Songyou Yin Combined With TACE In The Treatment Of Patients With Unresectable Hepatocellular Carcinoma:Clinical Study And Potential Mechanism

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Transcatheter arterial embolization (TAE) or transcatheter arterial chemoembolization(TACE) has been widely performed as a palliative treatment for unresectable HCC, which can prolong the survival of HCC patients. However, the high incidence of tumor recurrence and metastasis after TACE is still a major problem. A higher rate of pulmonary metastasis following TACE treatment has been reported in HCC patients.Epithelial-mesenchymal transition(EMT), a process important for embryonic development, may be involved in many pathological processes such as wound healing, tissue fibrosis and cancer progression. TACE treatment for HCC results in a more aggressive and invasive type of residual cancer. Studies have revealed that EMT plays a important role in this process.Thus, inhibiting or reversing the process of EMT may be beneficial for patients with TACE treatment.The Chinese herbal medicine formula "Songyou Yin," a dietary component authorized by the Chinese State Food and Drug Administration (Grant No. G20070160), was originally designed to promote vital energy and considered as a nontoxic therapeutic supplement for cancer patients. The mixture includes five Chinese medicinal herbal extracts, whose proportions (w/w) are as follows:Salvia miltiorrhiza Bge., China,14.3%; Astragalus membranaceus Bge., China,14.3%; Lycium barbarum L., China,23.8%; Crataegus pinnatiWda Bge., China,23.8%; and Trionyx sinensis Wiegmann, China,23.8%. Pilot study has demonstrated that in vitro, "Songyou Yin" caused dramatic attenuation of tumor proliferation by induction of apoptosis that was associated with caspase-3 activation, and inhibit invasiveness of MHCC97H via reducing matrix metalloproteinase-2 (MMP2) activity. In vivo, "Songyou Yin" prolonged the life span of nude mice bearing xenografts and the lung metastatic extent was decreased. Another research maintained that residual HCC after in vivo oxaliplatin treatment demonstrated significantly increased metastasis to the lung (10/12 vs.3/12) when re-inoculated into the livers of new recipient nude mice. Songyou Yin (4.2 and 8.4 g/kg) attenuated EMT and inhibited the enhanced metastatic potential of residual HCC in nude mice (6/15 vs.13/15 and 3/15 vs.13/15, respectively). However, the effect of Songyou Yin in the treatment of patients with unresectable hepatocellular carcinoma is unknown and the detailed understanding of the relationship between EMT arrest and SYY treatment in vitro deserves more exploring.In this study, firstly we established a clinical study to investigate the therapeutic effect of Songyou Yin combined with TACE in the treatment of patients with unresectable hepatocellular carcinoma. Secondly, we explored the mechanism whether and how Songyou Yin attenuated EMT in vitro.Part One Songyou Yin combined with TACE in the treatment of patients with unresectable hepatocellular carcinoma:a clinical studyPurpose:The study aimed to explore the therapeutic effect of Songyou Yin combined with TACE in the treatment of patients with unresectable hepatocellular carcinoma.Methods:A clinical trail was performed, in which 44 patients with unresectable hepatocellular carcinoma received TACE therapy.T he patients were randomized to receive Songyou Yin or control placebo. The effects of the two treatment arms were evaluated and compared according to objective response, survival and treatment-related adverse-effects.Results:In the randomized controlled trial, Overall median survival time were 16.5 months in the SYY group and 7 months in the control group. The survival rates of 6,9,12 month in SYY group and control group were 69.57%,60.87%,55.8%, and 56.12%,40.18%,40.18%, respectively. Partial response rates were 10.53% and 0 in the SYY group and control group, respectively, while 0 in the control group.Adverse effects were rare and no significant difference found between two groups.Conclusion:Songyou Yin may be benefit for patients with unresectable HCC treated by TACE, which probably offer a better clinical outcome. Further study with larger sample size is need to prove the clinical effectiveness of Songyou Yin in combination with TACE for unresectable HCC.Part Two Relationship between EMT arrest and Songyou Yin treatmentPurpose:We aimed to explore the molecular mechanism of whether and how Songyou Yin attenuated EMT in vitro.Methods:TGF-β was used to induce MHCC97L and MHCC97H to undergo EMT. Then, different concentrations of Songyou Yin were added for co-culture. After 48h, the EMT associated markers were tested by RT-PCR, Western Blot and ICC. Wound-healing assay and trans-well assay were used to evaluated migration and invasion of cells in vitro.Results:Wound-healing assay and trans-well assay demonstrated that Songyou Yin could inhibit migration and invasion of HCC cells. WB results demonstrated Songyou Yin could abolish the down-regulation of E-cadherin induced by TGF-β, but had no effects on the expression of Snail/Slug. ICC results demonstrated Songyou Yin attenuates the loss of cellular surface E-cadherin. RT-PCR results demonstrated Songyou Yin increased the mRNA of E-cadherin but had no effects on the main transcriptional factors(snail、slug、zeb1、zeb2、twist、E47、KLF8)Conclusion:Songyou Yin could abolish the down-regulation of E-cadherin induced by TGF-β and attenuated migration and invasion of HCC cells. However, the underlying mechanism remains unclear.Conclusions1 Songyou Yin may benefit patients with unresectable HCC treated by TACE, which probably offer a better clinical outcome.2 Songyou Yin could abolish the down-regulation of E-cadherin induced by TGF-p and attenuated migration and invasion of HCC cells. Songyou Yin may be used as complementary therapy for patients after TACE treatment.Novelty1 We firstly discovered that Songyou Yin in combination with TACE treatment may be beneficial for advanced HCC patients by prolonging life expectancy.2 We firstly demonstrated that Songyou Yin could abolish the down-regulation of E-cadherin induced by TGF-P and attenuated migration and invasion of HCC cells. We provided theoretical basis for its clinically usage.