The Study Of Jianpi Liqi Yiliu Formula Combined With Cytokine-Induced Killer In Treating Advanced Hepatocellular Carcinoma

Objectives1. Clinical study:Advanced hepatocellular carcinoma(HCC) patients were included and divided into two groups with the method of prospective randomized controlled study. Jianpi Liqi Yiliu Formula(JLYF) and Cytokine-Induced Killer (CIK) transfusion were given in the treatment group while dialectical decoction and CIK transfusion were given in the control group. The efficacy were observed and compared with related trial. This study aims to evaluate the effect of Chinese medicine combined with CIK in treating advanced hepatocellular carcinoma.2. Experimental study:Human Bel-7402 hepatocellular cancer cell line were used to inverstigate the inhibition of proliferation and underlying mechanism of JLYF and JLYF combined with CIK. This study aims to verify whether tranditional chinese medicine combined with CIK treatment have a synergistic effect.Methods1. Clinical study:Patients with hepatocellular carcinoma who had Child-pugh liver function class A or B, Eastern Cooperative Oncology Group performance status (ECOG PS) score 0-2 and TNM stage Ⅲc or Ⅳ were randomly assigned to receive either JLYF(150mL bid po, dl-30) and CIK transfusion((1-3) ×109 qd ivd, dl-3) or dialectical decoction(150mL bid po, d1-30) and CIK transfusion((1-3) X109 qd ivd, d1-3) for 2 or more than 2 cycles treatments. Primary outcome was overall survival. Secondary outcomes included time to progression (TTP), disease control rate (DCR) and safety. ECOG PS, Child-pugh liver function class, quality of life and immune function were also assessed.2. Experimental study:This study comprised 2 parts. ① MTS assay was performed to observe the effect of JLYF on proliferation of Bel-7402 cells. To quantitatively analyze the underlying mechanisms of JLYF, Annexin V/PI double staining and PI staining assay were examined to detect cell apoptosis and the cell cycle distribution, respectively. Western blotting analysis was also used to further explore the potential target proteins of the JLYF mechanism. ②The effect of JLYF and CIK on proliferation were confirmed by MTS assay. To inverstigate whether JLYF and CIK have a synergistic effect, the Combination Index(CI) and Dose-reduction index(DRI) were calculated by CalcuSyn soft. In addition, Western blotting analysis was employed to explore the potential mechanism of the synergistic effect of JLYF and CIK.Results1 Clinical study1.1 Patient characteristics60 cases, included from the In-patient Oncology Department of Guangdong Provincial Hospital of Tranditional Chinese Medicine from January 2011 to January 2014, were randomly assigned to treatment group and control group.No case was exclused or dropped out, all cases had been fatal, and follow-up were finished until the cut-off date May 31th 2014. The baseline characteristics, such as age, sex, ECOG PS, portal vein embolization, TNM staging, hepatitis B virus infection, Child-pugh liver function class, alpha fetal protein(AFP), syndrome differentiation of Chinese medicine, had no significant difference and was well balanced between 2 groups.1.2 Disease control rateIn the treatment group,11 cases(36.7%) were in stable diease(SD) and 19 cases(63.3%) were in progressive disease (PR). In the control group,9 cases (30.0%) were in stable diease(SD) and 21 cases (70.0%) were in progressive disease (PR). Disease control rate was 36.7% and 30.0% in the treatment and control group.1.3 Time to progressionUp to the last follow-up date, the median time to progression in the treatment group was 3.7 months, while in the control group was 2.5 months. There was statistical difference between two groups(P=0.006).1.4 Overall survivalUp to the last follow-up date, the median overall survival in the treatment group was 5.2 months, as compared with 4.6 months in the control group. There was no statistical difference between two groups(P=0.083).1.5 ECOG PS and Child-Pugh liver function classCompared to before treatment, the score of ECOG PS and Child-Pugh liver function class of both two groups were decreased after treatment. But only the ECOG PS in the treatment group between before and after treatment has statistical difference(P=0.03).1.6 Immune functionBefore treatment, there was no statistical difference between two groups of T lymphocyte subsets. Compared to before treatment, the CD3+, CD4+and NK+ of both two groups were increased after treatment and there was statistical difference between before and after treatment. Compared to before treatment, CD8+of the treatment group was increased after