[Study on impact of ethanol extracts from Sedum sarmentosum in inhibiting STAT-3 signaling and inducing apoptosis of human hepatocellular carcinoma cell line HepG2].

OBJECTIVE: To investigate the impact of ethanol extracts from Sedum sarmentosum (ESB) on STAT-3 signaling and its probable molecular mechanism in inducing apoptosis. METHOD: MTT assay was used to detect the impact of ESB on HepG2 cell proliferation. FITC-Annexin V-FITC /PI double-labeling were used to investigate the impact on hepatoma carcinoma cell apoptosis. Western blot analysis was used to test the expression levels of cell apoptosis-related proteins Caspase-3, Caspase-9, PARP, P-STAT-3 (Tyr705) , STAT-3, Bcl-2, Mcl-1. RESULT: ESB could notably inhibit proliferation of HepG2 cells, and induce HepG2 cell apoptosis, with the dose-dependent inhibitory effect. In addition, ESB could inhibit STAT-3 signaling, down-regulate Mcl-1 and Bcl-2 expressions, and induce degradation/activation of apoptosis-related proteins Caspase-3 and Caspase-9 and PARP degradation in a dose-dependent manner. CONCLUSION: ESB inhibits HepG2 cell proliferation and induces apoptosis by inhibiting STAT-3 signaling and Mcl-1 and Bcl-2 expressions.

Synergistic activities of MET/RON inhibitor BMS-777607 and mTOR inhibitor AZD8055 to polyploid cells derived from pancreatic cancer and cancer stem cells.

Tyrosine kinase inhibitor BMS-777067 is an inhibitor of RON/MET receptor tyrosine kinases currently under clinical trials. Here, we report the synergistic activity of BMS-777607 in combination with mTOR inhibitor AZD8055 in killing chemoresistant pancreatic cancer and cancer stem cells. Treatment of pancreatic cancer L3.6pl cells with BMS-777607 alone inhibited clonogenic growth and moderately induced apoptotic death. However, BMS-777607 caused extensive polyploidy in L3.6pl cells through inhibition of aurora kinase B activity, independent of RON expression. In contrast, L3.6pl-derived cancer stem cells were highly resistant to BMS-777607-induced growth inhibition and apoptosis. The effect of BMS-777607 on induction of cancer stem cell polyploidy was also weak. BMS-777607-induced polyploidy features a predominant cell population with 8N chromosome content in both L3.6pl and cancer stem cells. These cells also showed decreased sensitivity toward chemotherapeutics by increased survival of IC(50) values in response to doxorubicin, cisplatin, methotrexate, 5-fluorouracial, and gemcitabine. Among a panel of chemical inhibitors that target different signaling proteins, we found that BMS-777607 in combination with mTOR inhibitor AZD8055 exerted synergistic effects on L3.6pl and cancer stem cells. More than 70% of L3.6pl and cancer stem cells lost their viability when both inhibitors were used. Specifically, BMS-777607 in combination with inhibition of mTORC2, but not mTORC1, was responsible for the observed synergism. Our findings demonstrate that BMS-777607 at therapeutic doses exerts inhibitory activities on pancreatic cancer cells but also induces polyploidy insensitive to chemotherapeutics. Combination of BMS-777607 with AZD8055 achieves the maximal cytotoxic effect on pancreatic cancer and cancer stem cells.

Oncogenic variant RON160 expression in breast cancer and its potential as a therapeutic target by small molecule tyrosine kinase inhibitor.

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Small-molecule inhibitor BMS-777607 induces breast cancer cell polyploidy with increased resistance to cytotoxic chemotherapy agents.

The RON receptor tyrosine kinase is a therapeutic target for cancer treatment. Here, we report therapeutic effect and phenotypic change of breast cancer cells in response to BMS-777607, a RON tyrosine kinase inhibitor. Treatment of breast cancer cells with BMS-777607 at therapeutic doses inhibited cancerous clonogenic growth but had only minimal effect on cell apoptosis. Significantly, BMS-777607 induced extensive polyploidy with multiple sets of chromosomes in cancer cells. This effect is independent of RON expression. Knockdown of RON in T-47D and ZR-75-1 cells by specific siRNA did not prevent polyploid formation. Immunofluorescent analysis of α-tubulin and γ-tubulin expression in polyploid cells revealed that BMS-777607 disrupts bipolar spindle formation and causes multipolar-like microtubule assembly. Also, both metaphase equatorial alignment and chromosomal segregation were absent in polyploid cells. These results suggest that cellular mitosis arrests at prophase/pro-metaphase and fails to undergo cytokinesis. By analyzing kinase-inhibitory profiles, aurora kinase B was identified as the target molecule inhibited by BMS-777607. In BMS-777607-treated cells, aurora kinase B was inhibited followed by protein degradation. Moreover, BMS-777607 inhibited Ser10 phosphorylation of histone H3, a substrate of aurora kinase B. Chemosensitivity analysis indicated the resistance of polyploid cells toward chemotherapeutics. Treatment with doxorubicin, bleomycin, methotrexate, and paclitaxel significantly increased cellular IC50 values. These findings highlight the theory that BMS-777607 acts as a multikinase inhibitor at therapeutic doses and is capable of inducing polyploidy by inhibiting aurora kinase B. Increased resistance of polyploid cells to cytotoxic chemotherapeutics could have a negative impact on targeted cancer therapy using BMS-777607.