Apoptotic Mode Of Cell Death Research Articles

Antiproliferative and apoptotic effects of Sesbania grandiflora leaves in human cancer cells.

Natural phytochemicals and their derivatives are good drug candidates for anticancer therapeutic approaches against multiple targets. We report here the initial findings from our studies on the anticancer properties of the leaves of the medicinal plant Sesbania grandiflora. In the current study, five different solvent fractions from the leaves of S. grandiflora were tested on cancer cell lines such as MCF-7, HepG2, Hep-2, HCT-15, and A549. The methanolic fraction of S. grandiflora was found to exert potent antiproliferative effects especially in the human lung cancer cell line, A549. Caspase 3 was activated in the methanolic fraction treated A549 cells thereby leading to cell death by apoptosis. DAPI staining, DNA laddering, and decrease in mitochondrial membrane potential further confirmed the apoptotic mode of cell death. The high levels of ROS intermediates as evidenced by DCF-DA staining could have played a role in the apoptotic induction. Decrease in levels of cyclin D1 and decrease in the activation of NFkB were observed in A549 cells on treatment with methanolic fraction, giving a hint on the possible mechanism of action. These results prove that the medicinal plant S. grandiflora can be explored further for promising candidate molecules to combat cancer, especially lung cancer.

Mechanism of cytotoxicity by Psoralea corylifolia extract in human breast carcinoma cells.

Psoralea corylifolia has been widely used in herbal medicine, and a few studies show its anticancer activity. However, the detailed mechanism of the anticancer activity of P. corylifolia seed extract (PC extract) was not studied. This study evaluates the anticancer activity and underlying mechanism of PC extract in a human breast cancer cell line (MCF7). PC extract caused a concentration-dependent decrease in the proliferation of MCF7 cells and an increase in apoptotic death as measured by annexin-V-FITC and TUNEL assays. Increased cleavage of poly(ADP-ribose) polymerase in cells treated with PC extract further confirmed the apoptotic mode of cell death. There was a decrease (~2-fold) of mitochondrial membrane potential in cells treated with PC extract. In cells treated with PC extract, an increase in intracellular reactive oxygen species (ROS) and a decrease in mitochondrial ROS was observed. A significant decrease in ATP (~1.8-fold) was observed in extract-treated cells. Moreover, MCF7 cells treated with extract showed cleavage of caspase-9 and caspase-7, upregulation of Bax, release of cytochrome-c, and loss of mitochondrial integrity. Taken together, these results suggest the involvement of the mitochondrial pathway in PC extract-induced apoptosis in MCF7 cells.

Gliotoxin Isolated from Marine Fungus Aspergillus sp. Induces Apoptosis of Human Cervical Cancer and Chondrosarcoma Cells

Gliotoxin, a secondary metabolite produced by marine fungus Aspergillus sp., possesses various biological activities including anticancer activity. However, the mechanism underlying gliotoxin-induced cytotoxicity on human cervical cancer (Hela) and human chondrosarcoma (SW1353) cells remains unclear. In this study, we focused on the effect of gliotoxin induction on apoptosis, the activating expressions of caspase family enzymes in the cells. Apoptotic cell levels were measured through DAPI and Annexin V/Propidium Iodide (PI) double staining analysis. The apoptotic protein expression of Bcl-2 and caspase family was detected by Western blot in Hela and SW1353 cells. Our results showed that gliotoxin treatment inhibited cell proliferation and induced significant morphological changes. Gliotoxin induced apoptosis was further confirmed by DNA fragmentation, chromatin condensation and disrupted mitochondrial membrane potential. Gliotoxin-induced activation of caspase-3, caspase-8 and caspase-9, down-regulation of Bcl-2, up-regulation of Bax and cytochromec (cyt c) release showed evidence for the gliotoxin activity on apoptosis. These findings suggest that gliotoxin isolated from marine fungus Aspergillus sp. induced apoptosis in Hela and SW1353 cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death.

