Over-expression of APAF-1 and caspase-9 augments radiation-induced apoptosis in U-373MG glioma cells.

The p53 tumor-suppressor gene plays a critical role in radiation-induced apoptosis. Several genes, including Bax and Fas, are involved in p53-mediated apoptosis, and their over-expression enhances the degree of radiation-induced apoptosis. Apaf-1 and caspase-9 have been reported to be downstream components of p53-mediated apoptosis, suggesting that these genes play a role in radiation-induced apoptosis. In this study, we transduced U-373MG cells harboring mutant p53 with the Apaf-1 and/or caspase-9 genes via adenoviral (Adv) vectors concomitant with X-ray irradiation and evaluated the degree of apoptosis. The percentage of apoptotic cells in U-373MG cells co-infected with the Adv for Apaf-1 (Adv-APAF-1) and that for caspase-9 (Adv-Casp9) and treated with irradiation (24%) was much higher than that in cells co-infected with Adv-APAF-1 and Adv-Casp9 and not treated with irradiation (0.86%) and that in cells infected with either Adv-APAF-1 or Adv-Casp9 and treated with irradiation (2.0% or 2.6%, respectively). The apoptosis induced by co-transduction of Apaf-1 and caspase-9 and irradiation was repressed in cells that were co-infected with the Adv for Bcl-X(L) but not in cells co-infected with the Adv for Bcl-2. These results indicate that Apaf-1 and caspase-9 play a role in radiation-induced apoptosis in cancer cells harboring mutant p53. Bcl-X(L) may be critically involved in the radioresistance of cancer cells by repressing Apaf-1- and caspase-9-mediated apoptosis. Expression of Apaf-1 and caspase-9 in tumors may be an important determinant of the therapeutic effect of irradiation in cancer treatment.     

Degree of apoptosis induced by adenovirus-mediated transduction of p53 or p73alpha depends on the p53 status of glioma cells

It has been reported that U-87MG glioma cells with wild-type p53 are resistant to p53 replacement gene therapy. As some gliomas harbor wild-type p53, it would be important to override the resistance mechanism due to wild-type p53 in glioma gene therapy. In this study, we transduced U-87MG cells or U251 glioma cells harboring mutated p53 with the p53 or p73alpha gene (a homologue of p53, that differently induces some p53-responsive genes) via adenovirus vectors (Advs) at same multiplicities of infection (MOIs) into respective cells (U-87MG: MOI 1000, U251: MOI 100), and evaluated the degree of apoptosis. The results demonstrate that the degree of apoptosis induced by Adv-mediated transduction of p53 in U-87MG cells was lower than that in U251 cells, whereas that induced by Adv-mediated transduction of p73alpha in U-87MG cells was higher than that in U251 cells. Bax expression in U-87MG and U251 cells induced by Adv-mediated transduction of p53 was almost the same as that of p73alpha. On the other hand, Adv-mediated transduction of p73alpha induced caspase-9 at higher levels than that of p53 in both cells. The results indicate that Adv-mediated transduction of p73alpha might be beneficial to overcome the resistance mechanism of glioma cells harboring wild-type p53.     

Co-transduction of Apaf-1 and caspase-9 augments etoposide-induced apoptosis in U-373MG glioma cells.

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAF1) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAF1, and that in cells infected with both Adv-APAF1 and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X(L) in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.     

Co-transduction of Apaf-1 and Caspase-9 Augments Etoposide-induced Apoptosis in U-373MG Glioma Cells

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAFl) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAFl, and that in cells infected with both Adv-APAFl and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X_L in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.     

