LN229 and GBM8401 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 % fetal bovine serum (FBS), penicillin, and streptomycin. SVGp12 cells were grown in minimum essential medium (MEM) containing 10 % FBS, penicillin, and streptomycin. The cells were cultured at 37 °C and 5 % CO₂ to maintain their viability.

The cells were incubated in RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 0.1 % SDS, and 1 % Triton X-100) at 4 °C for 10 minutes for lysis. The resulting lysates were then centrifuged at 15,000 rpm for 10 minutes, and the supernatants were collected. Normal brain lysates were procured from Origene Technologies. Subsequently, 30 micrograms of cell lysate from each group were loaded onto a 10 % sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA), and then the membranes were blocked with 5 % skim milk in TBST at room temperature for 1 hour. Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; anti-N-cadherin), Abcam (Cambridge, MA, USA; anti-survivin, anti-O-6-methylguanine-DNA methyltransferase (MGMT)), Agilent (Santa Clara, CA, USA; anti-Ki-67), and Atlas Antibodies AB (Bromma, Sweden; anti-DLGAP5). The antibodies were diluted to the specified ratios using enhancer kits in accordance with the manufacturer's guidelines (anti-survivin: 1:1000; anti-Ki-67: 1:250; anti-N-cadherin: 1:1000; anti-DLGAP5: 1:1000; anti-MGMT: 1:2000). The bands were visualized by employing enhanced chemiluminescence reagents and X-ray film (GE Healthcare, Piscataway, NJ, USA).

To gauge cell viability, 3×10³ LN229, GBM8401, and SVGp12 cells/well were plated in 96-well plates. The next day, the cells were treated with momordicine I, kuguacin J, kuguaglycoside C, and momordicoside I aglycone at specific concentrations for 48 h. To conduct the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (G3581, Promega, Madison, WI, USA), twenty microliters of MTS reagent were added to the cells, and they were incubated for 2-4 hours based on the manufacturer's instructions. Next, the absorbance at 490 nm was quantified with a Varioskan LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA) (Tsai et al., 2019[41]).

LN229 and GBM8401 cells (1×10³) were plated in 6-well plates. The next day, the cells were treated with momordicine and incubated at 37 °C and 5 % CO₂ for 14 days before being stained with crystal violet. To quantify clonogenic cell growth, ImageJ software (NIH, Bethesda, MD) was used, and colonies larger than 0.5 mm in diameter were counted.

To evaluate cell proliferation, LN229 and GBM8401 cells (5×10⁴ cells in 6-well plates) were treated with momordicine I for 48 hours at the specified concentration and processed with FITC-BrdU Flow Kits following the instructions provided by the manufacturer (BD Biosciences). For the assessment of cell cycle distribution after momordicine I treatment, the DNA content was measured via fluorescence-activated cell sorting (FACS) as described previously (Pozarowski and Darzynkiewicz, 2004[30]). The experimental and control groups were both treated with 70 % ethanol at 4 °C and subsequently stored at -20 °C overnight. Subsequently, the cells were rinsed twice with chilled phosphate-buffered saline (PBS) and stained in the dark with a propidium iodide (PI) solution (consisting of 50 μg/ml PI in PBS, 1 % Tween 20, and 10 μg/ml RNase A) for 30 minutes. The DNA content was then quantified by FACS (BD Biosciences, San Jose, CA, USA) in two separate experiments.

To analyze cellular apoptosis, a 7-AAD/ PE Annexin V assay was conducted in accordance with a previous description (Andree et al., 1990[1]; Casciola-Rosen et al., 1996[4]; Chang et al., 2017[6]; Vermes et al., 1995[42]). In brief, cells were seeded in 6-well plates. The next day, the cells were treated with the indicated concentration of momordicine I for 48 h. Then, the cells were collected, and the protocol was performed in accordance with the instructions from the manufacturer (BD MitoScreen). A FACSCalibur flow cytometer (BD Biosciences) and Cell-Quest Pro software (BD Biosciences) were utilized to examine all samples.

