Expression Levels Of Anti-apoptotic Proteins Research Articles

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy.

Acidic polysaccharide of Panax ginseng regulates the mitochondria/caspase-dependent apoptotic pathway in radiation-induced damage to the jejunum in mice

Owing to its susceptibility to radiation, the small intestine of mice is valuable for studying radioprotective effects. When exposed to radiation, intestinal crypt cells immediately go through apoptosis, which impairs swift differentiation necessary for the regeneration of intestinal villi. Our previous studies have elucidated that acidic polysaccharide of Panax ginseng (APG) protects the mouse small intestine from radiation-induced damage by lengthening villi with proliferation and repopulation of crypt cells. In the present study, we identified the molecular mechanism involved. C57BL/6 mice were irradiated with gamma-rays with or without APG and the expression levels of apoptosis-related molecules in the jejunum were investigated using immunohistochemistry. APG pretreatment strongly decreased the radiation-induced apoptosis in the jejunum. It increased the expression levels of anti-apoptotic proteins (Bcl-2 and Bcl-XS/L) and dramatically reduced the expression levels of pro-apoptotic proteins (p53, BAX, cytochrome c and caspase-3). Therefore, APG attenuated the apoptosis through the intrinsic pathway, which is controlled by p53 and Bcl-2 family members. Results presented in this study suggest that APG protects the mouse small intestine from irradiation-induced apoptosis through inhibition of the p53-dependent pathway and the mitochondria/caspase pathway. Thus, APG may be a potential agent for preventing radiation induced injuries in intestinal cells during radio-therapy such as in cancer treatment.

Diazoxide and cyclosporin A protect primary cholinergic neurons against beta-amyloid (1-42)-induced cytotoxicity

Objective: Activation of mitochondrial (MitoKATP) channels was found to protect against anoxic and chemical stress in brain. This present study sought to investigate the ability of diazoxide and cyclosporin A to antagonize cytotoxicity induced by beta-amyloid peptide (A-beta1-42) in cultured rat primary basal forebrain cholinergic neurons.Methods: Cytotoxicity was induced by A-beta1-42 (2 μM) in the presence of either diazoxide (500 μM), a selective opener of the mitochondrial ATP-sensitive potassium channel (MitoKATP), or cyclosporin A (20 μM), an inhibitor of the mitochondrial permeability transition pore (MTP), or the combination of both the reagents. We determined cell morphology and cell viability using MTT assay and expression levels of anti-apoptotic protein (Bcl-2), pro-apoptotic proteins (Bax, cytochrome C, caspase-3 and cleaved caspase-3) using Western blotting at 24 hours and 72 hours.Results: Cell viability decreased markedly after exposure to A-beta1-42 for 72 hours with a decrease in the expression of Bcl-2 protein and cytochrome C and an increase in the caspase-3 and cleaved caspase-3 levels. Both diazoxide and cyclosporin A exerted significant protective effects on cell viability by ameliorating the decrease in Bcl-2 and the increase in cytochrome c and caspase-3 activity induced by A-beta1-42. The combination of both the reagents had a greater protective effect than either one alone.Conclusions: The present research demonstrates that activation of MitoKATP channels independently or in combination with inhibitors of the MTP can elicit a protective effect against primary cholinergic neuron cytotoxicity induced by A-beta1-42. These findings suggest new mitochondrial targets for the development of therapeutic agents against A-beta-induced cytotoxicity.

Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with other agents is a promising strategy to overcome TRAIL resistance in malignant cells. Wogonin, a flavonoid originated from Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL's antitumor activity in vivo has never been studied. In this study, the effect of combination of TRAIL and wogonin was tested in a non-small-cell lung cancer xenografted tumor model in nude mice. Consistent with the in vitro study showing that wogonin sensitized A549 cells to TRAIL-induced apoptosis, wogonin greatly enhanced TRAIL-induced suppression of tumor growth, accompanied with increased apoptosis in tumor tissues as determined by TUNEL assay. The expression levels of antiapoptotic proteins including long form of cellular FLICE-like inhibitory protein (cFLIPL), X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues after co-treatment with wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated by proteasomal degradation that involved intracellular reactive oxygen species (ROS), because wogonin robustly induced ROS accumulation and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins in cells co-treated with wogonin and TRAIL. These results show for the first time that wogonin enhances TRAIL's antitumor activity in vivo, suggesting this strategy has an application potential for clinical anticancer therapy.