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug, Gemcitabine

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking or reversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategies to reduce cancer related death. One such promising chemopreventive agent which has attracted considerable attention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. The present study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent, gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the results by nuclear morphological examination. Further we analyzed the modulation of expression of Bcl-2 and COX-2 on treatment of these cells with SFN by RT-PCR. SFN showed cytotoxic effects on MCF-7 cells in a dose- and time-dependent manner via an apoptotic mode of cell death. In addition, a combinational treatment of SFN and gemcitabine on MCF-7 cells resulted in growth inhibition in a synergistic manner with a combination index (CI) <1. Notably, SFN was found to significantly downregulate the expression of Bcl-2, an anti-apoptotic gene, and COX-2, a gene involved in inflammation, in a time-dependent manner. These results indicate that SFN induces apoptosis and anti-inflammatory effects on MCF-7 cells via downregulation of Bcl-2 and COX-2 respectively. The combination of SFN and gemcitabine may potentiate the efficacy of gemcitabine and minimize the toxicity to normal cells. Taken together, SFN may be a potent anti-cancer agent for breast cancer treatment.

Induction of HepG2 cell apoptosis by Irgarol 1051 through mitochondrial dysfunction and oxidative stresses

In this study, HepG2 cells were exposed to 0.04–40mg/L Irgarol 1051. Results show that Irgarol 1051 can damage cell morphology and cause a significant decrease in cell viability. Positive staining by Annexin V, caspase-3 activity enhancement, and the damage in cell ultrastructure indicated an apoptotic mode of cell death for 4.0mg/L Irgarol 1051 treatment. At the same time, caspase-9 was also significantly induced by 0.4 and 4.0mg/L Irgarol 1051 at 72h, which suggests that the intrinsic mitochondria pathway was involved in the apoptosis. The mitochondrial membrane potential decreased significantly after the HepG2 cells were exposed to Irgarol 1051 for 6 and 72h. Especially, the translocation of cytochrome c from mitochondria to cytosol was recorded, supporting the idea that the mitochondrial pathway was involved in the apoptosis signal pathways induced by Irgarol 1051. The significantly increased levels of intracellular reactive oxygen species (ROS) and an immediate ROS burst were also recorded. The results here may imply that Irgarol 1051 induces HepG2 cell apoptosis through mitochondrial dysfunction and oxidative stresses. Although it is possible that this chemical has no detrimental effects on human health at the environmentally relevant concentration, it may cause problems to top coastal predators due to bio-accumulation through the food chain.

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca2+ rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca2+ level and of their accumulation in lipid raft microdomains.

Photodynamic therapeutic activity of indocyanine green entrapped in polymeric nanoparticles

Photodynamic therapy (PDT) is a therapeutic modality involving the use of a photosensitizer agent activated by light of appropriate wavelength to selectively destroy tumor cells. Indocyanine green (ICG) is a promising photosensitive agent for PDT of tumor cells. The main disadvantage of using ICG in PDT is the instability of ICG in aqueous solutions. Encapsulating ICG dye in a biocompatible matrix based on PEBBLE technology showed an improvement of aqueous stability comparing with free ICG dye. The main objective of this study is to investigate the photodynamic effect of ICG-ormosil PEBBLEs on two different cell lines: human breast adenocarcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2). ICG-embedded ormosil PEBBLEs were synthesized based on a sol-gel process, and characterized by transmission electron microscopy and other fluorescence tests. The cell viability was evaluated by MTT and trypan blue assays. Apoptosis, necrosis, and DNA damage (comet assay), were evaluated by fluorescence microscopic tests. The results declared that ICG-ormosil PEBBLEs and free ICG both have the same cytotoxic and phototoxic effect on MCF-7 and HepG2 cell lines, where the apoptotic mode of cell death is preferentially occurred in case of PDT using ICG-ormosil PEBBLEs. Both ICG and ICG-ormosil PEBBLEs induced DNA damage after laser exposure. These results would suggest that entrapping ICG in Polymeric nanoparticles forming ICG-ormosil PEBBLEs improve the aqueous stability of the photosensitizer and in the same time retain its photodynamic activity, suggesting that it is preferred to use ICG-ormosil PEBBLEs instead of free ICG dye.

