Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.

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Current knowledge indicates that the mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, differentiation, and motility. Recently mSin1 has been identified as a critical component of mTORC2, which is essential for phosphorylation of Akt and other signaling molecules. Studies have shown that rapamycin inhibits phosphorylation of mSin1. However, the underlying mechanism is unknown. Here we found that rapamycin inhibited phosphorylation of mSin1 potently and rapidly. Expression of rapamycin-resistant mutant of mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mutant of mTOR (mTOR-TE), prevented rapamycin from inhibiting mSin1 phosphorylation, suggesting that rapamycin-induced dephosphorylation of mSin1 is mTOR-dependent. Surprisingly, ectopic expression of rapamycin-resistant and constitutively active p70 S6 kinase 1 (S6K1) did not confer resistance to rapamycin-induced dephosphorylation of mSin1. Furthermore, disruption of mTORC1 and mTORC2 by silencing raptor and rictor, respectively, or downregulation of S6K1 or Akt did not induce the dephosphorylation of mSin1 as rapamycin did. However, silencing mTOR or mLST8 mimicked the effect of rapamycin, inhibiting mSin1 phosphorylation. Our findings suggest that rapamycin inhibits mSin1 phosphorylation, which is independent of mTORC1 and mTORC2, but is possibly dependent on a new mTOR complex, which at least contains mTOR and mLST8.

Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death.

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Introduction: Resistance to apoptosis is a hallmark of tumor progression and therapeutic failure in cancer, including Pancreatic Cancer. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PDAC are largely ineffective. There is an urgent yet unmet need for development of therapeutic agents for the treatment of PDAC. In the present study, we investigated the effects of Tetrandrine (a bis-benzylisoquinoline alkaloid) derivative- TET, on growth and viability of pancreatic cancer in vitro and in Xenograft models in vivo. Methods: Pancreatic Cancer cell lines: PANC-1 (epitheloid carcinoma), BxPC3 (Pancreatic Ductal Adeno-Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in this study. Cytotoxicity was evaluated using the Crystal violet and MTT survival assays. Apoptosis was monitored by Flow cytometry following Annexin V/PI staining. Nuclear morphology was visualized by Immunofluorescence. Western Blot analysis was used to measure protein expression. Human pancreatic cancer (BXPC3) derived xenografts were generated in NOD/ SCID mice and TET (40 mg/kg body weight) was orally administered daily for four weeks. Tumor growth (measured as tumor volume by Vernier Caliper) and body weights were measured on alternate days. Tumor weight was measured at the end of the experimental period, prior to xenograft tissue harvesting. Results: TET inhibited growth and promoted cell death of pancreatic cancer cells in both dose and time dependent manner with an IC50 in the range of 5-10μM at 72 hr. The effects of TET were irreversible and there was progressive cell death with increasing time and at higher concentrations of TET. Treatment with TET resulted in nuclear condensation and apoptotic body formation, activation of caspase 3 and PARP cleavage, indicating apoptotic cell death. Moreover apoptosis was confirmed by flow cytometry after Annexin V and PI staining. TET administration not only halted the growth of BXPC3 derived xenografts, but also decreased tumor volume (TET treated vs PBS treated) over treatment period. Apoptosis (measured by TUNEL assay) was also observed in tumor tissues following TET treatment in vivo. Conclusion: These results show for the first time, that TET inhibits pancreatic cancer cell growth in vitro and induces pancreatic tumor regression in vivo in part by apoptosis. These results highlight the potential use of TET in treatment of pancreatic cancer. Acknowledgement: Financial support from following Sources is gratefully acknowledged: Carroll W. Feist endowed Chair Funds (Koul H), FWCC support and Chair commitment funds (Koul H) from the Chancellor and from the Dean School of Medicine- LSUHSC-Shreveport. All the members of Koul laboratory for their suggestions and help. Citation Format: Karnika Singh, Prakash Srinivasan Timiri Shanmugam, Sweaty Koul, Qin Dong, Neil Koul, Hari K. Koul. Tetrandrine promotes pancreatic cancer cell apoptosis in vitro and tumor regression in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3516.

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