Targeting hexokinase 2 to enhance anticancer efficacy of trichosanthin in HeLa and SCC25 cell models

Abstract

Background and purpose: Trichosanthin (TCS) is a plant-based ribosome-inactivating protein exhibiting a range of pharmacological properties, including abortifacient and anticancer. However, the routine clinical use in cancer treatment was hampered by its antigenicity. Hexokinase 2 (HK2) is a pivotal regulator of glycolysis, where aberrant expression is observed in many cancers. This study investigates the anticancer effects and mechanisms of TCS in combination with benserazide (Benz), a HK2 inhibitor, in Hela and SCC25 cancer models.

Experimental approach: MTT, colony-formation and cell cycle assays were performed to assess the cytotoxic effects of TCS and Benz in HeLa and SCC25 cells. Seahorse assay, western blotting, flow cytometry analysis and RNA sequencing were employed to investigate the pharmacological effects of the combo treatment. SCC25 cell xenograft mouse model was established for in vivo efficacy study.

Key results: Combined use of TCS and Benz exhibited synergistic anticancer effects in both Hela and SCC25 cell models. The observed synergistic effects were attributed to the modulation of glycolysis by targeting HK2, leading to reduced lactate production and increased ROS accumulation which further inhibited colony formation and cell cycle progression, as well as triggered apoptosis. Moreover, this combination effectively inhibited NFκB/ERK signalling pathways, which were found to be significantly activated upon single use of TCS. It was found that the combination significantly suppressed the tumour growth in SCC25 cell xenograft mouse model.

Conclusion: Our findings suggested that targeting HK2 and modulating glycolysis may offer a promising avenue for improving the therapeutic outcomes of TCS-based anticancer treatments.

Keywords: Ribosome-inactivating protein; cancer therapy; drug combination; glycolysis; synergistic effect.

Figure 1.

Investigation of HK2 expression and its potential clinical significance in malignant tumour s. (a) The relative mRNA expressions of HK2 in cervical carcinoma tissues and adjacent normal tissues. (b) Kaplan–Meier survival curves of patients with cervical cancer stratified by high and low HK2 transcription expression levels (P < 0.05). (c, d) The relative mRNA and protein expression levels of HK2 in HNSCC tissues and adjacent normal tissues. (e) Western blot detection of HK2 protein expression on three cell lines, including normal fibroblast, SCC25, and HeLa. (f) Quantified data were presented as mean ± SE (n = 3). (g) The hexokinase activities in HeLa and SCC25 cells under 25 and 50 μg/mL TCS treatments. (h) Chemical structure of HK2 inhibitor, benserazide hydrochloride. **represents P<0.01, **** represents P < 0.0001.

Figure 2.

Drug synergy of TCS and Benz combination in HeLa and SCC25 cells. (a) HeLa cell viability results after 72 h treatments of TCS or Benz alone and in combination. (b) Plots of CI values for combined treatments of TCS and Benz at different concentrations in HeLa cells. CI values < 0.9, 0.9 < CI < 1.1 or > 1.1 indicates drug synergism, additive, or antagonism, respectively. (c) Cell viability inhibition in SCC25 cells under 72 h treatments. (d) F_a-CI plots in SCC25 cells. (e) Evaluating the cell growth inhibition of combined TCS and Benz treatment in HeLa and SCC25 cancer cells compared to normal fibroblasts. Data were expressed as mean ± SE (n = 3); ** represents P<0.01, **** represents P< 0.0001.

Figure 3.

Changes in metabolic and energy parameters in HeLa and SCC25 cells exposed to TCS and Benz combined treatments in HeLa and SCC25 cells. (a, b) ECAR profiles of drug-treated HeLa and SCC25 cells under three sequential injections of glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (2-DG, 50 mM). ECAR values were normalized to cell numbers. (c, d, e) ECAR data were quantified to indicate basal glycolysis, glycolysis capacity, and glycolysis reserve in HeLa and SCC25 cells. (f) Measurement of cellular ATP amount in HeLa and SCC25 cells. Data were presented as mean ± SE (n = 3); ns indicates non-significant, * represents P< 0.05, ** represents P<0.01, ***represents P<0.001, **** represents P< 0.0001.

Figure 4.

Lactate secretion and colony formation in HeLa and SCC25 cell models. (a, b) Quantification of extracellular lactate level after 24 h treatment. HeLa cells were treated with 50 μg/mL TCS and 50 μM Benz, alone or in combined. SCC25 cells were treated with 25 μg/mL TCS and 25 μM Benz, alone or in combined. (c, d) Colony formation and quantification results. HeLa cells were treated with 12.50 μg/mL TCS and 5 μM Benz, alone or in combined. SCC25 cells were treated with 6.25 μg/mL TCS and 2.5 μM Benz, alone or in combined. After 72 h drug exposure, cells were maintained in drug-free culture medium to form colonies. Data were presented as mean ± SE (n = 3); ns indicates non-significant, * represents P< 0.05, ** represents P<0.01, ***represents P<0.001, **** represents P< 0.0001.

