. Briefly, the cells were fixed by 70% cold ethanol, and left at -20 0C for 2 hours. The cell pellet was then resuspended in PBS containing RNase A (1 mg/ml) on ice for 20 minutes in dark condition. Intracellular DNA was labeled with propidium iodide (PI) (1 mg/ml) and analyzed by flowcytometry using FACScan software (Cyan ADP, Dako, Denmark).

Enzyme-linked Immunosorbent Assay for Quantitative Detection of Human Bcl-2

The analysis was carried out using the Human Bcl-2 ELISA kit (Bender MedSystems, Austria). For the sample preparation, the cell was lysed with 1X Lysis Buffer. After 1 hour of incubation at room temperature, the sample was spun at 1000 x g for 15 minutes and the supernatant was taken. Briefly, 80 µl of Sample Diluent and 20 µl of sample was added to the wells coated with monoclonal antibody to human Bcl-2 after the wells were washed two times with the Wash Buffer. Next, 50 µl of Biotin-Conjugate was added to the wells. After 2 hours of incubation at room temperature on a microplate shaker, 100 µl of Streptavidin-HRP was added. The sample was incubated again for 1 hour at room temperature on a microplate shaker. The solution was withdrawn and the wells were washed 3X with the Wash Buffer. Immediately, 100 µl of TMB substrate solution was added. Finally, 100 µl of Stop Solution was added to each well to stop the enzymes reaction. The absorbance was read using an ELISA reader (Sunrise, Tecan) at 450 nm and 620 nm as a reference wavelength. The optical density was compared with the standard graph to determine the concentration of the protein.

Human Bax Enzyme Immunometric Assay

Bax protein concentration determination was carried out using the Human Bax Enzyme Immunometric Assay Kit (BD, USA). The lysate (sample) preparation was done according to the manual. Following centrifugation (16000 x g for 15 minutes), the cells were resuspended in Modified Cell Lysis Buffer 4 (0.5 µl/ml of Sigma Protease Inhibitor Cocktail and 1mM phenylmethylsulfonyl fluoride (PMSF)). Next, 100 µl of the sample was added to the wells coated with monoclonal antibody to human Bax-α in triplicate. The plate was tapped gently to mix the contents. The sample was incubated at room temperature on a plate shaker for 1 hour. The wells were emptied and washed 5X with the Wash Buffer. After the final wash, the plate was tapped gently on a lint free paper towel to remove any remaining Wash Buffer. The sample was incubated again for 1 hour at room temperature on a plate shaker after the addition of 100 µl of Yellow Antibody (biotinylated monoclonal antibody to Bax-α) into each well. The wells were washed again for 5X with the Wash Buffer and emptied. Next, 100 µl Blue Conjugate (streptavidin



conjugated to horseradish peroxidase) was added to each well. The sample was left on a plate shaker for 30 minutes at room temperature. The wells were washed again as the previous step. A solution of 3.3’,5.5’ tetramethylbenzidine (TMB) and hydrogen peroxide was added at 100 µl into each well. Finally, 100 µl of Stop Solution containing hydrochloric acid in water was added to stop the enzyme reaction. The optical density was read at 450nm and 570 nm as a reference wavelength by an ELISA reader (Sunrise, Tecan). The optical density was compared with the standard graph to determine the concentration of the protein.

Statistical analysis

Student’s t-test was performed to determine the significant difference between means of control and treated samples. A p-value of less than 0.05 (p<0.05) was considered as statistically significant.

FIGURE 1. CHEMICAL STRUCTURE OF NORDAMNACANTHAL (RAJENDRAN ET AL., 2004)

FIGURE 2. THE MORPHOLOGY OF (B) MCF-7 AND (D) MOLT-4 CELLS TREATED WITH NORDAMNACANTHAL

AT THE IC50 VALUE FOR 72 HOURS VIEWED UNDER AN INVERTED LIGHT MICROSCOPE. The treated cells showed apoptotic characteristics such as nuclear compaction (nc), chromatin

condensation (cc),membrane blebbing (mb) and the formation of apoptotic body (ab). Necrotic cells (nt) were observed (arrows). Controls were also included (A and C) (400X magnification).

