摘要
目的：
1.本研究对中医药联合吉非替尼治疗晚期非小细胞肺癌进行系统评价,以提供扶正抗癌方联合吉非替尼治疗非小细胞肺癌的研究可行性文献证据。
2.本研究对三种NSCLC细胞系(A549、H1650、PC9)进行药物敏感性及信号通路相关的实验,观察扶正抗癌方与吉非替尼联用的作用效果,并探讨其联合作用的机制。
方法：
1.计算机检索全国图书馆参考咨询联盟(2006.-2012)、中国知网(2006.-2012)、VIP(2006-2012)所收录的相关文献。收集以中医药联合吉非替尼为干预措施治疗非小细胞肺癌的随机对照试验。对纳入研究逐个进行质量评价,采用ReMan4.2.10软件对所提取资料进行Meta分析。
2.采用MTT增殖抑制试验,对三种非小细胞肺癌细胞：A549、H1650、PC9进行扶正抗癌方及吉非替尼单药药敏试验并观察其抑制效果。采用流式细胞术,检测扶正抗癌方、吉非替尼以及两药同时使用对三种细胞凋亡的调控作用。
3.用扶正抗癌方不同浓度及在不同时间点处理细胞,采用western blot的方法观察不同信号通路中几种蛋白磷酸化的改变情况。用扶正抗癌方、吉非替尼最佳浓度单独使用以及扶正抗癌方和吉非替尼联合使用处理三种细胞,采用western blot的方法观察不同信号通路中激酶蛋白分子磷酸化的改变情况
结果：
1.检索结果获得84篇文献,最终纳入9项随机对照实验,586例晚期非小细胞肺癌患者。治疗组(中医药+吉非替尼)和对照组(吉非替尼)比较疾病控制率、功能状态改善、皮疹、腹泻、恶心呕吐、肝功能状态,分别进行异质性检验,患者组间差异无统计学意义,进行meta分析相对获益系数(RR)分别为1.18、1.45、1.18、1.31、1.16、1.01。漏斗图分析不存在明显发表性偏倚,但是样本量普遍存在较小的弊端。
2.MTT增殖抑制试验结果
(1)吉非替尼单独使用时对不同细胞的增殖抑制作用。24H吉非替尼对不同细胞的影响：经两因素方差分析,吉非替尼对不同细胞增殖抑制率差异无统计学意义,F=0.15,P=0.7063。吉非替尼的不同剂量对细胞增殖抑制率差异有统计学意义,F=4.41,P=0.0031。48H吉非替尼对不同细胞的影响：经两因素方差分析,吉非替尼对不同细胞增殖抑制率差异有统计学意义,F=7.57,P=0.0101,对A549细胞较H1650细胞抑制率更为明显。吉非替尼的不同剂量对细胞增殖抑制率差异有统计学意义,F=3.53,P=0.0046,随着剂量的增加,抑制率增加。72H吉非替尼对不同细胞的影响：经两因素方差分析,吉非替尼对不同细胞增殖抑制率差异有统计学意义,F=40.11,P=0.0000,对A549细胞较H1650细胞抑制率更为明显。吉非替尼的不同剂量对细胞增殖抑制率差异具有统计学意义,F=6.27,P=0.0001,随着剂量的增加,抑制率增加。
(2)扶正抗癌方单独使用时对三种细胞的增殖抑制作用。24H扶正抗癌方对不同细胞的影响：经两因素方差分析,扶正抗癌方对不同细胞增殖抑制率差异有统计学意义,F=9.23,P=0.0004,扶正抗癌方对A.549细胞增殖抑制明显。扶正抗癌方的不同剂量对细胞增殖抑制率差异具有统计学意义,P=0.0000,随着剂量的增加,抑制率增加。48H扶正抗癌方对不同细胞的影响：经两因素方差分析,扶正抗癌方对不同细胞的抑制率差异有统计学意义,F=6.54,P=0.0031,扶正抗癌方对AA549及PC9细胞增殖抑制明显。扶正抗癌方的不同剂量对细胞增殖抑制率差异具有统计学意义,P=0.0000,随着剂量的增加,抑制率增加,对于PC9细胞,低剂量促进细胞增殖,高剂量抑制细胞增殖。72H扶正抗癌方对不同细胞的影响：经两因素方差分析,扶正抗癌方对不同细胞的抑制率差异不显著,F=0.44,P=0.6486,扶正抗癌方对AA549及PC9细胞增殖抑制明显。扶正抗癌方的不同剂量对细胞增殖抑制率差异具有显著性,P=0.0000,随着剂量的增加,抑制率增加,对于PC9细胞。
3.流式细胞术检测细胞凋亡单用扶正抗癌方或Gifitinib处理三种细胞48h均可促使其凋亡,AA549凋亡率分别为(4.633±0.666)%、(7.8±2.6963)%,H1650凋亡率分别为(5.7667±2.6274)%和(11.5±5.4286)%,PC9凋亡率分别为(13.6667±4.26654)%和(6.6667±2.3180)%,但经联合处理后,A549凋亡率为(12.6333±4.47698)%, H1650凋亡率为(25.4667±20.6011)%PC9凋亡率为(26.8333±10.4357)%,提示扶正抗癌方联合Gifitinib具有协同促进A549、H1650、PC9细胞凋亡的作用。
