The Study On Synergistic Effect Of FuZheng KangAi Decoction With Gefitinib In Treating Advanced Non-small-cell Lung Cancer

Objective1.Clinical study:We observed the progression-free survival(PFS) and median survival time (MST) of gefitinib plus FZKA(GF) group and gefitinib(G) group with the method of retrospective case-matching cohort study. This study aims to explore whether the PFS or MST of GF group is better than that of G group.2. In vivo study:The H1650cell lines which resistance to gefitinib were inoculated into the nude mice. Tumors were established by the injection of H1650cells, the nude mice received intragastric administration of gefitinib and FZKA. Then we evaluated the tumor inhibition rate of the two drugs. So as to verify whether traditional Chinese medicine combined with targeted drugs have a synergistic effect, and to explore the molecular mechanism.Methods1. Clinical study:We matched over50groups of stage Ⅲb or Ⅳ non-small cell lung cancer patients with the method of retrospective case-matching cohort study. And they were divided into the treatment group (GF) and control group(G). Patients in GF group received gefitinib(G,250mg/day orally) plus FZKA(F,250ml/bid/day orally), while in G group only received gefitinib(G,250mg/day orally). Gefitinib or FZKA was administered until progression of the disease, or development of unacceptable toxicities. CT scan was performed every6weeks to evaluate the effect. The primary endpoint was progression-free survival (PFS), secondary endpoints were median survival time (MST), objective response rate and safety.2. In vivo study:Tumors were established by the injection of H1650cells, the60mice were randomly divided into6groups of10each experiment: (1)Control group:0.9%NaCl;(2)Gefitinib(G) group:gefitinib5g/ml;(3)Lower dose of traditional Chinese medicine(LF) group:FZKA6.2g/ml;(4)Higher dose of traditional Chinese medicine(HF) group:FZKA12.4g/ml;(5)Gefitinib plus lower dose of traditional Chinese medicine(GLF) group:gefitinib5g/ml, FZKA6.2g/ml;(6)Gefitinib plus higher dose of traditional Chinese medicine(GHF) group:gefitinib5g/ml, FZKA12.4g/ml, and the mice received intragastric administration of the different treatment. At the end of experiment, tumor volume and weight were measured. Then we calculate the tumor volume and the inhibition rate of different groups, observe the biopsy of the tumor and target organs, detect the microvessel density and the proliferation index. Cell apoptosis was analyzed to caleulate the apoptosis index by TUNEL method, the expression of EGFR, ERK, AKT and the phosphorylated protein of them was assayed by immunohistochemistry SP method.Result1. Clinical study1.1Case-matching procedureFrom July2007to December2012,159subjects were engaged into the study.100patients received GF treatment from Guangdong Provincial Hospital of Traditional Chinese Medicine,59patients received G treatment from Guangdong Provincial Peoples Hospital. We used a matching procedure to match all the patients by gender, age, smoking status, performance status(PS) and pathological factors. Matching factors included sex, age categories (30-39,40-49,50-59,60-69,70-79), pathological stage (Ⅲb or Ⅳ ), smoking status (never or ever), pathology, and performance status. As a result,58pairs were matched successfully.1patient with the age of27years failed to be matched. Among the116patients,58patients accepted the treatment of traditional Chinese medicine combined with gefitinib and58patients accepted the single drug treatment with gefitinib. The mutation rate of EGFR(exons18,20,21mutation, exon19deletion) in G group was74.1%, but the mutation rate in GF group was10.3%. Both of the two groups included patients with EGFR wild type, the incident rate in GF group was4.9%while in GF group was8.6%.1.2Disease progression-free survival:Up to the last follow-up time, the median progression-free survival time in the G group was11.43months, while in GF group was13.1months. There was statistically difference between two groups(P=0.013). In the first-line treatment, the median progression-free survival time of the G group was11.43months and of the GF group was12.33months, there was statistically difference between two groups(P=0.024). Subgroup analyses were performed to compare progression-free survival between treatments in groups defined according to EGFR gene status, gender, smoking status and the pathologic types. The median progression-free survival time of the G group was11.80months and of the GF group was13.1months in the patients with EGFR mutations(exon19deletions or21exon mutation), there was statistically difference between two