O

O O

CH

OH

A

B D

ab

nc

mb

nt

cc

C



Results

Cytotoxicity of nordamnacanthal towards MCF-7 and MOLT-4: Nordamnacanthal exhibited cytotoxic properties towards the breast adenocarcinoma (MCF-7) and acute T-lymphoblastic leukemia (MOLT-4). Nordamnacanthal was more cytotoxic towards MOLT-4 than MCF-7 with the IC50 value of 3.8 µg/ml and 54 µg/ml, respectively (Table 1).

FIGURE 3. THE MORPHOLOGY OF (B) MCF-7 AND (D) MOLT-4 CELLS TREATED WITH

NORDAMNACANTHAL AT THE IC50 VALUE FOR 72 HOURS. Samples were stained with acridine orange and propidium iodide, and observed under a fluorescence microscope. The cells showed apoptosis characteristics such as nuclear

compaction (nc), chromatin condensation (cc) the membrane plasma blebbing (mb). The mitotic cell death (mt) and necrotic cells (nt) were also observed. Control was also included

(A) (200 X magnification).

Morphological changes of MCF-7 and MOLT-4 treated with nordamnacanthal: Majority of nordamnacanthal-treated MCF-7 and MOLT-4 cells showed morphological features of apoptosis observed under an inverted light microscope and fluorescence microscope following staining with AO/PI. The morphological characteristics include condensation of nuclear chromatin and cytoplasm, membrane blebbing and the formation of apoptotic bodies. The apoptotic bodies appeared to be round or oval masses of cytoplasm, smaller in size than the cell of origin. Interestingly, some of the MCF-7-treated cells exhibited enlargement of cell volume and formation of multinucleated cells. The control cells appeared to be shiny, clear and healthy. There were also small numbers of necrotic cells with ruptured plasma membrane observed (Figure 2 and Figure 3).

Fluorescence analysis of mode of cell death

It is confirmed that majority of MCF-7 and MOLT-4 cells underwent apoptosis as compared to necrosis. At the IC50 value, the percentages of apoptotic cells for MCF-7 and MOLT-4 were 30% and 31%, respectively. The percentages of necrotic cells were only 1.3% and 3.3%, for MCF-7 and MOLT-4, respectively (Figure 4).

A B mt

nt

nc

mb

D nt

mb

cc

C



Cell cycle analysis

Figure 5 illustrates the changes in the cell cycle distribution of MCF-7 and MOLT-4 cells treated with nordamnacanthal at the IC50 value as compared to the control. The population of MCF-7 cells at the G2/M phase increased significantly (p<0.05). Cell population at the G0/G1 and S, and sub-G0 phases decreased and increased, respectively. The differences were insignificant (p>0.05) compared to the control, yet for MOLT-4 cells, there was no significant increase in the cell population at any phases (p>0.05).

FIGURE 4. THE PERCENTAGE OF VIABLE, APOPTOTIC AND NECROTIC (A) MCF-7 AND (B) MOLT-4 CELLS TREATED WITH NORDAMNACANTHAL AT THE IC50 VALUE FOR 72 HOURS.

Controls were also included. The counts were done under a fluorescence microscope following staining with acridine orange and propidium iodide. Each data is presented

as mean + SEM.

0

10

20

30

40

50

60

70

80

90

100

control 54µg/ml

Concentration (µg/ml)

Cel

ls (

%) viable

apoptosis

necrosis

0

10

20

30

40

50

60

70

80

90

100

control 3.8µg/ml

Concentration (µg/ml)

Cell

s (%

)

viable

apoptosis

necrosis

TABLE 1. THE IC50 VALUE OF MCF-7 AND MOLT-4 CELLS TREATED WITH

NORDAMNACANTHAL FOR 72 HOURS AS DETERMINED USING MTT ASSAY AND TRYPAN

BLUE DYE EXCLUSION TEST. Each sample was run in triplicate. The result is presented as mean value + SEM

Cell line IC50 (µ g/ml)

MTT Trypan Blue Dye Exclusion Test

MCF-7 >110 54 + 0.333

MOLT-4 >30 3.8 + 2.028

A

1.2

%

30%

68%

3% 0.3

97%

B 65%

3% 1%

83%

16%

32%



Effect of nordamnacanthal on the level of expression of Bcl-2 and Bax in MCF-7 and MOLT-4: Bcl-2 and Bax were downregulated in nordamnacanthal-treated MCF-7 cells. On the other hand, expression of the proteins in MOLT-4 was not significantly different (p<0.05) from the control (Figure 6 and Figure 7).

