Novel API Coated Catheter Removes Amyloid-ß from Plasma of Patients with Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by the deposition of Amyloid-beta (Aß) plaques in the brain. We have previously developed Amytrap peptide (the active pharmacological ingredient, API) and linked it to a sepharose bead matrix by click chemistry to form Amytrapper matrix, which was able to bind and remove Aß from human sera and plasma spiked with biotinylated Aß42 (bio-Aß42) in vitro. To extend the logic of the previous studies, the current study investigates whether the Amytrap peptide coated inside a medically viable polycarbonate catheter (Amytrapper catheter) could bind and retain Aß from the human sera. The Amytrapper matrix and the novel Amytrapper catheter were able to bind and retain spiked bio-Aß42 from human sera or native Aß from plasma of AD patients. Additional characteristics of the Amytrapper catheter are evaluated and presented in this study. The results presented here provide a proof-of-principle for the first time that extracorporeal Amytrapper device aids clearance of native Aß (from plasma of AD patients). Thus, our device Amytrapper, either in the form of Sepharose matrix or catheter, could become a novel therapeutic strategy to remove Aß from circulation in AD patients.

Author Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.

Publisher Correction: AMP kinase promotes glioblastoma bioenergetics and tumour growth.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

AMP kinase promotes glioblastoma bioenergetics and tumour growth.

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

Constitutively activated ERK sensitizes cancer cells to doxorubicin: Involvement of p53-EGFR-ERK pathway.

The tumour suppressor gene p53 is mutated in approximately 50% of the human cancers. p53 is involved in genotoxic stress-induced cellular responses. The role of EGFR and ERK in DNA-damage-induced apoptosis is well known. We investigated the involvement of activation of ERK signalling as a consequence of non-functional p53, in sensitivity of cells to doxorubicin. We performed cell survival assays in cancer cell lines with varying p53 status: MCF-7 (wild-type p53, WTp53), MDA MB-468 (mutant p53, MUTp53), H1299 (absence of p53, NULLp53) and an isogenic cell line MCF-7As (WTp53 abrogated). Our results indicate that enhanced chemosensitivity of cells lacking wild-type p53 function is because of elevated levels of EGFR which activates ERK. Additionally, we noted that independent of p53 status, pERK contributes to doxorubicin-induced cell death.

FOXD1-ALDH1A3 Signaling Is a Determinant for the Self-Renewal and Tumorigenicity of Mesenchymal Glioma Stem Cells.

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219-30. ©2016 AACR.

Evolving Lessons on the Complex Role of AMPK in Normal Physiology and Cancer.

AMP kinase (AMPK) is an evolutionarily conserved enzyme required for adaptive responses to various physiological and pathological conditions. AMPK executes numerous cellular functions, some of which are often perceived at odds with each other. While AMPK is essential for embryonic growth and development, its full impact in adult tissues is revealed under stressful situations that organisms face in the real world. Conflicting reports about its cellular functions, particularly in cancer, are intriguing and a growing number of AMPK activators are being developed to treat human diseases such as cancer and diabetes. Whether these drugs will have only context-specific benefits or detrimental effects in the treatment of human cancer will be a subject of intense research. Here we review the current state of AMPK research with an emphasis on cancer and discuss the yet unresolved context-dependent functions of AMPK in human cancer.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

The AMPK inhibitor compound C is a potent AMPK-independent antiglioma agent.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor important for cell growth, proliferation, survival, and metabolic regulation. Active AMPK inhibits biosynthetic enzymes like mTOR and acetyl CoA carboxylase (required for protein and lipid synthesis, respectively) to ensure that cells maintain essential nutrients and energy during metabolic crisis. Despite our knowledge about this incredibly important kinase, no specific chemical inhibitors are available to examine its function. However, one small molecule known as compound C (also called dorsomorphin) has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor. In nearly all these reports including a recent study in glioma, the biochemical and cellular effects of compound C have been attributed to its inhibitory action toward AMPK. While examining the status of AMPK activation in human gliomas, we observed that glioblastomas express copious amount of active AMPK. Compound C effectively reduced glioma viability in vitro both by inhibiting proliferation and inducing cell death. As expected, compound C inhibited AMPK; however, all the antiproliferative effects of this compound were AMPK independent. Instead, compound C killed glioma cells by multiple mechanisms, including activation of the calpain/cathepsin pathway, inhibition of AKT, mTORC1/C2, cell-cycle block at G2-M, and induction of necroptosis and autophagy. Importantly, normal astrocytes were significantly less susceptible to compound C. In summary, compound C is an extremely potent antiglioma agent but we suggest that caution should be taken in interpreting results when this compound is used as an AMPK inhibitor.

Direct inhibition of retinoblastoma phosphorylation by nimbolide causes cell-cycle arrest and suppresses glioblastoma growth.

PURPOSE: Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica, controls glioblastoma growth. EXPERIMENTAL DESIGN: Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. RESULTS: We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro and in vivo. Azt caused cell-cycle arrest, most prominently at the G1-S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1-S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of glioblastoma multiforme growth. CONCLUSIONS: Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.

The direct inhibitory effect of dutasteride or finasteride on androgen receptor activity is cell line specific.

BACKGROUND: Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT. METHODS: A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes. RESULTS: Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited. CONCLUSIONS: The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.

