| Zhang Yujie,Yin Yushi,He Xiang,et al.Mechanisms of chaperon-mediated autophagy to promote radioresistance of colon cancer cells by degrading protein KRT6B[J].Chinese Journal of Radiological Medicine and Protection,2026,46(2):176-182 |
| Mechanisms of chaperon-mediated autophagy to promote radioresistance of colon cancer cells by degrading protein KRT6B |
| Received:September 25, 2025 |
| DOI：10.3760/cma.j.cn112271-20250925-00344 |
| KeyWords:Autophagy Radioresistance Colon cancer KRT6B |
| FundProject:国家自然科学基金(82574034);浙江省自然科学基金(LMS25H220002) |
| Author Name | Affiliation | E-mail | | Zhang Yujie | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | Yin Yushi | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | He Xiang | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | Lin Dehuan | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | Sun Yile | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | Ma Shumei | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | | | Liu Xiaodong | School of Public Health, Wenzhou Medical University, Wenzhou 325035, China | liuxd2014@126.com |
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| Abstract:: |
| Objective To explore the mechanisms by which chaperone-mediated autophagy (CMA) regulates the radiosensitivity of colon cancer cells. Methods Colon cancer cell line SW480 was selected and exposed to γ-ray irradiation at a dose of 8 Gy. At 4, 8, 12, 24, and 48 h post-irradiation, changes in the expression of CMA activity proteins, i.e., lysosome-associated membrane protein type 2A (LAMP2) and heat shock 70 kDa protein 8 (HSPA8), were detected using the Western blotting technique. Then, keratin 6B (KRT6B), a potential regulatory protein of CMA, was selected using proteomic mass spectrometry. Following the transfection of the overexpression plasmids of LAMP2, HSPA8, and KRT6B, the expression levels of KRT6B in the Vector control group, LAMP2-overexpression group, and HSPA8-overexpression group were detected. The mortality rate of cells was determined through trypan blue staining, and their radiosensitivity was detected using colony formation assays (CFAs). The interaction and binding sites of KRT6B with HSPA8 and LAMP2 were examined through co-immunoprecipitation (CO-IP) experiments. Results Ionizing radiation at a dose of 8 Gy promoted the expression of LAMP2, a key CMA protein in cells SW480 (t=3.15-3.72, P<0.05), while no significant changes were observed in the expression of HSPA8. Proteomic analysis indicates that KRT6B was up-regulated the most significantly following LAMP2 silencing and was down-regulated after ionizing radiation. Compared to the normal control (NC) group(15.8%), the ionizing radiation-induced cell mortality rate was significantly increased to 32.5% (t=9.42, P<0.05) by KRT6B overexpression. In contrast, the KRT6B silencing significantly reduced the cell mortality rate to 9.86% (t=3.73, P<0.05), which was partially reversed to 16.39% (t=6.18, P<0.05) by LAMP2 silencing. CFAs further confirmed that the KRT6B overexpression significantly increased the radiosensitivity of cells SW480 (F=179.70, P<0.001). CO-IP experiments verified that KRT6B could directly interact with LAMP2 and HSPA8, with ionizing radiation further enhancing their interactions. Both serum starvation and the LAMP2 and HSPA8 overexpression significantly inhibited the KRT6B expression. In addition, CO-IP analysis of mutant plasmids suggested that the binding sites of HSPA8 and KRT6B were located in the amino acid residues 204-208. Conclusions Ionizing radiation can enhance the CMA activity in colon cancer cells, leading to the degradation of protein KRT6B and further reducing the radiosensitivity of colon cancer cells SW480. |
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