FIGURE 5. CELL CYCLE DISTRIBUTION FROM FLOWCYTOMETRIC ANALYSIS OF (B) MCF-7

AND (D) MOLT-4 CELLS TREATED WITH NORDAMNACANTHAL AT THE IC50 VALUE FOR 72 HOURS. Controls were also included (A and C). (R2= sub-G0; R3= G0/G; R4= S; R5= G2/M).

FIGURE 6. THE CONCENTRATION OF BCL-2 OF MCF-7 AND MOLT-4

TREATED WITH NORDAMNACANTHAL AT THE IC50 VALUE FOR 72 HOURS. Controls were also included. Each data is presented as mean + SEM.

0

20

40

60

80

100

120

140

160

MCF-7 MOLT-4

Cell line

Co

ncen

trat

ion

(ng

/ml) control

IC50

Discussion

Nordamnacanthal was cytotoxic towards both of the cell lines (MCF-7 and MOLT-4) with the IC50 value of 54 µg/ml and 3.8 µg/ml, respectively. Natural products provide a great chemical structural diversity to cause cytotoxicity even at low concentrations (micrograms) (Nor Hadiani et al., 1997). It is interesting to note that there was a discrepancy in the IC50

Sub-G0 = 1.46 + 0.15 G0/G1= 75.35 + 0.27 S= 5.5 + 0.13 G2/M= 18.35 + 0.16

A

Sub-G0 = 2.93 + 0.17 G0/G1= 67.56 + 0.69 S= 4.73 + 0.22 G2/M= 25.63 + 0.62

B

Sub-G0 = 7.29 + 0.48 G0/G1= 51.64 + 1.49 S= 11.90 + 0.01 G2/M= 31.13 + 1.83

C

Sub-G0 = 9.07 + 0.74 G0/G1= 48.36 + 1.23 S= 11.92 + 0.17

G2/M= 32.47 + 0.90

D



value achieved from MTT and trypan blue dye exclusion test (Table 1). The IC50 from MTT assay was far higher compared to the one from the latter method. This phenomenon possibly occur as a result of different endpoints being assessed using the two different methods (McKim et al., 2005). The principle of MTT is mainly based on the intact mitochondria Krebs cycle activity (Mosmann, 1983) while the other one is based on the membrane integrity of the cell (Freshney, 1994). Based on the findings, we assume that MTT is not an appropriate assay for determination of cytotoxicity of nordamnacanthal. Higher reading in OD at the higher concentrations (>30 µg/ml) of the compound indicates nordamnacanthal-MTT interaction. Similar observation on certain chemotherapeutic drugs that interact directly with the MTT salt hence resulting in false negative result was reported previously (Ulukaya et al., 2004). Therefore, we decided to carry out further analysis based on the IC50 achieved from the trypan blue dye exclusion test.

FIGURE 7. THE CONCENTRATION OF BAX OF MCF-7 AND MOLT-4

TREATED WITH NORDAMNACANTHAL AT THE IC50 VALUE FOR 72 HOURS. Controls were also included. Each data is presented as mean + SEM.

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

MCF-7 MOLT-4

Cell line

Co

ncen

trati

on

(pg

/ml)

control

IC50

It has been found that nordamnacanthal was more potent towards MOLT-4 than MCF-7 due to the lower IC50. The higher potency of a compound will require a smaller amount to produce cytotoxicity, resulting in lower IC50 value (Inayat-Hussain and Thomas, 2004). Since nordamnacanthal exhibited cytotoxicity at the concentration less than 10 µg/ml, it is considered a potential one to be developed into anticancer chemotherapeutic drug for leukemia (Shier, 1991). The exact mechanisms on how nordamnacanthal from M. elliptica exerts its cytotoxicity are still unknown. The most possible one is by penetration into the cancer cells, reaching the nucleus, thus inhibiting the activity of DNA polymerase specifically pols α and ĸ, similar to the action of nordamnacanthal from M. citrifolia on the human colon cancer cells (HCT116 cells). This will eventually suppresses the cell growth (Kohei et al., 2010). In fact, Sakaguchi et al. (2002) have proven that pol’s inhibitors were cytotoxic and suppressed human cancer cell proliferation. Another mechanism could be due to the prominent chemical feature of quinones with ability to undergo redox cycling to generate reactive oxygen species which eventually cause damage to the tumor cell (Kalyanaraman et al., 1991; Schreiber et al., 1987).