4.扶正抗癌方(fzka)单独干预三种细胞dose-response及time-response实验结果
(1)扶正抗癌方对A549进行干预的dose-response实验中,P-EGFR、EZH2均呈剂量依赖的下降趋势,提示,扶正抗癌方对AA549这两种蛋白表达具有正向调节作用,且不同蛋白不同剂量之间,统计分析,p=0.0000,提示具有统计学意义。PPAR-gomma和P53均呈剂量依赖的上升趋势,不同剂量之间统计分析结果p=0.000,具有统计学意义,同样提示扶正抗癌方对A549这两种蛋白表达具有正向调节作用。扶正抗癌方对H1650进行干预的dose-response实验中, EZH2呈剂量依赖的下降趋势,提示扶正抗癌方对H1650这种蛋白表达具有正向调节作用,且不同剂量之间,统计分析,p=0.0077,提示具有统计学意义。对于P-EGFR,没有明显的剂量依赖趋势,但是药物浓度到达一定程度时,P-EGFR表达仍为下降。PPAR-gomma和P53均未呈剂量依赖的上升趋势,不同剂量之间统计分析结果p=0.000,具有统计学意义,提示扶正抗癌方对H1650这两种蛋白表达正向调节作用不明显。扶正抗癌方对PC9进行干预的dose-response实验中,EZH2呈剂量依赖的下降趋势,提示,扶正抗癌方对PC9的EZH2蛋白表达具有正向调节作用,且不同剂量之间,统计分析,p=0.0000,提示具有统计学意义。对于P-EGFR,没有明显的剂量依赖趋势,但是药物浓度到达一定程度时,P-EGFR表达仍为下降。PPAR-gomma和P53均呈剂量依赖的上升趋势,不同剂量之间统计分析结果p=0.000,具有统计学意义,同样提示扶正抗癌方对PC9这两种蛋白表达具有正向调节作用但到达一定浓度后对其变成负向调节。
(2)扶正抗癌方对A549细胞time-response干预实验中,P-erk呈现很好的时间相关性,且不同时间点之间统计分析p=0.0036,具有统计学意义。扶正抗癌方对H1650细胞time-response干预实验中,P-AKT、P-erk、P-P38呈现很好的时间相关性,且不同时间点之间统计分析p--0.0000、p=0.0130、p=0.000,具有统计学意义。扶正抗癌方对PC9细胞time-response干预实验中,P-AKT、P-P38呈现很好的时间相关性,且不同时间点之间统计分析p--0.0000、p=0.0046,具有统计学意义。
5.扶正抗癌方联合吉非替尼对三种非小细胞肺癌细胞干预目的蛋白和蛋白激酶的变化比较分析结果
(1)扶正抗癌方对A549的P-EGFR的单独效应F值为90.31,P值为0.0000,吉非替尼的单独效应F值为70.44,P值为0.0000,两者联合效应的F值为8.32,P值为0.0204,说明扶正抗癌方和吉非替尼对于P-EGFR蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-EGFR蛋白表达的下调影响更加显著,两者交互作用具有统计学意义。EZH2:扶正抗癌方干预后的单独效应F值为49.66,P值为0.0001,吉非替尼的单独效应F值为55.17,P值为0.0001,两者联合效应的F值为1.69,P值为0.2293,说明扶正抗癌方和吉非替尼对于EZH2蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于EZH2蛋白表达的下调影响更加显著,但两者交互作用不具有统计学意义。ppar-gomma:扶正抗癌方的单独效应F值为2.96,P值为0.1234,吉非替尼的单独效应F值为31.95,P值为0.0005,两者联合效应的F值为2.83,P值为0.1311,说明吉非替尼对于ppar-gomma蛋白表达具有影响,具有统计学意义,其蛋白表达上调,扶正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于ppar-gomma蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P53：扶正抗癌方的单独效应F值为80.63,P值为0.0000,吉非替尼的单独效应F值为2.04,P值为0.1913,两者联合效应的F值为26.77,P值为0.0008,说明扶正抗癌方对于P53蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达为下调的影响,但不具有统计学意义,二者联合用药对于P53蛋白表达的上调影响更加显著,两者有交互作用具有统计学意义。