Genistein促進trichostatin A的抗腫瘤效果：體內研究

Genistein enhances the antitumor effect of trichostatin A: an in vivo study

劉上宇(Shan-Yu Liou)

指導教授 ： 葉姝蘭

中山醫學大學/健康管理學院/營養學研究所/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00187

摘要

我們先前體外研究中利用抗癌藥物TSA合併genistein處理肺癌細胞A549，發現genistein能透過TNFR-1路徑及組蛋白乙醯化之改變，增強TSA對A549之細胞凋亡，但genistein在體內是否也有此效果並不清楚。因此，本研究利用免疫缺陷之裸鼠進行異種移植注射人類肺癌細胞A549，探討TSA合併genistein在體內之抗癌效果。首先我們觀察genistein在裸鼠體內分佈情形，genistein利用管餵(OL: 20 mg/kgw or OH: 100 mg/kgw, 3次/週)或腹腔注射(IPL:2 mg/kgw or IPH: 10 mg/kgw, 3 次/週)給予genistein，為期12週，動物犧牲後，取出並測量血漿、肺、肝臟中genistein總含量。結果顯示，管餵組中genistein總含量高於腹腔注射組，且有劑量效應。接著的第二部分研究，將人類肺腺癌細胞A549(細胞數：5 × 106 cells)以皮下注射方式，注入裸鼠背部，三週後將裸鼠隨機分為六組(5-7隻/組)，即tumor bearing，TSA，OG，IG，TSA+OG以及TSA + IG組，TSA組經由腹腔注射處理(0.5 mg/kgw， 2次/週)，genistein分別經由管餵(OG: 100 mg/kgw, 3次/週)或腹腔注射處理(IG: 10 mg/kgw, 3 次/週)，tumor bearing組則只給予normal-saline。研究結果發現，不同處理方式對於裸鼠體重無顯著影響，只有TSA+IG與tumor bearing組相比，能明顯降低腫瘤大小。給予genistein 2小時後，動物血漿中濃度以OG高於IG處理組，但在腫瘤組織中genistein濃度，則以IG高於OG處理組。我們以western blot方式觀察腫瘤組織中蛋白表現，發現TSA+IG中TNFR-1、acetyl histone H3、histone H4表現量高於tumor bearing以及TSA組，且有顯著差異，而TSA+OG對於這些蛋白表現影響並不顯著，或者效果低於TSA+IG組。我們也觀察裸鼠血漿與腫瘤組織中TNF-α以及TBARS (thiobarbituric acid reactive substances)含量，結果發現單獨TSA處理能明顯增加TNF-α以及TBARS含量，IG能明顯減少TSA誘導增加的TNF-α以及TBARS，但OG則無此效果。綜合以上，我們在動物模式中，觀察到經由腹腔注射genistein能增強TSA的抗腫瘤性，而其機制與增加腫瘤組織中TNFR-1和acetyl histone H3/H4的表現有關。

關鍵字

異黃酮 ； 肺癌 ； 腫瘤壞死因子

並列摘要

In our previous in vitro studies, we found that genistein enhanced the apoptosis induced by trichostatin A (TSA), a novel anticancer drug, in human lung cancer cells; through up-regulation of tumor necrosis factor receptor-1 (TNFR-1) and histone acetylation. However, whether genistein exerts such effects in vivo is unclear. Thus, in the present study, using nude mice we performed a mouse xenograft experiment to investigate the enhancing effects of genistein on the antitumor effect of TSA in vivo. First, we investigated the distribution of genistein in nude mice. Genistein was administered to nude mice by gavage (OL: 20 mg/kgw or OH: 100 mg/kgw, 3 times/week) or by intraperitoneal injection (IPL:2 mg/kgw or IPH: 10 mg/kgw, 3times/week) for 12 weeks. After sacrificed the concentration of total genistein were determined. The results showed that the concentrations of total genistein in the plasma, lungs and livers of nude mice among groups were in an order: OH> OL, IPH> IPL. Secondly, the nude mice were subcutaneously injected in the right flank with A549 cells at a dose of 5 × 106 cells. Three weeks after cell injection, the animals were then randomly assigned to the following six groups (n = 5-7/group) for 15 weeks: tumor bearing group, TSA group, OG group, IG group, TSA+OG group, and TSA + IG group. TSA was given by intraperitoneal injection at a dose of 0.5 mg/kgw (2 times/week). Genistein was given by gavage (OG; 100 mg/kgw, 3 times/week) or by intraperitoneal (IG; 10 mg/kgw, 3 times/week). Tumor bearing group was given the vehicle only. The results showed that various treatments did not significantly affect the body weight of nude mice. Only TSA+IG treatment significantly decreased tumor volume as compared with tumor bearing group. Two hours after genistein administration, the total genistein concentrations in plasma of animals with OG treatment were higher than those with IG treatment. However, the total genistein concentrations in tumors of animals with IG treatment were hither than those with OG treatment. Tumors from mice treated with TSA+IG had higher TNFR-1, acetyl histone H3 and H4 than did those from tumor bearing and TSA-treated mice; whereas, the effects of OG+TSA on these protein expression were not significant or lower than that of TSA+IG. TSA treatment significantly increased the levels of TNF-α in plasma and tumors as well as the level of thiobarbituric acid reactive substances (TBARs) in plasma. IG significantly decreased the rise of TNF-α and TBARs induced by TSA, while OG had no significantly effects. Taken together, these data demonstrated that genistein administered by intraperitoneal injection enhanced the antitumor effect of TSA in vivo. The mechanisms were associated with increasing the expression of TNFR-1 and acetyl histone H3/H4 in tumors.

並列關鍵字

genistein ； trichostatin A ； A549 ； TNFR-1

參考文獻

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