The intracellular production of ROS was assessed using a 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. We incubated the momordicine I-treated cells with 10 mM DCFH-DA at 37 °C for 30 min. Then, we harvested the cells and washed them twice with PBS. The 2',7'-dichlorofluorescein (DCF) fluorescence was analyzed by using a FACSCalibur flow cytometer (BD Biosciences) (Cheng et al., 2016[8]). To analyze cellular senescence via flow cytometry, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG; D2893, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was utilized as a substrate, which emits fluorescence and becomes membrane impermeable when cleaved by β-galactosidase. LN229 and GBM8401 cells were dissociated with trypsin-EDTA, washed with PBS, and incubated with 33 μM C₁₂FDG in 50 μl of PBS for 1 hour. Then, the cells were analyzed immediately using a FACSCalibur flow cytometer, and the fluorescence signal of C₁₂FDG was measured in an FL1 detector. The β-galactosidase activity was presented as the median fluorescence intensity (MFI) of the glioma population, and data were evaluated using FACSDiva software (Version 6.1, BD Biosciences, San Jose, CA, USA) according to previously described methods (Noppe et al., 2009[27]; Tsai et al., 2018[40]).

We analyzed total RNA from LN229 and GBM8401 cells with Human OneArray Plus microarrays (Phalanx Biotech Group, Hsinchu, Taiwan). Then, we determined gene expression using Agilent Technologies (Santa Clara, CA, USA) 0.1 XDR protocol. Fold changes were calculated as the ratios of the mean values in momordicine I-treated cells to the values in untreated cells. Then, the significant differentially expressed genes were subjected to Gene Ontology (GO) Biological Process analysis, which is an effective method for annotating genes and gene products and for revealing characteristic biological attributes based on high-throughput genomic or transcriptomic data (Gene Ontology Consortium, 2006[12]; Ashburner et al., 2000[2]). The mRNA microarray data are available at the NCBI Gene Expression Omnibus (accession no. GSE231550).

We used the CGGA (http://wwwcgga.org.cn) (Zhao et al., 2021[48]) online tool to perform customizable functional analyses, such as patient survival analysis, cancer and normal tissue differential expression analyses, and gene correlation analyses.

To measure cellular oxidative respiration, a Seahorse XF bioenergetic system was utilized in combination with a Seahorse Cell Mito Stress Test Kit (Seahorse Bioscience, North Billerica, MA, USA). LN229 and GBM8401 glioma cells were seeded into an XFp microplate in DMEM supplemented with 2 % FBS and treated with momordicine for 24 hours over the following two days. Next, the medium was replaced with sodium bicarbonate-free DMEM supplemented with 2 % FBS, and the OCR was measured at steady state. To determine the maximal and nonmitochondrial respiration rates, oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.5 μM), and a mixture of rotenone (0.5 μM) and myxothiazol (1 μM) were automatically added to the wells, as per the manufacturer's instructions, as part of the Seahorse Cell Mito Stress Test.

Differences between the experimental and control groups were estimated by Student's t test. Outcomes are reported as means ± SDs or as stated. Statistical significance was set at P less than 0.05.

To validate the antiglioma effect of isolated compounds in M. charantia L. extract, we purchased momordicine I, kuguacin J, kuguaglycoside C, and momordicoside I aglycone from ChemFaces Biotechnology (Wuhan, Hubei 430056, PRC). The molecular structure of each tested compound is shown in Figure 2a-d(Fig. 2) (top panels). To assess the cytotoxic properties of these four compounds in LN229 and GBM8401 glioma cells, we used an MTS assay to evaluate cell viability after treatment with specific concentrations for 48 h. SVGp12 astrocytes were used as normal controls. As shown in Figure 2a-d(Fig. 2) (bottom panels), momordicine I dose-dependently suppressed the proliferation of GBM8401 and LN229 glioma cells (Figure 2a(Fig. 2)). However, kuguacin J, kuguaglycoside C, and momordicoside I aglycone did not suppress the proliferation of GBM8401 and LN229 glioma cells (Figure 2b-d(Fig. 2)). The half-maximal inhibitory concentration (IC₅₀) of each compound in SVG-p12 astrocytes, GBM8401 glioma cells, and LN229 glioma cells is listed in Table 1(Tab. 1). Although high concentrations of momordicine I reduced astrocyte viability, no significant cytotoxic effects were observed on SVG-p12 astrocytes. Based on these results, we concluded that momordicine I exerted an inhibitory effect on glioma cell proliferation without serious cytotoxic effects on astrocytes.

