CAJ | 학술논문

目的观察分子靶向药物索拉非尼与常用化学治疗药物5-氟尿嘧啶(5-FU)合用模式对人肝癌细胞株MHCCLM3增殖的抑制作用,并初步探讨其机制.方法以人肝癌细胞系MHCCLM3细胞为靶细胞,用索拉非尼和(或)5-FU干预细胞.将细胞分4组,即索拉非尼组、5-FU组、两药联用组、空白对照组.使用CCK-8的方法检测药物对细胞的增殖抑制作用；参考中效原理的方法计算两药合用时的联合指数(CI);应用流式细胞技术检测药物对细胞周期的影响；用Westem blot方法检测增殖相关信号通路RAF/MEK/ERK和STAT3的相关蛋白,以及细胞周期相关蛋白cyclinD1的表达.两两比较使用最小显著差异法检验.结果 (1)与空白对照组比较,索拉非尼组、5-FU组、两药联用组对MHCCLM3细胞的抑制率分别为46.16％±2.52％、28.67％±6.16％、22.59％±6.89％；索拉非尼、5-FU分别单用或合用均能明显抑制MHCCLM3细胞的增殖.(2)索拉非尼与5-FU按一定的药物浓度比例(索拉非尼∶5-FU=2 μ mol∶1 mg/L)合用时,CI＞1,提示两药合用产生拮抗作用.(3) 5-FU单药对细胞的半数抑制浓度(IC50)为(102.86±27.84) mg/L;当与一定浓度(8μmol)的索拉非尼合用后,5-FU对细胞的IC50增加到(178.61±20.73) mg/L.提示与索拉非尼合用时,MHCCLM3细胞对5-FU的敏感性下降.(4)细胞周期检测结果显示,G1期细胞比例,空白对照组为44.73％±1.63％,索拉非尼组为65.80％±0.56％.S期细胞的比例,空白对照组为46.63％±0.65％,索拉非尼组为22.83％±1.75％.提示索拉非尼作用后,能把MHCCLM3细胞阻滞在G1期.(5)索拉非尼能明显阻断RAF/MEK/ERK和STAT3信号通路,下调cyclinD1蛋白的表达量,索拉非尼作用后,磷酸化C-RAF、磷酸化ERK1/2、磷酸化STAT3(Y705)和cyclinD 14种蛋白的表达量分别下降至对照组的(0.56±0.05)倍、(0.54±0.02)倍、(0.36±0.02)倍和(0.57±0.03)倍；而5-FU对这两条信号通路基本不起作用,但能上调cyclinD1的表达,5-FU作用后cyclinD1的表达量上调至对照组的(1.45±0.12)倍.结论索拉非尼和5-FU对肝癌细胞均有显著的抗肿瘤作用,但两药联合应用并没有产生协同作用.其可能的机制为,索拉非尼通过阻断RAF/MEK/ERK信号通路以及STAT3相关通路,抑制了细胞周期相关蛋白cyclinD1的表达,使肿瘤细胞阻滞在G1期,而S期细胞数目减少,从而使肿瘤细胞对5-FU的敏感性降低. 目的觀察分子靶嚮藥物索拉非尼與常用化學治療藥物5-氟尿嘧啶(5-FU)閤用模式對人肝癌細胞株MHCCLM3增殖的抑製作用,併初步探討其機製.方法以人肝癌細胞繫MHCCLM3細胞為靶細胞,用索拉非尼和(或)5-FU榦預細胞.將細胞分4組,即索拉非尼組、5-FU組、兩藥聯用組、空白對照組.使用CCK-8的方法檢測藥物對細胞的增殖抑製作用；參攷中效原理的方法計算兩藥閤用時的聯閤指數(CI);應用流式細胞技術檢測藥物對細胞週期的影響；用Westem blot方法檢測增殖相關信號通路RAF/MEK/ERK和STAT3的相關蛋白,以及細胞週期相關蛋白cyclinD1的錶達.兩兩比較使用最小顯著差異法檢驗.結果 (1)與空白對照組比較,索拉非尼組、5-FU組、兩藥聯用組對MHCCLM3細胞的抑製率分彆為46.16％±2.52％、28.67％±6.16％、22.59％±6.89％；索拉非尼、5-FU分彆單用或閤用均能明顯抑製MHCCLM3細胞的增殖.(2)索拉非尼與5-FU按一定的藥物濃度比例(索拉非尼∶5-FU=2 μ mol∶1 mg/L)閤用時,CI＞1,提示兩藥閤用產生拮抗作用.(3) 5-FU單藥對細胞的半數抑製濃度(IC50)為(102.86±27.84) mg/L;噹與一定濃度(8μmol)的索拉非尼閤用後,5-FU對細胞的IC50增加到(178.61±20.73) mg/L.提示與索拉非尼閤用時,MHCCLM3細胞對5-FU的敏感性下降.(4)細胞週期檢測結果顯示,G1期細胞比例,空白對照組為44.73％±1.63％,索拉非尼組為65.80％±0.56％.S期細胞的比例,空白對照組為46.63％±0.65％,索拉非尼組為22.83％±1.75％.提示索拉非尼作用後,能把MHCCLM3細胞阻滯在G1期.(5)索拉非尼能明顯阻斷RAF/MEK/ERK和STAT3信號通路,下調cyclinD1蛋白的錶達量,索拉非尼作用後,燐痠化C-RAF、燐痠化ERK1/2、燐痠化STAT3(Y705)和cyclinD 14種蛋白的錶達量分彆下降至對照組的(0.56±0.05)倍、(0.54±0.02)倍、(0.36±0.02)倍和(0.57±0.03)倍；而5-FU對這兩條信號通路基本不起作用,但能上調cyclinD1的錶達,5-FU作用後cyclinD1的錶達量上調至對照組的(1.45±0.12)倍.結論索拉非尼和5-FU對肝癌細胞均有顯著的抗腫瘤作用,但兩藥聯閤應用併沒有產生協同作用.其可能的機製為,索拉非尼通過阻斷RAF/MEK/ERK信號通路以及STAT3相關通路,抑製瞭細胞週期相關蛋白cyclinD1的錶達,使腫瘤細胞阻滯在G1期,而S期細胞數目減少,從而使腫瘤細胞對5-FU的敏感性降低.
