Studies On Radiosensitization Of Hepatocellular Carcinoma By B-cell Translocation Gene 2(BTG2)

BackgroundHepatocellular carcinoma(HCC) is the third leading cause of cancer-related death, and ranks sixth among the most common cancers in the world[1-3]. HCC is usually diagnosed at the advanced stage due to the lack of early detection means such as serum biomarkers, or clinical symptoms and signs; making surgical cure impossible. Overall, the prognosis of patients with HCC is very poor. Clinically, HCC patients are treated with surgical resection of tumor lesions, liver transplantation, chemotherapy or radiotherapy. Radiation therapy is being integrated with other treatment options into comprehensive treatment regimes of HCC, especially for localized hepatic tumors refractory to conventional therapy[4]. To date, controlling HCC using radiation therapy remains unsatisfactory due to the low radiation sensitivity and tolerance of the whole liver[5]. Thus, improving the therapeutic effect of radiation on hepatoma has important clinical significance and is much needed.Previously, tumor suppressor gene p53 has been used to enhance radiotherapy effectiveness on oral cancer patients after resection surgery of tumor lesions[6, 7]. B-cell translocation gene 2(BTG2) proteins were reported to be putative tumor suppressor genes, which is a p53-dependent component of the DNA damage cellular response pathway[8-10]. BTG2 is an early growth response gene and belongs to the APRO family [11-13]. Recent studies imply that BTG2 protein functions to regulate cell differentiation, development and apoptosis [14, 15]. BTG2 expressions have been shown to be reduced or lost in a variety of cancers [8, 16-20]. The restoration of BTG2 expressions in a medulloblastoma mouse model suppressed tumor formation and growth [21].ObjectiveThe aim of this study is to assess BTG2 expressions and its role in liver cancer; and expect to provide insightful information on the association of BTG2 with HCC prognosis and promotion in HCC cell apoptosis after radiotherapy in vitro and in a nude mouse model of transplanted hepatoma.Methods1. Patients and liver sample collection. All HCC tissue samples were collected from 44 patients in Daping Hospital(Chongqing, China). These patients were histologically diagnosed with HCC according to the World Health Organization(WHO) criteria. Tissue array and immunohistochemical analysis BTG2 expression. Stained sections were independently evaluated and scored under a microscope by two pathologists using the following four-point scale(positive cell count, grades 0-3): 0, no positive cells; 1, <25% positive cells; 2, 25-50% positive cells; 3, >50% positive cells. Low expression levels in the nuclei were defined as having an immunohistochemical staining score of 0-1, whereas high expression levels in the nuclei were defined as having an immunohistochemical staining score of 2-3.2. Cell line, culture and pEGFP-N1-BTG2 transfection. Human HCC cell line Huh7 was transfected with eukaryotic expression vector pEGFP-N1-BTG2 or empty vector as a control using Lipofectamine 2000(Invitrogen).Protein extraction and Western blot analysis protein expression. RNA extraction and PCR analysis RNA expression. Flow cytometric analysis of apoptosis and cell counting assay after radiation treatment. Experiments were independently repeated for at least three times.3. Animal experiments. Severe combined immune deficient(SCID) mice were purchased from the Chinese Academy of Medical Sciences(Beijing, China), and were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. For animal experiments, mice were first implanted with 1 x 107 Huh7 cells stably transfected with empty vector or pEGFP-N1-BTG2 vector. Twenty-one days after tumor cell injection, the tumor site was irradiated with 8 Gy on days 21, 24 and 27. Tumor volume was measured twice a week for four weeks; and at the end of the experiments, xenograft tissues were taken out and were subjected to other experiments. Western blot analysis Bax protein expression. Immunohistochemical analysis Ki67 expression. Apoptosis in xenograft tissues was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) method.Results1. 44 HCC tissue specimens were collected for tissue array and Immunohistochemical analysis of BTG2 expressions. Patients were divided into two groups(low expression and high expression) based on staining data. Our data revealed that patients with higher BTG2 expressions in the nucleus had better survival rates, compared to patients with low BTG2 expressions.2. In vitro effects of BTG2 expressions on HCC cells were assessed by stably overexpressing BTG2 in Huh7 cells. After stable gene transfection, Western blot data demonstrated that BTG2 expressions were restored at protein level by 3.6 folds(P<0.05) in stably transfected Huh7-BTG2 cells, compared to Huh7-Vector cells. Then, we explored the effects of BTG2 expressions on the regulation of radiation efficiency in Huh7 cells; and found that radiation treatment significantly reduced the number of Huh7-BTG2 cells, compared to Huh7-vector cells. Radiation treatment did not have any effect on Huh7-vector cell proliferation, compared to parental Huh7 cells. We then assessed the effects of radiation treatment on apoptosis in Huh7-BTG2 cells vs. Huh7-vector cells. Our results revealed a marked increase(approximately 2.5 folds) in the apoptosis of Huh7-BTG2 cells(55.9%) after radiation treatment, compared to Huh7-vector cells(24.6%). There was no difference in apoptosis in Huh7-vector cells(24.6%), compared to Huh7 cells(17.2%).3. To explore whether overexpression of BTG2 proteins can repress the formation and growth of HCC cell xenografts in vivo, we transplanted hepatoma cells into nude mice as previously described. Our data revealed that overexpression of BTG2 per se did not have obvious effects on tumor growth in nude mice 21 days after tumor injection. We then irradiated the mice on days 21, 24 and 27(for a total of three times), and monitored tumor volume. Data revealed that after three times of irradiation, tumor volume was significantly reduced in nude mice injected with Huh7-BTG2 cells, compared to nude mice injected with Huh7-vector cell injection(35% of the control, P<0.05). These results suggest that BTG2 overexpression reduced hepatoma tumor growth after radiation therapy in nude mice. Furthermore, we resected xenograft tissues and stained the xenograft sections for H&E, Ki67, BTG2 and TUNEL. Our data revealed that after radiation treatment, the growth of tumor xenografts was significantly repressed in BTG2-overexpressed hepatoma tumor, compared to vector-only hepatoma cell injection, as evident from Ki67(a proliferation marker) immunohistochemistry staining. A marked increase in TUNEL staining was observed in BTG2 overexpressing tumor sections(39.8%, P<0.05), as compared to vector expressing tumor sections. Bax expressions were also significantly increased in tumor tissues with BTG2 overexpression, compared with the group that had no or low BTG2 expression(1.5-fold, P<0.05).Conclusions1. Patients with higher BTG2 expressions in the nucleus had better survival rates, compared to patients with low BTG2 expressions.2. BTG2 overexpression sensitizes Huh7 cells to radiation-induced apoptosis in vitro.3. BTG2 overexpression sensitizes Huh7 cells to radiation-induced apoptosis in vivo.