Synthesis, characterization and cytotoxicity studies of platinum(II) complexes with amino acid ligands in various coordination modes

Reactions of [Pt(CO3)(PPh3)2]·CH2Cl2 (1) with non-substituted and alkyl substituted amino acids, NH(R)CH(R′)CO2H (R/R′=H/Me, L1; H/iPr, L2; H/CH2CHMe2, L3; Me/H, L4; Et/H, L5), in the presence of Tl[PF6] in methanol afforded with liberation of CO2 the formation of platinum(II) complexes of the type [Pt(PPh3)2{NHR–CHR′–C(O)O-κN,κO}][PF6] (R/R′=H/Me, 2; H/iPr, 3; H/CH2CHMe2, 4; Me/H, 5; Et/H, 6). Single-crystal X-ray diffraction analysis of complex 4 exhibited a square-planar coordination of the platinum atom having coordinated two triphenylphosphane ligands and a deprotonated κN,κO-coordinated leucine ligand (L3−H). On varying the pKa value of the amino group, platinum(II) complexes with different coordination modes of amino acid ligands were obtained. Thus, treatment of complex 1 with N-acetyl l-alanine (L6), possessing a comparatively highly acidic NH proton, in 1:1 ratio in methanol resulted in the formation of [Pt(PPh3)2{N(COMe)–CHMe–C(O)O-κN,κO}] (7), while reacting N-phenyl glycine (L7) having a moderately acidic NH proton with complex 1 afforded a mixture of complexes [Pt(PPh3)2{NPh–CH2–C(O)O-κN,κO}] (8) and [Pt(PPh3)2{NHPh–CH2–C(O)O-κO}2] (10). Treatment of complex 1 with two equivalents of L6/L7 in dichloromethane resulted in the formation of [Pt(PPh3)2{NHR–CHR′–C(O)O-κO}2] (R/R′=COMe/Me, 9; Ph/H, 10). An analogous reactivity was observed for l-lactic acid on treating with complex 1 in 1:1 and 2:1 ratio resulting in [Pt(PPh3)2{O–CHMe–C(O)O-κO,κO′}] (11) and [Pt(PPh3)2{HO–CHMe–C(O)O-κO}2] (12). The identities of all complexes have been proven by NMR (1H, 13C, 31P) spectroscopic and high-resolution ESI mass-spectrometric investigations. In vitro cytotoxicity studies against human tumor cell lines (8505C, A2780, HeLa, SW480, and MCF-7) showed the highest activities for the neutral complex 7. Furthermore, complexes 7 and 9 against the A2780 cell line induced an apoptotic mode of cell death, which was further supported by morphological investigation and DNA laddering. Cell cycle perturbation studies showed that both complexes induced faster cell death than cisplatin.

Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

Cucurbitacin induces autophagy through mitochondrial ROS production which counteracts to limit caspase-dependent apoptosis

Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

New Therapeutic Strategy for Sensitisation of CLL Cells with Inactivation of the DNA Damage Response by Targeting the Deubiquitylating Enzyme USP7-Dependent Pathways,