To understand the morphological change in glioma cells in the presence of momordicine I, we observed cell morphology after treatment with specific concentrations of momordicine I 48 h after treatment. The percentages of viable LN229 and GBM8401 cells decreased as the concentration of momordicine I increased (Figure 3a(Fig. 3)). To assess the long-term antiglioma effect of momordicine I, we performed colony formation assays using LN229 and GBM8401 cells treated with momordicine I. The findings revealed that momordicine I inhibited colony formation in both GBM cell lines in a concentration-dependent manner (Figure 3b(Fig. 3)).

To further examine the impact of momordicine I on cell proliferation, we performed a BrdU incorporation assay with flow cytometry. As shown in Figure 4a(Fig. 4), momordicine I dose-dependently inhibited proliferation in both glioma cell lines, as verified by BrdU flow cytometry analysis (with a decrease in the number of proliferating LN229 cells from 31.57 % to 0.14 % and 26.79 % to 0.74 % in GBM8401 cells). In addition, crude extracts of bitter melon induced apoptotic death in various cancer cell lines. Thus, we tested whether momordicine I induces apoptotic death in LN229 and GBM8401 cells by 7-AAD/PE Annexin V staining with flow cytometry (Figure 4b(Fig. 4)). Treatment with momordicine I (6-10 μM) for 48 h dose-dependently induced apoptotic death in both LN229 and GBM8401 cells (with an increase in the apoptosis rate from 7.40 % to 54.04 % in LN229 cells and 5.22 % to 72.82 % in GBM8401 cells). Moreover, we evaluated the expression of survivin and Ki-67 in LN229 and GBM8401 cells after momordicine I treatment by immunoblotting. Momordicine I suppressed the expression of these proteins (Figure 4c(Fig. 4)). These results indicated that momordicine I inhibited proliferation and induced apoptotic death in glioma cell lines.

Aggressive invasion and migration are important features of GBM and cause poor differentiation between normal brain tissue and tumor tissue. Therefore, we tested whether momordicine I has inhibitory effects on the migration and invasion of glioma cells. The results of the wound healing assay and transwell invasion assay confirmed that momordicine I suppressed human LN229 and GBM8401 glioma cell migration (Figure 5a(Fig. 5)) and invasive ability (Figure 5b(Fig. 5)). Moreover, a reduction in the expression of the mesenchymal marker Twist was found in momordicine I-treated glioma cells (Figure 5c(Fig. 5)).

M. charantia extracts can induce ROS-mediated cell death in human lung cancer and ovarian cancer (Chan et al., 2020[5]; Thiagarajan et al., 2019[39]). To understand the effect of momordicine I on ROS generation in glioma, we used Mito-SOX staining with flow cytometry. As shown in Figure 6a(Fig. 6), momordicine I dose-dependently increased intracellular ROS production. Furthermore, we performed a C₁₂FDG assay to determine whether momordicine I induces senescence in glioma cells. Senescence was enhanced in LN229 and GBM8401 cells treated with momordicine I (Figure 6b(Fig. 6)). Based on these results, we concluded that momordicine I increased intracellular ROS generation and senescence in glioma cells.

To understand the relationship between momordicine I and the metabolic profile of glioma cells, we used the Seahorse bioanalyzer to test whether momordicine I affects the OXPHOS capacity of glioma cells. Significant decreases in OXPHOS-related parameters, including the OCR, basal respiration rate, maximal respiratory capacity, and nonmitochondrial respiration rate, were observed in both LN229 (Figure 7a, b(Fig. 7)) and GBM8401 (Figure 7c, d(Fig. 7)) cells treated with momordicine I. These data show that momordicine I regulates the OCR and mitochondrial respiration. Thus, its antiglioma effect could be partially mediated through metabolic dysregulation.

