| BackgroundLiver cancer, most commonly hepatocellular carcinoma (HCC), is the fifth most frequently diagnosed cancer in men worldwide, but the second most frequent cause of cancer death. In clinical practice, fewer than30%of patients with HCC have the chance to be treated with curative options such as liver transplantation, surgical resection, and ablation therapy because HCC is typically confirmed at an advanced stage at diagnosis. As a result, transcatheter hepatic arterial chemoembolization (TACE) and systemic chemotherapy are frequently used although unfortunately the overall response rate to such treatments is poor. The emergence of the CSC theory provides insight into why treatment of tumors with chemotherapy often appears to show an initial response but ultimately results in treatment failure. Our previous study demonstrated that Songyou Yin (SYY, containing five herbs) inhibits molecular changes consistent with the epithelial-mesenchymal transition (EMT) in oxaliplatin-treated tumor tissues and cell lines. There is accumulating evidence that the EMT and CSCs form a coalition against cancer therapy. Thus, the objective of this study was to investigate whether SYY directly down-regulates the proportion of CSCs and inhibits stemness of HCC cells in tumor tissues and HCC cell lines, thus resulting in sensitization of HCC to oxaliplatin.Methods The human HCC cell lines MHCC97H with high metastatic potential and Hep3B with low metastatic potential were treated with oxaliplatin from low concentration to high concentration sequentially. Stable resistant cell line MHCC97H-OXA and Hep3B-OXA were investigated changes in CSC proportion and sternness characteristics such as invasion, motility, colony formation, tumorigenesis, and pulmonary metastasis in oxaliplatin-resistant cell lines and evaluated their changes in chemosensitivity which were also reconfirmed in vivo. MHCC97H and Hep3B cells cultured with2mg/mL SYY for4weeks were termed MHCC97H-SYY and Hep3B-SYY respectively. And the stemness characteristics such as invasion, motility, colony formation, tumorigenesis, and pulmonary metastasis were also investigated. Orthotopic xenografts, survival time and pulmonary metastasis of oxaliplatin and SYY co-treated group were determined. Expression profiling Chip was used to screen for differential gene and which was verified by IHC and Western blot in Oxaliplatin and/or SYY treated tissue. Knock down and over expression of target gene were used to do the functional verification.ResultsOxaliplatin treated hepatoma cell lines showed increased the percentage of apoptosis, decreased proliferation rate, enhanced invasion ability, colony forming ability and sensitivity to oxaliplatin. Biomarkers related to CSC and EMT such as E-cadherin was down regulated, while vimentin、slug、β-catenin、CD44、CD90、ALDH and OCT2were up regulated. Oxaliplatin treated nude mice showed the decreased body weight (P=0.0296), the increased number of metastatic lung nodules (P=0.0124). The re-inoculation of oxaliplatin treated hepatoma tissue also showed the enhanced tumorigenic ability (P=0.046), enhanced pulmonary metastasis ability, increased oxaliplatin resistant and intrahepatic dissemination ability. SYY-treated hepatoma cell lines showed weakened invasion ability, colony forming ability and decreased sensitivity to oxaliplatin. Biomarkers related to CSC and EMT such as E-cadherin was up regulated, while vimentin, CD90, CD24, EpCAM et al were down regulated. As to SYY-treated nude mice showed the decreased tumor weight (P=0.0069) and the number of metastatic lung nodules (P=0.014). Oxaliplatin and SYY combination treatment resulted in enhanced inhibition of tumor growth and reduced pulmonary metastasis. Gene chip showed the increased expression of ID1after oxaliplatin treatment, while, the expression of ID1decrease after treated by SYY. ID1over expression cell line by lentiviral showed increased chemosensitivity to oxaliplatin (P=0.0023), and increased Tumor Growth ability, while ID1knockdown cell line showed decreased chemosensitivity to oxaliplatin (P=0.0121) and the decreased tumor growth ability。Conclusion 1. HCC tissue and cell lines showed the increased "stemness" after chemotherapy by oxaliplatin.2. SYY could reduce the "stemness" of HCC tissue and cell lines, and also could reduce the increased "stemness" of HCC after chemotherapy by oxaliplatin.3. Down regulation of ID1in HCC tissue and cell lines is one of the most important mechanism of SYY.Potential application of this work1. It was confirmed that CSC of the residual hepatocellular carcinoma after chemotherapy by oxaliplatin was increased and "sternness" was enhanced, and which was important for targeted therapy to hepatoma CSC. Thereby, it was hopeful to reduce the recurrence of liver cancer.2. The traditional Chinese medicine SYY could reduce the proportion and "stemness" of the residual after treated by oxaliplatin, and which will make up for deficiencies of traditional treatment for HCC. And it will provide a theoretical basis for traditional Chinese medicine treatment of liver cancer.3. The combination therapy of traditional Chinese medicine SYY and oxaliplatin will be an effective clinical treatment of liver cancer.Originalities of this work1. For the first time, the liver cancer stem cell theory was used to explain the mechanism of traditional Chinese medicine.2. For the first time, it was reported that SYY could reduce the "stemness" of HCC tissue and cell lines, and also could reduce the increased "stemness" after chemotherapy by oxaliplatin3. For the first time, it was reported that down regulation of ID1in HCC tissue and cell lines is one of the most important mechanism of traditional Chinese medicine SYY... |
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