목적 관찰분자파향약물색랍비니여상용화학치료약물5-불뇨밀정(5-FU)합용모식대인간암세포주MHCCLM3증식적억제작용,병초보탐토기궤제.방법 이인간암세포계MHCCLM3세포위파세포,용색랍비니화(혹)5-FU간예세포.장세포분4조,즉색랍비니조、5-FU조、량약련용조、공백대조조.사용CCK-8적방법검측약물대세포적증식억제작용；삼고중효원리적방법계산량약합용시적연합지수(CI);응용류식세포기술검측약물대세포주기적영향；용Westem blot방법검측증식상관신호통로RAF/MEK/ERK화STAT3적상관단백,이급세포주기상관단백cyclinD1적표체.량량비교사용최소현저차이법검험.결과 (1)여공백대조조비교,색랍비니조、5-FU조、량약련용조대MHCCLM3세포적억제솔분별위46.16％±2.52％、28.67％±6.16％、22.59％±6.89％；색랍비니、5-FU분별단용혹합용균능명현억제MHCCLM3세포적증식.(2)색랍비니여5-FU안일정적약물농도비례(색랍비니∶5-FU=2 μ mol∶1 mg/L)합용시,CI＞1,제시량약합용산생길항작용.(3) 5-FU단약대세포적반수억제농도(IC50)위(102.86±27.84) mg/L;당여일정농도(8μmol)적색랍비니합용후,5-FU대세포적IC50증가도(178.61±20.73) mg/L.제시여색랍비니합용시,MHCCLM3세포대5-FU적민감성하강.(4)세포주기검측결과현시,G1기세포비례,공백대조조위44.73％±1.63％,색랍비니조위65.80％±0.56％.S기세포적비례,공백대조조위46.63％±0.65％,색랍비니조위22.83％±1.75％.제시색랍비니작용후,능파MHCCLM3세포조체재G1기.(5)색랍비니능명현조단RAF/MEK/ERK화STAT3신호통로,하조cyclinD1단백적표체량,색랍비니작용후,린산화C-RAF、린산화ERK1/2、린산화STAT3(Y705)화cyclinD 14충단백적표체량분별하강지대조조적(0.56±0.05)배、(0.54±0.02)배、(0.36±0.02)배화(0.57±0.03)배；이5-FU대저량조신호통로기본불기작용,단능상조cyclinD1적표체,5-FU작용후cyclinD1적표체량상조지대조조적(1.45±0.12)배.결론 색랍비니화5-FU대간암세포균유현저적항종류작용,단량약연합응용병몰유산생협동작용.기가능적궤제위,색랍비니통과조단RAF/MEK/ERK신호통로이급STAT3상관통로,억제료세포주기상관단백cyclinD1적표체,사종류세포조체재G1기,이S기세포수목감소,종이사종류세포대5-FU적민감성강저.
Objective To investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3.Methods The effects of somfenib and 5-FU,alone or in combination,on the proliferation of MHCCLM3 cells were evaluated by cell viability assays.Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay.Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting.Results Sorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells,with the following inhibition rates:somfenib:46.16％ ± 2.52％,5-FU:28.67％ ± 6.16％,and sorafenib + 5-FU:22.59％ ± 6.89％.The somfenib + 5-FU combination did not provide better results than treatment with either drag alone.The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1,indicating that the two agents induced antagonistic,instead of synergistic,effects on the MHCCLM3 cells.In addition,the MHCCLM3 cells were less semitive to 5-FU when administrated in combination with sorafenib,as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 ± 27.84) mg/L to (178.61 ± 20.73) mg/L (P =0.003).Sorafenib alone induced G1 phase arrest (increasing from 44.73％ ± 1.63％ to 65.80％ ± 0.56％; P ＜ 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63％ ± 0.65％ to 22.83％ ± 1.75％; P ＜ 0.01),as well as down-regulated cyclinD1 expression (0.57 ± 0.03-fold change vs.untreated control group; P ＜ 0.01).5-FU alone up-regulated cyclinD1 expression (1.45 ± 0.12-fold change vs.untreated control group; P ＜ 0.01).Moreover,somfenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways,with the fold-changes of p-C-RAF,p-ERK1/2 and p-STAT3 being 0.56 ±0.05,0.54 ± 0.02 and 0.36 ± 0.02,respectively (allP ＜ 0.01); 5-FU alone produced no significant effects on these pathways.Conclusion Administered alone,both somfenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells.The somfenib + 5-FU combination treatment produces antagonistic,rather than synergistic,effects.Somfenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G lphase arrest and S phase reduction,thereby reducing the cells' sensitivity to 5-FU.