Antiproliferative and Proapoptotic Effects of Labisia pumila Ethanol Extract and Its Active Fraction in Human Melanoma HM3KO Cells

Abstract

The present study was to determine the anticancer potential of Labisia pumila in in vitro models. Results from the study revealed that ethanol extract of L. pumila was more cytotoxic against HM3KO cells while having reduced effects on nonmalignant cells as compared to aqueous and hexane extracts. Thus, ethanol extract was selected to be further separated by using the bioassay-guided fractionation method to give an active fraction, SF2Lp. Results obtained from the flow cytometry analysis showed that SF2Lp was able to arrest the HM3KO cell cycle at the G1 phase, while morphological findings from AO-EB nuclear staining assays along with the Apoptotic Index confirmed the induction of apoptosis by SF2Lp in HM3KO cells. Results from the mechanistic study further revealed that SF2Lp treatment was able to concurrently increase the expression level of p53 and pro-apoptotic protein Bax and also reduce the expression level of anti-apoptotic protein BCl-2 in HM3KO cells, directly contributing to the increase in Bax/Bcl-2 ratio. These findings, therefore, suggested that L. pumila was able to inhibit HM3KO cell growth possibly by arresting the cell cycle at G1 phase and inducing apoptosis in HM3KO cells via the up- and down-regulation of Bax/Bcl-2 protein, mediated through a p53-dependent pathway.

Figure 1

Representative images from morphological observation under phase contrast of an inverted microscope of SF2Lp-treated and -untreated HM3KO cells. HM3KO cells were treated with 7.59 μg/mL SF2Lp for 24 (b), 48 (c), and 72 (d) hours. A population of 72 hours DMSO-treated HM3KO cells, which served as negative control, showed less apoptotic cells (a). Red arrows showed nuclear condensation and cells shrinkage due to apoptosis which occurred actively at the beginning of the treatment and the presence of apoptotic bodies (black arrows) after 72 hours of treatment. Magnification: 400x.

Figure 2

Representative images from morphological observation of SF2Lp-treated and -untreated HM3KO cells stained with Giemsa. HM3KO cells were treated with 7.59 μg/mL SF2Lp for 24 (b), 48 (c), and 72 (d) hours. DMSO-treated HM3KO cells served as negative control (a). SF2Lp-treated cells showed significant morphological changes including shrinkage of cytoplasm and compaction of the nucleus (red arrow), indicating that cells underwent apoptosis. Magnification: 400x.

Figure 3

Representative images from morphological observation of SF2Lp-treated and -untreated HM3KO cells stained with acridine orange-ethidium bromide (AO-EB). HM3KO cells were treated with 7.59 μg/mL SF2Lp for 24 (b), 48 (c), and 72 (d) hours. DMSO-treated HM3KO cells served as negative control (a). SF2Lp-treated cells showed significant morphological changes including nuclear condensation (blue arrows), membrane blebbing (yellow arrows), and apoptotic bodies (green arrows). Magnification for (a): 400x; (b)–(d): 1000x.

Figure 4

Induction of apoptosis in HM3KO cells by SF2Lp at the IC₅₀ concentration (7.59 μg/mL) at different exposure times (24, 48, and 72 h) as detected by AO-EB double staining. *P < 0.05 was taken as significantly different from control. Each value represents means ± SD from three independent experiments. SF2Lp-induced HM3KO cell death via apoptosis increased significantly in a time-dependent manner as compared to control.

Figure 5

Cell cycle distribution in HM3KO cells treated with SF2Lp at 3 μg/mL (SF2LP-3), 7.59 μg/mL (SF2LP-7.59), and 15 μg/mL (SF2LP-15) for 24 hours. DMSO-treated cells were used as control. The data represent the mean ± SD of 3 independent experiments. *Significantly different at P < 0.05 when the treated group was compared with the control.

Figure 6

The G1/S index in growing HM3KO cells treated with SF2Lp at 3 μg/mL (SF2LP-3), 7.59 μg/mL (SF2LP-7.59), and 15 μg/mL (SF2LP-15) for 24 hours. DMSO-treated cells were used as control. The data represent the mean ± SD of 3 independent experiments. *Significantly different at P < 0.05 when the treated group was compared with the control.

Figure 7

Representative Western blots showing the effect of SF2Lp at its IC₅₀ concentration (7.59 μg/mL) on expression level of p53 in HM3KO cells at different time intervals (3, 6, 12, and 24 hours). To confirm equal loading, the membrane was reprobed with β-actin. The data represent the mean ± SD of 3 independent experiments. *Significantly different at P < 0.05 over control group.

Figure 8

Representative Western blots showing the effect of SF2Lp at its IC₅₀ concentration (7.59 μg/mL) on the expression level of proapoptotic Bax in HM3KO cells at different time intervals (3, 6, 12, and 24 hours). To confirm equal loading, the membrane was reprobed with β-actin. The data represent the mean ± SD of 3 independent experiments. *Significantly different at P < 0.05 over control group.

Figure 9

Representative Western blots showing the effect of SF2Lp at its IC₅₀ concentration (7.59 μg/mL) on the expression level of antiapoptotic Bcl-2 in HM3KO cells at different time intervals (3, 6, 12, and 24 hours). To confirm equal loading, the membrane was reprobed with β-actin. The data represent the mean ± SD of 3 independent experiments. *Significantly different at P < 0.05 over control group.

Figure 10

Effects of L. pumila active fraction, SF2Lp, at is IC₅₀ concentration (7.59 μg/mL) on the Bax/Bcl-2 ratio in HM3KO cells at different time intervals (3, 6, 12, and 24 hours). The data represent the mean ±  SD of 3 independent experiments. *Significantly different at P < 0.05 over control group.