Potential of Novel Methyl Jasmonate Analogs as Anticancer Agents to Metabolically Target HK-2 Activity in Glioblastoma Cells

Abstract

Change in the energy metabolism of cancer cells, which display significant differences compared to normal cells, is a rising phenomenon in developing new therapeutic approaches against cancers. One of the metabolic enzymes, hexokinase-II (HK-II) is involved in glycolysis, and inhibiting the HK-II activity may be a potential metabolic target for cancer therapy as most of the drugs in clinical use act on DNA damage. Methyl jasmonate (MJ) is one of the compounds blocking HK-II activity in cancer cells. In a previous study, we showed that the novel MJ analogs inhibit HK-II activity through VDAC detachment from the mitochondria. In this study, to evaluate the potential of targeting HK-2 activity, through patient cohort analysis, we first determined HK-2 expression levels and prognostic significance in highly lethal glioblastoma (GBM) brain tumor. We then examined the in vitro therapeutic effects of the novel analogs in the GBM cells. Here, we report that, among all, compound-10 (C-10) showed significant in vitro therapeutic efficacy as compared to MJ which is in use for preclinical and clinical studies. Afterward, we analyzed cell death triggered by C-10 in two different GBM cell lines. We found that C-10 treatment increased the apoptotic/necrotic cells and autophagy in GBM cells. The newly developed analog, C-10, was found to be lethal against GBM by the activation of cell death authorities, mostly in a necrotic and autophagic fashion at the early stages of the treatment. Considering that possibly decreased intracellular ATP levels by C-10 mediated inhibition of HK-2 activity and disabled VDAC interaction, a more detailed analysis of HK-2 inhibition-mediated cell death can provide a deep understanding of the mechanism of action on the oncosis/necroptosis axis. These findings provide an option to design clinically relevant and effective novel HK-II inhibitors and suggest novel MJ analogs to further study them as potential anticancer agents against GBM.

Keywords: 2DG; apoptosis; autophagy; glioblastoma; hexokinase-II; methyl jasmonate; necrosis.

FIGURE 1

High expressors of HK-II display poor prognosis according to the glioma cohort analysis. Kaplan–Meyer analysis of HK-II high versus low expressors (A). Differentially expressed genes in HK-II high versus low expressors (B). Differentially regulated pathways in HK_II high versus low expressors (C).

FIGURE 2

Designed, synthesized and screened novel Methyl Jasmonate analogs as HK-2 inhibitors.

FIGURE 3

Response to the treatment with a panel of novel MJ analogs in GBM cells. Plot showing the percent viability of U-87 cells (A) and LN229 cells (B) treated with 10 different compounds at increasing doses (100 μM-10 mM) for 24 h. Data are expressed as mean ± SEM. Differences are considered significant at *p < 0.01 and **p < 0.001. Determination of the IC₅₀ values of the MJ analog C-10, which is the most effective among the 10 different compounds compared to MJ, known as an HK-II inhibitor, in U-87 (C) and LN229 cells (D).

FIGURE 4

Compound-10 (C-10)–mediated cell death analysis in GBM cells. Flow cytometry analysis of cell death in U87-MG and LN229 cells after 24 h of treatment with C-10 (5 mM dose). Annexin-V or propidium iodide staining indicates apoptotic and necrotic cell death, respectively (A,B). Detection of LC3I and LC3II, an autophagy marker, in GBM cells treated with C-10 by using the Western blot method (C). Plot shows quantification of LC3I and LC3II levels by ImageJ (LC3I and LC3II band intensities are normalized to β-actin) in U-87 (D) and LN229 cells (E).

FIGURE 5

Autophagy detection by staining LC3 and acidic compartments in C-10–treated GBM cells. Immunofluorescence staining of LC3B (known as autophagy marker) in LN229 cells treated with C-10 (A,B) and no treatment (C,D) for 24 h. Plot showing the relative increase of LC3B compared to the control group (E). Numbers of LC3B puncta were increased fivefold after C-10 treatment (F). U87-MG cells were treated with C-10 for 24 h (only C-10 solvent was used in the control group). Acridine orange staining was used to detect autophagic cell death of the C-10–treated cells (G-I) and the untreated controls (J-I). Ratiometric analysis (R/GFIR) was performed. Plot showing the rate of autophagic cell death compared to the control group (M). [(B’, D’, H’, and K’) are three fold relative magnification].