Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

Abstract

Background: Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Chemotherapy is crucial in NSCLC treatment, and targeting Wee1 kinase, a key regulator of the G2/M cell cycle checkpoint, may enhance the efficacy of cytotoxic agents. This study investigates the potential of the Wee1 inhibitor MK-1775 in combination with gemcitabine and pemetrexed to enhance cytotoxicity in NSCLC cell lines.

Methods: Human NSCLC cell lines H1975, HCC827, A549, and H460 were treated with MK-1775 and chemotherapeutic agents, both alone and in combination. Growth inhibitory effects were assessed using the CCK8 assay. Apoptotic markers were evaluated via Western blotting, and cell cycle distribution was analyzed using FACS. In vivo efficacy was assessed using xenograft mouse models with H1975 and H460 cells, monitoring tumor growth and treatment toxicity.

Results: MK-1775 combined with gemcitabine or pemetrexed significantly decreased cell survival rates and IC₅₀ values in A549 and HCC827 cell lines. Increased levels of phosphorylated cdc2, γ-H2AX, and PARP indicated enhanced apoptosis. Cell cycle analysis revealed G2/M phase arrest in p53-mutant HCC827 and H1975 cells treated with MK-1775 and gemcitabine. In xenograft models, the combination significantly inhibited tumor growth without significant toxicity.

Conclusions: MK-1775 enhances the cytotoxic effects of gemcitabine and pemetrexed in NSCLC cell lines and effectively inhibits tumor growth in vivo. These findings suggest that Wee1 inhibition by MK-1775, combined with chemotherapy, represents a promising therapeutic strategy for NSCLC treatment.

Keywords: Chemotherapy; MK-1775; Non-small cell lung cancer.

Fig. 1

MK-1775 in combination with gemcitabine increased the cytotoxic activity in NSCLC. The survival rate of gemcitabine alone or plus wee1 inhibitor in p53^wt NSCLC A549 (A) and H460 (B). The p53^mut NSCLC HCC827 (C) and H1975 (D) cells treated with the same drug therapy for 72 h. The IC₅₀ is displayed in bar graph (E) or detailed data in the table (F). The p values (mean ± SEM) presented are from three independent experiments. ∗p < 0.05 by Student's t-tests.

Fig. 2

Wee1 inhibition plus pemetrexed improved cell death in NSCLC except for HCC827 cells. The cell survival of pemetrexed only or in combination with MK-1775 in A549 (A), H460 (B), HCC827 (C), and H1975 (D). The bar graph (E) and the table (F) presented the IC₅₀ and p values (mean ± SEM) after NSCLC treated with the above monotherapy or combination therapy. The p values presented are from three independent experiments. ∗p < 0.05 by Student's t-tests.

Fig. 3

MK-1775 in combination with chemotherapy induced apoptosis in NSCLC. Western blot analyzed the expression of Wee1, cdc2, γ-H2AX, and PARP after MK-1775 monotherapy or combined with gemcitabine in A549 (A), H460 (B), HCC827 (C), and H1975 (D) or four cells treated MK-1775 plus pemetrexed for 24 h in E (The original uncropped images are provided in the Supplementary file 2).

Fig. 4

Wee1 inhibition by MK-1775 plus gemcitabine stimulated the G2/M phase in p53^mut HCC827. FACS evaluated the cell cycle after MK-1775 alone or plus gemcitabine in HCC827 (A), and H1975 (B) for 24 and 48 h. The mean ± SEM presented are from three independent experiments.

Fig. 5

Wee1 inhibition plus gemcitabine inhibits H1975 tumor formation. (A) Schema showing the experimental timeline. (B) There were no significant differences in the body weights of xenografted mice treated with vehicle, gemcitabine, MK-1775, or combined with these two drugs. (C) Tumor volume raised in the vehicle, gemcitabine, and MK-1775 groups but not in the gemcitabine plus MK1775 group. (D) The harvested tumors are shown. (E) The tumor weights show the mean values of D, along with the standard errors and p values. Data are expressed as mean ± SEM. Scale bar: 1 cm ∗, p < 0.05. Gem: gemcitabine.

Fig. 6

Wee1 inhibition plus pemetrexed inhibits H460 tumor formation. (A) Schema showing the experimental timeline. (B) There were no significant differences in the body weights of xenografted mice treated with vehicle, pemetrexed, MK-1775, or pemetrexed plus MK-1775. (C) Tumor volume raised in the vehicle and pemetrexed groups but not in the combination group. (D) The harvested tumors are shown. (E) The tumor weights show the mean values of D, along with the standard errors and p values. Data are expressed as mean ± SEM. Scale bar: 1 cm ∗, p < 0.05.