Diospyros rhodocalyx Kurz induces mitochondrial-mediated apoptosis via BAX, Bcl-2, and caspase-3 pathways in LNCaP human prostate cancer cell line

Reagents and chemicals

CellTiter 96^® Aqueous One Solution Cell Proliferation Assay containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS] was acquired from Promega, WI, USA. Cell culture media (RPMI 1640 and DMEM) and DPBS were purchased from Cytiva, Logan, USA. Penicillin-Streptomycin and fetal bovine serum (FBS) were obtained from Invitrogen, CA, USA. Propidium iodide solution (PI), dimethyl sulfoxide (DMSO), and RNase A were obtained from Sigma-Aldrich, St. Louis, MO, USA. FITC Annexin V Apoptosis Detection Kit with PI (cat. 640914) was from BioLegend, CA, USA. Rabbit anti-human BAX (D2E11) (cat. 5023), rabbit anti-human Bcl-2 (D55G8) (cat. 4223), rabbit anti-human cleaved caspase-3 (ASP175/5A1E) (cat. 9664), rabbit anti-human β-actin (13E5) (cat. 4970), and secondary anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (cat. 7074) were purchased from Cell Signaling Technology, Danvers, MA. Other analytical grade chemicals were obtained from RCI Labscan, Bangkok, Thailand.

Culture of LNCaP and WPMY-1 cell lines

The human prostate cancer cell line (LNCaP) and human prostatic stromal myofibroblast cell line (WPMY-1) were purchased from ATCC (VA, USA). LNCaP and WPMY-1 cells were cultured in the RPMI-1640 and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. All cell lines were supplemented with 10% FBS and 1% Penicillin-Streptomycin. The cells were maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO₂.

Ethyl acetate extract of D. rhodocalyx Kurz (EADR) preparation

D. rhodocalyx Kurz was procured from Suphan Buri, Thailand. Ethyl acetate served as the solvent for extracting phytochemical components that dissolve both polar and non-polar compounds (Ng, Samsuri & Yong, 2020; Snyder, 1974). Moreover, ethyl acetate exhibits low cytotoxicity towards cells (Abarca-Vargas, Peña Malacara & Petricevich, 2016). A total of 140 g of dried powder underwent maceration in ethyl acetate (0.5 L, 3 times) at 25 °C over 48 h. The extracted solvents were then collected and filtrated through Whatman no. 2 filter paper (Cytiva, Little Chalfont, UK). Subsequently, the filtrate was concentrated by evaporation using a vacuum rotary evaporator at 45 °C to yield crude extract. The percentage yield of crude extracts was calculated based on the weight (g) of the dry extract divided by the weight (g) of plant extract (Engida et al., 2013). Finally, the EADR was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 4 mg/mL and stored at −20 °C.

Determination of cytotoxicity by MTS assay

LNCaP and WPMY-1 cells ( $5\times {10}^3$cells/mL) were treated with varying final concentrations of EADR (5, 10, 20, 30, and 40 µg/mL) for 24 and 48 h. The EADR was dissolved in a medium containing DMSO, with a final concentration of 1% DMSO. Cells in the control group were exposed to medium containing 1% DMSO without EADR (vehicle control). According to the manufacturer’s protocol, 20 µL of reagent was added into each well, resulting in a final concentration of 317 µg/mL of MTS in each well. Subsequently, cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 2 h in the dark. The absorbance was measured at a wavelength of 490 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The half-maximal inhibitory concentration (IC₅₀) was calculated using GraphPad Prism version 8.0 (GraphPad Inc., San Diego, CA, USA).

Cell cycle investigation by flow cytometry

LNCaP and WPMY-1 cells ( $5\times {10}^5$cells/mL) were incubated with various concentrations of EADR (5, 10, and 15 µg/mL) for 24 h. Post-incubation, the cells were then washed twice with chilled DPBS and fixed in 70% ethanol for 48 h at −20 °C. Subsequently, the cells were treated with 100 µg/mL of RNase A for 30 min at 37 °C and stained with 50 µg/mL of PI. The cell cycle distribution was determined using a FACSCantroII flow cytometer and further analyzed using the ModFit LTTM software (Verity Software House, Topsham, ME).

Measurement of cell apoptosis by Annexin V-FITC and PI staining

The assessment of apoptosis induction by EADR was conducted through Annexin V-FITC and PI staining. According to the manufacturer’s protocol, briefly, LNCaP and WPMY-1 cells ( $1\times {10}^5$cells/mL) were treated with 5, 10, and 15 µg/mL of EADR for 24 h. Subsequently, cells were harvested, re-suspended in the binding buffer solution, and stained with 5 µL of Annexin V-FITC and 10 µL of PI at room temperature for 15 min in the dark. Then, 300 µL of Annexin-V binding buffer was added. The apoptotic cells were determined using FACSCantroII flow cytometer and analyzed with FlowJo v.10.8.1 software (FlowJo LLC, Ashland, OR, USA).

