Herbal Compound "Songyou Yin" Renders Hepatocellular Carcinoma Sensitive to Oxaliplatin through Inhibition of Stemness

Abstract

We investigated the effect of Chinese herbal compound Song-you Yin on HCC stemness. MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks, and their chemosensitivity to oxaliplatin was evaluated. The expression of CSC-related markers, cell invasion and migration, and colony formation were also examined. SYY-treated orthotopic nude mouse models of human HCC were developed to explore the effect of oxaliplatin on tumor growth, metastasis, and survival. The CSC-related molecular changes in vivo were also evaluated. The result showed that MHCC97H and Hep3B cells pretreated with SYY showed significantly increased chemosensitivity to oxaliplatin and the downregulation of CSC-related markers CD90, CD24, and EPCAM. SYY also attenuated cell motility, invasion, and colony formation in MHCC97H and Hep3B cell lines. The reduced tumorigenicity and pulmonary metastasis were observed in SYY-pretreated cell lines. Combination treatment with oxaliplatin and SYY significantly reduced tumor volume and pulmonary metastasis and prolonged survival compared with oxaliplatin treatment alone. Immunohistochemical analysis showed reduced expression of CD90, ABCG2, ALDH, CD44, EPCAM, vimentin, and MMP-9 and increased the expression of E-cadherin, in HCC cells following combination treatment. These data clearly demonstrate that SYY renders hepatocellular carcinoma sensitive to oxaliplatin through the inhibition of stemness.

Figure 1

The expression of the CSC-related markers in HCCLM3, MHCC97H, HepG2, SMMC7721, Hep3B and Huh7 HCC cell cultures.

Figure 2

SYY exhibited no significant cytotoxicity in HCC cell lines but increased their sensitivity to oxaliplatin: (a) MHCC97H cells treated with 2 mg/mL and 4 mg/mL SYY demonstrated no increase in LDH release indicating no acute cytotoxicity and (b) MHCC97H and Hep3B cell lines treated with 2 mg/mL SYY for 4 weeks showed increased chemosensitivity to oxaliplatin compared with parental cell lines.

Figure 3

Hepatoma cell lines MHCC97H and Hep3B treated with SYY (2 mg/mL) for 4 weeks exhibited decreased stemness. (a) Tumor cells that were treated with SYY for 4 weeks showed reduced expression of CSC-related markers compared with their parental cell lines by flow cytometry. (b) RT-PCR and Western blot analyses confirmed the decreased expression of CSC-related markers in SYY-treated cell lines. (c) The transwell assay demonstrated that MHCC97H-SYY and Hep3B-SYY cells migrated through the basement membrane less efficiently than the parental cell lines MHCC97H and Hep3B. (d) Colony formation assay demonstrated that MHCC97H-SYY and Hep3B-SYY had a significantly lower proliferation rate and colony-forming ability than untreated parental cells. (e) MHCC97H-SYY cell lines also showed diminished subcutaneous tumor growth capacity and pulmonary metastasis compared with parental cells in nude mouse models.

Figure 4

Oxaliplatin and SYY cotreatment resulted in a smaller tumor volume and a lower percentage of pulmonary metastasis than oxaliplatin treatment alone. (a) Treatment with low-dose SYY (2 g/kg) did not significantly inhibit tumor growth or decrease the incidence of pulmonary metastasis. Treatment with 4 g/kg SYY decreased the number of pulmonary metastasis nodules but did not inhibit tumor growth. In contrast, high-dose SYY (8 g/kg) treatment significantly inhibited tumor growth and pulmonary metastasis. (b) Cotreatment with oxaliplatin and SYY (4 g/kg) reduced tumor volume, decreased the percentage of pulmonary metastasis, and prolonged survival compared with oxaliplatin treatment alone.

Figure 5

Oxaliplatin and SYY cotreatment reduced the proportion of CSCs, decreased the expression of MMP-9, and reversed EMT with increased expression of E-cadherin compared with oxaliplatin treatment alone.