Abstract 3865Chronic Lymphocytic Leukaemia (CLL) is characterised by marked clinical heterogeneity and tumours with deletion or mutation of the TP53 and ATM genes on chromosomes 17p and 11q are associated with poor outcome. ATM is a protein kinase that, following induction of DNA double strand breaks (DSBs), phosphorylates a number of downstream targets and synchronises a network of cellular responses including p53 activation that leads to induction of pro-apoptotic genes and activation of apoptosis. Consequently, loss of integrity of ATM/p53 pathway results in apoptotic resistance, retention of cells with genomic damage, and tumour progression.Cellular p53 levels are regulated through a p53-Mdm2 regulatory loop whereby Mdm2, a ubiquitin ligase, facilitates p53 polyubiquitination and targeting of p53 for proteasome degradation. It was recently shown that stabilisation of p53 can be achieved by manipulation of this regulatory loop through use of small molecule inhibitors of this p53 degradation pathway, termed nutlins, which prevent p53 ubiquitination. More recently, a class of deubiquitinating enzymes (DUBs) highlighted an additional level of p53-Mdm2 regulation. In particular, a specific DUB, (USP7/HAUSP), has a high affinity for Mdm2 and functions by antagonizing Mdm2 ubiquitination. Unlike nutlins, USP7 also has been implicated in the regulation of cell cycle, mitosis and DNA damage response and we reasoned that USP7 inhibition may sensitise CLL tumours with ATM and TP53 defects.Our analysis of 25 primary CLL tumours with different ATM and TP53 status indicated that USP7 was robustly expressed in all CLL tumour cells tested. Through collaboration with Hybrigenics we obtained a specific USP7 inhibitor, HBX19818. To determine if cell killing could be induced in ATM or TP53 deficient tumours by inhibiting the USP7-Mdm2 pathway, we analysed the induction of cell death over a range of HBX19818 concentrations using both isogenic CLL cell lines with and without ATM and/or p53, as well as 18 primary CLL tumours. We observed a significant cytotoxic effect of HBX19818 in isogenic CLL lines, at concentrations between 1–10μM, irrespective of their ATM and TP53 status. Strikingly, the majority of primary CLL tumours were sensitive to HBX19818 concentrations between 8μM and 16μM to which non-tumour PBMCs were resistant.Western blotting was used to monitor the induction of p53, apoptosis (associated with caspase 7 and PARP1 cleavage), and also whether DNA damage was induced (as measured by H2AX phosphorylation) in response to HBX19818. Our analysis revealed that pharmacological inhibition of USP7 led to p53 upregulation in the p53 proficient CLL cells associated with a robust induction of p21 indicating that the stabilised p53 was active. This response was absent in Mec-1 cell line with non-functional p53. Interestingly, caspase 7 and PARP1 were only cleaved in the ATM wild type CLL cell lines suggesting that activated p53 was capable of inducing cell death. In contrast, the ATM and p53 deficient CLL cell lines did not exhibit any markers suggesting an apoptotic mode of cell death. Rather, these cell lines displayed elevated levels of phospho-H2AX, suggesting the induction of DNA damage, possibly caused by an underlying DNA repair defect. Consistent with our hypothesis, both p53 proficient and p53 non-functional CLL cell lines failed to induce the recruitment of the HR protein Rad51 to sites of IR-induced DNA double strand breaks.Taken together, our data implies that in addition to p53 activation, pharmacological inhibition of USP7 can exert a cytotoxic effect by further mechanisms, possibly by modulating DNA double strand break repair. This is consistent with previous reports suggesting that USP7 regulates monoubiquitination of transcription factor FOXO4 and is involved in the regulation of DNA repair and mitotic progression via its interactions with Claspin and Chfr respectively. We suggest that pharmacological inhibition of USP7 represents a promising target for the treatment of tumours with defective ATM and p53 signalling. Disclosures:No relevant conflicts of interest to declare.

English

Andrographolide (AGP), the major phytoconstituent isolated from&nbsp;Andrographis paniculata&nbsp;was found to exhibit growth inhibition and cytotoxicity against the hormone-independent (PC-3 and DU-145) and hormone-dependent (LNCaP) prostate cancer cell lines&nbsp;via&nbsp;the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards PC-3 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated PC-3 cells whereas Annexin V-FITC/PI flow cytometry analysis was carried out to confirm apoptosis induced by AGP in PC-3 cells. Cell cycle and apoptotic regulatory proteins were determined by western blot analysis. AGP was found to induce G2/M cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, AGP was found to downregulate CDK1 without affecting the levels of CDK4 and cyclin D1. Induction of apoptosis was associated with an increase in activation and expression of caspase 8 which then is believed to have induced cleavage of Bid into tBid. In addition, activation and enhancement of executioner caspase 9 and Bax proteins without affecting Bcl-2 protein levels were observed. &nbsp; Key words:&nbsp;Andrographolide, prostate cancer, cell cycle, cyclin-dependent kinases, apoptosis, caspase 8, caspase 9, Bcl-2 family.

