gx-15-070 and Esophageal-Neoplasms

Obatoclax, a pan-inhibitor of anti-apoptotic Bcl-2 proteins, exhibits cytotoxic effect on cancer cells through both apoptosis-dependent and -independent pathways. Here we show that obatoclax caused cytotoxicity in both cisplatin-sensitive and -resistant esophageal cancer cells. Although obatoclax showed differential apoptogenic effects in these cells, it consistently blocked autophagic flux, which was evidenced by concomitant accumulation of LC3-II and p62. Obatoclax was trapped in lysosomes and induced lysosome clustering. Obatoclax also substantially reduced the expression of lysosomal cathepsins B, D and L. Moreover, cathepsin knockdown was sufficient to induce cytotoxicity, connecting lysosomal function to cell viability. Consistent with the known function of autophagy, obatoclax caused the accumulation of polyubiquitinated proteins and showed synergy with proteasome inhibition. Taken together, our studies unveiled impaired lysosomal function as a novel mechanism whereby obatoclax mediates its cytotoxic effect in esophageal cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cathepsin B; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Enzyme Inhibitors; Esophageal Neoplasms; Humans; Indoles; Lysosomes; Pyrroles; Tumor Cells, Cultured

To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.. MTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.. The CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).. Obatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.

Topics: Apoptosis; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Esophageal Neoplasms; Histones; Humans; Indoles; Leupeptins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Pyrroles

Pan-Bcl-2 family inhibitor obatoclax has been demonstrated to be effective against various cancers, of which the mechanism of action is not fully understood. In this study, we demonstrate that obatoclax suppressed esophageal cancer cell viability with concomitant G1/G0-phase cell cycle arrest. At the tested concentrations (1/2 IC50 and IC50), obatoclax neither induced PARP cleavage nor increased the Annexin V-positive population, suggesting G1/G0-phase arrest rather than apoptosis accounts for most of the reduction of cell viability produced by obatoclax. Double knockdown of Bak and Bax by small interference RNA failed to block obatoclax-induced G1/G0-phase arrest, implying its role in cell cycle progression is Bak/Bax-independent. The cell cycle arresting effect of obatoclax was associated with up-regulation of p21(waf1/Cip1). Knockdown of p21(waf1/Cip1) significantly attenuated obatoclax-induced G1/G0-phase arrest. Although obatoclax stimulated phosphorylation of Erk, p38, and JNK, pharmacological inhibition of p38 but not Erk or JNK blocked obatoclax-induced G1/G0-phase arrest. Moreover, knockdown of p38 abolished the cell cycle arresting effect of obatoclax. In consistent with this finding, inhibition of p38 blocked obatoclax-induced p21(waf1/Cip1) expression while inhibition of Erk or JNK failed to exert similar effect. To conclude, these findings suggest that obatoclax induced cell cycle arrest via p38/p21(waf1/Cip1) signaling pathway. This study may shed a new light on the anti-cancer activity of obatoclax in relation to cell cycle arrest.

Topics: Antineoplastic Agents; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Esophageal Neoplasms; Humans; Indoles; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyrroles

Despite improvements in both surgical techniques and radio- and chemo-therapy regimens, the prognosis of esophageal cancer is poor. In pursuit of novel effective strategy, this study examined the effect of the BH3-mimetic GX15-070 on esophageal carcinoma cells. We discovered that GX15-070 inhibited the growth of esophageal cancer cells. There was synergism between GX15-070 and carboplatin or 5-fluorouracil. GX15-070 induced autophagy in esophagus cancer cell line EC9706 and osteosarcoma cancer cell line U2OS. 3-methyladenine and chloroquine, inhibitors of autophagy with distinct mechanisms, potentiated the cytotoxicity of GX15-070. In conclusion, GX15-070 inhibits growth of esophageal cancer cells.

Topics: Autophagy; Carboplatin; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Drug Therapy, Combination; Esophageal Neoplasms; Fluorouracil; Humans; Indoles; Osteosarcoma; Pyrroles; Signal Transduction