Bitter Melon (Momordica charantia) Extract Inhibits Tumorigenicity and Overcomes Cisplatin-Resistance in Ovarian Cancer Cells Through Targeting AMPK Signaling Cascade

Abstract

Objective Acquired chemoresistance is a major obstacle in the clinical management of ovarian cancer. Therefore, searching for alternative therapeutic modalities is urgently needed. Bitter melon (Momordica charantia) is a traditional dietary fruit, but its extract also shows potential medicinal values in human diabetes and cancers. Here, we sought to investigate the extract of bitter melon (BME) in antitumorigenic and cisplatin-induced cytotoxicity in ovarian cancer cells.

Methods: Three varieties of bitter melon were used to prepare the BME. Ovarian cancer cell lines, human immortalized epithelial ovarian cells (HOSEs), and nude mice were used to evaluate the cell cytotoxicity, cisplatin resistance, and tumor inhibitory effect of BME. The molecular mechanism of BME was examined by Western blotting.

Results: Cotreatment with BME and cisplatin markedly attenuated tumor growth in vitro and in vivo in a mouse xenograft model, whereas there was no observable toxicity in HOSEs or in nude mice in vivo Interestingly, the antitumorigenic effects of BME varied with different varieties of bitter melon, suggesting that the amount of antitumorigenic substances may vary. Studies of the molecular mechanism demonstrated that BME activates AMP-activated protein kinase (AMPK) in an AMP-independent but CaMKK (Ca(2+)/calmodulin-dependent protein kinase)-dependent manner, exerting anticancer effects through activation of AMPK and suppression of the mTOR/p70S6K and/or the AKT/ERK/FOXM1 (Forkhead Box M1) signaling cascade.

Conclusion: BME functions as a natural AMPK activator in the inhibition of ovarian cancer cell growth and might be useful as a supplement to improve the efficacy of cisplatin-based chemotherapy in ovarian cancer.

Keywords: AMPK activator; Momordica charantia; bitter melon; chemoresistance; cisplatin; ovarian cancer.

Figure 1.

BME impairs cell growth and induces cell cycle arrest and apoptosis in ovarian cancer cells. (A) Concentration-dependent inhibition of cell proliferation in A2780cp (P ≤ .05), A2780s (P ≤ .05), C13* (P < .05), and OV2008 (P ≤ .05) cells treated with Chinese BME for 5 days was seen using XTT assays, whereas no inhibition was observed in HOSE cells. (B) Focus formation assay demonstrating a significant reduction of the number of colonies formed in A2780cp and C13* cells treated with 0.25% and 0.5% (volume/volume [v/v]) Chinese BME, with no apparent inhibition of colony formation in HOSE cells. (C) Upper: A2780cp cells were rapidly treated with 10% (v/v) Chinese BME for 24 hours, and the extent of cell cycle arrest and apoptosis was estimated by propidium iodide–based flow cytometry. Lower: C13* and OV2008 cells were treated with 5% and 10% (v/v) Chinese BME for 24 hours, and Western blot analysis was performed to examine the effects on cleavage of caspase 3 and PARP, and phosphorylation of SAPK/JNK, c-Jun, p38, MAPKAPK-2, and HSP27. Representative cropped blots are presented. Abbreviations: BME, extract of bitter melon; HOSE, human immortalized epithelial ovarian cells.

Figure 2.

Bitter melon extract (BME) inhibits ovarian cancer cell migration and invasion. (A) Wound healing assays demonstrating that there was a marked retardation in wound closure rate of SKOV3 cells treated with 5% and 10% (v/v) Chinese BME compared with untreated controls over a time course of 12 hours (*P ≤ .001). The width of the wound is indicated by arrows in the photographs; 3 independent experiments were performed. (B) Transwell migration assay showed that SKOV3 cells with Chinese BME treatment (5% and 10%) exhibited a significant reduction in the number of cells penetrating through the membrane as compared with untreated controls (*P ≤ .001). (C) SKOV3 cells treated with Chinese BME (10%) showed less invaded cells through the matrigel as compared with the control (*P = .0006). The numbers of migrated or invaded cells in 3 randomly chosen fields were counted for 3 independent experiments, and the normalized numerical data were presented in bar charts with error bars.

Figure 3.

Bitter melon extract (BME) from different varieties of bitter melon displays significant antitumor effects in an ES2 xenograft mouse tumor model. (A) ES2 cells were subcutaneously injected into the right flank of female nude mice. On the seventh day after tumor cell injection, treatments with 10% (v/v) Taiwanese (TW) and Chinese BME (100 µL) were initiated and carried on every other day, with a total of 6 injections. Treatment with 10% (v/v) TW (*P = .01) and Chinese BME (**P = .0007) in PBS resulted in a slower rate of tumor growth as compared with PBS-treated controls. The tumor volume of each group was measured for 12 days and expressed as the mean ± SEM from 5 mice. (B) Chinese and Taiwanese BME markedly diminished (28%-71%) tumor weight, when compared with controls, on day 12. (C) Photographs illustrate that excised tumors from nude mice subcutaneously injected with ovarian cancer cell lines ES2 exhibited tumor stasis after BME injection, as evidenced by the smaller tumor sizes.