P-AKT:扶正抗癌方的单独效应F值为8.97,P值为0.0172,吉非替尼的单独效应F值为34.10,P值为0.0004,两者联合效应的F值为8.09,P值为0.0217,说明扶正抗癌方和吉非替尼对于P-AKT蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-AKT蛋白表达的下调影响显著,两者有交互作用具有统计学意义,吉非替尼的下调最为显著。P-erk:扶正抗癌方的单独效应F值为1.43,P值为0.2654,吉非替尼的单独效应F值为22.22,P值为0.0015,两者联合效应的F值为3.72,P值为0.0898,说明吉非替尼对于P-erk蛋白表达具有上调作用,具有统计学意义,扶正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于P-erk蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P-P38：扶正抗癌方的单独效应F值为8.66,P值为0.0186,吉非替尼的单独效应F值为29.58,P值为0.0006,两者联合效应的F值为0.97,P值为0.3544,说明扶正抗癌方及吉非替尼对于P-P38蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达上调较扶正抗癌方更显著,二者联合用药对于p-p38蛋白表达的上调显著,但两者交互作用不具有统计学意义。
(2)扶正抗癌方对于H1650的P-EGFR蛋白表达的单独效应F值为3.84,P值为0.0855,吉非替尼的单独效应F值为6.01,P值为0.0399,两者联合效应的F值为3.00,P值为0.1217,说明扶正抗癌方和吉非替尼对于P-EGFR蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-EGFR蛋白表达的下调影响更加显著,两者有交互作用具有统计学意义。EZH2：扶正抗癌方的单独效应F值为89.93,P值为0.0000,吉非替尼的单独效应F值为152.01,P值为0.0000,两者联合效应的F值为22.58,P值为0.0014,说明扶正抗癌方和吉非替尼对于EZH2蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于EZH2蛋白表达的下调影响更加显著,但两者交互作用不具有统计学意义。ppar-gomma:扶正抗癌方的单独效应F值为8.97,P值为0.0172,吉非替尼的单独效应F值为25.96,P值为0.0009,两者联合效应的F值为2.76,P值为0.1352,说明吉非替尼对于ppar-gomma蛋白表达具有影响,具有统计学意义,其蛋白表达上调,扶J正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于ppar-gomma蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P53：扶正抗癌方的单独效应F值为17.71,P值为0.0030,吉非替尼的单独效应F值为25.66,P值为0.0010,两者联合效应的F值为1.65,P值为0.2353,说明扶正抗癌方对于P53蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达为下调的影响,但不具有统计学意义,二者联合用药对于p53蛋白表达的上调影响更加显著,两者有交互作用具有统计学意义。P-AKT：扶正抗癌方的单独效应F值为44.61,P值为0.0002,吉非替尼的单独效应F值为2.75,P值为0.1358,两者联合效应的F值为6.90,P值为0.0303,说明扶正抗癌方和吉非替尼对于P-AKT蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-AKT蛋白表达的下调影响显著,两者有交互作用具有统计学意义,吉非替尼的下调最为显著。p-erk:扶正抗癌方的单独效应F值为5.50,P值为0.0471,吉非替尼的单独效应F值为10.08,P值为0.0131,两者联合效应的F值为10.19,P值为0.0128,说明吉非替尼对于P-erk蛋白表达具有上调作用,具有统计学意义,扶正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于P-erk蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P-P38：扶正抗癌方的单独效应F值为309.69,P值为0.0000,吉非替尼的单独效应F值为118.04,P值为0.0000,两者联合效应的F值为3.92,P值为0.0832,说明扶正抗癌方及吉非替尼对于P-P38蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达上调较扶正抗癌方更显著,二者联合用药对于P-P38蛋白表达的上调显著,但两者交互作用不具有统计学意义。