Co-transduction of p27Kip1 strongly augments Fas ligand- and caspase-8-mediated apoptosis in U-373MG glioma cells

BACKGROUND: p27Kip1 is a potential tumor suppressor gene. As malignant gliomas express Fas at high levels, the relationship between Fas-mediated apoptosis and p27Kip1 expression may improve therapeutic approaches for treating gliomas. MATERIALS AND METHODS: In this study, we transduced U-373MG glioma cells with the Fas ligand or caspase-8 genes using adenovirus vectors after transduction of the p27Kip1 gene to induce cell cycle arrest in U-373MG cells, and evaluated the degree of apoptosis. RESULTS: The results demonstrate that expression of p27Kip1 enhanced Fas ligand- or caspase-8-mediated apoptosis in U-373MG cells. Expression of apoptosis-related genes such as Bax, Bcl-X(L), Bcl-2 or caspase-8 were reduced by p27Kip1 transduction compared with that of beta-actin, whereas p27Kip1 transduction did not affect the expression level of Fas or the Fas ligand. CONCLUSION: Combined transduction of p27Kip1 with Fas ligand or caspase-8 would overide the resistance mechanism to apoptosis in malignant gliomas.     

Adenovirus-mediated transfer of p53 augments hyperthermia-induced apoptosis in U251 glioma cells

PURPOSE: Hyperthermia kills glioma cells by inducing apoptosis and is thereby an effective therapeutic modality for the treatment of malignant gliomas. However, cells harboring mutated p53 are refractory to hyperthermia-induced apoptosis. In this study, we assessed whether or not adenovirus (Adv)-mediated transduction of p53 overrides this resistant mechanism. METHODS AND MATERIALS: We transduced the p53 wild-type tumor suppressor gene into U251 glioma cells harboring mutated p53 using Adv vectors in combination with hyperthermia (43, 44.5 degrees C), and evaluated the degree of cell death and apoptosis. RESULTS: The percentage of cells that had died, as measured by trypan blue staining, among U251 cells infected with the Adv for p53 (Adv-p53) and treated with hyperthermia, was significantly higher than the percentage of cells that had died among U251 cells infected with Adv-p53 and not treated with hyperthermia, or those infected with the control Adv for dE (Adv-dE) and treated with hyperthermia. The degree of apoptosis, measured at 24 h after treatment, in hyperthermia-treated U251 cells infected with Adv-p53 (43 degrees C, 73%; 44.5 degrees C, 92%) was much higher than that infected with Adv-p53 (41%), or that infected with control Adv-dE and treated with hyperthermia (43 degrees C, 1.3%; 44.5 degrees C, 19%). Treatment with combined hyperthermia and Adv-p53 infection induced cleavage of caspase-3 in U251 cells. CONCLUSION: These results indicate that Adv-mediated transduction of p53 would render glioma cells highly sensitive to hyperthermia.     

5-aza-2'-deoxycytidine upregulates caspase-9 expression cooperating with p53-induced apoptosis in human lung cancer cells

Treating lung cancer cell lines using low-dose 5-aza-2'-deoxycytidine (DAC) caused an accumulation of procaspase-9 through mRNA upregulation, but the cells did not undergo apoptosis. However, when cells were treated with DAC and infected with a low dose of a recombinant wild-type p53 adenovirus vector (Ad-p53), a synergistic growth inhibitory effect was observed. Combination treatment induced Apaf-1 and procaspase-9 expression in which cytochrome c releases by Ad-p53 triggered the mitochondrial pathway of apoptosis. Selective blockage of caspase-9 activities by Z-LEHD-FMK completely attenuated DAC-induced enhancement of apoptosis mediated by Ad-p53 infection, and ectopic overexpression of procaspase-9 sensitized cells to Ad-p53-induced apoptosis in p53-null cells. In addition, DAC sensitized lung cancer cells to cisplatin and paclitaxel. Induction of the mitochondrial pathway of apoptosis using a slightly toxic dose of DAC may therefore be a strategy for treating lung cancer, and DAC treatment may have clinical implications when combined with chemotherapy or apoptosis-inducing gene therapy.     

The mechanism of ellipticine-induced apoptosis and cell cycle arrest in human breast MCF-7 cancer cells

Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here, we first report the molecular mechanism of ellipticine's apoptotic action in human breast MCF-7 cancer cells. Treatment of cells with ellipticine resulted in inhibition of growth, and G2/M phase arrest of the cell cycle. This effect was associated with a marked increase in the protein expression of p53 and, p21/WAF1 and KIP1/p27, but not of WAF1/p21. Ellipticine treatment increased the expression of Fas/APO-1 and its ligands, mFas ligand and sFas ligand, and subsequent activation of caspase-8. The mitochondrial apoptotic pathway amplified the Fas/Fas ligand death receptor pathway by Bid interaction. This effect was found to result in a significant increase in activation of caspase-9. Taken together, we have concluded that the molecular mechanisms during ellipticine-mediated growth inhibition and induction of apoptosis in MCF-7 cells were due to (1) cell cycle arrest and induction of apoptosis, (2) induction of p53 and KIP1/p27 expression, (3) triggering of Fas/Fas ligand pathway, (4) disruption of mitochondrial function, and (5) the apoptotic signaling was amplified by cross-talk between Fas death receptor and mitochondrial apoptotic pathway.     

