Overexpression Of HIF-1α In Osteosarcoma And Mechanism Of TSA Antitumor In Vitro

OBJECTIVETo explore the effects of overexpression of HIF-1αon malignant phenotype and matastesis of osteosarcoma and to investigate the anticancer activity and its mechanism of histone deacetylase inhibitor trichostatin A in vitro, thus providing a new approach and strategy for the treatment of osteosarcoma.METHODS1. The immunohistochemical test of the overexpression of HIF-1αin osteosarcoma tissue and its relationship with clinicopathological para- meterThe formalin-fixed, paraffin embeded osteosarcoma tissues, and para-tumor tissues in some of cases taken from a total of 39 cases of primary osteosarcoma with complete follow-up informations were collected and the expression of HIF-1α, VEGF, MVD were tested immunohistochemically. The results and the clinicopathological parameters of patients were analyzed statistically. Fresh frozen osteosarcoma specimens were taken from 15 casese and their mRNA expression of HIF-1αand VEGF were measured by means of RT-PCR. The relataionship between HIF-1αexpression and the angiogenesis and the significance of HIF-1αexpression in predicting the prognosis are also studied.2. Study on the effect of TSA on cell growth, apoptosis, cell cycle, and invasion of osteosarcoma MG-63 cell line in vitro.MG-63 cells were cultured in DMEM, treated with TSA in different concentrations at various times in vitro. Direct microscope, MTT, flow cytometry, Annexin V, TUNEL, electromicroscope, and Transwell ways were carried out to reveal the anticancer activities of TSA. And the morphological changes, growth, cell cycle, cell apoptosis, and invasive properties were detected, too.3. Mimic hypoxia induced the expression of mRNA and protein of HIF-1αand effect of TSA on the MG-63 cells in vitroMG-63 cells were cultured in vitro under the mimic hypoxia with desferrioxamine. The mRNA levels and proteins of HIF-1αand its target gene VEGF were tested through RT-PCR, Western blotting, and ELISA. The cells were exposed to TSA to investigate the effects of TSA on the expression of HIF-1αand its activities of anti-angiogenesis.RESULTS1. The HIF-1αwas overexpressed in osteosarcoma, with the 39 specimens, the immunolabelling being mainly located in the nuclei of cells, expression negative was found in 8 cases(20.5%), faint in 14(35.9%), moderate in 9(23.1%), strong in 8(20.5%), with an overall positive rate of 79.5%, while expression of HIF-1αas almost absent in para-tumor tissues.The difference between them was statistically (p<0.05). HIF-1αoverexpressed within the tumor, around the region of necrosis, or far away from microvessel. The positive VEGF expression were detected within the cytoplasm and on the cell membrane. In all 39 cases, expression was negative in 4 cases(10.3%), faint in 16(41%), moderate in 11(28.2%), strong in 8(20.5%), overall positive rate 89.7% and had a significance with para-tumor tissues (p<0.05). In all 39 cases, the average number of MVD in osteosarcoma (29.92±5.2) had statistically significant with the number of para-tumor tissues(12.41±3.7) (p<0.05).The consecutive slides staining showed that the positive signal of HIF-1α,VEGF, and MVD were detected in the same area. Spearman's related analysis also demonstrated that the expression of HIF-1αand VEGF had a positive relation with MVD. All results revealed that HIF-1αassociated with tumor angiogenesis.The cross-tabulations for clinicopathological parameters demonstr- ated that HIF-1a expression was significantly associated with surgical stage (ⅡA versusⅡB/Ⅲ) and percentage of dead cells (<90% versus≥90%) (p<0.05). Patient's surgical stage, percentage of dead cells and HIF-1a expression showed significant influence on DFS and OS (P<0.05,P<0.05,P<0.001, respectively) in univariate analysis. In multivariate analysis (Cox regression model) , the surgical stage(ⅡA versusⅡB/Ⅲ) and percentage of dead cells(<90% versus≥90%) were significant for DFS and OS (P=0.015,P=0.041,P=0.017,P=0.031, respectively),however, HIF-1a expression did not. Patients with HIF-1a moderate/strong expression showed significant shorter OS and DFS compared with HIF-1a negative/weak expression in Kaplan–Meier survive curves.The expression rate of mRNA of HIF-1a and VEGF were 93.3% (14/15) and 100%(15/15),respectively. mRNA half-quantitation results showed that the expression of HIF-1a and VEGF in tumor tissues more than para-tumor tissues(p<0.05).2. TSA inhibited the growth of MG-63 cells and had a dramatic effect on the morphology of the cells with the decrease in the amount of cytoplasm and appearance of numerous cytoplasmic projections, the increasing rate of suspension/adherence and the decreasing of cell viability rate(p<0.05) in dose- and time-dependent. TSA arrested the cell cycle in G2/M phase with the decrease amount