Effects of autophagy regulation of tumor-associated macrophages on radiosensitivity of colorectal cancer cells

摘要

Tumor-associated macrophages (TAMs), a major component of the tumor microenvironment, are crucial to the processes of tumor growth, infiltration and metastasis, and contribute to drug resistance. The importance of TAMs in radiation resistance of colorectal cancer remains unclear. To investigate the effects of autophagy regulation of TAMs on the radiosensitivity of colorectal cancer cells, the current study induced TAM formation from THP-1 monocyte cells. Sequential treatment of THP-1 cells with PMA for 72 h and human recombinant interleukin-4 for 24 h was used to stimulate THP-1 differentiation to TAMs. Expression of the cell surface markers CD68, CD204 and CD206, and changes to cell morphology were used to confirm successful differentiation. The TAMs were stimulated to promote or inhibit autophagy during co-culture with LoVo colorectal adenocarcinoma cells. The cells were irradiated, with subsequent measurement of LoVo colony formation and apoptosis. Additionally, the expression of p53, Bcl-2, survivin and Smac proteins was assessed by western blotting. Monodansylcadaverin staining was used to analyze the presence of autophagic vacuoles in TAM, and western blot analysis was used to assess the expression of Beclin-1, LC3B I and II, ATG-3, -5 and -7. The results demonstrated TAM autophagy to be markedly altered by rapamycin and bafilomycin A1 treatment. Following co-culture with TAMs, the colony formation rate and survival fraction of LoVo cells were significantly higher than those in the control group (P<0.05). It was further demonstrated that the regulation of autophagy in TAMs was able to inhibit the colony formation of LoVo colorectal cancer cells. Upregulation of TAM autophagy using rapamycin exhibited more effective inhibition of LoVo colony formation than autophagy downregulation. Notably, apoptosis was significantly increased in LoVo cells when co-cultured with TAMs only, or with rapamycin-mediated autophagy upregulated TAMs, compared with LoVo cells cultured alone (P<0.01). Expression of Bcl-2, survivin and p53 were reduced in LoVo cells co-cultured with TAMs, compared with the control group (P<0.05), whereas Smac expression was increased in the co-culture groups (P<0.01). It was demonstrated that rapamycin-mediated autophagy stimulation in TAMs led to reduced expression levels of survivin and Bcl-2, however, Smac expression was increased. The upregulation of autophagy in TAMs inhibited proliferation and induced apoptosis in colon cancer cells, and altered the expression of radiosensitivity-associated proteins. This data indicated that the radiosensitivity of colorectal cancer cells is associated with autophagy of TAM, and that stimulating TAM autophagy may increase the radiosensitivity of colorectal cancer cells.