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Introduction
Results Conclusions
Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 Roswell Park Summer Research Program
Andrea Schneider, Pragya Srivastava, Ravi Lella, Timothy Marlowe, Gabrielle Lovett, Raghu Gogada, Dhyan Chandra
Mechanisms of Neemoids Induced Apoptosis and Autophagy in Cancer Cells
Whole cell lysates preparation, subcellular fractionation and Western blotting-
Whole cell lysates were trypsinized and harvested by standard procedure. They were
lysed in NP-40 buffer, 1 mM dithiothreitol (DTT) and a mixture of protease
inhibitors. To prepare mitochondrial and cytosolic fractions, cells were harvested,
washed in ice-cold PBS, and then resusupended in homogenizing buffer. After a 30
min incubation on ice, cells were homogenized with a glass Pyrex homogenizer and
centrifuged at 1100 g for 5 min at 4°C to remove cell debris and unbroken cells. The
supernatant was then centrifuged at 12000 g for 12 min to obtain a mitochondrial
pellet. The resulting supernatant was then centrifuged at 100,000 g for 1 h to obtain
cytosolic fraction. Protein concentration was determined with a Micro-BCA kit.
Samples were prepared and subjected to standard Western blotting procedure.
Quantification of apoptosis and caspase activity measurement- Live and dead cells
were measured using trypan blue dye. DEVDase, LEHDase and IETDase activities
were measured by standard procedure. Production of AFC was monitored on a
spectrofluorimeter using an excitation wavelength of 400 nm and an emission
wavelength of 508 nm. The results were presented as fold activation over the
control.
Immunofluorescence- Cells were grown on coverslips and then treated with
neemoids, cells were treated with either DAPI (nuclei) or Mito- Tracker Orange
(mitochondria). Cells were fixed, permeabilized and immunolabeled for cytochrome
c. After mounting and sealing, images were captured under fluorescence
microscope.
Analysis of mtDNA content by real-time PCR- Genomic DNA was isolated from
untreated and neemoids treated HCT116 WT cells using the ZR Genomic DNA II
Kit. After quantification of DNA samples by the NanoDrop 8000, mtDNA content
was determined on the Applied Biosystems 7300 real-time PCR system. β-actin and
cytochrome c oxidase subunit II (COX II) were used for amplification of nuclear and
mitochondrial DNA respectively. Average threshold cycle number (Ct) values were
obtained by amplification of CoII (mtDNA-Specific) and β-actin (nDNA-specific).
mtDNA content was determined as 2^ΔCt, or fold difference of mtDNA from nDNA.
Statistical analysis- all data shown are a statistical analysis of at least 3 independent
experiments.
Figure 2: A. Neem induced time-dependent cell death in HCT
116 WT cells. B. LNCaP, PPC1, and MDA cells were treated
with neemoids at 36h.
independent apoptotic cell death
inhibition of autophagy enhances caspase activated
cell death
Bax activation and oligomerization
mitochondria fragmentation or PTP
cancer cell death
primarily target the mitochondria and the modulation
of multiple cell death pathways could have significant
potential in treatment of cancer.
Roswell Summer Research
The American Cancer Society
Figure 1: Apoptosis, above is a simplified diagram of the mitochondrial
and death-receptor pathways of apoptosis.
Growth of cancer cells is characterized by dysregulated proliferation and
evasion of cell death mechanisms. Apoptosis is the process by which cell
death occurs in a regulated manner and is critical for growth. Many of the
cancer therapeutics available today target the apoptosis pathway for
treatment. However, the need to find more effective therapy with
minimal toxicities remains paramount. One promising agent is
Azadirachta indica, more widely known as neem. Neem contains many
phytochemicals, called limonoids, which possess anticancer properties. It
has been shown to have anti-inflammatory, anti-angiogenic and anti-
oxidant effects. In this study, we show that treatment of multiple cancer
cell types with neem leads to the activation of caspase-8 and caspase-9
independent of Bax status, release of cytochrome c and apoptosis-
inducing factor (AIF) from the mitochondria to activate caspase
dependent as well as AIF-mediated apoptosis. Loss of p53 had no affects
on apoptosis. In HcT116-wt and -p53 null, treatment with neem resulted
in a decrease in cellular levels of Bim, Bid, Bax and Bak. Lack of Bax
oligomerization and depletion of Bax on mitochondria combined with
similar levels of caspase activities in WT and Bax-null cells supported
that neem induces caspase activation independent of Bax status. Neem-
induced caspase activation was not affected by cyclosporin A treatment,
hence, excluding a role for permeability transition pore. Treatment with
neem resulted in increased mitochondrial DNA content during apoptosis
suggesting that mitochondria are a primary target for neem induced cell
death. Interestingly, we found that neem treatment resulted in
accumulation of LC3-II in cancer cells, suggesting the involvement of
autophagy in neem-induced cancer cell death. We also show that
inhibition of caspases enhanced neem-induced autophagy.
3. Neem induces apoptotic cell death
4. Neem induces caspase-9 mediated mitochondrial apoptosis
A
was increased upon neem
treatment. B. z-VAD and
LY294002 treatments do not
drug treatments.
Figure 4: A. Cleavage of both caspase 8 and 9
is seen at 36h. B-C. There is an increase in
caspase 9 activity as well as increase in
caspase 8 activity. D-E. Caspase 9 cleavage is
seen earlier in the neem treatments then
cleavage of caspase 8. F. Caspase 3 cleavage
in caspase 9 knockdowns was decreased as
compared to caspase 3 cleavage in caspase 8
knockdowns.
5. Release of cytochrome c and AIF is accompanied by caspase-3 activation
Figure 5: A. Cytochrome c release from the mitochondria can be
seen beginning after 12 hrs of neem treatment. B. An increase in
the release of cytochrome c from mitochondria can be seen in the
immunolabeled neem treated cells.
7: Inhibition of autophagy enhances caspase activation and cell death
Figure 6: A. Significant cell death was seen even when z-Vad inhibited
caspase activity. B. Cleavage of LC3 II indicates autophagy.
Figure 7: A. Neem induced cell death was significant even when
combined with 3-MA a known autophagy inhibitor. B. Significant
increase of caspase 3 was seen when autophagy was inhibited. C.
LC3-I accumulation prevents cleavage to LC3-II. D. ATG5 and
Beclin-1 ShRNA knockdowns. E. Knockdown of ATG5 and
Beclin-1 in HCT116 cells results in increased cell death.
LC3
Autolysosome
Lysosome
Autophagosome
Phagophore
Figure 8: above is a schematic model of autophagy
Chandra D, Singh KK. Genetic insights into OXPHOS defect and its role in cancer. Biochim Biophys Acta. 2011 Jun;1807(6):620-5. Gogada R, Yadav N, Liu J, Tang S, Zhang D, Schneider A, Seshadri A, Sun L, Aldaz CM, Tang D, Chandra D. BIM, a proapoptotic protein, upregulated via transcription factor E2F1-dependent mechanism, functions as a prosurvival molecule in cancer. J Biol Chem. 2013 Jan 4;288(1):368-81. Srivastava P, Yadav N, Lella R, Schneider A, Jones A, Marlowe T, Lovett G, O'Loughlin K, Minderman H, Gogada R, Chandra D. Neem oil limonoids induces p53-independent apoptosis and autophagy. Carcinogenesis. 2012 Nov;33(11):2199-207. Green DR. Apoptotic pathways: the roads to ruin. Cell. 1998 Sep 18;94(6):695-8.