treatment and there was statistical difference between before and after treatment. There was statistical difference in the CD3+difference value of before and after treatment between two groups.1.7 Quality of lifeBefore treatment, there was no statistical difference between two groups of quality of life. Compared to before treatment, the five functional scales and the global health status were increased, while all of the symptom scales were decreased in both of two groups. There were statistical difference of the physical functioning, role functioning, emotional functioning, global health status, fatigue, nausea and vomiting, dyspnoea, appetite loss, constipation and diarrhea in the treatment group while the physical functioning and fatigue in the control group. Global health and fatigue have statistical difference in the difference value of before and after treatment between two groups.1.8 Sub-group analysis1.8.1 The difference of efficacy between two groups with the same ECOG PSThere were 6 cases, who have ECOG PS score 0-1, in the treatment group while 7 cases in the control group. The median time to progression was 7.3 months and 4.4 months, respectively, indicating statistical difference between two groups(P=0.02). The median overall survival was 7.3 months and 5.2 months, respectively, indicating no statistical difference between two groups(P=0.20).There were 24 cases, who have ECOG PS score 2, in the treatment group while 23 cases in the control group. The median time to progression was 3.5 months and 2.5 months, respectively, indicating statistical difference between two groups(P=0.03). The median overall survival was 5.0 months and 4.6 months, respectively, indicating no statistical difference between two groups (P=0.15).1.8.2 The difference of efficacy between two groups with the same Child-Pugh liver function classThere were 19 cases, who have Child-Pugh liver function class A, in the treatment group while 21 cases in the control group. The median time to progression was 4.6 months and 2.6 months, respectively, indicating statistical difference between two groups(P=0.02). The median overall survival was 6.6 months and 4.8 months, respectively, indicating no statistical difference between two groups(P=0.33).There were 11 cases, who have Child-Pugh liver function class B, in the treatment group while 9 cases in the control group. The median time to progression was 2.8 months and 2.5 months, respectively, indicating statistical difference between two groups (P=0.06). The median overall survival was 5.0 months and 4.4 months, respectively, indicating no statistical difference between two groups(P=0.20).1.9 Safety2 cases in the treatment group and 1 case in the control group appeared fever after received CIK transfusion. All cases of the blood culture were negative and the temperature were below 41℃. After received supportive treatment, the temperature can dropped gradually to normal range. There were no hematological toxicity, hapatotoxicity and nephrotoxicity in any cases during treatment. No case terminate the treatment because of adverse effect.2 Experimental study2.1 Effect of JLYF on proliferation and the underlying mechanism of human hepatocellular carcinoma Bel-7402 cells2.1.1 Effect of JLYF on Bel-7402 cell viability detected by MTS assayJLYF reduced the percentage of viable Bel-7402 cells in a dose-dependent manner. With time increased, the inhibition effect of JLYF decreased gradually, indicating the most effective timing was at 24h.2.1.2 Effect of JLYF on cell cycle distribution detected by PI staining assayAfter different concentrations of JLYF exposed for 24h, the G0/G1 phase of the Bel-7402 cells was increased while the M phase was decreased in dose-dependent manner. The G0/G1 phase and M phase of 4mg/mL and 8mg/mL have statistical difference to Omg/mL(P<0.05).2.1.3 Effect of JLYF on cell apoptosis by detected Annexin V/PI double staining assayAfter Bel-7402 cells was exposed to different concentrations of JLYF for 24h, the percentage of Annexin V positive Bel-7402 cells was not increased, indicating that JLYF could not induce early apoptosis. Meanwhile, the percentage of PI positive or Annexin V/PI double positive Bel-7402 cells was not increased too, indicating that JLYF could not induce late phase apoptosis or necrosis.2.1.4 Effect of JLYF on signal transduction pathway by detected western blotting analysisAfter Bel-7402 cells was exposed to 8mg/mL of JLYF for 1h,2h,4h and 8h, the phosphorylation of STAT3 was decreased while the phosphorylation of AKT was increased. The phosphorylation of ERK was increased in a time-dependant manner after