Abstract LB-370: Genetically engineered oncolytic Edmonston strain of measles strain harboring the wild-type N, P, L genes (MV-NPL) effectively target lung cancer stem cells

Abstract Increasing evidence suggests that only a small percentage of cancer cells in solid tumors have tumorigenic potential. These highly tumorigenic cells share with normal stem cells the ability to proliferate and give rise to diversified tumor cell types including those with the capacity for self-renewal. These cells are termed cancer stem cells (CSCs) and are believed to contribute to CSCs resistance to conventional therapies. Therefore, specific targeting of CSCs is an attractive and novel therapeutic approach. Measles virus Edmonston strain (MV-Edm) is thought to possess remarkable oncolytic activity that selectively destroys human tumor cells. Recently, we succeeded and reported the development of novel oncolytic measles virus (MV) by genetic engineering of MV-Edmonston strain (MV-Edm), that have demonstrated its remarkable oncolytic activity against wide ranges of human tumor cells, with the N, P, and L genes of wild-type MV (MV-NPL). The oncolytic activities of the MV-NPL on human renal cell carcinoma were superior to those of MV-Edm both in vitro and in vivo studies even in the presence of IFN[[Unsupported Character - ‐]]α[[Unsupported Character - ]](Xing, M. et. al. Mol Ther 2010). Here, we demonstrated that lung CSCs, side population fraction of human lung cancer cells of A549 (A549-SP), expressed high level of CD46 required for MV-NPL infection and MV-NPL had a superior oncolytic capacity against both A549-SP and A549-NSP (non SP) cells in a time and dose-dependent manner without damaging normal lung cells. Our results of pan-caspase inhibiton assays showed the partial contribution of caspases-dependent apoptosis in the oncolysis of A549-SP cells by MV-NPL, even though the expression levels of anti-apoptotic protein of both XIAP and Mcl-1 were higher in A549-SP cells than A549-NSP cells. Additionally, we demonstrated that an addition of either PI3K inhibitor or MEK inhibitor further enhanced the oncolytic effects of MV-NPL. Furthermore, our in vivo studies using nude mice bearing A549 xenografts revealed that serial intratumoral MV-NPL administrations exhibited significant antitumor effects without severe side effects. Collectively, these data suggested that genetically engineered MV-NPL could be a promising oncolytic agent to effectively eradicate lung CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-370. doi:1538-7445.AM2012-LB-370

Combining the chemotherapeutic effects of epigallocatechin 3-gallate with siRNA-mediated p53 knock-down results in synergic pro-apoptotic effects

Plant extracts and compounds are applied to a wide variety of diseases in which traditional drugs have proven ineffective. A quickly developing trend in biomedicine is the therapeutic use of siRNA (short interfering RNA) structures. The focus of this study was on evaluating the gene expression involved in the modulation of apoptosis, in cases of combinatorial treatment (−)-epigallocatechin-3-gallate (EGCG) and/or p53siRNA. EGCG in combination with p53siRNA exerts synergic pro-apoptotic effects that are greater than those of each agent taken individually. There is a cumulative antiproliferative effect, induced by EGCG and p53siRNA treatment, and it is mediated through the activation of a large number of pro-apoptotic genes and the inhibition of anti-apoptotic protein expression levels. p53siRNA promotes the convergence of the extrinsic and intrinsic pathways in a synergic manner with EGCG. The chemotherapeutic effects of EGCG in combination with p53siRNA therapy induced a synergic pro-apoptotic effect, indicating the potential for development of promising new anticancer therapies.