Figure 5.

Cell cycle analysis after 24 h of treatment. HeLa cells were treated with 25 μg/mL TCS and 25 μM Benz, either alone or in combination. SCC25 cells were treated with 12.5 μg/mL TCS and 12.5 μM Benz, either alone or in combination. (a) Flow cytometry results showing cell cycle distribution in HeLa cells. (b) The cell percentage of G0/G1, S and G2-M phases were quantified and plotted as histogram in HeLa cells. (c) Flow cytometry results depicting cell cycle distribution in SCC25 cells. (d) Quantification of cell cycle distribution in SCC25 cells. Results were presented as mean ± SE (n = 3); ns indicates non-significant, * represents P< 0.05, ***represents P<0.001, **** represents P< 0.0001.

Figure 6.

Cellular ROS, mitochondrial membrane potential (MMP), and apoptosis in HeLa and SCC25 cell models. HeLa cells were treated with 25 μg/mL TCS and 25 μM Benz, alone or in combined. SCC25 cells were treated with 12.5 μg/mL TCS and 12.5 μM Benz, alone or in combined. (a) Cellular ROS amounts were measured in both cell lines after 4 h drug treatment. (b) Cells were exposed to 24 h treatment, followed by flow cytometry assay to detect the MMP changes which were indicated by the ratio of JC-1 aggregate (PE-red)/JC-1 monomer (FITC-green) fluorescence signal. (c) Quantification results of MMP. (d) Flow cytometry to determine the percentage of apoptotic cells after 24 h treatment. Late apoptotic cells are shown in the upper right quadrant, and early apoptotic cells are shown in the lower right quadrant. (e) Quantification results of cell apoptosis percentage. (f, g) Western blotting to detect apoptotic protein Caspase 3 (Cas3) and its cleavage in HeLa and SCC25 cells. Data were presented as mean ± SE (n = 3); ns indicates non-significant, * represents P< 0.05, ** represents P<0.01, **** represents P< 0.0001.

Figure 7.

Effect of TCS treatment alone on NF-κB/ERK related gene/protein expressions. (a) KEGG enrichment analysis of RNA-sequencing data in HeLa cells with 80ng TCS treatment for 24h. (b) Western blot results of NF-κB, p-NF-κB, ERK, p-ERK protein expressions. HeLa cells were exposed to a serial of TCS treatments at 6.25, 12.5 and 25 μg/mL, respectively, for 24 h. (c) Quantification results of relative protein expression level in HeLa cells. (d) Western blot results of NF-κB, p-NF-κB, ERK, p-ERK protein expressions. SCC25 cells were exposed to a serial of TCS treatments at 3.13, 6.25 and 12.5 μg/mL, respectively, for 24 h. (e) Quantified results of relative protein expressions in SCC25 cells. Data were presented as mean ± SE (n = 3); ns indicates non-significant, ** represents P<0.01, ***represents P<0.001, **** represents P< 0.0001.

Figure 8.

Effect of TCS combined with Benz treatment on NF-κB/ERK related protein expression. HeLa cells were treated with 6.25 μg/mL TCS and 50 μM Benz, alone or in combined, for 24 h. SCC25 cells were treated with 3.13 μg/mL TCS and 25 μM Benz, alone or in combined, for 24 h. (a) Western blot results of NF-κB, p-NF-κB, ERK, p-ERK protein expressions in HeLa cells. (b) Quantification results of relative protein expression in HeLa cells. (c) Western blot results of NF-κB, p-NF-κB, ERK, p-ERK protein expressions in SCC25 cells. (d) Quantified results of relative protein expression in SCC25 cells. (e) Examination of nuclear NF-κB protein expression within nuclear extracts of HeLa and SCC25 cells. (f) Quantified results of nuclear NF-κB protein level in HeLa and SCC25 cells. Data were presented as mean ± SE (n = 3); ns indicates non-significant, * represents P< 0.05, ***represents P<0.001, **** represents P< 0.0001.

Figure 9.

The in vivo evaluation of anti tumour efficacy of TCS and Benz combination in SCC25 cell xenograft mouse model. Nude mice aged between 6 and 8 weeks were randomly divided into four groups, with each group consisting of six mice: the vehicle group (administered with PBS containing 10 % PEG), the TCS group (1 mg/kg), the Benz group (500 mg/kg), and the combination group (1mg/kg TCS + 500 mg/kg Benz). (a) The tumour growth curve was recorded for each administration group over a period of 19 days. (b) At the end of the drug administration period, photographs of the harvested tumour s were taken. (c, d) The volume and weight of the harvested tumour s were measured. (e) Changes in the body weight of the animals were monitored throughout the 19-day drug administration period. Data were presented as mean ± SD (n = 6); ***represents P<0.001, **** represents P< 0.0001.