Clinical And In Vitro Study Of Fuzheng Kangai Decoction With Gefitinib In Treating Non-smali-cell Lung Cancer

BackgroundLung cancer is currently one of the major reasons of cancer-related death, in which85-90%belongs to non-small-cell lung cancer (NSCLC). Most of the NSCLC cases were discovered at late stage, and the regular first-line chemotherapy does not achieve satisfactory result.The application of targeted drugs has significant progress in recent years, and EGFR tyrosine kinase inhibitors (EGFR-TKI) attract most attention. EGFR family members play important role in the development of lung cancer, and is over-expressed in40-80%of NSCLC. It is closely related to the growth, proliferation, migration, invasion and apoptosis of tumor cells. EGFR is a trans-membrane protein of HER/ErbB family. The integration with extracellular factors can affect a series of downstream pathways, such as Ras/Raf/MAPK, PI3K/Akt, and Jak/STAT3, and in turn affect downstream proteins like c-Fos, c-Jun, Bad, Caspase9, FOXO, etc, to promote cell division, increase cell proliferation, transformation and migration. There are single-targerted and multi-targeted EGFR-TKIs. The most widely used and representative drug in this class is gefitinib. Its mechanism is to compete with ATP for binding site on EGFR, in order to inhibit phosphorylation of EGFR, and thus the EGFR signaling pathway.It is currently agreed that gefitinib can be used in first-line treatment of NSCLC patients with EGFR point mutation. The effect of EGFR-TKI on those patients is better than standard chemotherapy, with less adverse side effect. Therefore, it can be used in elderly patients or patients with poor PS scoring. However, patients without EGFR mutation, or with unclear mutation status cannot benefit from this finding. In addition, it was observed that patients achieved acquired drug-resistance eventually after receiving EGFR-TKI. Therefore, although the progression free survival (PFS) is prolonged after EGFR-TKI treatment, the overall survival (OS) is similar to that of chemotherapy. The current treatment strategy for acquired resistance includesâ‘ usage of irreversible EGFR-TKI (including BIBW2992, EKB-569),â‘¡alliance of multiple drugs or single use of multi-targeted drugs, to inhibit different signaling pahtway at the same time (like vandetanib, BMS-690514), orâ‘¢second-line chemotherapy. Although some of the above drugs had entered clinincal trials, the effect is yet to prove.Traditional Chinese medicine (TCM) has been used in China for thousands of years, and is widely used in prevention and treatment of different diseases. It is often used together with Western medicine. In the treatment of NSCLC, TCM can boost immunity and promote recovery after surgery; can increase efficacy and reduce toxicity when use with radiotherapy and chemotherapy. It can also be used as maintenance therapy to improve overall condition in late stage patients with poor PS and cannot tolerate regular treatment. Currently TCM is mostly used to reduce adverse side-effects in patients receiving EGFR-TKI treatment. The most common adverse effect of geifitinib include acneform eruptions, which seriousness is related to efficacy. TCM is effective in relieving these eruptions but does not reduce efficacy. A few reports suggested that TCM can improve effect of EGFR-TKI on NSCLC, but there was no report on the sensitization of patients towards gefitinib by TCM.FuZheng KangAi decoction (FZKA) was used clinically in Guangdong Provincial Hospital of TCM for more than a decade. Studies suggest that most NSCLC patients are under a condition of deficiency and excess. Clinical syndromes are mainly qi-def iciency and yin-deficiency, together with phlegm, dampness and qi-stagnation. It was observed there are many NSCLC patients have "spleen-deficiency with phlegm and blood stasis" syndrome. After prolonged observation and practice, Dr Wu Wan-yin developed this decoction, consisting mainly of Radix Pseudostellariae, Astragalus monghol, Pseudobulbus Cremastrae seu Pleiones, Curcuma Zedoary, etc. This decoction mainly targeted in boosting vital qi and anti-tumor. The five drugs that can boost vital qi, have effect of vitalizing spleen and lung, promoting qi and reduxing phlegm. The seven anti-tumor herbs have effect of clearing toxic heat, resolving phlegm and blood stasis. It was said in TCM that "spleen is the source of phlegm, while lung is the storehouse of phlegm". Deficiency of spleen leads to ineffective use of essence of water and grain, dampness stagnated to phlegm and is stored in lung. Deficiency of qi leads to stagnation of blood, and further turns into blood stasis, therefore eventually resulting in "stagnation of phlegm and blood stasis". This decoction blocks the origin of phlegm and boosts the vital qi, therefore can achieve increasing effect and reducing toxicity when used with Western medicine. Current clinical observation suggested that FuZheng KangAi decoction with gefitinib as a second-line treatment for NSCLC has a better effect than second-line chemotherapy. Therefore, it is considered that this decoction can increase efficacy and sensitize NSCLC towards gefitinib.Objective1.Clinical study:To include NSCLC patients who refuse chemotherapy as first-line treatment, and offer FuZheng KangAi decoction with gefitinib, in order to investigate the combination of these two drugs as a first-line treatment for NSCLC patients, and to compare the efficacy with current reports of single drug gefitnib as first-line treatment. Therefore providing direction for following clinical treatment, and providing ground and guideline for the use of the two drugs.2. In vitro study:To use3NSCLC cell lines (PC9, A549, H1650) with