(3)扶正抗癌方对PC9细胞P-EGFR蛋白表达的单独效应F值为4.42,P值为0.0686,吉非替尼的单独效应F值为675.00,P值为0.0000,两者联合效应的F值为1.47,P值为0.2598,说明扶正抗癌方和吉非替尼对于P-EGFR蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-EGFR蛋白表达的下调影响更加显著,两者有交互作用具有统计学意义。EZH2：扶正抗癌方的单独效应F值为127.48,P值为0.0000,吉非替尼的单独效应F值为242.55,P值为0.0000,两者联合效应的F值为6.86,P值为0.0307,说明扶正抗癌方和吉非替尼对于EZH2蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于EZH2蛋白表达的下调影响更加显著,但两者交互作用不具有统计学意义。ppar-gomma:扶正抗癌方的单独效应F值为7.90,P值为0.0228,吉非替尼的单独效应F值为14.59,P值为0.0051,两者联合效应的F值为2.16,P值为0.1800,说明吉非替尼对于ppar-gomma蛋白表达具有影响,具有统计学意义,其蛋白表达上调,扶正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于ppar-gomma蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P53：扶正抗癌方的单独效应F值为12.95,P值为0.0070,吉非替尼的单独效应F值为49.45,P值为0.0001,两者联合效应的F值为1.76,P值为0.2208,说明扶正抗癌方对于P53蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达为下调的影响,但不具有统计学意义,二者联合用药对于P53蛋白表达的上调影响更加显著,两者有交互作用具有统计学意义。P-AKT:扶正抗癌方的单独效应F值为3.31,P值为0.1066。吉非替尼的单独效应F值为33.39,P值为0.0004,两者联合效应的F值为7.02,P值为0.0293,说明扶正抗癌方和吉非替尼对于P-AKT蛋白表达具有影响,具有统计学意义,其蛋白表达均下调,二者联合用药对于P-AKT蛋白表达的下调影响显著,两者有交互作用具有统计学意义,吉非替尼的下调最为显著。P-erk:扶正抗癌方的单独效应F值为23.06,P值为0.0014,吉非替尼的单独效应F值为53.75,P值为0.0001,两者联合效应的F值为4.77,P值为0.0605,说明吉非替尼对于P-erk蛋白表达具有上调作用,具有统计学意义,扶正抗癌方对于蛋白表达影响不显著,不具有统计学意义,二者联合用药对于P-erk蛋白表达的上调影响更加显著,但两者交互作用不具有统计学意义。P-P38：扶正抗癌方的单独效应F值为0.04,P值为0.8530,吉非替尼的单独效应F值为17.38,P值为0.0031,两者联合效应的F值为29.24,P值为0.0006,说明扶正抗癌方及吉非替尼对于P-P38蛋白表达具有影响,具有统计学意义,其蛋白表达上调,吉非替尼对于蛋白表达上调较扶正抗癌方更显著,二者联合用药对于P-P38蛋白表达的上调显著,但两者交互作用不具有统计学意义。
结论：
1.目前相关临床研究结果提示,在吉非替尼治疗基础上,联合中医药较单纯吉非替尼治疗非小细胞肺癌有一定的增效作用,在提高生存质量、减轻毒副作用、增强吉非替尼疗效有一定优势。但仍需多中心大样本随机对照试验提供有高质量的证据支持此结果。
2.扶正抗癌方单独对三种细胞进行干预,均能抑制三种细胞(A549、H1650、PC9)增殖。扶正抗癌方冻干粉在同一时间对三种细胞的抑制效果均呈现明显的剂量依赖关系。扶正抗癌方冻干粉对吉非替尼耐药的细胞A549和H1650抑制效果明显强于PC9,对耐药株细胞H1650细胞抑制效最优。扶正抗癌方干预细胞之后细胞之间24h、48h及72h比较分析差异有统计学意义。这也许可以给予我们临床中一点提示,在对Gifitinib不敏感(例如A549)或者耐药(H1650)的患者用药过程中,可以适当加大中药复方汤剂的使用,而对于Gifitinib敏感的患者,我们用药仍然要以Gifitinib为主,如皮疹或者腹泻严重,可适当给予中药复方减轻患者副反应症状,也非常有利于提高恢复患者继续治疗的信心。