Potent Anti-Leukemic Activity of a Novel Cdk9 Inhibitor in Preclinical CLL Mouse Models

Abstract 1789Active agents for the treatment of CLL are available. However, CLL eventually progresses to refractory disease. Defects in apoptosis, cytogenetic abnormalities and alterations in non-malignant cells of the micro-environment are likely to be the major causes of therapeutic resistance. Therefore, novel agents are required to abrogate apoptosis blocks and to alter the micro-environment of CLL. One approach targets cyclin-dependent kinases (Cdk). Specifically, Cdk9 promotes initiation and elongation steps in transcription maintaining expression levels of anti-apoptotic proteins with inherently rapid turnover rates. Inhibitors of cyclin-dependent kinases (Cdks) have been previously shown to exert cytotoxic effects on CLL cells by inhibiting Cdk7 and Cdk9. However, inhibitors used in these studies suffer from lack of selectivity, making unambiguous interpretation of results difficult, and leading to unspecific toxicity.Here, we studied the novel Cdk inhibitor LDC-9-A, which exhibits potent and selective inhibitory activity against Cdk9. The in vitro cytotoxicity of LDC-9-A was evaluated after 24 and 48 hours in CLL cases (n=8) and the CLL derived prolymphocytic cell line MEC-1. After a culture period of 48 hours LD50 values ranged between 0.1 and 1.9 μM for primary CLL cells and MEC-1 cells, respectively. Western blot analysis of protein extracts from MEC-1 cells showed that LDC-9-A down-modulates the anti-apoptotic proteins Bcl-2 and Mcl-1 in a dose- and time-dependent manner.In order to study the in vivo anti-CLL activity of LDC-9-A 9–12 month-old transgenic TCL1 mice (n=4) were treated for two consecutive days with an orally bioavailable LDC-9-A formulation (50 mg/kg body weight). Control groups consisted of TCL1 mice receiving vehicle only (n=3) and wild-type control littermates (n=4) receiving LDC-9-A. Numbers of peripheral blood leukemic cells (CD19+CD5+CD3−) and non-malignant T-cells (CD19−CD5+CD3+) were longitudinally evaluated by flow cytometry. Remarkably, LDC-9-A treatment of TCL1 transgenic mice led to a decrease of blood leukemic cells to 6.3±2.0% (mean±SEM) of baseline levels (p<0.0001) two days post treatment cessation. The numbers of T-cells decreased to 15.5±6.6% (p<0.01) in TCL1 mice while the blood T-cells of LDC-9-A treated wild-type controls did only decrease to 49.5±11.6% of baseline levels. Because LDC-9-A was expected to alter cellular Cdk9-mediated anti-apoptotic protein replenishment Bcl-2 levels were determined by flow cytometry analysis. Interestingly, cellular Bcl-2 levels of surviving leukemic cells as well as surviving non-malignant T-cells were significantly higher compared to levels before treatment (1.6- and 2.6-fold, respectively, p<0.05). These data implied that either LDC-9-A was not able to clear cells with high Bcl-2 expression or that LDC-9-A led to a compensatory up-regulation of Bcl-2 in residual cells.Finally, the anti-CLL activity of LDC-9-A was further validated by employing a recently developed adoptive transfer model of primary human CLL cells. In this model NOD/SCID/γcnull (NSG) mice were transplanted with CLL cells and treated with different doses of LDC-9-A for 3–5 sequential days. In line with the data generated with the TCL1 murine CLL model the LDC-9-A treatment of xenografted animals with 60 and 90 mg/kg body weight resulted in an 80–90% reduction of human CLL cell recovery from murine spleens as compared to vehicle treated controls.In summary, we demonstrated effective anti-CLL activity of the novel Cdk9 inhibitor LDC-9-A in two independent preclinical mouse models. The activity of LDC-9-A against CLL cells was achieved at least in part by modulating cellular anti-apoptotic protein levels. Furthermore, LDC-9-A interfered with non-malignant T-cells, which represent an important component of the CLL micro-environment. Disclosures:Eickhoff:Lead Discovery Center GmbH (LDC): Employment. Baumann:Lead Discovery Center GmbH (LDC): Employment. Unger:Lead Discovery Center GmbH (LDC): Employment. Schultz-Fademrecht:Lead Discovery Center GmbH (LDC): Employment. Dürig:Roche: Speakers Bureau; GSK: Speakers Bureau; Santaris Pharma: Consultancy, Research Funding; Celgene: Research Funding.

