Proanthocyanidins reduce cellular function in the most globally diagnosed cancers in vitro

Cell culture and treatments

The human cell lines used in this study are listed in Table 1. All cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in media (Table 1) containing 10% fetal bovine serum, 200 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, and maintained at 37 °C in a humidified incubator with 5% CO₂. Purified grape seeds oligomeric PACs were purchased from Sigma Chemical Co., catalog number (1298219) (St, Louis, MO, USA). Figure 1 shows the structural formula of the PACs obtained. PACs were dissolved in DMSO and diluted with culture media (Khairnar et al., 2018; Liberty et al., 2007; Neto, Amoroso & Liberty, 2008).

Cell viability assay (MTT assay)

Cells were seeded at 1 ×10⁴ cells/well in 96 well flat-bottom plates and cultured for 24 h to allow cells to attach and grow. The next day, the culture medium was exchanged with a serum-free medium to starve cells for 24 h. Then, 0, 5, 10, 50, 100, 200, 400 or 800 µg/mL PACs were added to the cells. The treated cells were incubated for 24 h, 48 h, or 72 h. After each incubation, cells were washed three times with phosphate-buffered saline (PBS). Then, 20 µL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT, 5 mg/mL, Sigma Chemical Co.) was added to wells and incubated at 37 °C for 3 h. The MTT was removed from each well, and 150 µL DMSO were added and incubated for 5 min at 37 °C. Absorbance was measured at 570 nm (A) using a microplate reader (Spectramax Plus 384, Molecular Devices, USA). The MTT assay was used to determine the IC₅₀ value for each cell type at each time point and to evaluate PAC effects on cell viability (Kumar, Nagarajan & Uchil, 2018).

Cell proliferation

Cells were seeded at 5 × 10³ cells/well in 12 well culture plates in complete medium and incubated at 37 °C in a 5% CO₂ incubator. PACs (0, 5, 10, 50, 100, or 200 µg/mL) were added to each cell type does concentrations, and the treated cells were incubated for 24 h, 48 h, and 72 h. After incubation, trypan blue exclusion was used to count cells. Briefly, 20 µL cell suspension were mixed with 20 µL trypan blue dye 0.4% (Gibco) and incubated at 25 °C for 3 min. 10 µL of the mixture was loaded into a counting slide and counted. Cells were also imaged and counted under bright field magnification after 72-h incubation with 100 µg/mL PACs.

Wound healing assay

Cells were seeded a density of 20,000 cells/well in 6 well plates and cultured until a monolayer was formed. Then, a straight line was scratched across the center of each well using a sterile 200 µL pipette tip. The culture medium was removed, and each well was rinsed with PBS to remove any cell debris. The cells were treated with PACs at their respective IC₅₀ doses. The migration response after scratching and treatment was evaluated. Cells were imaged using an EVOSTM FL cell imaging system (Life Technologies, USA) 0, 24, and 48 h after treatment (Rodriguez, Wu & Guan, 2005). ImageJ software was used to measure the scratch size. Each experiment was performed in triplicate.

Apoptotic nucleus morphology study

4′, 6′-diamidino-2-phenylindole (DAPI, Life Technologies) was used to observe apoptotic nucleus morphology. Briefly, 2 ×10⁵ cells/well were seeded into 6-well plates and cultured for 24 h. Then, cells were starved for another 24 h. Each cell line was treated with PACs at the respective IC₅₀ dose. Then, the cells were rinsed three times with cold PBS and fixed with 100% methanol for 30 min at 25 °C. Cells were stained with 10 µl DAPI (300 nM) for 5 min at 25 °C in the dark. Then, cells were washed several times with cold PBS. Nuclear morphology was observed with an EVOSTM FL cell imaging system (Life Technologies) after 24 h.

Flow cytometric analysis of apoptosis in HT-29, MCF-7, and PC-3 cancer cells

To evaluate the PACs effects in inducing apoptosis, Annexin V-FITC and propidium iodide (PI) dual staining was used to distinguish apoptotic cells. Annexin V-FITC apoptosis detection kit (BD Bioscience, Franklin Lakes, NJ, USA). In brief, 2 × 10⁵ cells/well were seeded into 6-well plates and cultured for 24 h; then, cells were starved for another 24 h. Each cell line was treated with PACs at the respective IC₅₀ concentration for 24 h, while untreated cells were used as control. Culture medium was then discarded, and cells were washed twice with cold PBS and collected, resuspended in a binding buffer. Approximately 100 µl of cell solution was transferred to FACS tube at a concentration of 1 × 10⁶ cells/ml. Approximately 5 µl annexin V-FITC and 5 µl PI were added to each tube and incubated 15 min at 4° C, in the dark; then, 400 µl binding buffer was added to each tube and analyzed immediately using flow cytometer system (FACSCANTO II, BD Biosciences). Weasel v3.6.2 software (Frank Battye, Melbourne, Australia) was used to analyze the results and dot plot the data where cells are described by sections as follows: the live cells are shown in the bottom left section, early apoptotic cells are shown in the bottom right section, necrotic cells are shown in the top left section, and the late apoptotic cells are shown in the top right section.

RNA isolation

Cells (10⁶ cells/well) were seeded in 6-well plates. Then, cells were treated with PACs as follows: HT-29 cells were treated with 96.62 µg/mL PACs; MCF-7 cells were treated with 115.4 µg/mL PACs; and PC-3 cells were treated with 87.78 µg/mL for 24 h. Untreated cells were used as controls. This PAC concentrations were based on the respective IC₅₀ doses determined by the MTT assay. Total RNA from each cell line was isolated with 700 µL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). A NanoDrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) was used to measure the RNA purity and yield.