3.扶正抗癌方联合吉非替尼促凋亡效果优于扶正抗癌方单药或吉非替尼单药,提示扶正抗癌方对吉非替尼治疗非小细胞肺癌有增效增敏作用。流式细胞仪检测凋亡结果提示A549细胞,各组之间差异具有统计学意义,扶正抗癌方及吉非替尼均有促进细胞凋亡的作用,二者联合用药作用下,细胞凋亡更明显,组间差异性具有统计学意义。H1650细胞,各组之间差异具有显著性,扶正抗癌方及吉非替尼均有促进细胞凋亡的作用,二者联合用药作用下,细胞凋亡更明显,但组间差异性不具有统计学意义。PC9细胞,各组之间差异具统计学意义,扶正抗癌方及吉非替尼均有促进细胞凋亡的作用,二者联合用药作用下,细胞凋亡更明显,组间差异性具有统计学意义。
扶正抗癌方单独对三种细胞进行干预,均能抑制三种非小细胞肺癌细胞(A549、H1650. PC9)增殖,与吉非替尼联用,对吉非替尼治疗非小细胞肺癌(无突变耐药,继发获得性耐药及敏感细胞)有增敏和增效的作用。
4.扶正抗癌方单独使用对A549的P-EGFR、EZH2、PPAR-gomma和P53四种蛋白表达具有正向调节作用。对H1650的EZH2具有正向调节作用,对于P-EGFR,没有明显的剂量依赖趋势,但是药物浓度到达一定程度时,P-EGFR表达仍为下降,而对PPAR-gomma和P53这两种蛋白表达正向调节作用不明显。扶正抗癌方对PC9的EZH2蛋白表达具有正向调节作用.扶正抗癌方对PC9的PPAR-gomma和P53这两种蛋白表达具有正向调节作用但到达一定浓度后对其变成负向调节。另外,扶正抗癌方对A549细胞通过P-erk调节进而影响以上目的蛋白的表达,对H1650细胞通过P-AKT、P-erk、P-p38调节进而影响以上目的蛋白表达,对PC9细胞通过P-AKT、P-p38调节进而影响以上目的蛋白表达。
扶正抗癌方联合吉非替尼对A549、H1650、PC9干预后,对比扶正抗癌方、吉非替尼单独干预组,对P-EGFR、EZH2蛋白表达下调,ppar-gomma、P53表达上调,均呈正向调节作用。在A549信号传导通路中,扶正抗癌方与吉非替尼联合使用对P-AKT、 P-erk、P-P38蛋白表达正向调节作用均优于扶正抗癌方、吉非替尼单独使用。在H1650信号传导通路中,扶正抗癌方与吉非替尼联合使用对P-AKT P-P38蛋白表达正向调节作用均优于扶正抗癌方、吉非替尼单独使用。在PC9信号传导通路中,扶正抗癌方与吉非替尼联合使用对P-erk、P-P38蛋白表达正向调节作用均优于扶正抗癌方、吉非替尼单独使用。
在吉非替尼治疗基础上,联合中医药较单纯吉非替尼治疗非小细胞肺癌有一定的增效作用。中医药联合吉非替尼治疗非小细胞肺癌中药复方不单单作用于一条或几条信号通路,亦不像现代提取单体药物作用于一种蛋白或蛋白靶点。肿瘤细胞的增殖、凋亡涉及许多更为复杂的信号通路,我们需要进一步对其进行研究,在已经得到阳性结果的信号传导通路,我们可以通过抑制其表达或者增强其表达,进一步观察下游的目地蛋白是否会有相应的变化,进而验证我们实验所得出的结果。
Objective
Lung cancer is the leading course of mortality in oncology. Non-small-cell lung cancer(NSCLC) is the major part of the lung cancer and most of the NSCLC patients were advanced when diagnosized. Patients with EGFR ge ne-non mutation or EGFR mutation are treated with chemotherapy based on platinum or epidermal growth factor receptor tyrosine kinased inhibitor (EGFR-TKI)respectively. Only30%of all the NSCLC patients who harboring the EGFR mutation have the opportunity of benefiting from EGFR-TKI while EGFR-TKI is an oral drug with less toxity. Even though, patients who are s ensitive to EGFR-TKI have a mean PFS of11months.