The anchorage-independent cell line (MOLT-4) was more sensitive to nordamnacanthal as compared to the anchorage-dependent cell line (MCF-7) as being reported previously (Ali et al., 2000). It is believed due to many factors in the microenvironment of solid tumor that are responsible for non-uniform and insufficient levels of anticancer agents being delivered. The abnormalities in the extracellular matrix (ECM) in tumors, for instance, lead to the deficiencies in interstitial transport (Wang and Yuan, 2006), which ultimately affect the bioavailability and efficacy of chemotherapeutic agents (Galmarini and Galmarini, 2003). The ECM is made up of a matrix of proteoglycans, collagens, and additional molecules, which are produced and assembled by stromal and tumor cells (Mow et al., 1984). In tumor, greater collagen content in the ECM required higher infusion



pressure to initiate flow in the tumor interstitium (McGuire et al., 2006), thus obstructs the transport of anti-cancer agent (Wang and Yuan, 2006).

Majority of nordamnacanthal-treated MCF-7 and MOLT-4 cells showed morphological features of apoptosis such as condensation of nuclear chromatin and cytoplasm, membrane blebbing and the formation of apoptotic bodies (Figure 2 and Figure 3). According to Salvesen and Dixit, (1997), caspase-3 was responsible for many of the morphological and biochemical features of apoptosis, whereby the process occurs in systematic and deliberate manner which involve series of activation of enzymes such as cysteine proteases (caspases) and endonucleases (Scott et al., 2008; Julius et al., 2007).

The induction of apoptosis by nordamnacanthal was further confirmed by staining the cells using AO/PI fluorescence dye. Assessments of membrane integrity with vital dye, such as trypan blue are nonspecific to determine the mode of cell death such as apoptotic cells unless morphological characteristics can be analyzed with the dyes (Todd et al., 1997). AO is a cell-permeable DNA-binding dye was used in combination with plasma membrane-impermeable DNA-binding dye PI. AO and PI excite a green and red fluorescence, respectively, when they are intercalated into DNA. AO is taken up by both viable and nonviable cells, while PI is excluded by cells with intact membrane (viable and apoptotic). PI fluoresce red predominantly for necrotic cells (Ciapetti et al., 2002). The examination of MCF-7 and MOLT-4 cells stained with AO/PI showed a combination of apoptotic cells (with dense green areas in the nuclei), necrotic cells (with red intact nucleus) and viable cells (with green intact nucleus). Control cells without treatment for both cell lines looked healthy with green colour of intact nucleus. Almost all the control cells for both cell types appeared to be round in shapes and similar sizes. In contrast, the treated cells for both cell types were irregular in sizes yet round in shape (Figure 3). Some of the cells were smaller in sizes as compared to the cell of origin. The cell shrinkage due to loss of cellular volume associated with cytoskeletal breakdown and plasma membrane blebbing also a hallmark of cell undergoing apoptosis (Ciapetti et al., 2002). The plasma and nuclear membrane were clearly observed. Other characteristics of apoptosis such as nuclear compaction, chromatin condensation and plasma membrane blebbing were clearly seen in both cells treated with nordamnacanthal. Caspase-mediated cleavage of specific substrates explains the special features of apoptosis. Cleavage of the nuclear lamins is required for nuclear shrinking and budding (Buendia et al., 1999; Rao et al., 1996). Loss of overall cell shape is probably due to the cleavage of cytoskeletal proteins such as fodrin and gelsolin (Kothakota, 1997) and finally the plasma membrane blebbing seems to be caused by the cleavage of PAK2, a member of the p21-activated kinases family (Rudel and Bokoch, 1997).

Increase in the cell population at the sub-G0 phase for both cell lines was observed but insignificant (p>0.05) (Figure 5). The visibility of “sub-G0” peak (hypodiploid DNA peak) by flow cytometry with other supporting information can be taken as definite evidence for the apoptotic cell population (Ormerod and Collins, 1992). The flow cytometric analysis showed that the compound induced cell cycle arrest at G2/M phase in MCF-7 cells. The cell death relative to G2/M blockage was correlated to increase DNA hypodiploid (sub-G0) rapidly after arrest at the G2/M transition.

