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Combining microtubule-targeting antimitotic drugs with targeted apoptosis potentiators is a promising new chemotherapeutic strategy to treat cancer. In this study, we investigate the cellular mechanism by which navitoclax (previously called ABT-263), a Bcl-2 family inhibitor, potentiates apoptosis triggered by paclitaxel and an inhibitor of kinesin-5 (K5I, also called a KSP inhibitor), across a panel of epithelial cancer lines. By using time-lapse microscopy, we showed that navitoclax has little effect on cell death during interphase, but strongly accelerates apoptosis during mitotic arrest, and greatly increases the fraction of apoptosis-resistant cells that die. By systematically knocking down individual Bcl-2 proteins, we determined that Mcl-1 and Bcl-xL are the primary negative regulators of apoptosis during prolonged mitotic arrest. Mcl-1 levels decrease during mitotic arrest because of an imbalance between synthesis and turnover, and turnover depends in part on the MULE/HUWE1 E3 ligase. The combination of Mcl-1 loss with inhibition of Bcl-xL by navitoclax causes rapid apoptosis in all lines tested. Variation in expression levels of Mcl-1 and Bcl-xL largely determines variation in response to antimitotics alone, and antimitotics combined with navitoclax, across our panel. We concluded that Bcl-xL is a critical target of Bcl-2 family inhibitors for enhancing the lethality of antimitotic drugs in epithelial cancers, and combination treatment with navitoclax and a spindle specific antimitotic, such as a K5I, might be more effective than paclitaxel alone., (©2011 AACR.)

In the course of screening for novel anticancer compounds, B1 [N-(2-(dimethylamino)ethyl)-2-aminothiazonaphthalimide], a novel amonafide analogue, was generated as a new anticancer candidate. In the present study, B1 displayed stronger antitumor effects than amonafide in HL60 cells. We further examined whether B1 overcomes the resistance conferred by Bcl-2, as overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that B1 overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells. Various apoptosis assessment assays showed that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3σ protein by B1, indicating that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells via 14-3-3σ. The analysis of chromatin immunoprecipitation assay indicated that MBD2 was associated with the methylated 14-3-3σ promoter-associated CpG island and thus interfered with transcriptional activity of the methylated promoter. Furthermore, knockdown of MBD2, using siRNA transfection, inhibited B1-induced apoptosis and overcame the resistance conferred by Bcl-2. Accordingly, these data showed the involvement of MBD2 in B1-induced apoptosis and overcoming the resistance conferred by Bcl-2, which suggested that MBD2 might guide the development of future anticancer agents., (©2010 AACR.)

Hepatocellular carcinomas (HCC) are drug-resistant tumors that frequently possess high telomerase activity. It was therefore the aim of our study to investigate the potential of telomerase-dependent virotherapy in multimodal treatment of HCC. In contrast to normal liver, HCC xenografts showed high telomerase activity, resulting in tumor-restricted expression of E1A by a telomerase-dependent replicating adenovirus (hTERT-Ad). Neither tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or chemotherapy alone nor the combined treatment with both agents resulted in significant destruction of HCC cells. Application of hTERT-Ad at low titers was also not capable to destroy HCC cells, but telomerase-dependent virotherapy overcame the resistance of HCC against TRAIL and chemotherapy. The synergistic effects are explained by a strong down-regulation of Mcl-1 expression through hTERT-Ad that sensitizes HCC for TRAIL- and chemotherapy-mediated apoptosis. To investigate whether down-regulation of Mcl-1 alone is sufficient to explain synergistic effects observed with virotherapy, Mcl-1 expression was inhibited by RNA interference. Treatment with Mcl-1-siRNA significantly enhanced caspase-3 activity after chemotherapy and TRAIL application, confirming that elimination of Mcl-1 is responsible for the drug sensitization by hTERT-Ad. Consistent with these results, heterologous overexpression of Mcl-1 significantly reduced the sensitization of hTERT-Ad transduced cells against apoptosis-inducing agents. Chemotherapy did not interfere with quantitative hTERT-Ad production in HCC cells. Whereas hTERT-Ad virotherapy alone was only capable to inhibit the growth of Hep3B xenografts, virochemotherapy resulted in vast destruction of the drug-resistant HCC. In conclusion our data indicate that telomerase-dependent virotherapy is an attractive strategy to overcome the natural resistance of HCC against anticancer drugs by elimination of Mcl-1.

Pancreatic cancer is lethal because of its invasiveness, rapid progression, and profound resistance to chemotherapy and radiation therapy. To identify the molecular mechanisms underlying this, we have examined the expression and potency of three major death receptors: tumor necrosis factor receptor (TNF-R), TNF-related apoptosis-inducing ligand receptor (TRAIL-R), and Fas in mediating cytotoxicity in four invasive pancreatic cancer cell lines. We have analyzed the expression of major antiapoptotic factors, cell cycle regulators and death receptor decoys (DcR) in comparison with normal pancreas tissues and five other human malignant tumor cell lines. We have found that different pancreatic cancer cell lines coexpress high-level TRAIL-R, Fas, and TNF-R1 but are strongly resistant to apoptosis triggered by the death receptors. DcR2 and DcR3 overexpression may partly contribute to the resistance of pancreatic cancer cells to TRAIL-R- and Fas-mediated cytotoxicity. Bcl-XL and Bcl-2 are predominantly overexpressed in pancreatic cancer cell lines, respectively. Bcl-XL is also predominantly overexpressed in prostate, colorectal, and intestinal cancer cells. The knockdown of the predominant Bcl-XL overexpression significantly reduces the viability of pancreatic cancer cells to TNFalpha- and TRAIL-mediated apoptosis by sublethal-dose single and combined antitumor drugs, including geldanamycin, PS-341, Trichostatin A, and doxorubicine. Geldanamyin and PS-341 synergistically block NFkappaB activation, suppress Akt/PKB pathway, and down-regulate Bcl-XL, Bcl-2, cIAP-1, and cyclin D1 expression. This combined regimen dramatically enhances TRAIL cytotoxic effects and breaks through chemoresistance. Bcl-XL plays a vital role in pancreatic cancer chemoresistance. Geldanamycin, PS-341, and TRAIL triple combination may be a novel therapeutic strategy for pancreatic cancer.