For a long time, using Chinese medicine as a treatment of non-small cell lung cancer has its own unique theory and methodology. It can be us ed alone to make the tumor smller than befor. It can give thepatients Ion g-term survival. Reduce chemotherapy side effects, improve patient treatm ent effects and prolong patients survival. There are so many clinical and basic experimental study has been reported in patients using chemotherap y combined with TCM. But weather the Chinese medicine in the treatment o f advanced non-small cell lung cancer patients is better than the using o f gefitinib alone or not has no answer. We speculate that combination of EGFR-TKI and FZKA freeze dried powder would be a useful strategy for pa tients who are resistant to EGFR-TKI.
1Evaluated systematically the studies of traditional Chinese medicin e combined with gefitinib in the treatment of advanced non-small cell lun g cancer were in order to provide the feasibility of documentary evidence of FuzhengKangai decoction combined with gefitinib in the treatment of p atients with non small cell lung cancer.
2Study of three kinds of NSCLC cell lines (A549, H1650, PC9) for dru g sensitivity test and related signaling pathways, observation the effect and discover the mechanism of the combination of Fuzhengkangai Decoction and Gifitinib in the singaling pathways in three different kinds of cel l lines
Methods
1We searched the National Digital Library Federation (http://www.ucdrs.n et/admin/union/index.do)(2006-2012)、CNKI(2006-2012)、VIP (2006-2012).Two re viewers independtly evaluated the quality of the included studies and ext racted the data. Meta-analyses were performed by RevMan4.0software.
2Cells were exposed to gefitinib, FZKA freeze dried powder or the co mbination and the effects of inhibition were analyzed with MTT assay and flow cytometry.
3Observing response of signaling pathway moleculars with the methods of western blotting.
Results
Nine randomized controlled trials (RCTs) involving528patients were included. The results of meta-analysis showed that:the combination group s howed remarkable advantage in disease control rate、the improvement of KPS, decreased diarrhea, no improvement of liver function damage, the decreased nausea and vomiting.
Cell lines were divided into gefitinib sensitive group and gefitinib resistant group by the OD value of MTT. ONE WAY ANOVA analysis was used t o analyze the inhinbit rates of different cells with the same treatment. Factor analysis was used to analyze the inhibit rates of different treatm ent in the same cell line.
For the gefitinib-resitant cell lines:A549and H1650, there are sigini ficant difference was observed between A549and H1650when using the same dose of gefitinib. F=7.57, P=0.0101. gefitinib make a superiority effect in A549to H1650.
For the three cell lines, fzka freeze dried powder make more remarkab le inhibit effects to A549than the other two cell lines(F=9.23, P=0.000
The combination group make more remarkable apatosis effects in three cells lines than the single drug used. A549(12.6333±4.47698)%, H1650(25.4667±20.6011)%,(26.8333±10.4357)%.
By the methods of western blotting, in the dose-response experiments of FZKA to intevent the A549, P-EGFR, EZH2showed a dose-dependent decline, PPAR-gomma and P53showed a dose-dependent rise, in the dose-response e xperiments of FZKA to intevent the H1650, EZH2showed the dose-dependent downward trend, suggesting that FZKA make the H1650has a positive protei n expression, For the P-EGFR, no dose-dependent trend showed. When the dr ug concentration reaches a certain level, P-EGFR expression will go down. PPAR-gomma and P53showed no dose-dependent rise, p=0.000in the stati stical analysis of the results, in the dose-response experiments of FZKA to intevent the PC9, EZH2showed the dose-dependent downward trend, FZKA has a positive regulating effection to EZH2expression of PC9. And betwee n the different doses, statistical analysis, p=0.0000. For the P-EGFR, showed no dose-dependent manner, when the drug concentration reaches a c ertain level, P-EGFR expression will go down. PPAR-gomma and P53showed a dose-dependent rise, statistical analysis of the results between differ ent doses of p=0.000, with statistical significance, The FZKA can inter vent the protein expression of PC9showed a positive trend when the dru g'concentration reaches a certain level.