Oncolytic adenovirus-mediated E1A gene therapy induces tumor-cell apoptosis and reduces tumor angiogenesis leading to inhibition of hepatocellular carcinoma growth in animal model

Oncolytic adenovirus (rAd)-mediated E1A gene therapy of cancer has become a novel therapeutic modality. In this study, we constructed a recombinant oncolytic adenovirus (rAd-E1A) expressing the tumor suppressor E1A gene. We demonstrated that the rAd-E1A replicated in HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cells but attenuated in the normal liver cell line HL-7702. It induced HCC cell apoptosis through upregulation of apoptosis-associated Bax, caspase-3, and Fas and downregulation of survivin and Bcl-2 in a p53-dependent pathway. It also downregulated the expression of angiogenesis- associated vascular endothelial growth factor (VEGF) and CD34 genes and reduced tumor vessel formation and angiogenesis. In mice bearing SMMC-7721 tumors, intratumoral injections of rAd- E1A significantly inhibited HCC growth. Therefore, the oncolytic adenovirus-mediated E1A gene therapy may be a useful therapeutic approach for HCC treatment.     

Adenovirus-mediated overexpression of Fas induces apoptosis of gliomas

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.     

Adenovirus-mediated transfer of bax with caspase-8 controlled by myelin basic protein promoter exerts an enhanced cytotoxic effect in gliomas

The transduction of Bax protein, which is up-regulated in radiation- and chemotherapy-induced apoptosis, augments the cytotoxicity of radiotherapy and chemotherapy for cancers. The cytotoxicity of Bax overexpression is caused primarily by mitochondrial dysfunction, which is also involved in the apoptosis triggered by caspase-8. In this study, we transduced the Bax gene in combination with caspase-8 gene to evaluate whether or not this approach induces effective cytotoxicity in glioma cells. In terms of cancer gene therapy, it is critically important to induce cytotoxic genes in a cancer-specific manner. Therefore, we used the myelin basic protein promoter to drive cytotoxic genes. The expression level controlled by the myelin basic protein promoter was relatively low in gliomas. In U251 and U-373 MG glioma cells, adenovirus-mediated transduction of the Bax gene combined with caspase-8 gene induced enhanced apoptosis and cell death as determined by morphological analysis and assay for dead cells, hypodiploid cells, and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling method). This therapeutic modality would be useful to induce a specific and enhanced cytotoxic effect for gliomas.     

p33ING1b增强骨肉瘤细胞U2OS对足叶乙甙的敏感性

背景与目的作为一个新的抑癌基因,ING1与p53有许多相似的生物学功能,如细胞生长抑制、DNA修复、凋亡和化疗敏感性等.本实验目的在于研究p33ING1b对骨肉瘤细胞药物敏感性的影响并探讨其作用机制.方法瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,用足叶乙甙(etoposide,VP-16)处理24 h,然后采用台盼蓝拒染法计数活细胞数并计算细胞生长抑制率,流式细胞仪、DAPI染色等方法检测细胞凋亡率,Western blot技术检测p53、p21WAF1、MDM2和Bax蛋白的表达.结果台盼蓝染色结果表明,瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24 h,细胞生长抑制率明显增加[(63.1±5.1)%].流式细胞计数和DAPI染色检测结果表明,细胞凋亡率明显增加(62.7%).Western blot检测结果显示,外源性p33 ING1b高表达明显提高了p53及其下游基因p21WAF1和Bax蛋白的表达水平,转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24 h,p53、p21和Bax蛋白表达增加.在各实验组中,MDM2的蛋白表达没有明显变化.结论p33ING1b能够上调p53蛋白,并上调p53下游因子--p21WAF1和Bax的表达水平,通过p53依赖性凋亡信号通路,提高骨肉瘤细胞U2OS对VP-16的敏感性.     

Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas

Various therapeutic approaches toward killing glioma cells by inducing apoptosis have been developed, but these approaches are often hampered by anti-apoptotic mechanisms. In this study, we attempted to develop a technique that overrides the resistance toward apoptosis in glioma cells. To date, p53- and Fas-mediated apoptotic pathways have been shown to be different. Therefore, we carried out a gene therapy that combines the pro-apoptotic effect of these two different pathways. The recombinant adenoviruses (Advs) for p53 and Fas ligand (FL) (Adv-p53 and Adv-FL, respectively) were constructed. Transfecting the p53 gene into glioma cell lines (A-172 and U251 glioma cells) led to overexpression of Bax, a protein that induces permeability transition; at the same time, this transfection brought about an overexpression of Fas. To intensify Fas-mediated apoptosis, we transferred the FL gene together with the p53 gene by Adv-mediated gene transduction into A-172 and U251 cells. Coinfecting Adv-p53 and Adv-FL into A-172 cells, which are relatively resistant to apoptosis by infection with Adv-p53 or Adv-FL alone (Adv-p53, multiplicity of infection (MOI) of 100: 8.5 +/- 0.7%; Adv-FL, MOI of 100: 3.0 +/- 0.1%) resulted in a drastic enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 24.2 +/- 0.9%). Coinfection with Adv-p53 and Adv-FL in U251 cells resulted in a similar enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 59.0 +/- 2.3%) compared with that seen in U251 cells transfected with Adv-p53 or Adv-FL alone (Adv-p53, MOI of 30: 3.1 +/- 0.3%; Adv-FL, MOI of 30: 18.1 +/- 0.3%). Regardless of whether a cell line is resistant or sensitive to FL- and p53-mediated apoptosis, coinfection with Adv-p53 and Adv-FL dramatically enhanced the degree of apoptosis of glioma cells. Our results indicate that the coinfection approach can be used as a modality for the gene therapy of gliomas, overriding the apoptosis-resistant mechanisms in glioma cells.     

Changes in p21WAF1, pRb, Mdm-2, Bax and Bcl-2 expression in cervical cancer cell lines transfected with a p53 expressing adenovirus

The aim of this study was to provide some insights into the molecular mechanisms involved in p53-dependent apoptosis and growth arrest. Changes in the levels of p53 protein and proteins regulated by p53 were studied in relation to events of the cell cycle and apoptosis in cervical cancer cell lines upon transfection with a p53 expressing adenovirus (Ad5-p53). The post-transfection level of p53 protein in SiHa cells was found to be unchanged during the 24-48 h period. In contrast, the level of p21WAF1 protein was shown to increase to its highest level at 24 h, and decreased gradually up to 48 h after the Ad5-p53 transfection. We further noted that the increase of p21WAF1 was accompanied by G1 arrest at 24 h and the decrease of p21WAF1 was associated with apoptosis at 36-48 h after transfection. An anti-p21WAF1 antibody cross-reactive protein band of approximately 14 kDa was observed in HeLa and C-33A cells when these cells were committed to apoptosis upon Ad5-p53 transfection. In SiHa cells, phosphorylation of pRb was inhibited during the early stage of Ad5-p53 transfection. This was followed by the cleavage of pRb. However, Ad5-p53 transfection did not change the levels of Bax and Bcl-2 proteins. Our results suggested that, Bax and Bcl-2 may not be important for the apoptosis of these cells, whereas cleavage of Rb, and the decrease of p21WAF1 could play important roles in p53-dependent apoptosis.     

Enhanced radiosensitization of p53 mutant cells by oleamide

PURPOSE: Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. METHODS AND MATERIALS: NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. RESULTS: Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. CONCLUSIONS: Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.     