Iron enhances generation of free radicals by Artemisinin causing a caspase-independent, apoptotic death in Leishmania donovani promastigotes

An increasing incidence of unresponsiveness to antimonials in Leishmaniasis has led to identification of plant-derived anti-leishmanial compounds like Artemisinin. Since iron-mediated generation of free radicals sustains the anti-malarial activity of Artemisinin, this study investigated whether similar mechanisms accounted for its activity in Leishmania promastigotes. Artemisinin effectively disrupted the redox potential via an increased generation of free radicals along with a decrease in levels of non-protein thiols. Attenuation of its IC50 by a free radical scavenger N-acetyl l-cysteine and an iron chelator desferoxamine established the pivotal role of free radicals and of the potentiating effect of iron. An enhanced Fluo-4 fluorescence reflected Artemisinin-induced mobilization of intracellular calcium, which triggered apoptosis. However, the absence of any detectable caspase activity indicated that the leishmanicidal activity of Artemisinin is mediated by an iron-dependent generation of reactive intermediates, terminating in a caspase-independent, apoptotic mode of cell death.

Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species

The present study investigated the apoptosis and DNA damage inducing potential of chlorpyrifos (CP) in Drosophila melanogaster. Third instar larvae of Drosophila were treated with different concentrations of CP (0.015–15.0μg/L) for 2–48h. Reactive oxygen species (ROS) generation, oxidative stress markers, DNA damage and apoptotic cell death end points were measured in them. A significant increase in DNA damage was concomitant with apoptotic mode of cell death in 15.0μg/L CP-treated organisms for 24 and 48h. Depolarization in mitochondrial membrane potential and increased casapase-3 and caspase-9 activities in these organisms indicated both as potential targets of CP. A significant positive correlation was observed among ROS generation, apoptosis and DNA damage. The study suggests that (i) ROS may be involved in inducing apoptosis and DNA damage in the CP-exposed larvae of Drosophila and (ii) D. melanogaster may be used as an alternative in vivo animal model for xenobiotics hazard assessment.

Small structural changes of pentacyclic lupane type triterpenoid derivatives lead to significant differences in their anticancer properties

In the present investigations five new derivatives of betulinic and betulonic acid were synthesized and the effect of this structural variations on anticancer activity was studied and discussed. The antiproliferative activity of betulinic and betulonic acid derivatives was studied against eight tumor cell lines of different histogenic origin. The derivatives exerted a dose dependent antiproliferative action at micromolar concentrations toward target tumor cell lines. The apoptotic mode of cell death on colon cancer cell line HT-29 was induced by the most active compounds 5, 2-amino-3-hydroxy-2-(hydroxymethyl)propyl (3- O-acetyl)betulinate, and 9, 2-amino-3-hydroxy-2-(hydroxymethyl)propyl betulonate. Treatment of HT-29 cells with 5 and 9 induced apoptosis, as observed by dye exclusion test (trypan blue) and by the appearance of a typical ladder pattern in the DNA fragmentation assay and FITC annexin V assay. Cell cycle perturbations caused by compound 5 are also presented.

Rimonabant-induced apoptosis in leukemia cell lines: Activation of caspase-dependent and -independent pathways