Activation of apoptosis signaling eliminates CD34+ progenitor cells in blast crisis CML independent of response to tyrosine kinase inhibitors

Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34(+) BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34(+) progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34(+) progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34(+) progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34(+) progenitor cells in BC CML, independent of response to TKIs.

Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1–expressing myeloma cells

AbstractDependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-xL/Bcl-2 dependence. Surprisingly, analysis of Mcl-1–dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-xL in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

Nutlin-3 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through up-regulation of death receptor 5 (DR5) in human sarcoma HOS cells and human colon cancer HCT116 cells

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. The expressions of DR4 and DR5, Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, are regulated by p53. In this study, the combined effects of nutlin-3 and TRAIL on apoptosis were investigated in HOS and HCT116 cells, which express wild-type p53. Nutlin-3 and TRAIL synergistically enhanced apoptosis owing to their intrinsic and extrinsic pathway signals, respectively. The increase in the Bid expression level and the decrease in the expression levels of anti-apoptotic proteins, c-FLIP and XIAP, were involved in this apoptosis enhancement. Furthermore, nutlin-3 activated the DR5 promoter and increased the expression levels of DR5 at mRNA and protein levels. These results indicate that the combination, treated with nutlin-3 and TRAIL, is useful for apoptosis induction in malignant cells expressing wild-type p53.

Akt Inhibitor Perifosine-Induced Cytotoxicity Is Associated with Significant Downregulation of Survivin in Human Multiple Myeloma (MM) Cells.

Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkylphospholipid, a new class of anti-tumor agents which potently inhibits Akt (PKB) activity. Our previous studies have shown that Perifosine induces significant cytotoxicity in MM cells triggered by c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage even in the presence of cytokines (ie, IL-6 and IGF-1) or bone marrow stromal cell (BMSCs). Importantly, MEK inhibitor and bortezomib enhance Perifosine-induced cytotoxicity. It has also shown significant anti-tumor activity in a human MM cell xenograft mouse model (Hideshima et al. Blood 2006, 107:4053–4062). In this study, we further delineated molecular mechanisms whereby Perifosine triggers cytotoxicity as a single agent and in combination with bortezomib in MM cells. In most MM cell lines, the IC50 for Perifosine-induced cytotoxicity is 5–10 μM range assessed by MTT assay at 24h; however, apoptosis assessed by APO2.7 staining, varied in each cell line. Moreover, neither the degree of JNK phosphorylation nor caspase-8/9/PARP cleavage correlated with Perifosine-induced cytotoxicity. Therefore we further examined expression level of anti-apoptotic proteins in MM cell lines and found that survivin, which has a crucial role in regulation of caspase-3 activity, was markedly downregulated by Perifosine treatment in a time- and dose-dependent fashion, without affecting expression of other anti-apoptotic proteins (ie, cIAP, XIAP, Bcl-2, Bcl-xL). Since survivin is a known downstream protein of β-catenin/TCF-4 cascade, we next hypothesized that Perifosine may inhibit β-catenin activity. As expected, Perifosine significantly downregulated both phosphorylation and protein expression of β-catenin, associated with downregulation of survivin and enhanced caspase-3 cleavage. Real-time PCR confirmed that gene expression of survivin was suppressed 35% and 55% after 3h and 6h Perifosine treatment, respectively. Since β-catenin is a substrate of proteasomes, we further examined whether bortezomib could augment survivin expression by blocking its degradation. Importantly, bortezomib significantly upregulated β-catenin and survivin, which was blocked in the presence of Perifosine. These results suggest that inhibition of bortezomib-induced survivin expression, at least in part, accounts for enhanced bortezomib-induced cytotoxicity by Perifosine. Based upon these preclinical studies, a rational combination trial of bortezomib with Perifosine to treat relapsed refractory MM is currently ongoing.

