Tanshinone IIA抑制 C6神經膠瘤細胞基質 ?屬蛋白 ?-9的表現

基質金屬蛋白 ?(matrix metalloproteinases ， MMPs) 是調控神經膠瘤細胞 (glioma cells)入侵能力的重要因子之ㄧ。 Tanshinone IIA 是從一種中藥草 - 丹蔘 (Danshen ， Radix Salvia miltiorhiza Bunge) 中所純化出來的一種活性成分，對抑制腫瘤的生長和入侵有相當好的效果。本論文利用 C6 glioma cells 探討 tanshinone IIA 是否會抑制酯多醣 (lipopolysaccharide ； LPS) 和 PMA (phorbol myristate acetate) 誘導 MMP-9 的表現。實驗結果發現，LPS 和 PMA 處理 C6 glioma cells 會增加 MMP-9 的蛋白量和活性，而先以 tanshinone IIA 處理細胞，則會抑制 MMP-9 的蛋白量和活性。我們也發現 tanshinone IIA 會增加細胞中血基質氧化酵素 -1(heme oxygenase-1 ； HO-1) 的表現，但是預先以活性氧自由基(reactive oxygen species ； ROS) 的抑制劑 l-NAC (l-N-acetylcysteine) 和 PI 3-K 抑制劑 (LY294002) 處理細胞，則皆會抑制 tanshinone IIA 所誘導的 HO-1 表現。 Tanshinone IIA也會增加 Akt/PKB 的磷酸化，而且 tanshinone IIA 所增加的 Akt/PKB 磷酸化會被 l-NAC所減少。接下來以 SnPP (tin protoporphyrin) 去抑制 HO-1 表現，發現可以減輕 tanshinone IIA 抑制 MMP-9 的蛋白量和活性，顯示 HO-1 在此抑制效應中扮演關鍵的角色。因為HO-1 可催化一氧化碳 (CO) 的產生，而以 CO donor (tricarbonyl dichlororuthenium ( ) diⅡmer) 處理細胞，則可以抑制誘導型一氧化碳合成 ?(iNOS) 和 MMP-9 的表現，反之，以CO 清除劑血紅素 (hemoglobin) 處理細胞，則會減輕 tanshinone IIA 對 MMP-9 的抑制作用。由於 NOS 的抑制劑 l-NAME 會抑制 LPS 和 PMA 誘導 MMP-9 的蛋白量和活性，很可能 tanshinone IIA 是透過誘導 HO-1 表現而達到抑制 MMP-9 的效果，使 HO-1 催化 CO 產生，導致 iNOS 被抑制，進而減少 MMP-9 的蛋白量和活性。

Tanshinone IIA inhibits matrix metalloproteinase-9 expression in C6 glioma cells.

Matrix metalloproteinases (MMPs) have been implicated as important factors in the control of the invasive capability of glioma cells. Tanshinone IIA, an active ingredient purified from the Chinese herb Danshen (Radix Salvia miltiorhiza Bunge), is known to exhibit potent effects on tumor progression and invasion. In the present study, we investigated whether tanshinone IIA inhibits LPS (lipopolysaccharide) and PMA (phorbol 12-myristate 13-acetate)-induced MMP-9 gene expression in C6 glioma cells. Treatment of C6 glioma cells with LPS and PMA increased the MMP-9 expression and the expression was inhibited by pretreatment of cells with tanshinone IIA at a concentration that is not toxic to C6 glioma cells. Incubation of C6 glioma cells with tanshinone IIA increased heme oxygenase-1 (HO-1) expression, which was inhibited by pretreatment of cells with l-N-acetylcysteine (l-NAC) prior to addition of tanshinone IIA, suggesting reactive oxygen species (ROS) are involved. Inhibition of PI 3-K/Akt pathway by LY294002 or wortmannin reduced the HO-1 protein expression by tanshinone IIA. In agreement, treatment of cells with tanshinone IIA increased phosphorylation of Akt/PKB, which was attenuated by l-Nac suggesting activation of PI3-kinase–Akt is secondary to ROS production. The inhibition of MMP-9 expression by tanshinone IIA was reversed by tin protoporphyrin (SnPP) suggesting tanshinone IIA may exert this inhibitory effect through HO-1. Increase of HO-1 expression catalyzes carbon monoxide (CO) production. Addition of CO donor mimicked the tanshinone IIA effect on suppressing inducible nitrite oxygen synthease (iNOS) and MMP-9 expression. Scavenge of CO by hemoglobin (Hb) reversed the inhibition due to tanshinone IIA. Inhibition of nitric oxide (NO) production by l-NAME inhibited MMP-9 expression due to tanshinone IIA. Taken together, these results suggest that tanshinone IIA exerts its inhibitory effect through induction of HO-1 expression. HO-1 catalyzes the formation of CO, which in turn inhibit iNOS induction. Inhibition of iNOS expression subsequently reduces the MMP-9 protein level and activity.