Synergistic immunotherapeutic effects of Lycium barbarum polysaccharide and interferon-α2b on the murine Renca renal cell carcinoma cell line in vitro and in vivo

Abstract

Novel therapeutic strategies to improve clinical efficacy in patients with renal cell carcinoma (RCC) are required. The possibility of combination therapy with Lycium barbarum polysaccharides (LBP) and recombinant interferon (IFN)‑α2b remains to be elucidated in RCC. The present study investigated the putative synergistic immunotherapeutic roles of LBP and IFN‑α2b against RCC in vitro and in vivo. The mouse RCC cell line, Renca, was used for in vitro experiments. Treatment of the cells with a combination of LBP and IFN‑α2b markedly inhibited cell proliferation, retarded cell cycle growth and promoted apoptosis in the Renca cells. Western blot analysis revealed that LBP and IFN‑α2b synergistically downregulated the expression levels of cyclin D1, c‑Myc and Bcl‑2, and upregulated the expression of the antiapoptotic protein, Bax. Myeloid‑derived suppressor cells (MDSCs) were markedly upregulated during tumour progression and promoted tumour growth by inhibiting the T‑cell‑mediated immune response. In vivo, a marked reduction in the MDSC ratio and tumour volume was observed in a group receiving combined treatment with LBP and IFN‑α2b in a xenograft tumour model. In conclusion, the present study suggested that the combination of LBP and IFN‑α2b is likely to be more effective in treating murine RCC compared with the less pronounced immunotherapeutic effects of administering LBP or IFN-α2b alone.

Figure 1

Effect of IFN-α2b in combination with LBP on the suppression of Renca cell viability. The effect of IFN-α2b plus LBP on cell viability was measured using an MTT assay. The Renca cells were treated with IFN-α2b (4,000 IU/ml) plus LBP (200 µg/ml) for 24, 48 and 72 h. IFN-α2b in combination with LBP significantly inhibited Renca cell viability at 48 h in a time-dependent manner. The data are expressed as the mean ± standard deviation of three independent experiments (^**P< 0.01). IFN-α2b, interferon-α2b; LBP, L. barbarum polysaccharides.

Figure 2

IFN-α2b, in combination with LBP, promotes apoptosis in Renca cells. The Renca cells were (A) non-treated or treated with (B) IFN-α2b (4,000 IU/ml), (C) LBP (200 µg/ml) or (D) IFN-α2b plus LBP for 48 h prior to staining with fluorescein isothiocyanate-annexin V and PI. The percentage of surviving cells was indicated in the lower left panel of the quadrant. The percentages indicated in the lower right and upper right quadrant of the histograms represent the early and late apoptotic cells, respectively. (E) The percentages of cells undergoing apoptosis induced by IFN-α2b (4,000 IU/ml), LBP (200 µg/ml) and IFN-α2b plus LBP were quantified. The data are expressed as the mean ± standard deviation (^*P<0.05, vs. control; ^**P<0.01, vs. the IFN-α2b or LBP groups alone). IFN-α2b, interferon-α2b; LBP, L. barbarum polysaccharides; PI, propidium iodide.

Figure 3

Effect of IFN-α2b alone or in combination with LBP on the Renca cell cycle. (A) The non-treated cells (control) exhibited a normal cell cycle pattern. The flow cytometric analysis revealed the presence of an increased number of cells in the G0/G1 phase and a decreased number in the S and G2 phases in Renca cells stimulated with (B) IFN-α2b alone (4,000 IU/ml) or (C) LBP alone (200 µg/ml) for 48 h compared with the control. However, the number of Renca cells arrested in the G0/G1 and G2 phases increased, whereas those in the S phase decreased, following stimulation with (D) IFN-α2b and LBP in combination for 48 h compared with the control group. G, growth phase; IFN-α2b, interferon-α2b; LBP, L. barbarium polysaccharides; S, synthesis phase.

Figure 4

Effect of IFN-α2b and LBP on protein expression levels. (A) The effects of IFN-α2b (4,000I U/ml) and LBP (200 µg/ml) on the expression levels of cell cycle-associated proteins (cyclin D1 and c-Myc) and cell apoptosis-associated proteins (Bcl-2 and Bax) were investigated by western blot analysis. The data are representative of three independent experiments. (B–E) The intensity of the bands was normalized against the internal standard, GAPDH, and the results are shown for (B) Bcl-2, (C) BAX, (D) cyclin D1 and (E) c-Myc as a ratio against the control. The data are expressed as the mean ± standard deviation (^*P<0.05, ^**P<0.01, vs. the IFN-α2b alone and LBP alone groups). GAPDH, glyceraldehyde-3-phosphate dehydrogenase, IFN-α2b, interferon-α2b; LBP, L. barbarium polysaccharides.

Figure 5

LBP and IFN-α2b inhibit the growth of Renca cell tumour xenografts. The images of the excised tumours were captured from the (A) control, (B) IFN-α2b, (C) LBP and (D) IFN-α2b plus LBP-treated groups. (E) A graph representing the average tumour volumes of Renca xenografts from the control, IFN-α2b alone, LBP alone and IFN-α2b plus LBP groups. The data are presented as the mean ± standard deviation. IFN-α2b, interferon-α2b; LBP, L. barbarum polysaccharide

Figure 6

Effect of LBP and IFN-α2b on the CD11b⁺Gr-1⁺ cells in the bone marrow of tumour-bearing mice. (A) CD11b⁺Gr-1⁺ markers on marrow suspension cells were determined using confocal laser scanning microscopy (magnification, ×800). (B–E) The ratios of CD11b⁺Gr-1⁺ MDSCs were determined using fluorescent-activated cell sorting. (F) The percentages of CD11b⁺Gr-1⁺ bone marrow cells were clearly reduced in the treatment groups compared with the control group. This reduction was more marked in the IFN-α2b plus LBP group compared with the IFN-α2b and LBP alone groups. No significant reduction was observed between the IFN-α2b and LBP alone groups. The data are expressed as the mean ± standard deviation (^*P<0.05, vs. control; ^**P<0.01 vs. IFN-α2b alone or LBP alone group. FITC, fluorescein isothiocyanate; CD, cluster of differentiation; IFN-α2b, interferon α2b; LBP, L. barbarium polysaccharides; MDSC, myeloid-derived suppressor cell.