| Objective:To evaluate the effect of Pien Tze Huang (PZH) on metastasis of colorectal cancer (CRC) and VEGF-C mediated lymphangiogenesis, so as to provide new insight into the molecular mechanism of the anti-metastatic activity of PZH in CRC, and to provide theoretical basis for the clinical application of PZH.Methods:In vivo study:24 male BALB/c mice were transplanted with xenograft tissues into the cecum, and then randomly assigned to receive one of two treatments (n=12/group). The groups of mice were intragastrically administered with a 250 mg/kg/day dosage of either PZH or saline daily. The animals were sacrificed 2 weeks after tumor implantation. Tumor tissues were removed and weighed. All the macroscopic tumor deposits and abnormalities in the liver were recorded to evaluate the effect of PZH on CRC metastasis. The expression of LYVE-1 in orthotopic implanted tumors was detected by Immunohistochemistry (IHC), to estimate the effect of PZH on lymphangiogenesis of CRC.In vitro study:(1) Human metastatic CRC cell line HCT-8, HCT-116 and SW620 were treated with different concentration of PZH for a certain period of time, cell counting by Trypan Blue Exclusion was examined to estimate cell survival, Wound Healing was used to evaluate cell migration, and ELISA and Western Blot was performed to detect the secretion level or the expression level of VEGF-C in the culture medium or in the cytoplasm. (2) Human lymphatic endothelial cells (HLECs) were received different concentration of PZH treatment for a certain timepoint, then microscopy was used to observe cell morphology, Annexin V/PI Staining and Cell DNA Content detected by Flow Cytometry (FCM) were used to estimate cell apoptosis and proliferation, Transwell assay was performed to evaluate cell migration, Tube Formation assay was used to evaluate lymphatic vessel formation ability, and Western Blot was used to determine the expression of VEGF-C, VEGFR-3, MMP-1 and MMP-3/10 in HLECs. (3) Exogenous- VEGF-C -stimulated HLECs were treated with different concentration of PZH, to study the effect of PZH on VEGF-C-mediated lymphangiogenesis, by MTT assay to estimate cell viability, Transwell assay to evaluate cell migration, Tube Formation assay to evaluate lymphatic vessel formation ability and Western Blot to determine the expression of VEGFR-3, MMP-1 and MMP-3/10.Result:In vivo study:(1) Size and weight of the orthotopic implanted tumors showed that PZH did not significantly affect CRC growth in that experimental period, while PZH treatment significantly reduced the number of tumor liver metastases as compared with the control group (P<0.05). (2) IHC determination suggested that PZH significantly decreased the protein expression of LYVE-1, the specific marker of lymphatic endothelial cells (P<0.05).In vitro study:(1) Trypan Blue Exclusion indicated that PZH could remarkablely decrease the amount of alive cells of HCT-8, HCT-116 and SW620 (P<0.05) in dose- and time-dependent manners. Wound healing suggested that PZH significantly inhibited the migration of the CRC cell lines. ELISA and Western Blot showed that PZH could markedly reduce the secretion level or the expression level of the lymphangiogenesis facilitated factor-VEGF-C in the culture medium or in the cytoplasm (P<0.05). (2) Microscopic observation showed that low concentration of PZH (<0.5 mg/ml) did not reduce cell density of HLECs, Annexin V/PI staining indicated that low concentration of PZH (<0.5 mg/ml) did not have significant effect on cell apoptosis, Cell DNA Content determination showed that low concentration of PZH (<0.5 mg/ml) did not have significant effect on cell cycle, while hige concentration of PZH (0.75 mg/ml) could reduce cell density, promote cell apoptosis and increase the percentage of G0/G1-phase and decrease the percentage of S-phase in HLECs. Transwell assay suggested that PZH markedly inhibit cell migration of HLECs in a dose-dependent manner. Tube Formation assay showed that PZH remarkablely suppressed the lymphatic vessel formation ability. Western Blot showed that PZH significantly down-regulated the protein expression of VEGF-C, VEGFR-3 (the specific receptor of VEGF-C), MMP-1 (collagenase) and MMP-3/10 (stromelysin) in HLECs. (3) MTT, Transwell and Tube Formation assays showed that the exogenous-VEGF-C-stimulated HLECs got increased cell viability, enhanced migration and lymphatic vessel formation abilities, while PZH treatment could significantly ameliorate the situations. Western Blot indicated that the expression of VEGFR-3, MMP-1 and MMP-3/10 were up-regulated after exogenous VEGF-C stimulated, and down-regulated by PZH treatment.Conclusion:PZH remarkably inhibits tumor liver metastasis and lymphangiogenesis in the orthotopic CRC mouse model, and significantly suppresses growth and migration in different CRC cell lines, and also reduces migration and lymphatic vessel formation abilities in HLECs, which indicates that PZH possesses significant anti-metastatic activity. PZH suppressing lymphangiogenesis via the donw-regulation of VEGF-C may be one of the molecular mechanisms that PZH inhibits metastasis in CRC. |
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