Research On The Effect Of XiaoJiYin As A Maintenance Therapy For Advanced Non-small Cell Lung Cancer And The Effect On The PI3K/AKT/mTOR Signal Transduction Pathway

ObjectivesTo investigate a treatment regime of Traditional Chinese Medicine (TCM) as a maintenance therapy for advanced Non-small cell Lung Cancer (NSCLC), to deepen the discovery in the academic content of renowned old TCM practitioner Liu Wei-sheng, and to further investigate the molecular mechanism of XiaoJiYin in suppressing lung cancer growth through the PI3K/AKT/mTOR pathway.Methods1 Clinical studySixty-four patients of advanced NSCLC were included according to the inclusion criteria, and were randomly divided into treatment group and control group, with 32 patients respectively. In the treatment group, XiaoJiYin was given as a maintenance therapy (60ml/m2, po, qd), while only follow-up and observation was given in the control group (Best support care). After 3 months treatment, the response rate, clinical syndrome scores, KPS scores, overall survival (OS) and progression-free survival (PFS) were compared between the two groups. Variance analysis was performed for the clinical characteristics between groups, while Kaplan-Meier analysis was conducted for the comparison of OS and PFS.2 Experimental studyHuman A549 lung cancer cell line was selected. It was cultivated in RPMI-1640, and XiaoJiYin containing rat serum of low, medium and high concentrations, and fetal bovine serum (FBS) as a control, were added to the cells in logarithmic phase for cell induction. MTS method was used for the suppressive effect of XiaoJiYin in A549 cell proliferation. Western-blot was used for the detection of p-AKT protein in PI3K/AKT pathway, and RT-PCR was used in detecting the expression of downstream mRNA, such as BAD, Caspase-9, FKHR, mTOR and NF-K B in the PI3K/AKT pathway. Variance analysis was performed between groups.Results1 Clinical study1.1 Patient characteristicsThe 64 cases of stage IIIB/IV NSCLC patients with PSO-2 were recruited from the In-patient Oncology Department of Guangdong Provincial Hospital of TCM. Patients were enrolled during September 2008 and December 2010. The pre-enrolment baseline characteristics, such as sex, smoking history, age, staging, KPS score and chemotherapy regimens received, had no significant difference and was balanced between groups (P>0.05). Four cases were drop-off, in which the 2 cases in treatment group withdrew due to discontinuation of treatment, while the 2 cases in the control group withdrew because of change in self-decision. There were 60 evaluable cases, 41 cases deceased, while follow-up was conducting. According to the plan, through statistical evaluation of sample number, the requirement of therapeutic evaluation was reached.1.2 Short-term EfficacyTherapeutic evaluation was conducted after 3 months of treatment. The response rate (RR) was 76.6% in the treatment group, while the disease control rate (DCR) was 43.3% in the control group. There was significant difference between the 2 groups (P=0.00).1.3 SurvivalUp to the last follow-up, the overall survival (OS) was 6.43 months and 3.27 months in the treatment and control groups respectively. Intra-group comparison suggested that the OS was extended for 3.16 months in the treatment group, indicating significant difference between the 2 groups (P=0.00). The progression-free survivals (PFS) were 5.07 months and 2.53 months in the treatment group and control group respectively. Intra-group comparison suggested that the PFS was extended for 2.54 months in the treatment group, indicating significant difference between the 2 groups (P=0.00). 1.4 Clinical syndrome differentiationThe clinical syndrome evaluation improvement was elevated for 43.3% in the treatment group when compared with control, indicating significant statistical difference (Z=-2.362, P=0.009).1.5 Physical statusThe KPS score in the treatment group was elevated for 26.6% compared with control group, with statistical difference, (Z=-2.128, P=0.033).1.6 Safety evaluationThe safety evaluation suggested that the blood routine, liver and kidney function and ECG examination showed no significant different before and after XiaoJiYin treatment. According to the WHO RESIST standard, there was no drop-off case due to intolerable adverse event. There were no hematotoxicity, gastrointestinal symptoms, alopecia or severe cardio, renal or hepato toxicity. Nausea of WHO toxicity grade 1 was observed on the second day of treatment for 3 patients in the treatment group. The symptom disappeared after 10 minutes without handling. Slight diarrhea was observed in on patient after administration of drug for 30 minutes, and was relieved without handling. No treatment-related death was observed.2 Experimental study2.1 Effect of different concentration of XiaoJiYin on cell proliferation by MTS methodDifferent concentration of XiaoJiYin containing serum can suppress proliferation of A549 lung cancer cells. The 72 hours anti-proliferation rate of high concentration XiaoJiYin containing serum on A549 had a significant difference compared with control group.2.2 The effect of XiaoJiYin in regulating the p-PI3K/p-AKT/mTOR gene expression in human lung cancer cellsThe gene expression quantity was recorded after adding different concentration of XiaoJiYin containing serum to A549 cells for 48 and 72 hours. The mRNA expression on BAD and Caspase-9 genes were significantly increased. The 2-??Ct in the high concentration serum for 48 hours group was significantly elevated compared with the control group and had statistical significance (P<0.01). There was no significant difference between experimental groups. In addition, the mRNA expression of mTOR, FKHR and NF-K B was also obviously elevated, and had statistical difference compared with control group (P<0.01) There was no significant difference between experimental groups.2.3 The effect of XiaoJiYin on the expression of PTEN, PI3K, ATF2 and p-AKT protein through Western BlottingThrough image processing analysis, using GAPDH grey scale as reference, the protein expression of PTEN and phosphor-AKT reduced, PI3K increased yet. Low and medium concentration led to p-PTEN down-regulating, showing significant difference compared with control group (P<0.01). Low and high concentration of XiaoJiYin led to up-regulation of p-PI3K, showing significant difference compared with control group (P<0.01). Medium and high concentration of XiaoJiYin led to down-regulation of p-AKT, showing significant difference compared with control group (P<0.01).Conclusion1 Clinical studyThrough a prospective randomized control trial (RCT), it was found that the response rate, overall survival and progression-free survival were obviously elevated in XiaoJiYin group compared with the control. The clinical symptoms and physical status were obviously improved, with slight adverse side effects and high safety. Therefore, XiaoJiYin as a maintenance therapy for advanced NSCLC can improve the survival and quality of life.2 Experimental studyXiaoJiYin containing serum can effectively suppress the proliferation of human lung cancer cell line A549 in vitro, and has certain cytotoxicity. XiaoJiYin can effectively suppress the AKT point in the PI3K/AKT signal transduction pathway, increasing the mRNA expression of downstream molecules like BAD and Caspase-9, enhancing apoptosis of A549 and therefore effectively suppress the growth of A549.