Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.     

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC4-Adr and its parental doxorubicin-sensitive line GLC4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC4-Adr. In addition, GLC4-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC4-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC4. Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.     

Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines.

BACKGROUND: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. PURPOSE: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. METHODS: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. RESULTS: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. CONCLUSIONS: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.     

Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.     

Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines.

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.     

Tumor necrosis factor-related apoptosis-inducing ligand cooperates with anticancer drugs to overcome chemoresistance in antiapoptotic Bcl-2 family members expressing jurkat cells.

PURPOSE: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-x(L) overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model EXPERIMENTAL DESIGN: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-x(L) overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC(6) and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. RESULTS: Bcl-2 and Bcl-x(L) overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-x(L) Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation CONCLUSIONS: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.     

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.     

Erucylphosphocholine-induced apoptosis in chemoresistant glioblastoma cell lines: involvement of caspase activation and mitochondrial alterations

Intrinsic chemoresistance constitutes a major problem in the therapy of malignant gliomas. In vitro experiments with four astrocytoma/glioblastoma (AC/GBM) cell lines revealed that the chemoresistance of two cell lines, A172 and T98G, to cisplatin and etoposide was due to resistance to drug-induced apoptosis. In contrast, all the AC/GBM cell lines tested were sensitive to treatment with the lipophilic ether lipid erucylphosphocholine, ErPC. ErPC-induced apoptosis was independent of wild-type p53-signaling and triggering of the CD95/CD95 ligand (CD95L) system. Inhibition of protein and RNA synthesis by cycloheximide and actinomycin D did not abrogate ErPC-induced apoptosis. However, expression of members of the bcl-2 protein family was modulated during ErPC treatment. Activation of caspase 3 and mitochondrial alterations were central to ErPC-induced apoptosis. We conclude that ErPC-induced activation of the mitochondrial pathway enables cell death in the chemoresistant AC/GBM cells.     

Inhibition of glutathione synthesis overcomes Bcl-2-mediated topoisomerase inhibitor resistance and induces nonapoptotic cell death via mitochondrial-independent pathway

Bcl-2 protein plays a critical role in inhibiting anticancer drug-induced apoptosis. We found that Bcl-2 overexpression is associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after treatment with etoposide or SN-38, a derivative of camptothecin, in leukemia 697 cells with wild-type p53. Treatment of Bcl-2-overexpressing 697 cells (697-Bcl-2) with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, reduced cellular glutathione levels and completely abolished Bcl-2-mediated drug resistance. Morphologic studies revealed that nonapoptotic cell death was induced in 697-Bcl-2 cells after treatment with BSO plus etoposide or SN-38. Activation of caspase-3/7 and cytochrome c release could not be detected in 697-Bcl-2 cells after these drug treatments. Notably, we showed that proteasome-mediated down-regulation of Puma and Noxa proteins occurs in 697-Bcl-2 cells after treatment with BSO plus topoisomerase inhibitor, although there is an increase in the protein levels of p53 in these 697-Bcl-2 cells. In contrast, parental 697 cells underwent typical apoptosis with up-regulation of Puma and Noxa proteins, followed by cytochrome c release and caspase-3/7 activation after treatment with topoisomerase inhibitor in the presence or absence of BSO. Our data suggest that BSO may possess a unique activity to overcome Bcl-2-mediated drug resistance by stimulating the signals that can bypass mitochondrial process in Bcl-2-overexpressing cells.     

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells

Antiapoptotic genes such as bcl-2 or xIAP may be responsible for resistance to apoptosis induced by cytotoxic drugs. The aim of this study was to investigate if downregulation of bcl-2 or xIAP by RNA interference (RNAi) would sensitize MCF-7 cells to etoposide and doxorubicin. FITC-siRNAs uptake was verified by fluorescence microscopy and downregulation of Bcl-2 or XIAP was confirmed by Western Blotting. Both siRNAs reduced the number of viable cells and increased cellular apoptosis. Treatment with siRNAs followed by treatment with etoposide or doxorubicin further reduced the number of viable cells, when compared to either of the treatments alone. Therefore, downregulation of bcl-2 or xIAP by RNAi enhances the effects of etoposide and doxorubicin.     

