Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway

Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer.

Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 mu M CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFN gamma through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPAR gamma receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2 alpha, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2 alpha induced by LPS/IFN gamma. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation. Cell Death and Disease (2012) 3, e331; doi:10.1038/cddis.2012.71; published online 28 June 2012

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

[EN] Background: Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. Methods: Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results: Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. Conclusions: A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

[EN] Background: DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods: Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results: Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions: An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

Prediction of clinical toxicity in locally advanced head and neck cancer patients by radio-induced apoptosis in peripheral blood lymphocytes (PBLs)

[EN] Head and neck cancer is treated mainly by surgery and radiotherapy. Normal tissue toxicity due to x-ray exposure is a limiting factor for treatment success. Many efforts have been employed to develop predictive tests applied to clinical practice. Determination of lymphocyte radio-sensitivity by radio-induced apoptosis arises as a possible method to predict tissue toxicity due to radiotherapy. The aim of the present study was to analyze radio-induced apoptosis of peripheral blood lymphocytes in head and neck cancer patients and to explore their role in predicting radiation induced toxicity. Seventy nine consecutive patients suffering from head and neck cancer, diagnosed and treated in our institution, were included in the study. Toxicity was evaluated using the Radiation Therapy Oncology Group scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Radiation-induced apoptosis increased in order to radiation dose and fitted to a semi logarithmic model defined by two constants: α and β. α, as the origin of the curve in the Y axis determining the percentage of spontaneous cell death, and β, as the slope of the curve determining the percentage of cell death induced at a determined radiation dose, were obtained. β value was statistically associated to normal tissue toxicity in terms of severe xerostomia, as higher levels of apoptosis were observed in patients with low toxicity (p = 0.035; Exp(B) 0.224, I.C.95% (0.060-0.904)). These data agree with our previous results and suggest that it is possible to estimate the radiosensitivity of peripheral blood lymphocytes from patients determining the radiation induced apoptosis with annexin V/propidium iodide staining. β values observed define an individual radiosensitivity profile that could predict late toxicity due to radiotherapy in locally advanced head and neck cancer patients. Anyhow, prospective studies with different cancer types and higher number of patients are needed to validate these results.

Prediction of clinical toxicity in localized cervical carcinoma by radio-induced apoptosis study in peripheral blood lymphocytes (PBLs)

[EN] Background: Cervical cancer is treated mainly by surgery and radiotherapy. Toxicity due to radiation is a limiting factor for treatment success. Determination of lymphocyte radiosensitivity by radio-induced apoptosis arises as a possible method for predictive test development. The aim of this study was to analyze radio-induced apoptosis of peripheral blood lymphocytes. Methods: Ninety four consecutive patients suffering from cervical carcinoma, diagnosed and treated in our institution, and four healthy controls were included in the study. Toxicity was evaluated using the Lent-Soma scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24, 48 and 72 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide to determine early and late apoptosis. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Results: Radiation-induced apoptosis (RIA) increased with radiation dose and time of incubation. Data strongly fitted to a semi logarithmic model as follows: RIA = βln(Gy) + α. This mathematical model was defined by two constants: α, is the origin of the curve in the Y axis and determines the percentage of spontaneous cell death and β, is the slope of the curve and determines the percentage of cell death induced at a determined radiation dose (β = ΔRIA/Δln(Gy)). Higher β values (increased rate of RIA at given radiation doses) were observed in patients with low sexual toxicity (Exp(B) = 0.83, C.I. 95% (0.73-0.95), p = 0.007; Exp(B) = 0.88, C.I. 95% (0.82-0.94), p = 0.001; Exp(B) = 0.93, C.I. 95% (0.88-0.99), p = 0.026 for 24, 48 and 72 hours respectively). This relation was also found with rectal (Exp(B) = 0.89, C.I. 95% (0.81-0.98), p = 0.026; Exp(B) = 0.95, C.I. 95% (0.91-0.98), p = 0.013 for 48 and 72 hours respectively) and urinary (Exp(B) = 0.83, C.I. 95% (0.71-0.97), p = 0.021 for 24 hours) toxicity. Conclusion: Radiation induced apoptosis at different time points and radiation doses fitted to a semi logarithmic model defined by a mathematical equation that gives an individual value of radiosensitivity and could predict late toxicity due to radiotherapy. Other prospective studies with higher number of patients are needed to validate these results.

Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim axis and reveals glutathione-S-transferase P1 as Achilles' heel

Glutathione-S-transferase of the Pi class (GSTP1) is frequently overexpressed in a variety of solid tumors and has been identified as a potential therapeutic target for cancer therapy. GSTP1 is a phase II detoxification enzyme and conjugates the tripeptide glutathione to endogenous metabolites and xenobiotics, thereby limiting the efficacy of antitumor chemotherapeutic treatments. In addition, GSTP1 regulates cellular stress responses and apoptosis by sequestering and inactivating c-Jun N-terminal kinase (JNK). Thiazolides are a novel class of antibiotics for the treatment of intestinal pathogens with no apparent side effects on the host cells and tissue. Here we show that thiazolides induce a GSTP1-dependent and glutathione-enhanced cell death in colorectal tumor cell lines. Downregulation of GSTP1 reduced the apoptotic activity of thiazolides, whereas overexpression enhanced it. Thiazolide treatment caused strong Jun kinase activation and Jun kinase-dependent apoptosis. As a critical downstream target of Jun kinase we identified the pro-apoptotic Bcl-2 homolog Bim. Thiazolides induced Bim expression and activation in a JNK-dependent manner. Downregulation of Bim in turn significantly blocked thiazolide-induced apoptosis. Whereas low concentrations of thiazolides failed to induce apoptosis directly, they potently sensitized colon cancer cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic drug-induced cell death. Although GSTP1 overexpression generally limits chemotherapy and thus antitumor treatment, our study identifies GSTP1 as Achilles' heel and thiazolides as novel interesting apoptosis sensitizer for the treatment of colorectal tumors.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Human mast cells undergo TRAIL-induced apoptosis

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions.

p53-induced apoptosis occurs in the absence of p14(ARF) in malignant pleural mesothelioma

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.