AMPK-mediated autophagy is a survival mechanism in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia.

The present study was designed to investigate (i) the role of AMPK activation in inducing autophagy in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia, and (ii) whether autophagy offers a survival advantage under these harsh conditions. Low androgen and low oxygen are two co-existing conditions frequently found in prostate cancer tissue following surgical or medical castration. In LNCaP cells, androgen deprivation and hypoxia together boosted AMPK activation to a higher level than that seen with either condition alone. The augmented AMPK response was associated with improved viability and the induction of autophagy. These observations suggest that a threshold of AMPK activity has to be attained in order to trigger autophagy, since neither androgen deprivation nor hypoxia by itself was capable of pushing AMPK activity past that threshold. Beclin-1 was identified as a potential downstream target of AMPK in turning on the autophagic cascade. If autophagy was blocked by chemical inhibition or RNA interference of key regulators, e.g., AMPK or beclin-1, more cells would die by apoptosis. The occurrence of autophagy is thus a survival mechanism for androgen-dependent prostate cancer cells to escape from an androgen-deprived and hypoxic subsistence.

The antiandrogenic effect of finasteride against a mutant androgen receptor.

Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild type AR. When PC-3 cells, which are AR-null, were transfected with either the wild type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.

Androgen receptor-mTOR crosstalk is regulated by testosterone availability: implication for prostate cancer cell survival.

BACKGROUND: Signaling between androgen receptor (AR) and mTOR may be crucial for prostate cancer cells to endure the low androgen and suboptimal nutrient conditions produced by androgen deprivation therapy. MATERIALS AND METHODS: AR and mTOR cross-talk was examined in LNCaP cells exposed to either high or low testosterone. AR and mTOR activities were modified separately using either siRNA knockdown or specific chemical inhibitor. The biological significance of the reciprocal communication was assessed by susceptibility to glucose deprivation-induced cell death. RESULTS: AR positively regulated mTOR activity in both low and high testosterone levels. TSC1 and TSC2, the two negative regulators of mTOR, may be involved since both were up-regulated by AR knockdown. Sub-baseline mTOR increased AR protein levels. However, this effect only occurred with low testosterone. More cells underwent apoptosis if AR function was inhibited during glucose deprivation, which significantly depressed mTOR activity. CONCLUSION: The compensatory increase of AR function due to a repressed mTOR signal is advantageous for survival. Disrupting this loop at the time of initiation of androgen deprivation therapy may delay, or even prevent, the recurrence of prostate cancer.

Survival advantage of AMPK activation to androgen-independent prostate cancer cells during energy stress.

Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.

Prx1 enhances androgen receptor function in prostate cancer cells by increasing receptor affinity to dihydrotestosterone.

Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype.

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation.

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.

Bystander killing of breast cancer MCF-7 cells by MDA-MB-231 cells exposed to 5-fluorouracil is mediated via Fas.

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.

Methyl-beta-cyclodextrin enhances the susceptibility of human breast cancer cells to carboplatin and 5-fluorouracil: involvement of Akt, NF-kappaB and Bcl-2.

The response rates of extensively used chemotherapeutic drugs, carboplatin (Carb) or 5-fluorouracil (5-FU) are relatively disappointing because of considerable side effects associated with their high-dose regimen. In the present study, we determined whether treatment with a cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), enhances the weak efficacy of low doses of Carb or 5-FU in human breast cancer cells. Data demonstrate that pretreatment with MCD significantly potentiates the cytotoxic activity of Carb and 5-FU in both MCF-7 and MDA-MB-231. Furthermore, we explored the molecular basis of enhanced cytotoxicity, and our data revealed that low-dose treatment with these drugs in MCD pretreated cells exhibited significantly decreased Akt phosphorylation, NF-kappaB activity and down-regulation in expression of anti-apoptotic protein Bcl-2. In addition, MCD pretreated cells demonstrated an increased intracellular drug accumulation as compared to cells treated with drugs alone. Taken together, our data provide the basis for potential therapeutic application of MCD in combination with other conventional cytotoxic drugs to facilitate reduction of drug dosage that offers a better chemotherapeutic approach with low toxicity.

DNA damaging drugs-induced down-regulation of Bcl-2 is essential for induction of apoptosis in high-risk HPV-positive HEp-2 and KB cells.

DNA damaging chemotherapeutic agents like carboplatin (Carb) and 5-fluorouracil (5-FU), whose effects are mediated through diverse intracellular targets, induce apoptosis in various cancer cells including human papillomavirus (HPV) positive HEp-2 and KB cells. The present work reports the involvement of Bcl-2 in response to the exposure of HEp-2 and KB cells to Carb or 5-FU. We demonstrate that both these drugs are potent inducers of apoptosis. Apoptosis was preceded by decrease in Bcl-2 protein level accompanied by caspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage without altering Bax expression. Further analysis revealed down-regulation of Bcl-2 mRNA as well as protein in drugs treated cells. Ectopic expression of Bcl-2 protected cells against drugs mediated DNA damage-induced apoptosis. Overall, data indicates that genotoxic stress leads to down-regulation of Bcl-2 in HEp-2 and KB cells, which plays a decisive role in the outcome of stress in these cells.