Doxorubicin sensitivity pattern in a panel of small-cell lung-cancer cell lines: correlation to etoposide and vincristine sensitivity and inverse correlation to carmustine sensitivity

Summary The aim of our investigations is to evaluate whether the sensitivity patterns of small-cell lung-cancer (SCLC) cell lines in vitro can be used in evaluating new drugs and in selecting drugs for the optimization of combination therapy. In our attempts to obtain a panel of cell lines demonstrating differential patterns in sensitivity, we have developed three SCLC lines exhibiting different types of multidrug resistance (MDR). In the present investigations we compared the sensitivity patterns shown by five wild-type SCLC lines and three MDR lines in response to six different types of drugs: doxorubicin, cytarabine, carmustine, cisplatin, vincristine, and etoposide. In the wild-type SCLC cell lines, the range of variation in sensitivity to all drugs was within a factor of 10. Cell lines showing low sensitivity to doxorubicin also exhibited low sensitivity to etoposide and vincristine, and vice versa. In contrast, the pattern of sensitivity to carmustine was almost the opposite of that to doxorubicin. A tendency to an inverse relationship between doxorubicin and carmustine sensitivity was also observed when doxorubicin sensitivity was reduced in near stationary cells and in cells exposed to the metabolic inhibitor 2-deoxy-d-glucose. In agreement with the pattern observed for the wild-type lines, all of the MDR sublines demonstrated collateral sensitivity to carmustine. As to cytarabine, the wild-type lines expressed a sensitivity pattern similar to that shown in response to doxorubicin. Interestingly, the opposite pattern was found in the MDR lines, as all three demonstrated cytarabine hypersensitivity. The combination of alkylating agents and MDR drugs are of proven clinical benefit in the treatment of solid tumors, as is the combination of anthracycline and cytarabine in acute myeloid leukemia. The experimentally derived sensitivity data on cytarabine, alkylating agents, and MDR drugs (i.e., etoposide, doxorubicin, vincristine) thus resemble the clinical experience with these drugs, and we conclude that the use of a clonogenic asay on the described panel of SCLC cell lines can give valuable information for the selection of agents for combination therapy.This work was supported by grants from the Danish Cancer Society, the Danish Medical Research Council, and the Lundbeck Foundation     

Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells.

PURPOSE: Many melanoma cell lines and primary cultures are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In this study, we investigated the molecular mechanisms that control melanoma cell resistance and searched for chemotherapeutic drugs that could overcome the TRAIL resistance in melanoma cells. EXPERIMENTAL DESIGN: We examined 21 melanoma cell lines and 3 primary melanoma cultures for their sensitivity to TRAIL-induced apoptosis, and then tested cisplatin, chemptothecin, and etoposide for their synergistic effects on TRAIL sensitivity in resistant melanoma cells. RESULTS: Of 21 melanoma cell lines, 11 showed various degrees of sensitivity to TRAIL-induced apoptosis through caspase-8-initiated cleavage of caspase-3 and DNA fragmentation factor 45. The remaining cell lines and primary cultures were resistant to TRAIL, but cisplatin, chemptothecin, and etoposide sensitized the resistant cell lines and primary cultures to TRAIL-induced apoptosis, which also occurred through the caspase-8-initiated caspase cascade. Of the two TRAIL death receptors (DR4 and DR5), melanoma cells primarily expressed DR5 on cell surface. Cisplatin treatment had no effects on cell surface DR5 expression or intracellular expression of Fas-associated death domain and caspase-8. Instead, cisplatin treatment down-regulated intracellular expression of the short form of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP) and inhibited phosphorylation of the long form of c-FLIP. CONCLUSIONS: The results presented here indicate that cisplatin inhibits c-FLIP protein expression and phosphorylation to restore TRAIL-induced caspase-8-initiated apoptosis in melanoma cells, thus providing a new combined therapeutic strategy for melanomas.     

