ABSTRACT
mTOR is aberrantly activated in hepatocellular carcinoma (HCC) and plays pivotal roles in tumorigenesis and chemoresistance. Rapamycin has been reported to exert antitumor activity in HCC and sensitizes HCC cells to cytotoxic agents. However, due to feedback activation of AKT after mTOR complex 1 (mTORC1) inhibition, simultaneous targeting of mTORC1/2 may be more effective. In this study, we examined the interaction between the dual mTORC1/2 inhibitor OSI-027 and doxorubicin in vitro and in vivo. OSI-027 was found to reduce phosphorylation of both mTORC1 and mTORC2 substrates, including 4E-BP1, p70S6K, and AKT (Ser473), and inhibit HCC cell proliferation. Similar to OSI-027 treatment, knockdown of mTORC2 induced G0-G1 phase cell-cycle arrest. In contrast, rapamycin or knockdown of mTORC1 increased phosphorylation of AKT (Ser473), yet had little antiproliferative effect. Notably, OSI-027 synergized with doxorubicin for the antiproliferative efficacy in a manner dependent of MDR1 expression in HCC cells. The synergistic antitumor effect of OSI-027 and doxorubicin was also observed in a HCC xenograft mouse model. Moreover, AKT was required for OSI-027-induced cell-cycle arrest and downregulation of MDR1. Our findings provide a rationale for dual mTORC1/mTORC2 inhibitors, such as OSI-027, as monotherapy or in combination with cytotoxic agents to treat HCC. Mol Cancer Ther; 14(8); 1805-15. ©2015 AACR.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antibiotics, Antineoplastic/pharmacology , Cell Cycle Checkpoints/drug effects , Doxorubicin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Multiprotein Complexes/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Synergism , Gene Knockdown Techniques , Humans , Imidazoles/pharmacology , Inhibitory Concentration 50 , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Multiprotein Complexes/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Triazines/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND AND OBJECTIVE: Hepatic sinusoidal obstruction syndrome (HSOS) is characterized by painful hepatomegaly, ascites, increased body weight, and jaundice. Gynura segetum (Compositae), a plant widely used in Chinese traditional medicine, often leads to the development of HSOS. However, the mechanism is unclear. The aim was to study the role of matrix metalloproteinase-9 (MMP-9) in the onset of HSOS induced by Gynura segetum. METHODS: Twenty-five male Sprague-Dawley rats were randomly divided into two groups. Twenty were exposed to 600 mg/kg daily Gynura segetum extract solution for three weeks; five control rats were exposed to tap water alone. Liver sections were evaluated by light microscopy with a modified scoring system. Routine transmission electron microscopy (TEM) methods were used to evaluate the ultrastructual features of fixed liver tissue, and blood samples were collected to determine liver enzyme concentrations. MMP-9 expression was assessed by both immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) methods. RESULTS: A stable and reproducible rat model of HSOS was achieved by long-term exposure to Gynura segetum extract. The treated rats presented clinical symptoms and the histopathological manifestation of HSOS, including abnormal liver enzyme concentrations (alanine aminotransferase (ALT): (84.8±13.62) vs. (167.0±72.63) U/L, P<0.05; aspartate aminotransferase (AST): (27.6±6.31) vs. (232.8±108.58) U/L, P<0.05). Hematoxylin and eosin (H&E) staining and TEM together revealed deposition of red blood cells, the damage and destruction of hepatic sinusoidal endothelial cells, collapse of hepatic sinusoids, hemorrhage of subendothelial cells, atrophy and destruction of hepatocytes, etc. Compared with controls, the expression of MMP-9 in the blood sample, the lung and liver tissues of HSOS rats was increased. CONCLUSIONS: MMP-9 may have an important role in early pathological changes of HSOS, and thus the onset of the disease.
Subject(s)
Drugs, Chinese Herbal/toxicity , Hepatic Veno-Occlusive Disease/enzymology , Matrix Metalloproteinase 9/biosynthesis , Plant Extracts/toxicity , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Asteraceae/chemistry , Hepatic Veno-Occlusive Disease/chemically induced , Hepatic Veno-Occlusive Disease/pathology , Immunohistochemistry , Male , Matrix Metalloproteinase 9/metabolism , Microscopy, Electron, Transmission , Plant Roots/chemistry , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
Doxorubicin-based therapy is not effective for the treatment of hepatocellular carcinomas (HCCs), which often undergo epithelial-mesenchymal transition (EMT) during tumor progression. Activation of signal transducer and activator of transcription 3 (STAT3) is associated with chemosensitivity and may contribute to EMT during HCC chemotherapy. Low doses of NSC 78459 (a novel STAT3 inhibitor) have little effect on HCC cell proliferation, but efficiently inhibit STAT3. HuH-7, Hep3B, and HepG2 cells, with epithelial phenotypes, show significantly enhanced doxorubicin cytotoxicity following co-treatment with NSC 74859, whereas mesenchymal SNU-449 cells show no such enhancement. NSC 74859 inhibits STAT3 activity and suppressed doxorubicin-induced EMT in epithelial HCC cells. siRNA-mediated STAT3 knockdown resulted in EMT inhibition, which led to attenuation of NSC 74859-mediated chemosensitivity. Our data indicate NSC 74859 co-administration enhances doxorubicin cytotoxicity by inhibiting STAT3 in epithelial HCC cells. STAT3 deactivation and associated EMT attenuation contribute to the synergistic anti-tumor effects of combined NSC 74859/doxorubicin therapy.