Mitochondrial membrane potential (MMP) determination by JC-1 staining

The alterations in MMP were assessed with a JC-1 mitochondrial membrane potential detection kit (Biotium, Hayward, CA, USA) according to the manufacturer’s instructions. JC-1 emits either green or red fluorescence depending on the MMP; the green signal indicates depolarized mitochondria, while the red signaling indicates polarized mitochondria. Consequently, the transition from red to green fluorescence served as a reliable indicator of a reduction in MMP (Sivandzade, Bhalerao & Cucullo, 2019). LNCaP cells ( $1\times {10}^5$ cells/mL) were exposed to various concentrations of EADR (5, 10, and 15 µg/mL) for 24 h. Following treatment, the cells were harvested, suspended in a 1X JC-1 reagent working solution, and incubated at 37 °C with 5% CO₂ for 15 min in the dark. Subsequently, the excess dye was washed twice using PBS. The MMP was determined using FACSCantroII flow cytometer and data analysis was performed using FlowJo v.10.8.1 software (FlowJo LLC, Ashland, OR, USA).

Western blot analysis

LNCaP cells ( $3\times {10}^5$ cells/mL) were treated with 5, 10, 15, and 20 µg/mL of the EADR for 24 h. Following the harvest process, the cells were washed with PBS. Subsequently, whole-cell lysates were prepared by scrapping the cells into ice-cold RIPA buffer supplemented with a protease inhibitor cocktail (Millipore, Burlington, MA, USA). The lysates from each sample were centrifuged at 14,000 g for 10 min at 4 °C, and protein concentration in the supernatant was determined using a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Richmond, CA, USA), following the manufacturer’s instruction. Equal amounts of protein lysate (30 µg) from each sample were separated by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% gel. Mini protein tetra system (Bio-Rad Laboratories, Richmond, CA, USA) was utilized to electrotransfer the separated proteins onto a polyvinylidene fluoride (PVDF) membrane (Merck, Darmstadt, Germany). The blot was then blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween 20, pH 7.4 under constant rotation for 2 h. Subsequently, the membranes were incubated overnight with monoclonal antibodies against BAX, Bcl-2, cleaved caspase-3, and β-actin at 4 °C. A secondary anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody was used to immunoblot the membranes at room temperature for 90 min. The protein bands were visualized using Immobilon ECL Ultra Western HRP Substrate (Millipore, Stafford, VA, USA), and chemiluminescence was detected by the ChemiDocTM MP Imaging System. The relative intensity of each band was quantified using Image J software (NIH, USA) with normalization to β-actin as a loading control.

Determination of metabolic profiling of EADR by GC-MS

To determine the phytochemical components present in EADR, 10 mg/mL of EADR was filtrated through a 0.45 µm membrane filter and dried by blowing nitrogen gas until the extract was fully dried. Subsequently, 300 µL of acetonitrile (Millipore, Stafford, VA, USA) was added to the dried extract and transferred to GC vials with inserts. The GC-MS profiling was performed using an Agilent 7010B GC/TQ with double HP-5MS UI capillary column (15 m long, 250 µm internal diameter, 0.25 µm file thickness). The injector, transfer line, and detector were maintained at 280 °C. The quadrupole mass spectrometer scanned across the range of 40–350 with the ionizing voltage of 70 eV. and ion source temperature of 300 °C. The GC operating conditions were set as follows: initial temperature 60 °C, 1 min isothermal, ramp at 30 °C min⁻¹ up to 150 °C, and ramp at 5 °C min⁻¹ up to 310 °C. Helium was utilized as the carrier gas at a flow rate of 1 mL/min and analysis was performed in spitless mode. The GC-MS system was controlled by the Enhanced MassHunter. Identification of phytochemical compounds was conducted by comparative analysis the spectrum of unknown compounds with those in the National Institute of Standard and Technology version 2020 (NIST20) and Wiley Search Libraries 11th Edition, 2017 (W11N17) library. The compound name, CAS number, molecular formula, and compound nature of each compound were retrieved from NIST20 and W11N17 libraries. The percentage of each compound was calculated based on its relative peak in the chromatogram (Lalitha et al., 2021). The collective sum of all compound areas was standardized to constitute 100% of the chromatogram area (Mangas Marín et al., 2018). Only compounds with a similarity index of 80% or higher were considered to be well-matched with the databases, indicating accuracy identification and likely presence in the library, as recommended by NIST (Labs, 2023).

Statistical analysis

The statistical analysis was performed using Graph Pad Prism version 8.4.3 (GraphPad Software, San Diego, CA, USA). The findings were presented as the mean ± standard deviation (SD) derived from three independent experiments. The statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Statistical significance was defined as a p-value < 0.05.