Glucocorticoid cotreatment induces apoptosis resistance toward cancer therapy in carcinomas

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Glucocorticoids (GCs) are frequently used as cotreatment because they may have potent proapoptotic properties and reduce nausea, hyperemesis, and acute toxicity on normal tissue. In contrast to the proapoptotic effect of GCs in lymphoid cells, resistance toward cancer therapy-mediated apoptosis was induced in solid tumors of human cervix and lung carcinomas. Filter hybridization, expression data, as well as functional assays identified multiple core apoptosis molecules, which are regulated by GCs in a pro- or antiapoptotic manner. Both antiapoptotic genes such as FLIP and members of the Bcl-2 and IAP family as well as proapoptotic elements of the death receptor and mitochondrial apoptosis pathways were down-regulated in carcinomas resulting in a decreased activity of caspase-8, caspase-9, and caspase-3. In contrast, death receptor and mitochondrial apoptosis signaling as well as caspase activity was enhanced by dexamethasone in lymphoid cells. To restore apoptosis sensitivity in dexamethasone-treated carcinomas, caspase-8 and caspase-9 were transfected. This resensitized tumor cells in vitro and xenografts in vivo to cisplatin induced cell death. These data therefore raise concern about the widespread combined use of GCs with antineoplastic drugs or agents in the clinical management of cancer patients.     

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.     

The p53 tumor suppressor gene in anticancer agent-induced apoptosis and chemosensitivity of human gastrointestinal cancer cell lines

Purpose: While the target of many anticancer agents has been identified, the processes leading to killing of the cancer cells and the molecular basis of resistance to the drugs are not well understood. We used human gastrointestinal cancer cell lines and examined how anticancer agents induced cell killing and how the chemosensitivity of these lines was determined. Methods: Twelve gastrointestinal cancer cell lines were examined for the presence of either a wild-type or mutant p53 gene by direct sequencing. We also determined whether or not cell killing would occur when the cell lines were exposed to anticancer drugs. The sensitivity to the anticancer agents was determined based on colony formation. Results: All 12 gastrointestinal cancer cell lines carried either a wild-type or mutant p53 gene. Three lines, MKN45, MKN74 and COLO320, carried the wild-type p53 gene, and nine carried the mutant p53 gene. When three lines were exposed to the anticancer agents etoposide, doxorubicin (DXR) or 5-fluorouracil (5-FU), cell death ensued. In these cells, the population of cells in G₁ phase increased after exposure to high-dose anticancer agents, but cells in G₂ phase increased when exposed to low-dose anticancer agents. Our observations support the concept that cells carrying the wild-type p53 gene tend to be sensitive to etoposide and DXR and, in particular, deletion of the p53 function results in a greater resistance to anticancer agents. Conclusion: Based on our findings, human gastrointestinal cancer-related cell death apparently occurs via a p53-dependent pathway. A relationship was observed between the induction of cell death and chemosensitivity. Received: 22 December 1997 / Accepted: 1 April 1998     

Optimization of Circulating Biomarkers of Obatoclax-Induced Cell Death in Patients with Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive disease in which, after initial sensitivity to platinum/etoposide chemotherapy, patients frequently relapse with drug-resistant disease. Deregulation of the Bcl-2 pathway is implicated in the pathogenesis of SCLC, and early phase studies of Bcl-2 inhibitors have been initiated in SCLC. Obatoclax is a small-molecule drug designed to target the antiapoptotic Bcl-2 family members to a proapoptotic effect. Preclinical studies were conducted to clarify the kinetics of obatoclax-induced apoptosis in a panel of SCLC cell lines to assist with the interpretation of biomarker data generated during early phase clinical trials. In vitro, obatoclax was synergistic with cisplatin and etoposide, and “priming” cells with obatoclax before the cytotoxics maximized tumor cell death. Peak levels of apoptosis, reflected by cleaved cytokeratin 18 (CK18) levels (M30 ELISA) and caspase activity (SR-DEVD-FMK), occurred 24 hours after obatoclax treatment. A phase 1b-2 trial of obatoclax administered using two infusion regimens in combination with carboplatin and etoposide has been completed in previously untreated patients with extensive-stage SCLC. Circulating pharmacodynamic biomarkers of cell death, full-length and/or cleaved CK18, and oligonucleosomal DNA were studied in the phase 1b trial. All SCLC patients classified as “responders” after two cycles of treatment showed significantly increased levels of full-length and cleaved CK18 (M65 ELISA) on day 3 of study. However, the preclinical data and the absence of a peak in circulating caspase-cleaved CK18 in trial patients suggest suboptimal timing of blood sampling, which will need refinement in future trials incorporating obatoclax.     

Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.