of G1 phase and the increase of S and G2/M phase. Annexin V showed the average apoptosis rate of MG-63 cells exposed to TSA for 24 hours in 100,200,300,500nM concentration were 13.50%±1.73,22.50%±1.45,42.05%±2.47,63.23 %±2.77, respectively. The apoptosis increased significantly in absent or present of TSA (p<0.05). The TUNEL demonstrated that the apoptosis rate increased gradually in dose- and time-dependent and had significant with control group(p<0.05). TSA induced cells apoptosis with the appearance of condense of cytoplasm and apoptosis body in electromicro- scope. Also, TSA inhibited the invasive rate of MG-63 cells in 50, 100, 200, 300, 500nM concentration (p<0.05).3. The mRNA and protein of HIF-1a and VEGF were induced both hypoxia and mimic hypoxia. The mRNA levels of HIF-1a had no change both normoxia and hypoxia, whereas the protein increased dramaticly under hypoxia. The mRNA and protein of VEGF were detected in normoxia and increased significantly in hypoxia (p<0.05). It is meant that the transcription activity of HIF-1a occurred at the protein levels. The mimic hypoxia had same inducing effect on HIF-1a, and the mRNA and protein of VEGF expression depended on the transcription activity of HIF-1a.The expression of HIF-1a and VEGF induced with mimic hypoxia were inhibited by TSA in time- and dose-dependent. After cells were treated with TSA 300nM for 24 hours in mimic hypoxia, mRNA of VEGF and protein of HIF-1a and VEGF decreased significantly (p<0.05). Exposed to TSA 50,100,200,300,500nM for 24 hours in hypoxia, MG-63 cells had no changes with mRNA of HIF-1a(p>0.05), however, mRNA of VEGF and protein of HIF-1a and VEGF decreased significantly compared with absence of TSA(p<0.05)。When MG-63 cells were treated with TSA for 4 hours in mimic hypoxia,, the protein of HIF-1a had no changes, however, decreased for 8 hours; accordingly, the protein of VEGF decreased for 8 hours and 4-fold times decrease in TSA 500nM concentration(p<0.05).CONCLUSION1. HIF-1a is overexpression in most osteosarcoma and has association with tumor angiogenesis. The occurrence of protein of HIF-1a have a close relationship with poor prognosis, although its effect on prognosis is less than the surgical stage and percentage of dead cells.2. Hypoxia can induce the expression of HIF-1a and its target gene VEGF. The expression was inhibited by histone deacetylase inhibitor trichostatin A in dose- and time-dependent manner.3. TSA have potent anticancer activities of inhibiting MG-63 cells growth and invasion, induced apoptosis, arrested the cell cycle in G2/M phase in dose- and time-dependent manner.4. HIF-1a play a important role in invasion and metastasis of osteosarcoma. HIF-1a might regard as a useful biomarker of high malignant phenotype in osteosarcoma. The present study support HIF-1a as a molecular target for anticancer therapy in osteosarcoma. TSA can be considered as a novel therapeutic strategy for osteosarcoma. INNOVATIONThis dissertation explored the role of HIF-1a in invasion and metastasis of osteosarcoma and its relationship with tumor angiogenesis from hypoxia and angiogenesis aspect. In the first time, we reveal the overexpression of HIF-1a in osteosarcoma and association with poor prognosis in patients. In the first time, we also study the TSA effect on invasion of osteosarcoma in vitro, and showed its potent inhibition. There are supporting evidences to prove the association between expression of HIF-1a and tumor angiogenesis in clinical and in vitro. Hypoxia can induce the expression of HIF-1a and VEGF and inhibited by TSA.SIGNIFICANCEAt present, the treatment of osteosarcoma have many tough difficultes, including lung metastasis at early stage, local recurrence, and poor response to chemotherapy. The hypoxia within tumor exists and can explain following two question: the first, the sources of metastasis and local recurrence are hypoxia cells which they metabolize in anaerobic manner and are away from microvessel; the second, the reason of poor response to chemotherapy is that the proliferation of these hypoxia cells is slowly and can not be killed by chemotherapy drug, and then can not reach the effective anti-tumor drug concentration because of a distance from microvessel. The present study investigated the role of HIF-1a in invasion and metastasis of osteosarcoma and correlation with tumor angiogenesis from hypoxia aspect in clinical research and in vitro. The overexpression of HIF-1a have association with poor prognosis in osteosarcoma patients; Hypoxia can induce the expression of HIF-1a and VEGF and inhibited by TSA. This study partly revealed the role of hypoxia-induced HIF-1a in invasion and metastasis of osteosarcoma, providing a new target and therapeutic strategy for oetsosarcoma. HDACIs can be considered as a novel drug for osteosarcoma.