組蛋白去乙醯酶6抑制劑減緩增生及毒殺神經膠質瘤細胞之探討

Antiproliferative and cytotoxic effects of histone deacetylase (HDAC 6) inhibitors on giloma cells: in vitro and in vivo studies

張菀茹(Wan-Ju Chang)

指導教授 ： 王家儀

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

惡性神經膠質瘤是最常見的腦瘤，過去文獻指出，經由外科手術或是放射線等治療之後，有相當大的比率病患會再復發。相關研究發現，組蛋白去乙醯酶抑制劑可以停止細胞周期、促進細胞分化及細胞凋亡，以及抑制腫瘤轉移。而在十一種組蛋白去乙醯酶之中，第六亞型(組蛋白去乙醯酶6，HDAC 6)被發現和神經保護有關，且在神經膠質瘤中，組蛋白去乙醯酶6的表現量有顯著增加的情形。因此我們選擇組蛋白去乙醯酶6進行探討。我們利用C6大鼠神經膠質瘤細胞株做為研究材料，來探討組蛋白去乙醯酶6抑制劑是否具有抑制神經膠質瘤增生或誘發腫瘤細胞死亡以及抑制血管新生的能力，而達到抗癌之效果。

　　在先前惡性神經膠質瘤的研究中發現，組蛋白去乙醯酶6抑制劑Suberoylanilide hydroxamic acid (SAHA, Vorinostat) 能有效抑制癌細胞的增生及促使癌細胞進行細胞凋亡。在本實驗中我們發現，給予同為組蛋白去乙醯酶6抑制劑之MPT0B291或SAHA，在低濃度( < 3 μM )情況下兩者都有細胞增生率上升的情形，而給予較高濃度的MPT0B291 (10-30 μM ) 處理後，發現隨著給藥濃度愈高，細胞增生率下降幅度愈大。利用高濃度之SAHA或MPT0B291 (30 μM) 處理神經膠質瘤細胞後，兩者都隨著給藥時間愈長，抑制增生能力愈強及毒殺細胞能力愈強。另外，MPT0B291及SAHA皆具有停止細胞周期及誘發細胞凋亡的能力，且兩者間沒有顯著差異。我們利用神經膠質瘤細胞 (107cells / 20μL) 注射到Sprague-Dawley大鼠腦中做為動物模式，並且也發現到，給予 MPT0B291 (10 μM /10 μL)或SAHA (10 μM /10 μL) 皆可使神經膠質瘤體積變小，並且抑制腫瘤增生、促進癌細胞進行細胞凋亡以及抑制腫瘤血管新生。由即時聚合酶鏈鎖反應的結果可知，MPT0B291能減緩因罹患神經膠質瘤而表現量上升的p53 mRNA表現量。基於以上的結果，我們推論MPT0B291在抑制神經膠質瘤的增生效果大於SAHA，而在促使細胞周期停止、細胞凋亡以及抑制血管新生方面則沒有差異。

關鍵字

神經膠質瘤 ； 組蛋白去乙醯酶細胞增生 ； 組蛋白去乙醯酶6抑制劑 ； 細胞培養 ； 動物模式 ； 細胞凋亡 ； 細胞週期停止

並列摘要

Malignant astrocyte gliomas are the most common type of primary brain tumor in adults. Multimodality treatments include surgery, radiation and chemotherapy. However, no curative therapy today and the prognosis for many patients with gliomas is poor. New therapeutic approaches are desperately needed. Malignant astrocyte gliomas are characterized by heterogeneity and dysregulation of several signaling pathways, and studies have demonstrated that this occurs by epigenetic mechanisms. Histone deacetylase (HDAC) inhibitors have been demonstrated to induce cell cycle arrest, promote cell differentiation or apoptosis, and inhibit metastasis. HDAC inhibitors have thus emerged as a new class of anti-tumor agents for various types of tumors. Previous studies showed that suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, suppresses the growth of gliomas. Among the 11 isoforms of HDAC, HDAC 6 has been shown to be neuroprotective. Increased expression of HDAC6 in glioma cells was observed. We therefore chose HDAC6 as the target for study. In the present study, we explored the anti-tumor effect of SAHA and MPT0B291, both are selective HDAC 6 inhibitors, in C6 rat glioma cells in vitro and in vivo.
Using MTT assay to evaluate cell proliferation in vitro, we found that treatments with low concentrations (< 10 μM) of MPT0B291 or SAHA increased C6 cell proliferation. However, higher concentrations MPT0B291 (10-30 μM) for 24 hr concentration-dependently supressed proliferation of C6 glioma cells (from 96 % to 58 % of control). Treatment with SAHA (10-30 μM) for 24 hr the effect was not significant (from 115% to 94% of control). We also found that MPT0B291 (30 μM) or SAHA (30 μM) time-dependently induced growth arrest and cell death. Flow cytometry analysis demonstrated significant inhibition of cell cycle progression (G2-M arrest) when C6 glioma cells were treated by MPT0B291 (30 μM) or SAHA (30 μM) for 24 h. In addition, apoptosis were observed when C6 glioma cells were treated with MPT0B291 (30 μM) or SAHA (30 μM) for 24 h and even to a greater degree at 48hr after drug treatment. We also established an animal model of glioma by intracerebral injection of C6 glioma cells (107 cells /20 μl) in the rat. Histological staining of brain tissue sections demonstrated glioma growth in the brain at day 7. Using this animal model, we examined the effect of MPT0B291 or SAHA on glioma growth in vivo. Our results indicated that MPT0B291 (10 μM /10 μL) or SAHA (10 μM /10 μL) injected into the tumor site at day 3 and day 5, reduced the glioma volume. This reduction in tumor volume was associated with an increase in apoptosis, a decrease in the proliferation and angiogenesis. Reverse transcription (RT) – qPCR results demonstrated that elevated p53 mRNA expression in brain tissue injected with C6 glioma cells. Treatment with MPT0B291 or SAHA also attenuated the elevation of p53 mRNA expression. There results the antiproliferative and cytotoxic effects of SAHA or MPT0B291 on glioma cells might be attributed to multiple mechanisms including cell cycle arrest, pro-apoptotic effect, anti-angiogenesis mechanisms and downregulation of tumor suppressor gene p53.

並列關鍵字

Glioma ； HDAC ； tumor growth ； HDAC6 inhibitor ； cell culture ； animal model ； proliferation ； apoptosis ； cell cycle arrest

參考文獻

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