α-Tomatine 在人類白血病細胞中之抗癌機轉

Abstract

α-Tomatine 是從茄科番茄屬植物番茄（Lycopersicon esculentum）中萃取出來的純化物，已經有研究文獻指出 α-tomatine 具有抗菌的作用，包括多種的真菌以及人類的病原菌。近年來，有文獻指出 α-tomatine 在直腸癌以及肝癌細胞上都具有抑制生長效果，但目前機轉卻依然不明。因此，本實驗的主旨便是評估 α-tomatine在 in vivo以及in vitro對於血癌的治療效果。藉由 MTT assay（3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide）的方法來評估 α-tomatine 對於 HL60 及 K562 細胞增生的影響，發現給予 α-tomatine 24 小時之後具有濃度相關性抑制細胞生長作用，於HL60 及 K562 的 IC50 分別是1.92 μM 和1.51 μM 。而不論是 HL60 或是 K562 ，給予 α-tomatine 之後，其細胞週期並無顯著的改變，並且 α-tomatine 在兩株細胞中皆非經由 caspase 的活化來造成細胞死亡。然而，α-tomatine 卻能有效地抑制 survivin 的表現以及促進 AIF 進入細胞核。此外，給予α-tomatine之後會影響粒線體膜電位的改變、促進Bak的活化以及增加 Mcl-1 short form（Mcl-1s）的表現。實驗更進一步證實，在 HL60 的異位移植動物模式中，α-tomatine （5 mg/Kg）可以有效的抑制腫瘤生長且不影響小鼠的體重。利用免疫組織染色及西方點墨法，在給予 α-tomatine 的組織中，也的確能看到 survivin表現量較低。綜合以上結果，我們得知 α-tomatine藉由活化Bak及Mcl-1s，造成細胞粒線體膜電位的改變，因而促進 AIF 進核，同時也會抑制 survivin的表現，但卻不經由影響細胞週期以及活化 caspase 的路徑來造成細胞凋亡，所以 α-tomatine 具有潛力發展成新的抗血癌藥物。

α-Tomatine, a tomato glycoalkaloid, has been reported to possess antibiotic properties against a variety of fungi and human pathogens. It also inhibited the growth of human colon and liver cancer cells. Although α-tomatine has been displayed anticancer activity in the previous studies, the mechanism of action remains unclearly. In this study, we evalulate the therapeutic potential of α-tomatine against leukemia cells in vitro and in vivo. Inhibition of cell proliferation is determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide （MTT） assay. In this assay, it shows an IC50 of 1.92 μM and 1.51 μM, in HL60 and K562, respectively, after 24 hours of treatment. Neither in HL60 nor K562 cells, the distribution of cell cycle does change using flow cytometry. By Western blotting, we also find that α-tomatine induces cell death independent on caspase activation in both cell lines. Nevertheless, pro-apoptotic protein survivin expression is obviously down-regulated, after α-tomatine treatment. α-Tomatine also triggers apoptosis inducing factor（AIF） into the nucleus. Moreover, α-tomatine can lead to lose the mitochrondrial membrane potential, and activate Bak and Mcl-1 short form（Mcl-1s）protein level. Furthermore, α-tomatine（5 mg/kg）can significantly inhibit tumor xenograft growth and doesn’t affect the body weight of mice. Then, tumors were analyzed for survivin alterations 3 by immunohistochemistry and immunoblotting. Indeed, α-tomatine can decrease survivin protein level in the tumors. Taken together, our findings indicate that α-tomatine can increase the activation of Bak or Mcl-1s, leading to lose mitochondrial membrane poptential and induction of AIF translocation to the nulceus. Meanwhile, α-tomatine can also inhibit survivin and cause apoptosis by cell cycle- and capase-independent pathway. All these findings suggest that α-tomatine could be served as a new candidate in leukemia cancer treatment.