Amygdalin promotes the activity of T cells to suppress the progression of HBV-related hepatocellular carcinoma via the JAK2/STAT3 signaling pathway

Abstract

Background: Hepatitis B virus (HBV) infection is a high-risk factor of hepatocellular carcinoma (HCC). Cellular immune responses are essential for HCC development, and the CD4+ and CD8+ T subtypes are identified as the primary anti-tumor immune cells. In the study, we investigated the effect and mechanism of amygdalin in the cellular immune response in HBV-related HCC and HCC progression.

Methods: The cell proliferation was examined by MTT analysis. Cells metastasis ability was detected by Invasion and migration assays. Quantification of apoptotic cells was performed with Flow cytometer assay. The protein levels of p-STAT3, STAT3, p-JAK2, JAK2, caspase-3, cleaved caspase-3 were detected by performing immunoblotting assays.

Results: We demonstrate that amygdalin treatment could rescue the HBV-T cell viability and IFN-γ and TNF-αproduction. In HBV-T cells, the MFI levels of CD8⁺ are lower than that in NC-T cells. Moreover, the phosphorylation levels of STAT3 and JAK2 are higher in HBV-T cells, compared to those in NC-T cells, and then reduced by amygdalin treatment. Co-culture with HBV-T cells could reduce IFN-γ and TNF-α, production while increase IL-6 and IL-10 production in HepG2.2.15 cells; these alterations could be partially reversed by amygdalin pretreatment. Finally, co-culture with HBV-T cells significantly promoted the cell viability, inhibited the apoptosis, and promoted the migration of HepG2.2.15 cells, and these alterations could be partially reversed by amygdalin treatment.

Conclusion: Our findings provide a rationale for further studies on the functions and mechanism of amygdalin inhibiting HBV-related HCC cell proliferation, invasion, and migration via T cell-mediated tumor immunity.

Keywords: Amygdalin; Hepatitis B virus (HBV); Hepatocellular carcinoma (HCC); T cell; The JAK2/STAT3 signaling.

Fig. 1

Effect of amygdalin on normal (NC-T) and HBV-related HCC T cells (HBV-T). a T cells isolated from the peripheral blood from healthy donors (NC-T) were stimulated with 5 μg/ml Con A and treated with a series of doses of amygdalin (0, 5, 10, 15, and 20 μg/ml) and examined for cell viability using MTT assay. b The cell viability of NC-T and T cells isolated from the peripheral blood from HBV-related HCC patients (HBV-T) was examined by MTT assay with or without amygdalin treatment (10 μg/ml). c-f The production of IFN-γ, TNF-α, IL-6, and IL-10 in NC-T and HBV-T cells was determined by ELISA with or without amygdalin treatment (10 μg/ml). n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, compared to NC-T group, #p < 0.05, ##p < 0.01, ###p < 0.001, compared to HBV-T group

Fig. 2

Effect of amygdalin on T cell activation and the phosphorylation of STAT3 and JAK2 in T cells. a, b The mean fluorescence intensity (MFI) of CD4⁺ and CD8⁺ T cells in total CD3⁺ T cells with or without amygdalin treatment determine by Flow cytometry analysis. n = 3. c, d The average mean fluorescence intensity (MFI) of p-STAT3 in CD4⁺ and CD8⁺ NC-T and HBV-T cells with or without amygdalin treatment determine by Flow cytometry analysis, n = 3. e The mRNA levels of STAT3 and JAK2 in NC-T and HBV-T cells with or without amygdalin treatment determined by real-time PCR, n = 3. f The protein levels of p-STAT3, STAT3, p-JAK2, and JAK2 in NC-T and HBV-T cells with or without amygdalin treatment determine by Immunoblotting, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared to NC-T group, #p < 0.05, ##p < 0.01, compared to HBV-T group

Fig. 3

Co-culture with T cells affects the cytokine production in HepG2.2.15 cells. a-d HepG2.2.15 cells were co-cultured with NC-T, HBV-T cells or amygdalin treated HBV-T cells and examined for the production of IFN-γ, TNF-α, IL-6, and IL-10 using ELISA with or without amygdalin treatment, n = 5. *p < 0.05, **p < 0.01, compared to NC-T group, #p < 0.05, ###p < 0.001, compared to HBV-T group

Fig. 4

Effects of amygdalin on HepG2.2.15 cell viability, apoptosis, invasion, and migration. a-d HepG2.2.15 cells were co-cultured with NC-T, HBV-T cells or amygdalin treated HBV-T cells and examined for the cell viability, apoptosis, caspase-3 cleavage, invasion, and migration of HepG2.2.15 cells using MTT (a), Flow cytometry (b), immunoblotting (c), and Transwell assays (d, e) n = 5 for MTT assay, n = 3 for flow cytometry, transwell assays and immunoblotting. *p < 0.05, **p < 0.01, compared to HepG2.2.15 + NC-T group, #p < 0.05, ##p < 0.01, compared to HepG2.2.15 + HBV-T group