different sensitivity towards gefitinib, to further investigate:â‘ whether FuZheng KangAi decoction can improve efficacy and increase sensitivity towards gefitinib;â‘¡the effect and mechanism of the two drugs on NSCLC with different EGFR mutation status;â‘¢to provide theoretical ground for further clinical and in vivo studies.Method1. Clinical study:Fifty-one cases of NSCLC patients who refuse to receive chemotherapy as first-line treatment were included according to the inclusion criteria. They were of PS0-3, without brain metastasis, and with normal bone marrow, heart, liver and kidney function. FuZheng KangAi decoction250ml po bid and gefitinib250mg po qd were given to those patients. Short-term efficacy such as overall response rate (ORR) was recorded after3months of treatment. The patients without disease progression continued treatment and follow-up until progression or death. SPSS16.0was used for statistical analysis. Descriptive analysis was used for patients' characteristics, short-term efficacy and1year survival. Kaplan-Meier method was used for progression free survival (PFS) and and median survival time (MST) analysis.2. In vitro study:Human lung cancer cell lines PC9, A549and H1650were used in this study. PC9is a cell line with high EGFR-TKI sensitivity, A549is a cell line with low EGFR-TKI sensitivity, and H1650is a cell line with EGFR-TKI resistance. Different concentrations of FZKA and gefitinib medicated rat serum were prepared. Medicated serum of different concentrations were added to the cells at different time intervals. CCK8was used to test the inhibition of lung cancer cell proliferation by FZKA and gefitinib medicated serum. Flow cytometry was used to test the change in cell cycle and apoptosis of cancer cells by FZKA and gefitinib medicated serum. Western Blot was used to test the change in expression of EGFR, Akt and MAPK and their phosphorylated proteins by FZKA and gefitinib medicated serum.Result1. Clinical Study1.1Clinical CharacteristicsFifty-one cases of stage â…¡/â…£ NSCLC patients with PS0-3were recruited from Guangdong Provincial Hospital of TCM Oncology Department, during July2009and September2011. Eight cases drop-out due to difficulty in follow-up. There were43cases of evaluable cases, in which there were16cases of death and27cases of survival with follow-up. The majority were female, elderly and adenocarcinoma.1.2Short-term efficacyEvaluation was made after3months of treatment. The overall response rate (ORR) reached37.2%and the disease control rate (DCR) reached72.1%.1.3Survival timeUp to the latest follow-up, the progression free survival (PFS) was10.78months, median survival time (MST) was10.0months and the1-year survival rate was55.56%.2. In vitro study2.1CCK8method for anti-proliferation effect of different concentration of FZKA and gefitinib medicated serum on lung cancer cellsThe anti-proliferation effect of gefitinib on A549and H1650at different concentration and time-interval was poor, while the effect on PC9at72h was fair. Different extend of anti-proliferation effect was achieved by different concentrations of FZKA on A549, PC9and H1650at72h. The anti-proliferation effect of combination of FZKA and gefitinib on A549and H1650at72h was better than single drug gefitinib, while the effect on PC9at72h was similar to that of single gefitinib. FZKA and gefitinib medicated serum had anti-proliferation on A549, PC9and H1650at different concentration and time interval, and achieved best result mostly at72h. It was suggested that FZKA may have sensitization effect towards gefitinib on A549and H1650cell lines.2.2Flow cytometry for change in cell cycle under different concentration of FZKA and gefitinib medicated serum on lung cancer cellsFZKA and gefitinib medicated serum can affect the cell cycle of A549, PC9and H1650to different extend, through stopping the cell cycle at G0/G1.2.3Flow cytometry for change in apoptosis under different concentration of FZKA and gefitinib medicated serum on lung cancer cellsFZKA and gefitinib medicated serum can affect the apoptosis of A549, PC9and H1650to different extend, in which pro-apoptotic effect was slight on H1650and PC9, and stronger on A549.2.4Western Blotting for change in expression of EGFR, Akt, MAPK and their phosphorylated proteins under different concentration of FZKA and gefitinib medicated serum on lung cancer cellsThe addition of FZKA and gefitinib medicated serum to A549and H1650cells resulted in down-regulation of p-EGFR and p-MAPK, suggesting the anti-proliferation effect on FZKA and gefitinib was acted through inhibiting the activation of EGFR and its downstream pathway Ras/Raf/MAPK. The addition of FZKA and gefitinib medicated serum to PC9cells resulted in down-regulation of p-Akt, suggesting the anti-proliferation effect on FZKA and gefitinib was acted through inhibiting the activation of EGFR downstream pathway PI3K/Akt.Conclusion1. Clinical study:FZKA with gefitinib can extend the PFS when used as first-line treatment for NSCLC patients instead of single geifitinib. The ORR was slightly better that of similar Asian studies.2. In vitro study:Different concentration of FZKA with gefitinib medicated serum had different extend of anti-proliferation effect on lung cancer cells at72h, and was better than that of single gefitinib. Different concentrations of FZKA with gefitinib medicated serum showed different extend of change in cell cycle on lung cancer cells. Different concentrations of FZKA with gefitinib medicated serum showed slight apoptotic effect on lung cancer cells. The mechanism of FZKA and gefitinib on A549and H1650cells may work through Ras/Raf/MAPK pathway, while may work through PI3K/Akt pathway on PC9cells.