Enhancement of apoptosis in irradiated glioblastoma cells by pentoxifylline

The p53 gene is frequently mutated in human glioblastomas, which are highly resistant to radiation and chemotherapy. Pentoxifylline has recently been shown to sensitize breast cancer cells in which the wild-type p53 function was abrogated to cisplatin-induced apoptosis. In this study, the effect of pentoxifylline on three irradiated human glioblastoma cell lines, U87MG, which has the wild-type p53; U251, which has a mutant p53; and LN-Z308, which has no p53, was examined. After radiation, even at a dose as high as 20 Gy, typical apoptotic cells were rarely produced. G(2) cell cycle arrest was observed with all three cell lines. Significant G(1) cell population was only observed with U87MG cells, in which increased levels of p21/WAF1/Cip1 occurred. Pentoxifylline increased the amount of radiation-induced DNA fragmentation and apoptosis in p53-defective U251 and LN-Z308 cells. Colony formation assay demonstrated that pentoxifylline moderately enhanced the cell killing effect of irradiation in these cells.     

Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells

Cisplatin-centered chemotherapy is a key treatment for ovarian cancer, but resistance to chemotherapeutic agents remains a major cause of treatment failure. Multiple factors are known to contribute to the development of this chemoresistance. Although it has been demonstrated that X-linked inhibitor of apoptosis protein (Xiap) prevents apoptosis by inhibiting effector caspases, if and how it is important in chemoresistance in ovarian cancer has not been studied. The effects of Xiap down-regulation and/or restoration of wild type p53 by recombinant adenovirus infection were examined on four ovarian epithelial cancer cell lines [C13*, A2780-s (wild type p53), A2780-cp (mutant p53), and SKOV3 (null p53)]. Apoptosis and protein expression (e.g., Xiap, caspase-3, p53, MDM2, and p21waf1) were assessed by Hoechst 33258 stain and Western blot, respectively. We demonstrated that Xiap down-regulation following adenoviral antisense expression induces apoptosis in the wild-type p53 cells, but not in the mutated or null cells. Xiap down-regulation resulted in caspase-3 activation, caspase-mediated MDM2 processing, and p53 accumulation. Restoration of wild type p53 in the p53-mutated or -null cells significantly enhanced the proapoptotic effect of Xiap antisense expression. Down-regulation of Xiap induced apoptosis in chemoresistant ovarian cancer cells, a process dependent on p53 status.     

Rituximab modifies the cisplatin-mitochondrial signaling pathway, resulting in apoptosis in cisplatin-resistant non-Hodgkin's lymphoma.

PURPOSE: Rituximab (chimeric anti-CD20) can reverse the cisplatin-resistant phenotype of AIDS-related non-Hodgkin's lymphoma cell lines and results in cisplatin-mediated apoptosis. The mechanism by which apoptosis is achieved by the combination treatment was examined. EXPERIMENTAL DESIGN: The AIDS-related lymphoma (ARL) cell line 2F7 was treated with rituximab, cisplatin, and a combination of the two and analyzed by Western blot analyses for signaling proteins involved in the death receptor-mediated and mitochondrial pathways. RESULTS: Rituximab selectively inhibited the expression of Bcl-2 in the ARL cells. However, other proteins analyzed [namely, Apaf-1, Bax, Bid, caspase-3, caspase-8, caspase-9, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2, cytochrome c, Fas, Fas ligand, FLIP, p53, and poly(ADP-ribose) polymerase] were not affected by either rituximab or cisplatin. Treatment with cisplatin induced the generation of mitochondrial reactive oxygen species, specifically intracellular peroxides. Furthermore, cisplatin alone was unable to induce the mitochondrial apoptotic events; however, the rituximab-cisplatin combination was able to synergistically induce significant apoptosis and mitochondria-mediated apoptotic events [mitochondrial membrane depolarization (DeltaPsi(m)), cytochrome c release from mitochondria, and caspase-3 and -9 activation]. The combination treatment facilitated the down-regulation of Bcl-2 by rituximab at an early time point. Decreased expression of additional proteins (Apaf-1, cIAP-1, cIAP-2, and XIAP) paralleled apoptosis detected at 24 h. CONCLUSIONS: These findings show that the selective down-regulation of Bcl-2 by rituximab leading to apoptosis in ARL cells by cisplatin is through the mitochondria-dependent caspase pathway.