It is interesting to mention that some of nordamnacanthal-treated MCF-7 cells showed the characteristics of cells undergoing mitotic cell death such as enlargement of cell volume, formation of multinucleated cells (Figure 3), which then will be followed by mitotic catastrophe and subsequent nuclear fragmentation (Bursch et al., 1990). Cells die after G2/M arrest has been specifically described as “mitotic cell death”. The phenomenon involves G2/M arrest associated with incomplete or defective mitosis. The characteristics of cells undergoing mitotic catastrophe is associated with the abnormal segregation of chromosomes and aberrant cytokinesis, resulting in cells with abnormal size and DNA content (Hendry and West, 1997). This form of cell death has been defined as loss of reproductive integrity after inappropriate entry into mitosis, and is frequently characterized by the emergence of cells containing multinuclear fragments or “micronuclei” (Muller et al., 1996). They showed no chromatin condensation or



cytoplasmatic condensation and fragmentation, indicating that the nuclear remnants were undergoing karyolysis (nuclear dissolution) (Ming-Jie et al., 2004). The mitotic-arrested cells eventually progresses into apoptosis (Ming-Jie et al., 2004; Jordan et al., 1996). Nevertheless, the mechanisms on how cytotoxic stress caused MCF-7 cells to undergo apoptosis at the G2/M checkpoint are still unclear. A study done by Hengartner (2000) drives a hypothesis that nordamnacanthal may be able to interact with the major component of microtubule assembly. Another speculation is that the compound induces inappropriate alteration in the expression or/and activation of Cdks and regulators, leading to blockage of cell cycle progression and induction of apoptosis (Antonio et al., 2009).

Bcl-2 and Bax were downregulated in nordamnacanthal-treated MCF-7 cells (Figure 6 and Figure 7). Down-regulation of Bcl-2 indicates that nordamnacanthal is inducing apoptosis in the cells (Che-Jen et al., 2008). Bcl-2 is presumably involved in the G2/M arrest (Figure 5). Bcl-2 has been demonstrated to interact with a variety of proteins involved in regulating the G2/M transition (Dong-Oh et al., 2008). The reason why Bax protein was downregulated is still not clear. On the other hand, the expression of Bcl-2 and Bax in the MOLT-4 cells treated with nordamnacanthal was not different from the control. It is suggested that other Bcl-2 family proteins of the anti-apoptotic members (example; Bcl-XL and Bcl-W) and the pro-apoptotic members (example; Bad, Bak and Bik) are involved (Cory and Adams, 2002). Besides that, the correct ratio of anti-apoptotic/pro-apoptotic protein is also important which is proportional to the response of a cell to apoptotic stimuli (Huang et al., 2008). Since nordamnacanthal induced apoptosis, it stands a chance to be used for cancer therapy by switching on the cell death machinery in cancer cells (Kohei et al., 2010).

Conclusion

Nordamnacanthal was more cytotoxic towards MOLT-4 than MCF-7 cell line. The compound induced apoptosis in both cell lines, but with G2/M arrest and the involvement of Bcl-2 and Bax only in MCF-7.

References

Ali, A., Ismail, N., Mackeen, M., Yazan, L. et al., 2000. “Antiviral and antimicrobial activities of anthraquinone isolated from

the roots of Morinda elliptica,” Pharmaceutical Biology, Vol.38, pp.298-301.

Antonio, C., Antonio, G., Francisco, A., Tomas-Barberan et.al., 2009. “Availability of polyphenols in fruit beverages

subjected to in vitro gastrointestinal digestion and their effects on proliferation, cell-cycle and apoptosis in human colon

cancer Caco-2 cells,” Food Chemistry, Vol.114, pp.813-20.

Buendia, B., Santa-Maria, A., Courvalin, J., 1999. “Caspase-dependent proteolysis of integral and peripheral proteins of

nuclear membranes and nuclear pore complex proteins during apoptosis”, Journal of Cell Science, Vol.112, pp.1743-

753.

Bursch, W., Paffe, S., Putz, B., Barthel, G., Schulte-Hermann, R., 1990. “Determination of the length of the histological

stages of apoptosis in normal liver and in altered hepatic foci of rats”, Carcinogenesis, Vol.11, pp.847-53.

