Mitomycin C treatment induces resistance and enhanced migration via phosphorylated Akt in aggressive lung cancer cells

Abstract

Since 1984, mitomycin C (MMC) has been applied in the treatment of non-small-cell lung cancer (NSCLC). MMC-based chemotherapeutic regimens are still under consideration owing to the efficacy and low cost as compared with other second-line regimens in patients with advanced NSCLC. Hence, it is important to investigate whether MMC induces potential negative effects in NSCLC. Here, we found that the malignant lung cancer cells, CL1-2 and CL1-5, were more resistant to MMC than were the parental CL1-0 cells and pre-malignant CL1-1 cells. CL1-2 and CL1-5 cells consistently showed lower sub-G1 fractions post MMC treatment. DNA repair-related proteins were not induced more in CL1-5 than in CL1-0 cells, but the levels of endogenous and MMC-induced phosphorylated Akt (p-Akt) were higher in CL1-5 cells. Administering a p-Akt inhibitor reduced the MMC resistance, demonstrating that p-Akt is important in the MMC resistance of CL1-5 cells. Furthermore, we revealed that cell migration was enhanced by MMC but lowered by a p-Akt inhibitor in CL1-5 cells. This study suggests that in CL1-5 cells, the activity of p-Akt, rather than DNA repair mechanisms, may underlie the resistance to MMC and enhance the cells' migration abilities after MMC treatment.

Keywords: CL1-0; CL1-5; cell migration; mitomycin C; phosphorylated Akt.

Figure 1. The effects of MMC on CL1-0, CL1-1, CL1-2, and CL1-5 cells

A. Colony numbers of CL1-0, CL1-1, CL1-2, and CL1-5 cells at the indicated concentration of MMC treatment (n = 3). B. The percent of sub-G1 was measured by propidium iodide staining and flow cytometry following 1 h treatment with 3 μM MMC in CL1-0, CL1-1, CL1-2, and CL1-5 cells (n = 3). C. Immunoblot analysis of full-length and cleaved caspase 3 products in CL1-0, CL1-1, CL1-2, and CL1-5 cells at 48 or 72 h after 3 μM MMC treatment. C: control, cells without MMC treatment. Loading control: actin.

Figure 2. Cell cycle distribution of synchronized CL1-0 and CL1-5 cells post 3 μM MMC treatment

A. Time scale of double thymidine block and MMC treatment. B. Cell cycle distribution of CL1-0 and CL1-5 cells at the indicated time points post MMC (3 μM) treatment in synchronized cells. C. Sub-G1 analysis following 1 h treatment with 3 μM MMC in synchronous or asynchronous CL1-0 and CL1-5 cells (n = 3).

Figure 3. Western blot analysis of DNA damage responses in Cl1-0 and CL1-5 cells post 3 μM MMC treatment

A. Immunoblot analysis of XPC, DDB2, and p-Rad18 (S403) in CL1-0 and CL1-5 cells at the indicated time points post 3 μM MMC treatment. C: control, cells without MMC treatment. Loading control: actin. B. Immunoblot analysis of p-ATM (S1981), p-DNA-PKcs (S2056), p-Akt (S473), and Akt in CL1-0 and CL1-5 cells at different time points post 3 μM MMC treatment. C: control, cells without MMC treatment. Loading control: actin. C. Immunoblot analysis of FANCD2 in CL1-0 and CL1-5 cells at the indicated time points post 3 μM MMC treatment.FANCD2-Ub: ubiquitinated FANCD2. Loading control: actin. D. Representative images of CL1-0 or CL1-5 cells with or without 3 μM MMC treatment for FANCD2 nuclear foci. Arrows: FANCD2 foci. Green: anti-FANCD2 antibody. Blue: DAPI.

Figure 4. The cell proliferation rates of CL1-0 and CL1-5 cells without p-Akt after MMC treatment

A. The activity of p-Akt was measured by immunoblot analyses in MK-2206-treated or MK-2206-untreated CL1-0 and CL1-5 cells with or without MMC treatment. Loading control: actin. B. The cell proliferation rates of CL1-0 and CL1-5 cells that were untreated, treated with MK-2206, treated with MMC, or treated with a combination of MK-2206 and MMC. *P < 0.05, n = 3. C. Colony formation of CL1-0 and CL1-5 cells that were untreated, treated with MK-2206, treated with MMC or with a combination of MK-2206 and MMC. **P < 0.01, n = 4. All values are represented as mean +/− SEM.

Figure 5. The migration of CL1-0 and CL1-5 cells without p-Akt after MMC treatment

A. The cell migration of CL1-0 and CL1-5 cells that were untreated, treated with MMC, or treated with MK-2206. *P < 0.05, n = 3. B. Immunoblot analysis for confirming the decrease in p-Akt by MK-2206 treatment in CL1-0 and CL1-5 cells. Loading control: actin. All values are represented as mean +/− SEM.