Cytotoxicity effect of EADR on LNCaP cells and WPMY-1 cells

To investigate the cytotoxicity of EADR on PCa, the viability of LNCaP and WPMY-1 cells post-EADR treatment were observed using MTS assay. As shown in Fig. 1A, the exposure to EADR ranging from 5–40 µg/mL significantly decreased LNCaP cell viability in a dose-dependent manner after 24 and 48 h. The IC₅₀ values for EADR were determined as 15.43 and 12.35 µg/mL at 24 and 48 h, respectively. Exposure of WPMY-1 cells to EADR also exhibited cytotoxicity after 24 and 48 h (Fig. 1B), with IC₅₀ values of 34.61 and 19.93 µg/mL at 24 and 48 h, respectively. The comparative results of the EADR-treated group and the control group are presented in Table 1. Interestingly, at a concentration below 20 µg/mL of EADR treatment for 24 h, no cytotoxicity on WPMY-1 cells was observed, whereas significant cytotoxicity was evidenced in LNCaP cells. This finding suggests that the treatment of EADR at low concentrations only exhibits a cytotoxic effect on prostate cancer cells.

Apoptosis induction effect of EADR on LNCaP and WPMY-1 cells

The effect of EADR on cell cycle distribution was examined using PI staining and flow cytometry analysis. Results revealed that EADR did not alter the cell cycle distribution in either LNCaP or WPMY-1 cells. Nevertheless, the results demonstrated that EADR significantly increased the percentage of cells in the sub-G1 phase, particularly in LNCaP cells. Specifically, treatment with 15 µg/mL of EADR led to a substantial increase in the sub-G1 population of LNCaP cells, reaching 36.49% (Figs. 2A and 2B). Conversely, there was no significant alteration observed in treated WPMY-1 cells (Figs. 2C and 2D). These findings suggest that EADR has the potential to induce cell death specifically in prostate cancer cells.

Further evaluation of the apoptosis induced by EADR was conducted using Annexin V-FITC/PI staining combined with flow cytometry analysis. Results showed that the EADR treatment significantly elevated the percentage of apoptotic LNCaP cells, accompanied by a simultaneous decrease in viable cells (Figs. 3A and 3C). Specifically, treatment with EADR at concentrations of 5, 10, and 15 µg/mL resulted in apoptotic cell percentages of 22.58%, 34.8%, and 43.90%, respectively (Fig. 3C). Notably, no significant differences were observed in the number of apoptotic and viable cells in WPMY-1 cells across all concentrations of EADR treatment. The percentages of apoptotic cells were 16.99%, 17.05%, and 18.23% for EADR treatments at concentrations of 5, 10, and 15 µg/mL, respectively (Figs. 3B and 3D). Therefore, these findings collectively indicate that EADR induces apoptosis in prostate cancer cells.

Decrease of the mitochondrial membrane potential (MMP) induced by EADR

Since mitochondria play a crucial role in apoptosis regulation, we investigated the impact of EADR on the alteration of MMP in LNCaP cells to explore its mechanism of action. Our results revealed the shift of JC-1 red (indicative of aggregates) to JC-1 green (monomeric form) in the EADR-exposed cells, as illustrated in Fig. 4A. This shift corresponded to a notable reduction in the JC-1 red/green ratio compared to the untreated control (Fig. 4B), indicating the reduction in MMP within the cells. Upon treatment with EADR at various concentrations of 5, 10, and 15 µg/mL, the JC-1 green fluorescence in cells increased to 6.85%, 14.7%, and 34.5%, respectively (Fig. 4A). Furthermore, the application of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a positive control elevated JC-1 green fluorescence to 98.4% (Fig. 4A). Collectively, these findings indicate the impact of EADR on MMP, suggesting its potential involvement in driving apoptosis in prostate cancer cells.

Effect of EADR on apoptotic-related proteins

To further elucidate the mechanisms driving EADR-induced cell death, Western blot analysis was conducted to assess the expression levels of crucial markers. These markers include pro-apoptotic BAX, anti-apoptotic Bcl-2, and the effector caspase, specifically cleaved caspase-3. As shown in Figs. 5A and 5B, treatment of LNCaP cells with varying concentrations of EADR (5, 10, 15, and 20 µg/mL) resulted in increased expression of BAX, exhibiting fold changes of 1.89, 1.99, 2.08, and 2.56 relative to the control, respectively. Conversely, EADR treatment led to a decrease in Bcl-2 expression, exhibiting fold changes of 1.04, 1.03, 0.72, and 0.67 compared to the control, respectively. Moreover, the expression of cleaved caspase-3 in LNCaP cells was elevated, demonstrating fold changes of 1.37, 2.61, 6.64, and 13.86 with EADR treatment at concentrations of 5, 10, 15, and 20 µg/mL, respectively. Therefore, these findings substantiate that EADR induces apoptosis by upregulating BAX, downregulating Bcl-2, and consequently activating the caspase-3 pathway in LNCaP cells.

Conducting phytochemical profiling of EADR using GC-MS

The compound profiling of EADR, along with the biological activities of the identified compounds, is detailed in Table 2. A total of thirty-eight compounds were identified through spectral matching with NIST20 and W11N17, accompanied by their molecular formulas and references to previously reported biological activities. Notably, among the compounds present in EADR, epilupeol, specifically 20(29)-Lupen-3alpha-ol (isomer 2), exhibited the highest relative percentage of the area at 44.8975%, followed by lup-20(29)-en-3-one at 37.5088%.