Che-Jen, H., Tsia-Kun, L., Ya-Ling, C., Ling-Wei, H. et al., 2008. “WRC-213, an L-methionine conjugated mitoxantrone

derivative, displays anticancer activity with reduced cardiotoxicity and drug resistance: Identification of topoisomerase

II inhibition and apoptotic machinery in prostate cancers,” Biochemical Pharmacology, Vol.75, pp.847-56.

Ciapetti, G., Granchi, D., Savarino, L., Cenni, E. et al., 2002. “In vitro testing of the potential for orthopedic bone cements

to cause apoptosis of osteoblast-like cells,” Biomaterials, Vol.23, pp.617-27.

Cory, S., Adams, J.M., 2002. “The Bcl2 family: regulators of the cellular life or death switch”, Nature Review Cancer, Vol.2,

pp.647-56.

Dong-Oh, M., Mun-Ock, K., Yung, H., Nam, D. et al., 2008. “Bcl-2 overexpression attenuates SP600125-induced apoptosis

in human leukemia U937 cells,” Cancer Letters, Vol.264, pp.316-25.

Freshney, I., 1994. Culture of animal cells: A manual of basic technique (3rd Ed.), New York, Wiley-LISS.



Galmarini, C., Galmarini, F., 2003. “Multidrug resistance in cancer therapy: role of the tumor microenvironment”, Current

Opinion Investigation Drugs, Vol.4, pp.1416-421.

Gorman, A., McCarthy, J., Finucane, D., Reville, W., Cotter, T., 1996. “Morphological assessment of apoptosis,” In: Cotter,

T.G., Martin, S.J. (Eds.), Techniques in apoptosis. A user guide, Portland Press Ltd, London, pp.1-20.

Hendry, J., West, C., 1997. “Apoptosis and mitotic cell death: Their relative contributions to normal-tissue and tumour

radiation response”, International Journal of Radiation Biology, Vol.71, pp.709-19.

Hengartner, M., 2000. “The biochemistry of apoptosis,” Nature, Vol.407, pp.770-76.

Inayat-Hussain, S.H., Thomas, N.F., 2004. “Recent advances in the discovery and development of stilbenes and lactones in

anticancer therapy”, Expert Opinion on Therapeutic Patents, Vol. 14, pp.1-16.

Jasril, L., Abdullah, M., Sukari, M., Ali, A., 2003. “Antitumor promoting and antioxidant activities of anthraquinones isolated

from cell suspension culture of Morinda elliptica,” Asia Pacific Journal of Molecular Biology and Biotechnology,

Vol.11, pp.3-7.

Jordan, M., Wendell, K., Gardiner, S., Derry, W., Copp, H., Wilson, L., 1996. “Mitotic block induced in hela cells by low

concentrations of paclitaxel (taxol) results in abnormal mitotic exit and apoptotic cell death,” Cancer Research, Vol.56,

pp.816-25.

Julius, L., Long, H., Ya, C., Shu, H. et al., 2007. “Apoptosis induction by avian reovirus through p53 and mitochondria

mediated pathway,” Biochemical and Biophysical Research Communications, Vol.356, pp.529-35.

Kalyanaraman, B., Morehouse, K., Mason, R., 1991. “An electron paramagnetic resonance study of the interactions between

the adriamycin semiquinone, hydrogen peroxide, iron-chelators, and radical scavengers,” Archives of Biochemistry and

Biophysics, Vol.286, pp.164-70.

Klucar, J., al-Rubeai, M., 1997. “G2 cell cycle arrest and apoptosis are induced in Burkitt’s lymphoma cells by anticancer

agent oracin,” FEBS Letters, Vol.400, pp.127-30.

Kohei, K., Wakako, H., Shogo, T., Ken, H., Toshiko, S., Yuko, K.Y., Hiromi, Y. and Yoshiyuki, M. 2010. “Inhibitory effect

of anthraquinones isolated from the Noni (Morinda citrifolia) root on animal A-, B- and Y-families of DNA

polymerases and human cancer cell proliferation,” Food Chemistry, Vol.118, pp.725-30.

Kothakota, S., 1997. “Caspase-3-generated fragment of gelsolin: Effector of morphological change in apoptosis”, Science,

Vol. 278, pp.294-98.

McGuire, S., Zaharoff, D., Yuan, F., 2006. “Nonlinear dependence of hydraulic conductivity on tissue deformation during

intratumoral infusion,” Annals of Biomedical Engineering, Vol.34, pp.1173-181.

