The mechanism of Tanshinone ⅡA on regulating EMT in cutaneous melanoma A375 cells through TAMs autophagy

Abstract

Objective To further clarify the role of TAMs autophagy in regulating the EMT and migration and invasion of A375 cells, the effects of TAMs autophagy changes on the biological behaviors of A375 cells co-cultured with TAMs were studied. Methods Human monocyte THP-1 cells were induced into M2 type by PMA and IL-4 for 72 h. The expressions of CD68, CD204 and CD206 on M2 surface were determined by flow cytometry and immunofluorescence, and the polarization efficiency of TAMs was determined. TAMs was treated with rapamycin, an autophagy modulator, for 24 h, and the expressions of LC3-Ⅱ and Beclin-1 were detected by Western blot and immunofluorescence after the removal of autophagy modulator intervention for 48 h. The autophagy level of TAMs after drug intervention was determined. Non-contact co-culture was conducted between TAMs after rapamycin intervention and human melanoma A375 cells. Routine culture in the above incubator was conducted by adding low concentration TanⅡA group (1 mg/L) and high concentration TanⅡA group (4 mg/L). After 48 hours of culture, Western blot was used to detect the expression of EMT-related factors in A375 cells, and transwell was used to detect the invasion and migration of A375 cells. Results Human monocyte (THP-1) was treated with PMA and IL-4 for 72 h. Flow cytometry and immunofluorescence detection showed that the expressions of CD68, CD204 and CD206 on M2 surface were significantly increased compared with M0 group, and the difference was statistically significant (all P<0.05). After treated with rapamycin, the expressions of LC3-Ⅱ and Beclin-1 were significantly up-regulated by immunofluorescence and Western blot analysis, with statistical significance (all P<0.05). After non-contact co-culture of TAMs and A375 cells for 48 h, the cells were treated with different concentrations of TanⅡA. Western Blot analysis showed that compared with the blank group, the protein expressions of Beclin1, LC3-Ⅱ and E-cadherin in the low-concentration TanⅡA group and the high-concentration TanⅡA group were significantly increased. The expression of N-cadherin protein was significantly decreased, and the differences between groups were statistically significant (all P<0.05). Transwell experiment showed that the migration and invasion abilities of A375 cells after treatment in the low-concentration TanⅡA group and the high-concentration TanⅡA group were decreased, and the differences between these groups were statistically significant (all P<0.05). Conclusion Rapamycin can regulate the occurrence of cutaneous melanoma EMT by inducing TAMs autophagy, and TanⅡA can regulate the invasion and metastasis of cutaneous melanoma through TAMs autophagy, thus affecting the biological behavior of melanoma.

Keywords

• tams
• autophagy
• skin melanoma
• tanshinone ⅱa
• emt