GBM cells acquire chemoresistance to TMZ by expressing a DNA repair enzyme, MGMT. This is an important factor linked to the poor survival of patients with glioma (Hegi et al., 2005[16]). The development of drug resistance is associated with metabolic reprogramming (Strickland and Stoll, 2017[35]). Since momordicine I decreased the OXPHOS capacity of glioma cells, we further tested whether momordicine I can inhibit the growth of TMZ-resistant GBM cells derived from clinical samples (GBM1, GBM2, GBM3). As shown in Figure 8a(Fig. 8), we measured the expression of the DNA repair enzyme MGMT to assess whether the glioma cells had TMZ resistance. MGMT expression was higher in TMZ-resistant LN229 and GBM8401 cells and in GBM1, GBM2, and GBM3 cells than in control LN229 and GBM8401 cells. Next, we used a tumor sphere formation assay to test whether momordicine I inhibits the growth of TMZ-resistant GBM cells. As shown in Figure 8b(Fig. 8), momordicine I significantly suppressed tumor sphere formation by TMZ-resistant human GBM cells. Based on these data, momordicine I could overcome the tumorigenicity of TMZ-resistant GBM cells.

The preliminary results verified that momordicine I has antiglioma effects. To elucidate the downstream molecular pathways, we utilized gene microarray analysis to identify the downstream targets and pathways most strongly correlated with this effect. LN229 and GBM8401 cells cultured with momordicine I for 48 h were investigated. Differentially expressed genes were identified as those with a greater than 3-fold difference in expression between momordicine I-treated and untreated cells; 70 upregulated and 186 downregulated genes were identified in LN229 cells, and 49 upregulated and 131 downregulated genes were identified in GBM8401 cells (Figure 9a(Fig. 9), top panel). The Venn diagram demonstrates that the expression of 142 genes was influenced by momordicine I in both glioma cell lines (Figure 9a(Fig. 9), bottom panel). Next, we used GO enrichment analysis to identify the top 10 enriched biological process terms, which included cell cycle, cell cycle process, and mitotic cell cycle (Figure 9b(Fig. 9)). Cell cycle analysis by flow cytometry revealed that momordicine I led to G1 arrest in GBM cells (Figure 9c(Fig. 9)). The top 5 up- and downregulated genes are shown in Figure 6d(Fig. 6). After reviewing the literature and searching the CGGA (http://cgga.org.cn/, accessed on 25 December 2021), we confirmed that DLGAP5 expression was correlated with advanced grade and poor survival in glioma patients (Figure 9e(Fig. 9)). Momordicine I suppressed DLGAP5 protein expression in LN229 and GBM8401 glioma cells (Figure 9f(Fig. 9)), implying that this protein is the likely downstream target. These results indicated that momordicine I inhibited glioma cell survival through cell cycle modulation and that DLGAP5 is probably the downstream target of momordicine I in glioma cells.

See also the Supplementary data.

Kao and Tsai conceived and designed this research. Kao, Chou, Huang and Tsai collected the data. Kao, Huang, and Tsai carried out the experiments. Kao, Chou, Huang, and Tsai analyzed the results. Kao and Tsai wrote the first draft of the paper. Tsai revised the paper. All authors read and approved the final manuscript.

The data that support the findings of this study are available on request from the corresponding author.

The study was funded by the Ministry of Science and Technology of Taiwan (grant numbers MOST 110-2314-B-016-035 and 111 2314-B-016-054), the Ministry of National Defense Medical Affairs Bureau (MND-MAB-D-112107), and the Tri-Service General Hospital (grant numbers TSGH-E-111229 and TSGH-E-112231).

The Ethical Review Board of Tri-Service General Hospital (Taiwan) approved the clinical studies (TSGHIRB No: B-110-57).

The authors declare that there are no conflicts of interest related to this article.