Long-term interferon-α treatment suppresses tumor growth but promotes metastasis capacity in hepatocellular carcinoma

Abstract

Purpose: To elucidate the effect of IFN-α treatment on tumor growth and metastasis of hepatocellular carcinoma (HCC).

Methods: IFN-α administration was conducted in nude mice using an orthotopic implantation model of human HCC, and the key molecular markers in the IFN-α treatment was detected by immunohistochemistry staining and PCR array.

Results: Up to 12 weeks of IFN-α treatment significantly suppressed tumor growth of HCC, but relatively increased the number of circulating tumor cells, which might be due to the enhanced tumor hypoxia as well as up-regulation of metastasis-related genes, such as HIF-1α, c-met, u-PA, PDGF-A, and IL-8. However, IFN-α had no direct effect on migration and invasion of HCC cells.

Conclusions: IFN-α has janus face of consistently suppressing HCC growth, however, promoting tumor metastasis capacity, which is of clinical indication for the scientific administration of IFN-α and the similar antiangiogenesis drugs for their dual effect on tumor growth and metastasis.

Fig. 1

Long-term administration of IFN-α inhibited tumor growth and improved survival in mice with orthotopic HCC model. a Tumor growth was significantly inhibited by long-term administration of IFN-α for up to 12 weeks. Tumor size was scrutinized twice a week by B ultrasound. Bars, SEM. b No significant body weight loss was observed between the IFN-α and control groups. c Significantly better overall survival occurred in the long-term administration group than the NS group (log-rank, P = 0.0067). d In the 6-week treatment experiments (orthotopic model using RFP-HCCLM3 cell lines), a much smaller tumor size was observed in the IFN-α group. Pictures of tumor size at 2, 4 and 5 weeks of tumor age. (Upper) NS group; (Lower) IFN-α group

Fig. 2

Severe tumor hypoxia was induced by IFN-α at a later tumor age. (a and b) Time-course changes of tumor angiogenesis (CD31) and proliferation (Ki-67), measured by immunohistochemistry staining. (c and d) Tumor hypoxia, measured by HIF-1α and pimonidazole staining, in the control group was gradually reduced throughout the tumor growth, whereas IFN-α induced more severe hypoxia than the control group at 7 weeks of tumor age. (e) Immunohistochemistry staining of CD31, ki-67, HIF-1α, and pimonidazole at 7 weeks of tumor age. (Left) The representative immunohistochemistry staining in control (NS) group and IFN-α treatment group. Black bars, 50 μm. (Right) Column represented staining, *** P < 0.001, ** P < 0.01, * P < 0.05

Fig. 3

IFN-α administration in vitro had no direct effect on tumor proliferation, migration and invasion and the up-regulation of metastasis related factors. a No inhibition of HCC-LM3 cell proliferation by IFN-α was observed in vitro, whereas inhibition on HUVECs by IFN-α was observed in vitro; bars, SEM. ** P < 0.01, * P < 0.05. b IFN-α did not attenuate HCC-LM3 cell migration in vitro for 24 and 48 h. The wound-healing assay was measured by the healing percentage of wound area at the original time point (0 h); bars, SEM. White bars, 50 μm. c Impact on HCC-LM3 motility of IFN-α was determined by Transwell assay. IFN-α did not reduce the HCC-LM3 cell migration for 24 and 48 h, and MMP-9 expression level (real-time PCR) was also not reduced by IFN-α. Black bars, 50um. (D) After incubation of IFN-α for 48 h, IFN-α in vitro did not induce the higher expression of metastasis-related factors such as uPA, c-met, or IL-8 in HCC-LM3 cells, which was confirmed with quantitative real-time PCR. Column was expressed by 2-^ΔCT, standardized to human GAPDH. Bars, SEM

Fig. 4

HCC growth is angiogenesis dependent, and IFN-α could exert its down-regulation of the angiogenesis factors. a IFN-α reduced the angiogenesis peak at the 4 week of tumor age. Comparison of relative RNA levels of angiogenesis—genes between total RNA isolated from tumors harvested from mice treated with IFN-α/control at the 4 week of tumor age. Black bars, relative changes more than 1.5 in ratio to control-treated tumors. Expression levels have been standardized to the probe/primer sets specific for human GAPDH. b Comparison of relative RNA levels of PECAM between total RNA isolated from tumors harvested from both IFN-α-treatment group and control group. Expression levels have been standardized to the probe/primer sets specific for human GAPDH

Fig. 5

Six-week administration of IFN-α induced up-regulation of metastasis-related genes. a and b Quantitative real-time PCR and Western blotting confirmed the up-regulation of the representative genes compared with the level of homochronous control group. (Left upper) Column was expressed by ratio of 2-^ΔCT in the homochronous group. (Left lower) Specific protein expression levels were normalized to the GAPDH protein. *** P < 0.001, ** P < 0.01. (Right) Pictures of Western blotting. c Immunohistochemistry staining confirmed the up-regulation of representative genes in tumor tissue. (Left) The representative staining in the NS group and IFN-α treatment group. Black bars, 50 μm. (Right) Column represented staining, *** P < 0.001, ** P < 0.01, * P < 0.05