Sonicated Extract from the Aril of Momordica Cochinchinensis Inhibits Cell Proliferation and Migration in Aggressive Prostate Cancer Cells

Abstract

Momordica cochinchinensis or gac fruit has been reported to have several biological activities, including antioxidation, anti-inflammatory, and anticancer activities. However, the effect on cancer cell metastasis has not been extensively studied. With this aim, the extract from the aril part was selected and investigated for prostate cancer cell migration. The aril extracts were prepared as boiled extract, sonicated extract, ethanol extract, and HAE (hexane:acetone:ethanol; 2 : 1 : 1) extract, while the prostate cancer cell models were PC-3 and LNCaP cells. An MTT assay was performed to compare the antiproliferative effect between prostate cancer cells and normal Vero cells. As a result, the sonicated extract had the highest efficiency in PC-3 cells, with IC₅₀ values of 2 mg/mL and 0.59 mg/mL for 48 and 72 h, respectively, while it had less of an effect in LNCaP cells and was not toxic to normal cells. Cell damage was further confirmed using LDH and cell cycle analysis. As a result, the sonicated extract did not cause cell damage or death and only inhibited cell proliferation. The effect on cancer metastasis was further examined by wound healing, transwell migration assays, and western blotting. The results demonstrated that the sonicated extract inhibited PC-3 cell migration and decreased MMP-9 but increased TIMP-1 expression. All these results support that gac fruit is a valuable source for further development as an anticancer agent for prostate cancer patients.

Figure 1

The antiproliferative effect of crude aril extracts in PC-3 cells. Cells were cultured in 96-well plates and treated with various concentrations of different crude extracts (0–5 mg/mL). (a) Boiled extract, (b) sonicated extract, (c) EtOH extract, and (d) HAE extract, and incubated for 24, 48, and 72 h. Cell viability was determined by MTT assay. Data are presented as the means ± SEM (n = 3) (p < 0.05^∗, p < 0.01^∗∗, and p < 0.001^∗∗∗ were considered significant compared to control) of three independent experiments.

Figure 2

The antiproliferative effect of various crude extracts from aril parts: (a–d) LNCaP cells; (e–h) Vero cells. (a–h) Cells were cultured in 96-well plates, treated with various concentrations of different extracts (0–5 mg/mL), and incubated for 24, 48, and 72 h, respectively. The cell viability of each extract was determined by MTT assay. Data are presented as the means ± SEM (n = 3) (p < 0.05^∗, p < 0.01^∗∗, and p < 0.001^∗∗∗ were considered significant compared to the control) of three independent experiments.

Figure 3

The cell damage effect of sonicated extract on PC-3 cells. PC-3 cells were treated with sonicated extracts and incubated for 24 h, and LDH released into the media was measured at 490 nm using a microplate reader. The graph presents the LDH release in medium collected from sample groups. Data are presented as the means ± SEM (n = 3). (p < 0.001^∗∗∗ was considered significant compared between the negative control and positive group, positive and treated control) of three independent experiments.

Figure 4

Cell cycle analysis in prostate cancer cells treated with sonicated extract. (a) PC-3 cells were treated with sonicated extract for 24 h, and cell cycle analysis was performed by flow cytometry using propidium iodide (PI) staining. (b) The percentages of cells in sub-G1, G1, S, and G2/M phases are indicated. Data are presented as the means ± SEM (n = 3). p < 0.05^∗ was compared with the control group by one-way ANOVA (Dunnett's test).

Figure 5

Inhibitory effect of the sonicated extract on PC-3 cell migration. PC-3 cells were treated with sonicated extract at 0, 2, 4, and 6 mg/mL and incubated for 24 h. (a) The migration of PC-3 cells was measured using a wound healing assay. After 0 and 24 h of migration, the scratches were photographed. (b) The migration of PC-3 cells was measured using the transwell insert migration assay. (c) The migration distance was calculated. (d) The relative migration of PC-3 cells was determined as described in the methods. All experiments were repeated three times. Data are presented as the means ± SEM (n = 3), p < 0.01^∗∗, p < 0.01^∗∗∗ versus the control group.

Figure 6

The effect of the sonicated extract on the expression of protein regulators in cancer cell migration. PC-3 cells were treated with the sonicated extracts at 0, 2, 4, and 6 mg/mL for 24 h. The protein expression levels of MMP-9 and TIMP-1 were evaluated using a Western blot assay. (a) The expression of MMP-9 and TIMP-1 normalized to the GAPDH loading control. (b–c) The relative band intensity was quantified by Image software. Data are presented as the means ± SEM (n = 3). p < 0.05^∗ was compared with the control group by one-way ANOVA (Dunnett's test).