In the dose-response experiments of FZKA to intevent the A549P-erk r endered good time correlation between different time points and the stati stical analysis showed:p=0.0036, Its has statistical significance. In the dose-response experiments of FZKA to intevent the H1650, P-AKT, P-er k, P-P38showed good time correlation between the different time points a nd the statistical analysis showed p=0.0000, p=0.0130, p=0.000, Th e data showed statistical significance. In the dose-response experiments of FZKA to intevent the PC9, P-AKT, P-P38showed good time correlation b etween different time points and statistical analysis p=0.0000, p=0.0046, with statistical significance.
The FZKA's singal effect to P-EGFR of A549, F value is90.31. P=0.0000. The Gifitinib's singal effect to P-EGFR of A549,49, F value is70.44, P=0,00The combination effect F value is8.32, P=0.0204. These date indicate that the two singal drug both has effect to the expression of P-EGFR. When these two drugs use together, they can make more significant influence to the expression of P-EGFR. The FZKA's singal effect to EZH2of A549, F value is49.66,p=0.000. The Gifitinib's singal effect to EZH2of A549, F value is55.17, P=0.0000. The combination effect F value is1.69. P=0.2293. These date indicate that the two singal drug both has effect to the expression of EZH2. When these two drugs use together, they can make more significant influence to the expression of EZH2. The FZKA's singal effect to ppar-gamma of A549, F value is2.96, P=0.000. The Gifitinib's singal effect to ppar-gamma of A549, F value is31.95, P=0.000. The combination effect F value is2.83.P=0.1311. These date indicate that Gifitinib has effect to the expression of ppar-gamma and FZKA has no effect to it. When these two drugs use together, they can make significant influence to the expression of ppar-gamma. The FZKA's singal effect to P53of A549, F value is80.63, P=0.000. The Gifitinib's singal effect to P53of A549, F value is2.04,P=0.1913, The combination effect F value is26.77.P=0.0008. These date indicate that FZKA has up regulation effect to the expression of P53and Gifitinib has down regulation effect. When these two drugs used together, they can make more significant influence to the expression of P53. The FZKA's singal effect to P-AKT of A549, F value is8.97, P=0.0172. The Gifitinib's singal effect to P-AKT of A549, F value is34.10, P=0.0004. The combination effect F value is8.09. P=0.0217. These date indicate that the two singal drug both has effect to the expression of P-AKT. When these two drugs use together, they can make more significant influence to the expression of P-AKT. The FZKA's singal effect to P-erk of A549, F value is1.43, P=0.2654, The Gifitinib's singal effect to P-erk of A549, F value is22.22, P=0.0015. The combination effect F value is3.72. P=0.0898. These date indicate that Gifitinib has up regulation effect to the expression of P-erk and FZKA has no effect. When these two drugs used together, they can make more significant influence to the expression of P-erk. The FZKA's singal effect to P-P38of A549, F value is8.66, P=0.0186, The Gifitinib's singal effect to P-P38of A549, F value is29.58, P=0.0006. The combination effect F value is0.97. P=0.3544. These date indicate that Gifitinib and FZKA both has up regulation effect to the expression of P-P38When these two drugs used together, they can make more significant influence to the expression of P-P38.
