Category: Clinical Pharmacology
Purpose: Glioblastoma (GBM) is one of the deadliest malignant central nervous system (CNS) tumours with a median survival rate of around 12.5 months in spite of huge advances in neurosurgery, radiation and chemotherapy1. Over the years, researchers worldwide have dedicated their efforts to invent new drugs to solve the current limitations and however, unfortunately limited drug response remains the main obstacle to significant clinical benefit. While the standard of care for GBM is temozolomide (TMZ), 50% of patients do not respond to TMZ, raising a drug resistance concern2. Frequent genetic alterations have been identified in human glioblastoma. Among these, RAF/MEK1/2/ERK signaling pathway has been demonstrated to be important in the development and progression of glioblastoma3. Furthermore, a comprehensive analysis of patient data revealed that an increased level of mitogen-activated protein kinase 2 (MEK2) expression activates many drug resistance genes and limits responses to TMZ treatment3.
Herein, we seek to elucidate whether the levels of MEK1, as the isoform of MEK2, could also act as a prognostic marker and to differentiate their roles in chemosensitisation, as a growing body of evidence reveals that despite the fact that MEK1 and MEK2 are 85% identical in sequence, knockout of MEK1 leads to embryonic death during gestation, whereas MEK2 deficient mice develop normally4.
Methods: U87MG cells were transfected with scrambled short-interfering RNA (Scr RNA) or siRNA against either MEK1 or MEK2 by using Lipofectamine 2000 or Lipofectamine RNAi MAX as the transfection reagent. Relevant protein downregulation efficiency was determined by Western Blotting, normalized by the level of GAPDH. Cell proliferation was determined using the MTS assay. Enhanced chemosensitization was evaluated by comparing the IC50 of TMZ in the U87mg cell line after siRNA mediated down regulation of either the MEK1or MEK2 genes versus scrambled siRNA. Cell cycle analyses (DNA content quantified by propidium iodide) and apoptosis (Annexin V assay) were measured by Flow Cytometry.
Results: As shown in Figures 1A&1B, after selective knockdown of MEK1 and MEK2, glioma cells displayed a significant slowdown in proliferation between Day 3 and Day 5 (****P< 0.0001, *P< 0.05 respectively). Similarly, MEK1 or MEK2 deficient cells showed a noticeable enhanced chemosensitisation with decreasing IC50 from from 774 µM to 223 µM (****p < 0.0001) (Figure 1C). A subsequent attempt was made to understand the underlying mechanism of enhanced TMZ sensitivity observed in the MEK1 or MEK2 silenced glioma cells. TMZ generally works by alkylating DNA, which leads to cell cycle arrest at the G2/M phase5. Cell cycle arrest at the G2/M phase allows DNA repair, as a way to maintain cellular homeostasis, or allows tumor cells to eliminate alkylated residues, which eventually promotes tumor cell survival and confers the drug resistance5. We thus investigated the cell cycle status to provide mechanistic insights of MEK1 and MEK2 mediated chemosensitization. MEK1-deficient glioma cells alone displayed a predominant accumulation effect at G1/G0 phase, when compared with MEK2 silenced groups or scrambled siRNA treated (control) groups (****P< 0.0001) and could completely abolish the G2/M phase arrest induced by TMZ (Figure 1D). Conversely, MEK2 downregulation alone showed only a slight arrest effect in the G1/G0 phase (*P< 0.05) and in effect MEK2 silenced glioma cells were still arresting in the G2/M phase following TMZ treatment (****P< 0.0001). This difference in MEK1 attenuating G2/M arrest by TMZ is also reflected in the expression of the apoptosis marker (Annexin V). Specifically, since MEK1 silenced groups attenuated the G2/M phase arrest by TMZ, when compared with MEK2 silenced treatment, we observed a significantly higher percentage of positive Annexin V cells in MEK1 silenced cells treated with TMZ in comparison with MEK2-deficient cells treated with TMZ (19.2 + 0.9 vs 10.8 + 1.2%, *P< 0.5 ) (Figure 1E). To conclude, cell cycle arresting at the G2/M phase to eliminate TMZ seems to be the main mechanism for the acquired drug resistance and silencing either MEK1 or MEK2 abolishes G2/M arrest such that glioma cells had little chance of mitigating the cytotoxicity effects by TMZ, leading to an enhanced chemosensitization.
Conclusion: Taken together, it is clear that MEK1 and MEK2 mediate different biological functions in glioma cells. We have revealed that down regulation of MEK1 or MEK2 leads to distinct chemosensitisation to TMZ by attenuating the G2/M phase arrest induced by TMZ, followed by triggering more apoptosis. This finding offers the potential of new validated treatments being developed for glioma patients.
1 Yao, Maojin, et al. Cellular & molecular immunology (2018):15, 737-739.
2 Lee, Sang Y. Genes & Diseases (2016): 3-3, 198-210.
3 He, Hua, et al. Cellular & molecular immunology (2016): 13-5, 658.
4 Ussar, Siegfried, et al. Journal of Biological Chemistry (2004): 279-42 ,43861-43869.
5 Hirose, Yuichi, et al. Cancer research (2001): 61-5,1957-1963.