McKim, J., Wilga, P., Pregenzer, J., Petrella, D., 2005. “A biochemical approach to in vitro toxicity testing,” Pharm. Discov.

[Internet] [cited 2009 Jun 6] [about 6 p.]. Available from http://www.ceetox.com/downloads/pharma_discovery.pdf

Ming-Jie, L., Zhao, W., Yong, J., Jiang-bing, Z., Yu, W., Ricky, N.S.W., 2004. “The mitotic-arresting and apoptosis-inducing

effects of diosgenyl saponins on human leukemia cell lines”, Biological and Pharmaceutical Bulletin, Vol. 27, pp.1059-

1065.

Mosmann, T., 1983. “Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity

assays,” Journal of Immunological Methods, Vol.65, pp.55-63.

Mow, V., Mak, A., Lai, W., Rosenberg, L., Tang, L., 1984. “Viscoelastic properties of proteoglycan subunits and aggregates

in varying solution concentrations,” Journal of Biomechanics, Vol.17, pp.325-38.

Muller, W., Nusse, M., Miller, B., Slavotinek, A. et al., 1996. “Micronuclei: A biological indicator of radiation damage,”

Mutation Research, Vol. 366, pp. 163-169.

Nor Hadiani, I., Abdul, M., Norio, A., Mariko, K. et al., 1997. “Anthraquinones from Morinda Elliptica,” Phytochemistry,

Vol. 8, pp. 1723-1725.

Ormerod, M.G., Collins, M.K.L., Rodriguez-Tarduchy, G., Robertson, D., 1992. “Apoptosis in interleukin-3 dependent

haemopoietic cells: Quantification by two flow cytometric methods,” Journal of Immunological Methods, Vol.153,

pp.57-65.

Rajendran, M., Johnson, J., Gandhidasan, R., Murugesan, R., 2004. “Photodynamic action of damnacanthal and

nordamnacanthal,” Journal of Photochemistry and Photobiology A: Chemistry, Vol. 162, pp.615-23.

Rao, L., Perez, D., White, E., 1996. “Lamin proteolysis facilitates nuclear events during apoptosis,” Journal of Cell Biology,

Vol.135, pp.1441-455.

Rudel, T., Bokoch, G., 1997. “Membrane and morphological changes in apoptotic cells regulated by caspase-mediated

activation of PAK2,” Science, Vol.276, pp.1571-574.

Salvesen, G., Dixit, V., 1997. “Caspases: intracellular signaling by proteolysis”, Cell, Vol.91, pp.443-46.



Schreiber, J., Mottley, C., Sinha, B., Kalyanaraman, B., Mason, R., 1987. “One-electron reduction of daunomycin,

daunomycinone, and 7- deoxydaunomycinone by the xanthine/xanthine oxidase system: detection of 16 semiquinone

free radicals by electron spin resonance,” Journal of the American Chemical Society, Vol.109, pp.348-51.

Scott, H., Sun-Hee, L., Wei, M., David, A. et al., 2008. “Apoptosis-associated caspase activation assays,” Methods, Vol.44,

pp.262-72.

Shier, W., 1991. “Mammalian cell culture on $5 a day: A laboratory manual of low cost methods,” Los Banos: University of

Philiphines.

Todd, R., William, J., Devendra, I., Agrawal., 1997. “Morphological and biochemical characterization and analysis of

apoptosis,” Journal of Pharmacological and Toxicogical Methods, Vol.37, pp.215-28.

Ulukaya, E., Colakogullari, M., Wood, E.J., 2004. “Interference by anticancer chemotherapeutic agents in the MTT-tumor

chemosensitivity assay,” Chemotherapy, Vol.50, pp.43-50.

Wang, Y., Yuan, F., 2006. “Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and

strategies for improvement”, Annals of Biomedical Engineering, Vol.34, pp.114-27.

Westendorf, J., Marquardt, H., Poginsky, B., Dominiak, M. et al., 1990. “Genotoxicity of naturally occurring

hydroxyanthraquinones,” Mutation Research, Vol.240, pp.1-12.

Wijnsma, R., Verpoorte, R., 1986. “Anthraquinones in Rubiaceae,” Fortschritte Chemie Organischer Naturstoffe, Vol.49,

pp.79-149.

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