Rimonabant (SR141716), a cannabinoid CB1 receptor antagonist known for anti-obesity activity, has more recently been shown to inhibit tumor cell growth. Here we demonstrated the antitumor potential of SR141716 in leukemia-derived cell lines and its low toxicity in normal cells (PBMC). SR141716 (1–20μM range of doses) reduced Jurkat and U937 cell number by activating death signals as well as affecting cell cycle progression. The most prominent response in U937 to SR141716 was a G0/G1 block, while in Jurkat cells there was activation of cell death processes. SR141716-treated cells exhibited the morphological and biochemical features of apoptosis and to some extent necrosis. Apoptotic mode of cell death was confirmed in both cell lines by analysis of cell morphology, phosphatidylserine exposure and DNA fragmentation. Moreover, the drug was found to induce an early and robust mitochondrial membrane depolarization. In Jurkat cells the apoptotic process was typically caspase-dependent, while in U937 caspase-independent pathways were also activated. The contribution of PARP activation to SR141716-induced apoptosis in U937 was suggested by protein PARylation, AIF release and apoptosis reversal by PARP inhibitors. Moreover, SR141716 negatively modulated, especially in U937, the PI3K/AKT pathways. In conclusion, our data indicate that SR141716 elicits alternative response and/or cell death pathways depending on the cell type affected.

Abstract 5550: Autophagic potentiation of radiosensitization by vitamin D3 and the analog, EB1089

Abstract Introduction: While chemotherapy and radiation treatment may be initially effective at suppressing breast cancer growth, disease recurrence and tumor metastasis are major problems in the clinic. A number of strategies have been developed in an attempt to enhance the response to radiation and prevent tumor cell recovery by interfering with cytoprotective signaling. Our current studies are designed to investigate the nature of cell death when 1-25 Vitamin D3 (1,25D3) is utilized to enhance sensitivity to fractionated radiation (FIR) in the MCF-7, BT474, and Hs578t breast cancer cell lines. Results: Previous studies in our laboratory indicate that a 72 hour exposure of MCF-7 cells to 100nM of the Vitamin D3 analog, EB1089, prior to 5×2Gy FIR resulted in an 80% reduction in viable cell number over 96 hours post-irradiation. EB1089 also sensitized MCF-7 cells to FIR in terms of clonogenic survival. Although apoptosis, mitotic catastrophe and senescence are all evident under these experimental conditions, our studies strongly suggest that autophagy plays a central role in the radiosensitization effects of EB1089 as well as attenuation of the proliferative recovery that is observed with irradiation alone. To further define the role of autophagy in radiation sensitization by EB 1089, cells were exposed to the autophagy inhibitor, chloroquine, prior to EB1089 and irradiation. Chloroquine had no effect on sensitivity to radiation alone and enhanced sensitivity to tamoxifen, consistent with the cytoprotective effects of autophagy in the case of this estrogen antagonist. Inhibition by chloroquine of autophagy induced by EB1089 + FIR did not interfere with the apparent radiosensitization by EB1089, as the cells appeared to shift to an apoptotic mode of cell death. Studies are in progress utilizing genetic knockdown of the expression of autophagy related genes such as ATG5 and Beclin-1 to confirm that interference with autophagy drives the cells towards alternative modes of cell death. Additional studies in progress are designed to evaluate the effects of 1,25D3 treatment on radiation sensitivity in breast tumor cells that are resistant to irradiation such as Hs578t breast cancer cells, in addition focusing on breast tumor cells that over express Her-2/neu (MCF7/HER2, isogenic with the parental MCF-7 cell line, as well as p53 mutant BT474 cells with endogenous over expression of Her-2). Conclusions: Vitamin D irreversibly commits MCF7 breast tumor cells towards cell death upon subsequent exposure to irradiation. Inhibition of autophagy drives the cell towards alternative modes of cell death, primarily apoptosis (but possibly mitotic catastrophe). EB1089 and 1,25D3 may prove to also promote sensitivity to radiation (and chemotherapy) in breast tumor cells that are intrinsically resistant to treatment through the overexpression of cytoprotective signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5550.

