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. 2022 Jun 13:2022:3992660.
doi: 10.1155/2022/3992660. eCollection 2022.

Comparative Study of Antiproliferative Activity in Different Plant Parts of Phaleria macrocarpa and the Underlying Mechanism of Action

Yuyun Ika Christina  1 , Muhaimin Rifa'i  1 , Nashi Widodo  1 , Muhammad Sasmito Djati  1
Affiliations

Comparative Study of Antiproliferative Activity in Different Plant Parts of Phaleria macrocarpa and the Underlying Mechanism of Action

Yuyun Ika Christina et al. ScientificWorldJournal. .

Abstract

Phaleria macrocarpa is a medicinal plant widely used in Indonesian folk medicine to treat several diseases, including cancer. However, the comparative evaluation of various plant parts of P. macrocarpa has not been studied for their anticancer properties on breast cancer. The study aimed to assess the antiproliferative activity of the ethanol extract of various parts of Phaleria macrocarpa against T47D human breast cancer cell lines. Several parts of P. macrocarpa, including pericarp, mesocarp, seed, and leaf, were used to determine the most potent plant part to inhibit the growth of T47D cells. The cytotoxic effects of each plant part were evaluated by WST-1 assay. The apoptotic level of T47D cells was determined by annexin V-FITC-PI and DNA fragmentation assay. Propidium iodide staining and the CFSE assay were used to examine the effect of each extract on cell cycle distribution and cell division, respectively. The relative number of caspase-3, Bax, and Bcl-2 was analyzed by flow cytometry technique. WST-1 assay revealed that P. macrocarpa leaves exhibited the most potent antiproliferative activity (p < 0.05) compared to other plant parts with selectivity only to T47D cells. P. macrocarpa leaves extract effectively induced apoptosis, inhibited proliferation, and arrested the cell cycle of T47D cells. The relative number of caspase-3 was significantly (p < 0.05) increased after being treated with P. macrocarpa leaf extract. P. macrocarpa leaf extract also leads to the dose-dependent accumulation in the Bax/Bcl-2 ratio due to upregulation of Bax and downregulation of Bcl-2. The overall results indicated that P. macrocarpa leaves could inhibit the proliferation of T47D cells and trigger apoptosis through caspase-3 activation and Bax/Bcl regulation. Therefore, P. macrocarpa leaves can be used for breast cancer therapy.

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Conflict of interest statement

The authors report no potential conflicts of interest.

Figures

Figure 1
Figure 1
Several plant parts of P. macrocarpa. (a) Whole fruits; (b) pericarps; (c) mesocarps; (d) seeds; (e) leaves.
Figure 2
Figure 2
Effect of ethanol extract of various parts of P. macrocarpa on T47D and TIG-1 cell viability. T47D cells were treated with ethanol extract of (a) pericarp; (b) mesocarp; (c) seed; (d) leaves for 24 h TIG-1 cells treated with ethanol extract of (e) pericarp; (f) mesocarp; (g) seed; (h) leaves for 24 h; (i) T47D cells treated with ethanol extract of leaves at the different durations of treatment (24, 48, and 72 h as measured by WST-1 assays. Data are shown as mean ± SD of three independent experiments.
Figure 3
Figure 3
Ethanol extract of P. macrocarpa leaves triggered more apoptosis of T47D cells. (a) Dot plot diagrams showed the percentage of T47D cell populations in different stages (necrosis, early apoptosis, late apoptosis, and live cells) after being treated with IC50 value of each part of P. macrocarpa extract for 24 and 48 h and evaluated by annexin V-FITC/PI and flow cytometry analysis. (b) The representative bar showed the percentage of cells undergoing apoptosis. The value was expressed as mean ± SD (n = 3). The different subsets indicated statistical differences (p < 0.05). Necrotic cells (upper left quadrant: annexin V−PI+); late apoptotic (upper right quadrant: annexin V+PI+); early apoptotic (lower right quadrant: annexin V+PI−); and live cells (lower left quadrant: annexin V−PI−).
Figure 4
Figure 4
Representative photomicrograph showed morphological changes in T47D cells after being treated with ethanol extract of each part of P. macrocarpa at IC50, 2x IC50 (2-fold of IC50), and 4x IC50 (4-fold of IC50).
Figure 5
Figure 5
DNA fragmentation of T47D cells treated different parts of P. macrocarpa for 24 and 48 h. (a). Pericarp; (b) seed; (c) mesocarp; (d) leaves. From left lane: M (DNA ladder 1 kb), C (untreated control cells), Cis (treated cells with 1 µg/mL of cisplatin), Lane 1–3 (treated cells with different concentrations (IC50, 2x IC50/2-fold of IC50, and 4x IC50/4-fold of IC50) for 24 h, and Lane 4–6 (treated cells with different concentrations (IC50, 2x IC50/2-fold of IC50, and 4x IC50/4-fold of IC50) for 48 h.
Figure 6
Figure 6
Effect of ethanol extract of various parts of P. macrocarpa on T47D cell division. The fluorescence intensity of carboxyfluorescein succinimidyl ester (CFSE) was recorded by flow cytometry. (a, b) Overlay histogram and the representative graph showed the percentage of cell division after treatment with all extracts of P. macrocarpa for 24 and 48 h. (c) Further investigation in cell division after 24 and 48 h treatment with P. macrocarpa leaves with three different concentrations (IC50, 2x IC50/2-fold of IC50, and 4x IC50/4-fold of IC50). (d) The representative graph demonstrated the percentage of cell division in all treatment group. Data are expressed as mean ± SD of three independent experiments (p < 0.05). The significant result (p < 0.05) was indicated by a different subset.
Figure 7
Figure 7
Evaluation of cell cycle of T47D cells treated with various parts of P. macrocarpa (IC50 concentration) for 24 and 48 h. (a) The histogram showed the distribution of the cell cycle phase. (b) The representative bar showed the percentage of each cell cycle phase from each sample. The values are presented as mean ± SD of three independent plates. A different subset indicated a significant result (p < 0.05).
Figure 8
Figure 8
P. macrocarpa leaves induce cell cycle arrest of T47D cells. (a) T47D cells were treated with three different concentrations (IC50, 2x IC50/2-fold of IC50, and 4x IC50/4-fold of IC50) of P. macrocarpa leaf extract, followed by PI staining and analyzed using flow cytometer. The cell cycle distribution pattern was represented by a histogram. (b) The representative bar showed the percentage of each cell cycle phase from each sample. Data are presented as mean ± SD (n = 3). The significant result (p < 0.05) was indicated by a different subset.
Figure 9
Figure 9
Effect of P. macrocarpa leaf extract on the relative number of caspase-3, Bax, and Bcl-2 in T47D cells as assessed by flow cytometry analysis. T47D cells were treated with P. macrocarpa leaf extract at IC50, 2x IC50/2-fold of IC50, and 4x IC50/4-fold of IC50 for 24 h. The representative histogram and graph showed the percentage of T47D cells which expressed (a) caspase-3, (b) Bax, and (c) Bcl-2. Data are presented as mean ± SD (n = 3). p < 0.05. ∗: significant compared with the untreated control group; ∗∗: significant compared with cisplatin group; ∗∗∗: significant compared with cisplatin and IC50 group; ns: no significant difference.
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