apogossypolone and Liver-Neoplasms

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.

Topics: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis Regulatory Proteins; Autophagy; bcl-X Protein; Beclin-1; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Gossypol; Hep G2 Cells; HMGB1 Protein; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Phosphorylation; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases

To investigate the in vitro and in vivo activities and related mechanism of apogossypolone (ApoG2) alone or in combination with adriamycin (ADM) against human hepatocellular carcinoma (HCC).. The IC50 of ApoG2 in vitro was tested by WST assay, and the synergistic effect was analyzed using the CalcuSyn method. Cell apoptosis was determined using 4',6-diamidino-2- phenylindole staining and flow cytometric analysis. Western blotting was used to determine the expression of apoptosis-related proteins. In vivo activity was evaluated in the xenograft model in nude mice, and apoptosis in tumor tissues was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay.. The IC50 of ApoG2 in HCC cells was 17.28-30.63 micromol/L. When ApoG2 was combined with ADM, increased cytotoxicity and apoptosis were observed in SMMC-7721 cells compared to treatment with ApoG2 alone. The Western blotting results indicated that the ApoG2 induced apoptosis in SMMC-7721 cells by downregulating anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL, up-regulating pro-apoptotic protein Noxa, and promoting the activities of caspases-9 and -3. The tumor growth of xenograft SMMC-7721 was inhibited in nude mice when ApoG2 was administered orally without causing damage to the normal tissues. The in vivo study also indicated an increasing anti-tumoral effect when ApoG2 at 100 or 200 mg/kg dosages were used together with ADM at 5.5 mg/kg, with relative tumor proliferation rate (T/C) values of 0.456 and 0.323, respectively. Apoptosis induced in vivo by ApoG2 alone or combined with ADM was confirmed by TUNEL assay in tumor tissues.. ApoG2 is a potential non-toxic target agent that induces apoptosis by upregulating Noxa, while inhibiting anti-apoptotic proteins and promoting the effect of chemotherapy agent ADM in HCC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Doxorubicin; Drug Therapy, Combination; Gossypol; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Mice; Mice, Nude; Molecular Structure; Neoplasm Transplantation; Proto-Oncogene Proteins c-bcl-2; Transplantation, Heterologous