SRJ23, A NEW INVESTIGATIONAL ANTI‐PROSTATE CANCER AGENT

In our drug discovery programme, from natural products andrographolide (AGP), the major phytoconstituent of Andrographis paniculata, was identified to have anticancer activity in vitro and in vivo. For improvement of the anti‐cancer potential of AGP, a series of semisynthetic derivatives of this compound was synthesized and tested for in vitro anticancer activity. From the National Cancer Institute (NCI) of USA in vitro anticancer screen, SRJ23 was found to exhibit selective anti‐cancer activity against prostate cancer cell lines (Jada et al., 2008. Br J Pharmacol 155: 641–654). The microculture tetrazolium (MTT) assay was utilized in assessing the in vitro growth inhibition and cytotoxicity of SRJ23 against three different human prostate cancer cell lines, PC‐3, DU‐145 and LNCaP. Subsequently, fluorescence microscopy was performed to determine the morphological cell death. Flow cytometry was used to analyze the cell cycle distribution of treated cells. SRJ23 at low micromolar concentrations exhibited growth inhibition and cytotoxicity in all three prostate cancer cell lines. The activity of the compound was selective towards PC‐3 cells and induced G2/M arrest which led to predominantly apoptotic mode of cell death. Interestingly, SRJ23 induced G1 cell cycle arrest in LNCaP and DU‐145 cell lines. Presently, SRJ23 is being considered a lead compound in the discovery of clinical anti‐prostate cancer agent. This research project is supported by Universiti Putra Malaysia.

Apoptosis in HepG2 cells exposed to high glucose

Hyperglycemia which characterizes diabetes, leads to several abnormalities in the cellular pathways. We examined the toxicity of glucose in human hepatoma HepG2 cells. HepG2 cells when incubated with 50 mM glucose for 72 h showed altered morphology i.e. presence of detached and shrunken rounded cells. Glucose treated HepG2 cells also exhibited a significant decrease in viability. Caspase-3 activity and Annexin V staining were significantly increased in glucose treated HepG2 cells, suggesting an apoptotic mode of cell death. Glucose induced apoptosis in HepG2 cells was a consequence of increased oxidative stress as evidenced by the increased reactive oxygen species (ROS) level, lipid peroxidation, protein carbonyl and 3-nitrotyrosine adduct formation. The intracellular antioxidant glutathione was found to be increased in HepG2 cells treated with glucose, possibly to aid the cells to overcome the persistent oxidative stress elicited by glucose in HepG2 cells. N-Acetyl cysteine, a precursor of glutathione and an antioxidant was effective in reversing the morphological changes, increasing the viability, decreasing the ROS level and 4-hydroxynonenal and 3-nitrotyrosine adduct formation, thus validating the role of oxidative stress as a major mechanism for glucose induced apoptosis in HepG2 cells. These results suggest that glucose induces apoptosis in liver cells through increased oxidative stress.

Chemical constituents and in vitro anticancer activity of Typhonium flagelliforme (Araceae)

Aim of the study Typhonium flagelliforme is an indigenous plant of Malaysia and is used by the local communities to treat cancer. This study aims to identify the chemical constituents of Typhonium flagelliforme particularly those which have antiproliferative properties towards human cancer cell lines. Materials and methods Purification of the chemical constituents by various chromatographic procedures was guided by the antiproliferative activity. Identification of the chemical constituents was carried out by various spectroscopic techniques including high resolution MS and NMR. The antiproliferative activity was assayed using MTT on NCI-H23 (lung cancer) and HS578T (breast cancer) cell lines. Microscopic observation and DeadEnd™ colourimetric TUNEL assay was used to identify the apoptotic mode of cell death. Results and conclusion Four pheophorbide related compounds, namely pheophorbide-a, pheophorbide-a′, pyropheophorbide-a and methyl pyropheophorbide-a were identified in the most active fraction, D/F19. These constituents exhibited antiproliferative activity against cancer cells and the activity increased following photoactivation. However, the greater antiproliferative activity exhibited by D/F19 itself compared to the pheophorbides and its other subfractions suggests some form of synergistic action between the constituents. The inhibitory effect of D/F19 and the pheophorbides was apoptotic in the absence of light. Other chemical constituents that have been identified in this study include hexadecanoic acid, oleic acid, linoleic acid, linolenic acid, campesterol, stigmasterol and β-sitosterol. Most of the chemical constituents identified in this plant have not been reported previously.