Bel-7402 cells was exposed to 8mg/mL of JLYF within 24h and the change was most significant in 24h. Compared to Oh, the expression of P53 was increased in 24h and the expression of Bcl-xL was decreased since 8h to 24h dramatically.2.2 Effect of JLYF combined with CIK on proliferation and the underlying mechanism of human hepatocellular carcinoma Bel-7402 cells2.2.1 Effect of JLYF combined with CIK on Bel-7402 cell viability detected by MTS assayAt each time point and the same concentration of JLYF, the viability of Bel-7402 was decreased in a dose-dependent manner when the ratio of effect cell and target cell (E:T) was increased. At 48h and 72h, the viability of Bel-7402 cells have statistical difference between E:T=20:1 and E:T=0.When the ratio of effect cell and target cell was 5:1, the viability of Bel-7402 cells was decreased as the concentration of JLYF increased at the same time point (P<0.05). When the ratio of effect cell and target cell was 10:1 and 20:1, the viability of Bel-7402 cells was not changed as the concentration of JLYF increased at the same time point (P>0.05).When the ratio of effect cell and target cell was 20:1 and was affected by the same concentration of JLYF, the viability of Bel-7402 cells was slightly decreased at 48h compared to 24h (P<0.05) and was similar to the viability at 72h. When the ratio of effect cell and target cell was 5:1 and 10:1, the viability of Bel-7402 cells was decreased at 48h and increased at 72h. The difference from 24h to 48h and the difference from 48h to 72h have no statistical difference.The combination index of JLYF and CIK was lower than 0.7 when 2mg/mL or 4mg/mL of JLYF combined with different ratio of CIK at 24h. Combination index of JLYF and CIK increased as the concentration of JLYF and the action time increased.The dose-reduction index of CIK increased as the concentration of JLYF increased at the same time point and the same ratio of CIK. This trend was most dramatic when the ratio of effect cell and target cell was 5:1 at 24h. The dose-reduction index of JLYF increased as the the ratio of effect cell and target cell increased at the same time point and the same concentration of JLYF. This trend was most dramatic when the concentration of JLYF was lower than 8mg/mL at 48h.2.2.1 Effect of JLYF combined with CIK on signal transduction pathway detected by Western Blotting analysisWhen the Bel-7402 cells were treated by the combination of JLYF and CIK, the phosphorylation of STAT3 increased in time-dependant manner and the phosphorylation level was similar to that affected by CIK alone.The phosphorylation of AKT did not decrease until the Bel-7402 cells were treated by the combination of JLYF and CIK for 24h, indicating statistical difference between Oh and 24h.The phosphorylation of ERK1/2 did not decrease until the Bel-7402 cells were treated by the combination of JLYF and CIK for 24h, indicating statistical difference between Oh and 24h.When the Bel-7402 cells were treated by the combination of JLYF and CIK for 24h, the expression of P53 increased dramatically and was higher than that affected by JLYF or CIK alone, indicating statistical difference between Oh and at 24h.When the Bel-7402 cells were treated by the combination of JLYF and CIK for 4h and 24h, the expression of Bcl-xL decreased dramatically. There was statistical difference between the expression of Bcl-xL at Oh and at 24h. Conclusion1 Clinical study1.1 For advanced HCC patients, tranditional chinese medicine combined with CIK treatment can prolong TTP, increase the ECOG PS, promote the immune function and improve the quality of lif. JLYF combined with CIK treatment was more effective than dialectical decoction.1.2 JLYF combined with CIK treatment is a better therapeutic regimen for advanced HCC patients who had ECOG PS 0-2 or Child-pugh liver function class A.2 Experimental study2.1 JLYF exhibited a dose-dependant growth inhibition effect on Bel-7402 cells through arrested the cell cycle at GO/G1 phase via down-regulated the phosphorylation of STAT3 and the expression of Bcl-xL, and up-regulated the phosphorylation of AKT, phosphorylation of ERK1/2 and the expression of P53. The inhibition effects may induct by multi protein and multi signal transduction pathway.2.2 JLYF combined with CIK exhibited growth inhibition effect on Bel-7402 cells. The combination effects displayed a synergistic effect in the case of particular concentration of JLYF and particular ratio of effective cell and target cell. JLYF and CIK might mediate synergistic effect through up-regulated the expression of P53 and down-regulated the expression of Bcl-xL.