groups(P=0.016). The median progression-free survival time in G group was11.43months while in GF group was12.00months for the female patients, there was statistically difference between two groups (P=0.009). The median progression-free survival time of the G group was11.80months and of the GF group was13.10months for the no smoking patients, there was statistically difference between two groups(P=O.007). The median progression-free survival time of the G group was11.80months and of GF group was13.10months for the patients with adenocarcinoma, there was statistically difference between two groups(P=0.022).1.3The overall survival:The median survival time in G group was18.7months while in group was22.83months up to the last follow-up time. There was statistically difference between two groups(P=0.049). Subgroup analyses were performed to compare the median survival time between treatments in groups defined according to EGFR gene status, gender, smoking status and the pathologic types. The median overall survival time of the G group was20.27months and in GF group was23.73months in patients whose EGFR gene statuses were unknown, there was statistically difference between two groups (P=0.018). The median survival time of the G group was18.70months and of the GF group was23.73months for the female patients, there was statistically difference between two groups(P=0.027). The median survival time in G group was18.87months while in group was23.73months for no smoking patients, there was statistically difference between two groups(P=0.044). The median survival time of the G group was18.87months and of the GF group was22.83months for the patients with adenocarcinoma, there was statistically difference between two groups(P=0.065).1.4The objective response rateThe objective response rate (CR+PR) in the G group was82.76%, while in GF group was74.14%. There was no statistically difference between two groups (P=0.527).1.5Adverse events:The most common adverse events were rash and diarrhea. The second were stomatitis and aminotransferase elevation. The incidence of rash in G group was41.38%, while in GF group was24.14%. There was statistically difference between two groups(P=O.048).2.In vivo study2.1The influence of the tumors volume and weight of implanted tumor by different treatments on nude mouse xenograft tumor model of H1650cellThe average tumor volume in GLF and GHF groups were significantly slower than the control group, there was statistically difference between treatment groups and control group. The average tumor volume in GLF group were significantly slower than the G group, there was statistically difference between two groups(P=0.001), GHF group were significantly slower than the G group (P=1.53E-4), too. The average tumor volume in GLF group were significantly slower than the LF group, there was statistically difference between two groups(P=1.53E-4). The average tumor weight in GHF group were significantly slower than the control group, there was statistically difference between two groups(P<0.01), and there was no statistically difference between other groups.2.2The inhibitory effect of proliferation by different treatments on nude mouse xenograft tumor model of H1650cellThe nuclear of proliferating cells showed brown-yellow after Ki67staining. The brown-yellow proliferating cells of treatment group significantly reduced compared to control group, cell proliferation rates were (63.94+8.19)%(control),(57.11±7.58)%(G),(54.39±8.85)%(LF),(55.94±12.26)%(HF),(50.61±8.81)%(GLF) and (48.72±11.63)%(GHF), respectively, which showed a lowered tendency. The cell proliferation rates in GLF group and GHF group decreased significantly than the G group (P<0.05), there was statistically difference between groups. The cell proliferation rates in GHF group decreased significantly than the HF group(P=0.029), there was statistically difference between the two groups.2.3The induced effect of apoptosis by different treatments on nude mouse xenograft tumor model of H1650cellThe cell apoptosis rates were seperately(13.00±7.86)%(control),(16.30+9.06)%(G),(19.00±7.75)%(LF),(18.50±5.36)%(HF),(26.00±3.46)%(GLF) and(26.70±16.50)%(GHF), which showed a increased tendency. The cell apoptosis rates in GLF group and GHF group increased significantly than the control group(P<0.05), there was statistically difference between groups. The cell apoptosis rates in GLF group and GHF group increased significantly than the G group(P<0.05), too. And cell apoptosis rates in GLF group increased significantly than the LF group (P<0.05), there was statistically difference between the two groups.2.4The influence of