apogossypolone and Nasopharyngeal-Neoplasms

Nasopharyngeal carcinoma (NPC) is a major cause of cancer deaths. Concurrent administration of radiation and chemotherapy is the treatment of choice for advanced NPC. Previously, we showed that apogossypolone (ApoG2) induced apoptosis by blocking the binding of Bcl-2 to Bax, arresting the cell cycle in the S phase, in turn inhibiting proliferation of NPC cells both in vitro and in vivo. In the present study, we showed that ApoG2 inhibited the proliferation of NPC cells in a dose-dependent manner. We treated CNE1, CNE2 and SUNE1 cells with ApoG2 for 72 h, and calculated the IC50 values as 2.84, 5.64 and 2.18 µM, respectively. Normal NP69 cell proliferation was not significantly inhibited. ApoG2 treatment induced significant autophagy, demonstrated by an increase in LC3-II protein expression, reduced protein p62 expression, and accumulation of punctuate GFP-LC3 in the cytoplasm of CNE1 or CNE2 cells. Sh-Atg5 attenuated the autophagy induced by ApoG2, indicating that Atg5 was required for ApoG2-induced autophagy. In addition, ApoG2 treatment blocked the binding of Bcl-2 to Beclin 1 protein, releasing pro-autophagic Beclin 1, which in turn triggered the autophagic cascade. Colony formation assays indicated that ApoG2 enhanced radiosensitization of CNE2 cells. In the ApoG2-plus-radiation combination group, more ring-shaped structures were evident in CNE1 and CNE2 cultures. LC3-II expression was enhanced and that of p62 reduced, compared to the ApoG2-only, radiation-only and control groups. ApoG2 enhanced the radiosensitivity of CNE2 xenografts in nude mice as measured by (C-T)/C ratios (as percentages); the values for the ApoG2 and radiation groups were 46.89% and 19.34%, respectively. The ApoG2-plus-radiation group exhibited greater antitumor activity (the inhibitory rate was 61.64%). Immunohistological staining showed that LC3-II expression became gradually upregulated in the ApoG2-plus-radiation group. Together, the results suggest that ApoG2 inhibits the binding of Bcl-2 to Beclin 1, inducing autophagy and radio-sensitizing NPC cells both in vitro and in vivo.

Topics: Animals; Autophagy; Carcinoma; Cell Line, Tumor; Cell Proliferation; Chemoradiotherapy; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Gossypol; Humans; Mice; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasms, Experimental; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

Apogossypolone (ApoG2), a novel derivative of gossypol, has been shown to be a potent inhibitor of antiapoptotic Bcl-2 family proteins and to have antitumor activity in multiple types of cancer cells. Recent reports suggest that gossypol stimulates the generation of cellular reactive oxygen species (ROS) in leukemia and colorectal carcinoma cells; however, gossypol-mediated cell death in leukemia cells was reported to be ROS-independent. This study was conducted to clarify the effect of ApoG2-induced ROS on mitochondria and cell viability, and to further evaluate its utility as a treatment for nasopharyngeal carcinoma (NPC). We tested the photocytotoxicity of ApoG2 to the poorly differentiated NPC cell line CNE-2 using the ROS-generating TL/10 illumination system. The rapid ApoG2-induced cell death was partially reversed by the antioxidant N-acetyl-L-cysteine (NAC), but the ApoG2-induced reduction of mitochondrial membrane potential (MMP) was not reversed by NAC. In the presence of TL/10 illumination, ApoG2 generated massive amounts of singlet oxygen and was more effective in inhibiting cell growth than in the absence of illumination. We also determined the influence of light on the anti-proliferative activity of ApoG2 using a CNE-2-xenograft mouse model. ApoG2 under TL/10 illumination healed tumor wounds and suppressed tumor growth more effectively than ApoG2 treatment alone. These results indicate that the ApoG2-induced CNE-2 cell death is partly ROS-dependent. ApoG2 may be used with photodynamic therapy (PDT) to treat NPC.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Gossypol; Humans; Light; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Photochemotherapy; Reactive Oxygen Species; Singlet Oxygen; Tumor Burden

apogossypolone (ApoG2) is a novel derivate of gossypol. We previously have reported that ApoG2 is a promising compound that kills nasopharyngeal carcinoma (NPC) cells by inhibiting the antiapoptotic function of Bcl-2 proteins. However, some researchers demonstrate that the antiproliferative effect of gossypol on breast cancer cells is mediated by induction of cell cycle arrest. So this study was aimed to investigate the effect of ApoG2 on cell cycle proliferation in NPC cells.. We found that ApoG2 significantly suppressed the expression of c-Myc in NPC cells and induced arrest at the DNA synthesis (S) phase in a large percentage of NPC cells. Immunoblot analysis showed that expression of c-Myc protein was significantly downregulated by ApoG2 and that the expression of c-Myc's downstream molecules cyclin D1 and cyclin E were inhibited whereas p21 was induced. To further identify the cause-effect relationship between the suppression of c-Myc signaling pathway and induction of cell cycle arrest, the expression of c-Myc was interfered by siRNA. The results of cell cycle analysis showed that the downregulation of c-Myc signaling pathway by siRNA interference could cause a significant arrest of NPC cell at S phase of the cell cycle. In CNE-2 xenografts, ApoG2 significantly downregulated the expression of c-Myc and suppressed tumor growth in vivo.. Our findings indicated that ApoG2 could potently disturb the proliferation of NPC cells by suppressing c-Myc signaling pathway. This data suggested that the inhibitory effect of ApoG2 on NPC cell cycle proliferation might contribute to its use in anticancer therapy.

Topics: Cell Cycle; Cell Line; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Gossypol; Humans; Molecular Structure; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; RNA Interference; Signal Transduction; Transplantation, Heterologous