A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC.

Methodology/principal findings: A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor.

Conclusions/significance: Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.

Figure 1. The effect of SAC on proliferation and colony-forming ability of MHCC97L cells.

(A) MTT assay at day 2, 3 and 4. (B) Representative plates of colony-forming assay (C) Bar-chart of colony-forming assay. Statistical analysis was performed by comparing to the value of 0 mM SAC treatment. **, p<0.01.

Figure 2. The effect of SAC on the expressions of Ki-67 and PCNA of MHCC97L cells.

(A) Representative immunofluorescent analysis of Ki-67 protein after SAC treatment for 48 hours. X-axis represents Ki-67 intensity and Y-axis represents cell number. (B) Bar-chart of immunofluorescent analysis of Ki67 protein under SAC treatment. (C) Quantitative real-time RT-PCR and (D) Western blot analyses of PCNA mRNA and protein respectively after SAC treatment for 48 hours. Statistical analysis was performed by comparing to the value of 0 mM SAC treatment. **, P<0.01.

Figure 3. The effect of SAC of apoptosis of MHCC97L cells.

(A) Flow cytometry analysis of MHCC97L cells. X-axis represents Annexin V label and Y-axis represents PI label. (B) Summary of apoptosis assay. (C) Western blot analysis of apoptosis proteins under SAC treatment. Statistical analysis was performed by comparing to the value of 0 mM SAC treatment. *, p<0.05; **, P<0.01.

Figure 4. The effect of SAC of cell cycle of MHCC97L cells.

(A) Representative histograms of cell cycle analysis. Y-axis represent number of cells; X-axis represents DNA content (PI intensity). (B) Distribution of G2, S and G1 phases of MHCC97L cells under SAC treatment. (C) Western blot analysis of S phase regulated proteins. Statistical analysis was performed by comparing to the value of 0 mM SAC treatment. *, p<0.05; **, P<0.01.

Figure 5. The effect of SAC on migration and invasion of MHCC97L cells.

(A) Wound-healing assay of MHCC97L cells under SAC treatment. (B)Invasion assay. Quantitative real-time RT-PCR analyses of (C) E-cadherin and (D) VEGF mRNA. Statistical analysis was performed by comparing to the value of 0 mM SAC treatment. *, p<0.05; **, P<0.01.

Figure 6. The Effect of SAC of in vivo tumor growth and metastasis of MHCC97L cells.

(A) In vivo images of rats by Xenogen IVIS® imaging system at week 6 after implantation. (B) Tumor-bearing liver tissues (C) In vivo images of lung tissues by Xenogen IVIS® imaging system.