the tumors microvessel density of implanted tumor by different treatments on nude mouse xenograft tumor model of H1650cellThe tumors microvessel density were16.40±3.10(control),7.20±2.15(G),10.40±2.80(LF),8.00±1.33(HF),8.60±1.58(GLF) and6.40±2.46(GHF), respectively, which showed a lowered tendency. The tumors microvessel density in treatment group decreased significantly than the control group (P<0.001), there was statistically difference between groups.2.5The influence on the expression of AKT, EGFR, ERK, p-AKT, p-EGFR and p-ERK of implanted tumor by different treatments on nude mouse xenograft tumor model of H1650cellThe mean density of AKT expression were0.327±0.024(control),0.234±0.026(G),0.275±0.020(LF),0.301±0.041(HF),0.292±0.024(GLF) and0.288±0.047(GHF), respectively. The mean density of AKT expression in treatment groups decreased significantly than the control group(P<0.001), there was statistically difference between groups. The mean density in GLF group and GHF group decreased significantly than the G group (P<0.001), there was statistically difference between groups.The mean density of EGFR expression were0.367±0.042(control),0.34±0.038(G),0.318±0.038(LF),0.326±0.041(HF),0.341±0.042(GLF) and0.327±0.015(GHF), respectively. There was no statistically difference between groups.The mean density of ERK expression were0.362±0.025(control),0.312±0.019(G),0.305±0.024(LF),0.300±0.005(HF),0.336±0.037(GLF) and0.327±0.015(GHF), respectively. The mean density of ERK expression in treatment groups decreased significantly than the control group(P<0.001), there was statistically difference between groups. The mean density in GHF group decreased significantly than the G group(P=0.001), and GHF group also decreased significantly than the HF group(P=0.019), there was statistically difference between groups.The mean density of p-AKT expression were0.334±0.026(control),0.289±0.084(G),0.282±0.075(LF),0.317±0.078(HF),0.311±0.078(GLF) and0.280±0.078(GHF), respectively, there was no statistically difference between groups.The mean density of p-EGFR expression were0.364±0.053(control),0.372±0.010(G),0.287±0.034(LF),0.292±0.051(HF),0.281±0.052(GLF) and0.281±0.051(GHF), respectively. The mean density of p-EGFR expression in treatment groups decreased significantly than the control group (P<0.05), there was statistically difference between groups.The mean density of p-ERK expression were0.336±0.033(control),0.291±0.022(G),0.237±0.014(LF),0.308±0.023(HF),0.243±0.018(GLF) and0.217±0.018(GHF), respectively. The mean density of p-EGFR expression in treatment(G, LF, GLF, GHF) groups decreased significantly than the control group(P<0.05), there was statistically difference between groups. The mean density in GLF group and GHF group decreased significantly than the G group(P <0.001), there was statistically difference between groups. The mean density in GHF group decreased significantly than the HF group (P<0.001), there was statistically difference between groups.Conclusion1. Clinical study1.1The progression-free survival and overall survival in GF group are longer than that in G group, which suggests that Fuzheng Kang’ai decoction can improve the effect of gefitinib.1.2The objective response rate of gefitinib combined with Fuzheng Kang’ai decoction is similar to gefitinib in patient with advanced non-small cell lung cancer. The incidence of rash in GF group was lower than that in G group. The result suggests that Fuzheng Kang’ai decoction can decrease toxicity of gefitinib.2. In vivo study2.1The inhibitory effect of gefitinib combined with Fuzheng Kang’ai decoction on nude mouse xenograft tumor model of H1650cell is superior than gefitinib or Fuzheng Kang’ai decoction. Especially in the high doses of Fuzheng Kang’ai decoction combined with gefitinib.2.2The inhibitory effect of proliferation by gefitinib combined with Fuzheng Kang’ ai decoction on nude mouse xenograft tumor model of H1650cell is superior than gefitinib or Fuzheng Kang’ ai decoction. Meanwhile, the induced effect of cell apoptosis is superior than gefitinib or Fuzheng Kang’ ai decoction, too.2.3Gefitinib, Fuzheng Kang’ ai decoction and gefitinib combined with Fuzheng Kang’ ai decoction could inhibit the tumor angiogenesis.2.4Both gefitinib and Fuzheng Kang’ ai decoction could down regulate the expression of p-EGFR、AKT、ERK and p-ERK, and gefitinib combined with Fuzheng Kang’ ai decoction could down regulate the expression of AKT、 ERK and p-ERK more than gefitinib or Fuzheng Kang’ ai decoction alone.2.5Fuzheng Kang’ ai decoction has synergistic effect on gefitinib by down regulating the expression of p-EGFR、AKT、ERK and p-ERK.