The FZKA's singal effect to P-EGFR of H1650, F value is3.84. P=0.0855. The Gifitinib's singal effect to P-EGFR of H1650,, F value is6.01, P=0.0399The combination effect F value is3.00, P=0.1217. These date indicate that the two singal drug both has down regulate effect to the expression of P-EGFR. When these two drugs use together, they can make more significant influence to the expression of P-EGFR. The FZKA's singal effect to EZH2of H1650, F value is89.93. P=0.0000. The Gifitinib's singal effect to EZH2of H1650,, F value is152.01, P=0.0000. The combination effect F value is22.58, P=0.0014. These date indicate that the two singal drug both has down regulate effect to the expression of EZH2. When these two drugs use together, they can make more significant influence to the expression of EZH2. The FZKA's singal effect to ppar-gomma of H1650, F value is8.97.P=0.0172. The Gifitinib's singal effect to ppar-gomma of H1650,,F value is25.96, P=0.0172. The combination effect F value is25.96, P=0.0009. These date indicate that Gifitinib has up regulate effect to the expression of ppar-gomma. When these two drugs use together, they can make more significant influence to the expression of ppar-gomma. The FZKA's singal effect to P53of H1650, F value is17.71.P=0.0030. The Gifitinib's singal effect to P53of H1650,,F value is25.66, P=0.0010. The combination effect F value is1.65, P=0.2353. These date indicate that these two drugs both has up regulate effect to the expression of P53. When these two drugs use together,they can make more significant influence to the expression of P53. The FZKA's singal effect to P-AKT of H1650, F value is44.61. P=0.0002. The Gifitinib's singal effect to P-AKT of H1650,, F value is2.75, P=0.1358. The combination effect F value is6.90, P=0.0.0303. These date indicate that these two drugs both has down regulate effect to the expression of P-AKT. When these two drugs use together,they can make more significant influence to the expression of P-AKT. The FZKA's singal effect to p-erk of H1650, F value is5.50. P=0.0471. The Gifitinib's singal effect to p-erk of H1650,, F value is10.08, P=0.0131. The combination effect F value is10.19, P=0.0128. These date indicate that Gifitinib has up regulate effect to the expression of p-erk. When these two drugs use together,they can make more significant influence to the expression of p-erk. The FZKA's singal effect to P-P38of H1650, F value is309.69. P=0.0000. The Gifitinib's singal effect to P-P38of H1650,, F value is118.04, P=0.0000. The combination effect F value is3.92, P=0.0832. These date indicate that Gifitinib has up regulate effect to the expression of P-P38. When these two drugs use together, they can make more significant influence to the expression of P-P38.
The FZKA's singal effect to P-EGFR of PC9, F value is4.42. P=0.0686.The Gifitinib's singal effect to P-EGFR of A549,49, F value is675.00, P=0.0000The combination effect F value is1.47, P=0.2598. These date indicate that the two singal drug both has effect to the expression of P-EGFR. When these two drugs use together, they can make more significant influence to the expression of P-EGFR. The FZKA's singal effect to EZH2of PC9,F value is127.48. P=0.0000. The Gifitinib's singal effect to EZH2of PC9, F value is242.55, P=0.0000The combination effect F value is6.86, P=0.0307. These date indicate that the two singal drug both has effect to the expression of EZH2. When these two drugs use together, they can make more significant influence to the expression of EZH2. The FZKA's singal effect to ppar-gomma of PC9, F value is7.90. P=0.0228. The Gifitinib's singal effect to ppar-gomma of PC9, F value is14.59, P=0.0051The combination effect F value is2.16, P=0.1800. These date indicate that the Gifitinib has the up regulate effect of ppar-gomma. When these two drugs use together, they can make more significant influence to the expression of ppar-gomma. The FZKA's singal effect to P53of PC9, F value is12.95. P=0.0070. The Gifitinib's singal effect to P53of PC9, F value is49.45, P=0.0001The combination effect F value is1.76, P=0.2208. These date indicate that the Gifitinib has the up regulate effect of P53. When these two drugs use together, they can make more significant influence to the expression of P53. The FZKA's singal effect to P-AKT of PC9, F value is3.31. P=0.1066. The Gifitinib's singal effect to P-AKT of PC9, F value is33.39, P=0.0004The combination effect F value is7.02, P=0.0293. These date indicate that the Gifitinib has the most significant down regulate effect to the expression of P-AKT. The FZKA's singal effect to P-erk of PC9, F value is23.06. P=0.0014. The Gifitinib's singal effect to P-erk of PC9, F value is53.75, P=0.0001The combination effect F value is4.77, P=0.0605. These date indicate that the Gifitinib has the most significant up regulate effect to the expression of P-erk. The FZKA's singal effect to P-P38of PC9, F value is0.04. P=0.8530. The Gifitinib's singal effect to P-P38of PC9, F value is17.38, P=0.0031The combination effect F value is29.24, P=0.0006. These date indicate that the two singal drug both has effect to the expression of P-P38. When these two drugs use together, they can make more significant influence to the expression of P-P38.
Conclusion
The present clinical study results suggest the cobination of TCM and gefitinib shows more superiority for non-small cell lung cancer, and its c linical application is worthy to be advocated.
The combination treatment of geitinib and the Chinese medicine powder achieved dose dependent enhanced apatosis effects. For A549cell, the Chi nese powder alone showed stonger inhibit effencicy than the other two cel l lines.
The combination treatment could effectively inhibit P-EGFR and EZH2t hrough inhibit the P13K/akt and RAS/Raf/MAPK/ERK pathway.
引文
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