根據行政院衛生署民國95 年統計資料,口腔癌已成為台灣男性十大癌症發生率的第五位,且發生率和死亡率都有逐年增加的趨勢,目前已經成為年增率最高的癌症。儘管近年來在診斷及治療技術上有進步,但口腔癌病人存活率依舊沒有顯著的改善,因此迫切需要有新的療法來解決。先前我們發現在口腔上皮變異及口腔癌有survivin基因的過度表現,但正常口腔上皮組織卻無表現。最近研究發現, silibinin 可以抑制許多種癌細胞的 survivin 表現,因此silibinin或許可做為未來治療口腔癌的潛力新藥物。本研究以人類口腔癌細胞株 SAS 及 Ca9-22 來探討 silibinin 對口腔癌細胞的影響,並進一步了解其可能的機制。 結果顯示, 150 與300 μM silibinin 處理 SAS 及 Ca9-22 細胞可以明顯抑制 survivin 的表現。 此外,以150與300 μM silibinin 處理 SAS 及 Ca9-22 細胞 24 及 48 小時, 可以明顯抑制其細胞生長,且濃度愈高或作用時間愈長,抑制效應就愈明顯 (48小時 IC50 分別為150 μM 和275 μM )。藉由流式細胞儀分析細胞之細胞週期結果顯示,以 150 與 300 μM silibinin 處理 SAS 及 Ca9-22 細胞株 48 小時後,會明顯增加 G1 期細胞數目百分比。 在西方墨點法分析中, silibinin 會增加 SAS 與 Ca9-22 細胞 PARP cleavage 表現量上升,可見得 silibinin 也可引起人類口腔癌細胞的細胞凋亡。 我們在 Ca9-22 和 SAS 細胞中加入 caspase-8 和 -9 的抑制劑(Z-LEHD-FMK, Z-IETD-FMK),發現皆可降低 silibinin 誘導的細胞凋亡現象發生,顯示 silibinin 可以活化內在和外在兩條不同的細胞死亡路徑。西方墨點法實驗中, silibinin 會增加 SAS 與 Ca9-22 細胞中 DR5, p-FADD 與 Bax 蛋白質的表現量,且活化 caspase-8 和 caspase-9,並造成 cyto-c 的釋放。 我們進一步將 SAS,Ca9-22 細胞前處理抗氧化劑 N-acetylcysteine ( NAC ) 後可降低 silibinin 所誘發的細胞凋亡反應,顯示自由基(ROS)在 silibinin 引起的人類口腔癌細胞細胞凋亡中扮演了重要的角色。另外,利用20 ng 的 TRAIL與 150 μM 的 silibinin 共同處理細胞,發現在 24 小時就有大量的PARP 被蛋白裂解,以流式細胞儀分析可發現其造成細胞凋亡的能力具有加成的效果。初步顯示 silibinin 在未來口腔癌治療與預防是具有潛力的。未來仍需要再以更精確的動物實驗及臨床試驗作更深入的評估其抗癌的功效。
Oral cancer is the fifth leading cause of cancer-related deaths in male population in Taiwan. Despite recent advances in radiotherapy and chemotherapy, the survival of patients with oral cancer has not improved significantly. Continued investigation of new chemotherapeutic agents is in urgent need. Our recent studies have shown that survivin is overexpressed in oral cancer, but not in normal tissues. It have shown that silibinin, a flavonoid isolated from Silybum marianum, inhibits survivin expression in various human cancer cell lines. We also found silibinin inhibited survivin expression in human oral cancer cell lines SAS and Ca9-22, supporting the idea that silibinin might develop as a specific strategy for cancer treatment. We further investigated the effects and mechanisms of silibinin on SAS and Ca9-22 cells. Silibinin significantly inhibited the proliferation of SAS and Ca9-22 cells in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that silibinin treatment induced apoptosis following G1 arrest. Western blotting showed silibinin treatment induced DR5, FADD, Bax, caspase-8, -9 activation and cytochrome C released . Silibinin-induced apoptosis was also inhibited by caspase 8 inhibitor (Z-LEHD-FMK) or caspase 9 inhibitor (Z-IETD-FMK) in SAS and Ca9-22 cells. The result showed that silibinin-induced apoptosis by activating intrinsic- and extrinsic-apoptosis pathway. Pretreatment of cells with N-acetyl cysteine ( NAC ) inhibibited the increase of DR5, Bax expression and PARP cleavage. ROS played an important role for silibinin-induced apoptosis. We further evaluated the potential synergistic effect of silibinin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in OSCC cell lines. Compared with either TRAIL (20 ng/ml ) or silibinin (150 μM)treated alone, co-administration of both drugs synergistrically induces apoptosis in both SAS and Ca9-22 cell lines. Our results indicated that silibinin sensitizes OSCC cells to TRAIL-mediated apoptosis. This may be regarded as a novel strategy for treatment of OSCC.
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