Allicin, a major component of garlic, inhibits apoptosis of macrophage in a depleted nutritional state

Objective Allicin is believed to be the main component responsible for the biological activity of garlic. The regulation of cell death might have therapeutic potential in many diseases, and previous studies have demonstrated that allicin stimulates the functional activity of macrophages. Therefore, this study examined the effects of allicin on the apoptosis of macrophages induced by serum- and amino acid-free culture. Methods The apoptosis of peritoneal macrophages was examined after pretreating them with allicin and incubating them in a depleted nutritional state. The rate of apoptosis was determined using propidium iodide staining analysis using flow cytometry, DNA fragmentation, and a caspase-3 assay. Western blot analysis was used to examine the changes in the pro- or antiapoptotic protein expression levels. Results DNA fragmentation and propidium iodide staining analyses revealed that allicin decreased the malnutrition-induced apoptosis of macrophages. The level of Bax expression, the amount of cytochrome-c released from the mitochondria, and the caspase-3 activity were also lower in the allicin-treated macrophages than in the untreated macrophages in a depleted nutritional state. Moreover, the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase) inhibitor suppressed the allicin-induced inhibition of apoptosis in a depleted nutritional state and allicin increased the level of ERK1/2 phosphorylation. Conclusion Allicin inhibits the apoptosis of macrophages in a depleted nutritional state through the MEK/extracellular signal-regulated kinase pathway.

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of smac/DIABLO from the mitochondria

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.

Casein kinase II (CK2) enhances death-inducing signaling complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines

Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli, as well as being elevated in many human cancers examined. We have demonstrated that CK2 is a key survival factor that protects human colon carcinoma cells from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. We determined that inhibition of CK2 phosphorylation events by DRB (5,6-dichlorobenzimidazole) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis, in the absence of effects in normal cells. Sensitization was caspase dependent, and independent of regulation via NF-kappaB. Further, inhibition of phosphorylation by CK2 did not modify the expression level of antiapoptotic proteins. Analysis of TRAIL-induced death-inducing signaling complex (DISC) formation demonstrated enhanced formation of the DISC, enhanced cleavage of caspase-8 and cleavage of Bid in the presence of DRB, thereby facilitating the release of proapoptotic factors from the mitochondria with subsequent downregulation of the expression of XIAP and c-IAP1. Further, silencing of CK2alpha in HT29 cells following transfection of CK2alpha shRNA abrogated CK2 kinase activity while simultaneously increasing TRAIL sensitivity. These findings demonstrate that CK2 plays a critical antiapoptotic role by conferring resistance to TRAIL at the level of the DISC.

Cisplatin induces apoptosis in oral squamous carcinoma cells by the mitochondria-mediated but not the NF-κB-suppressed pathway

Cisplatin (CDDP) is a potent DNA-damaging anticancer agent, and its cytotoxic action is exerted by the induction of apoptosis. However, activation of the transcription factor NF-κB results in protection against apoptosis. We examined the molecular mechanisms involved in the induction of apoptosis by CDDP as regards both suppression of NF-κB and activation of caspases. Human oral squamous carcinoma cells (B88) were employed in this study. We found that CDDP treatment affected neither NF-κB activity nor the expression levels of antiapoptotic proteins, including TRAF-1, TRAF-2, and cFLIP, in B88 cells. However, two apoptosome molecules, cytochrome c and Apaf-1, were significantly augmented in the cytoplasm by CDDP treatment. Further, the activation of caspase-9 and caspase-3, downstream molecules leading to mitochondria-mediated apoptosis, were detected after treatment with CDDP. Finally, apoptosis was also clearly observed, as evidenced by cleavage of PARP through the activation of caspase-3. These findings suggest that CDDP exerts its apoptotic action by the mitochondria-mediated activation of caspases but not by the activation of caspases due to the inhibition of NF-κB activity that follows the suppression of antiapoptotic proteins.