Figure 4.

BME synergistically enhanced cisplatin-mediated cell cytotoxicity of chemoresistant ovarian cancer cells. (A) XTT cell proliferation assay demonstrated that the growth rates of A2780cp and C13* cells were significantly reduced after treatment with BME. The growth inhibitory effect was more obvious when cells were treated with BME plus cisplatin, rather than cisplatin or BME alone. (B) Different varieties of bitter melon—Taiwanese (TW) and Thailand (Thai)—when administered with cisplatin (1.5 µg/mL), exerted similar synergistic growth inhibitory effects on the viability of the chemoresistant ovarian cancer cell lines, A2780cp and C13*. (C) BME could synergistically enhance cell cytotoxicity of taxol (1 ng/mL) in chemoresistant ovarian cancer cells. (D) BME displayed antitumor growth of an ES2 mouse xenograft tumor model. ES2 cells were inoculated subcutaneously in the right flank of female nude mice. On the seventh day of tumor cell injection, BME and cisplatin (3 mg/kg body weight) were intraperitoneally injected into the nude mice on alternate days for a total of 6 injections each. BME administration not only significantly reduced tumor growth but also enhanced cisplatin-induced cytotoxicity, as evidenced by a smaller tumor size.

Figure 5.

BME functions as a natural CaMKKβ/AMPK activator, repressing mTOR signaling activity in an AMP-independent manner. (A) Western blot analysis revealed that phosphorylation of Thr172 on AMPK increased in response to Chinese BME (1%, 2.5%, and 5%, volume/volume [v/v]) in both WT and RG HEK-293 cells. Representative cropped blots are presented for the 2 groups. (B) As in (A) but showing a time course with 5% (v/v) BME. (C) A CaMKKβ inhibitor blocked BME-mediated Thr172 phosphorylation in WT and RG cells. Treatment with STO-609 (10 µM) blocked Thr172 phosphorylation induced by BME and A23187 (2 µm; 8 hours). Representative cropped blots are presented for the 2 groups. (D) Western blot analysis showing that the phosphorylation of mTOR and p70S6K was decreased in ovarian cancer cell lines—that is, A2780cp, C13*, and OV2008—treated with 1%, 5%, and 10%, v/v Chinese BME for 24 hours. Representative cropped blots are presented. Abbreviations: BME, bitter melon extract; CaMKKβ, Ca²⁺/calmodulin-dependent protein kinase-β; AMPK, AMP-activated protein kinase; WT, wild-type, AMP-sensitive AMPK-γ2; RG, AMP/ADP-insensitive R531G mutant AMPK-γ2.

Figure 6.

BME acts as a natural AMPK activator to repress FOXM1 expression via impairment of the AKT/ERK/FOXM1 signaling cascade. (A) Western blot analysis showing that the phosphorylation of Thr172 on AMPK was increased, whereas the levels of FOXM1 and the phosphorylation of AKT and ERK were reduced in ovarian cancer cell lines—that is, A2780cp and C13*—treated with Chinese BME (1%, 5%, and 10%, volume/volume [v/v]) for 24 hours. Representative cropped blots processed in parallel are presented. (B) AMPK was activated after treatment of SKOV3 cells with Thai BME (2.5% and 5%, v/v). (C) Expression of FOXM1 was decreased significantly and phosphorylation of AKT and ERK was reduced after treatment of OV2008 cells with Taiwanese BME (10%, v/v). (D) qPCR analysis demonstrating that FOXM1 mRNA level of A2780cp, C13*, and OV2008 cells was markedly reduced when treated with Chinese BME (5%, v/v) for 24 hours; 18S RNA was used as an internal control. (E) Suppression of AMPK activity relieves AMPK-mediated FOXM1 inhibition. Western blot analysis showing that AKT/ERK/FOXM1 signaling increased when A2780cp cells were treated by the AMPK inhibitor, compound C (10 µM), whereas the level of AKT/ERK/FOXM1 signaling decreased when A2780cp cells were treated with Chinese BME (10%, v/v). Representative cropped blots are presented for the 2 groups. (F) Depletion of the endogenous AMPKα1 (α1KD) by siRNA knockdown approach in ovarian cancer cell lines such as A2780cp and ES2; NC represents the scrambled control. (G) XTT cell proliferation assay demonstrated that the growth rate of A2780cp cells with AMPKα1 knockdown was not suppressed by the treatment of BME (1%, v/v) as compared with the scrambled control. Additionally, the growth inhibitory effect was more obvious in A2780cp scrambled control cotreated with Chinese BME (1%, v/v) and cisplatin (2 µg/mL), whereas there was no significant reduction of cell growth in AMPKα1 knockdown A2780cp cells when cotreated with the same condition. (H) Western blot analysis showing that AKT/ERK/FOXM1 signaling slightly increased when AMPKα1 knockdown A2780cp cells (α1 KD) were treated with/without Chinese BME (10%, v/v) for 24 hours, as compared with the scrambled control (NC). Abbreviations: BME, bitter melon extract; AMPK, AMP-activated protein kinase; qPCR, reverse transcription polymerase chain reaction.