Reverse transcription-polymerase chain reaction (RT-PCR)

An Access RT-PCR System (Promega Corporation, Madison, WI, US) was used to synthesize complementary DNA (cDNA). Target genes were amplified using 1 µM primers specific for BCL2 associated X (Bax), BCL2 apoptosis regulator (Bcl-2), and β-actin (ACTB) (Macrogen Inc, Korea, Table 2). β-actin was used to normalized target gene expression.

Components of RT-PCR mix: 1 X reaction buffer, 0.2 mM dNTP mix, 1 mM MgSO₄ AMV reverse transcriptase 0.1 u/µL, 0.1 u/µL Tfl DNA Polymerase, 1 µg RNA template, and up to 50 µL of nuclease free water. Cycling conditions consisted of two steps: first to synthesis cDNA (1 cycle at 45 °C for 45 min and 1 cycle at 94 °C for 2 min) and second for PCR amplification for 40 cycles of denaturation (94 °C for 30 s), an annealing (52-−53 °C) for 1 min, and an extension step (68 °C) for 2 min. ΔΔCt method was used to calculate the mRNA expression level of both treated cells and control.

Caspase enzyme activity assay

A Caspase-3, 8, and 9 colorimetric assay kits (GeneTex, Inc., Irvine, CA, USA) were used to evaluate caspase activity. Briefly, 10⁶ cells/well were seeded into 6 well plates and cultured for 24 h. Then, the cells were starved for another 24 h. Cell lines were treated with PACs at their respective IC₅₀ doses for 24 and 48 h. Then, cells were harvested and resuspended in 50 µl chilled cell lysis buffer (this buffer is included in the caspase assay kit) and incubated on ice for 10 min. The cells were centrifuged at 10,000g for one min and the supernatant was transferred into a new tube. Total protein was measured using a BCA protein assay, and 2 mg/mL BSA was used to generate a standard curve. Approximately 100 µg protein (in the sample supernatant) was diluted in 50 µL cell lysis buffer. Then, 50 µL 2X reaction buffer was added to each sample. Next, 5 µL of 4 mM caspase p-nitroaniline (pNA)-conjugated substrate was added to each sample and incubated at 37 °C for 2 h. Absorbance was measured at 400-405 nm using a microplate reader (Spectramax Plus 384, Molecular Devices).

Statistical analysis

GraphPad Prism 6 software (La Jolla, CA, USA) was used to perform all statistical analyses. Data are expressed as mean ±  standard deviation (SD) of three biological replicates. The PAC cytotoxicity data in HT-29, MCF-7, and PC-3 cancer cell lines were obtained using non-linear regression analysis. Cellular proliferation and wound healing data were analyzed by two-way ANOVA. Dunnett’s multiple comparisons tests were used to compare the mean of each treatment in each cell line at 0, 24, 48 and 72 h. The wound healing assay data were analyzed using ImageJ software. Apoptotic nucleus morphology, caspase 3 absorbance, and relative gene expression were analyzed using Student’s t-tests. Differences between values were considered statistically significant at p ≤ 0.05.

PAC cytotoxicity in HT-29, MCF-7, and PC-3 cancer cell lines

Cell viability was estimated by MTT assays to investigate the cytotoxic effects of PACs on cancer cell lines. HT-29, MCF-7, and PC-3 cells were exposed to 0, 5, 10, 50, 100, 200, 400 or 800 µg/mL PACs for 24, 48 and 72 h. PACs inhibited cellular viability in a dose- and time-dependent fashion in all investigated cells (Fig. 2). The IC₅₀ of PACs was calculated to choose the appropriate treatment dose and time for subsequent experiments. PACs showed stronger cytotoxicity against PC-3 cancer cells (IC₅₀ = 87.78) than against HT-29 cancer cells (IC₅₀ = 96.62) and showed low cytotoxicity against MCF-7 cancer cells (IC₅₀ = 115.4). Figure 3 shows that in all three type of cancer cells when treated with PACs for 24, 48 and 72 h there was a major induction of cell cytotoxicity in a concentration- and time-dependent manner.

PACs inhibit HT-29, MCF-7, and PC-3 cancer cell proliferation

Cellular proliferation was detected by the trypan blue exclusion method to examine the effects of PACs on the cellular proliferation of HT-29, MCF-7, and PC-3 cells. PAC treatment significantly decreased cell proliferation in all investigated cells p < 0.001 (Figs. 4A–4C). Further, PACs altered cell morphology after 72 h at their respective IC₅₀ doses. We observed that the cells lost typical morphology, becoming fragmented, round, and vacuolated (Figs. 4D–4I). Our results demonstrate that PACs have significant anti-proliferative properties on cancer cell growth.

Wound healing assay in HT-29, MCF-7, and PC-3 cancer cell lines

We next investigated wound healing to evaluate cellular migration. Each cell line was treated with 100 µg/mL PACs. All tested cells showed a similarly decreased migration response after 24 and 48 h, indicated by an unhealed wound. Untreated cells showed complete closure of the wound after 48 h (Fig. 5). In all cells, there was no significant cell migration at 0 h. However, untreated cell migration significantly increased after 24 h, compared to HT-29 (^∗∗p < 0.01), MCF-7, and PC-3 cells (^∗p < 0.05). After 48 h, untreated cell migration was significantly increased compared to HT-29 (^∗∗∗p < 0.001), MCF-7 (^∗∗p < 0.01), and PC-3 cells (^∗p < 0.05).

PACs induce nucleus apoptosis in HT-29, MCF-7, and PC-3 cancer cells

DAPI staining was used to evaluate apoptotic nucleus morphology in each cell lines after 24 h of PACs at their respective IC₅₀ doses. We observed a significant increase in the apoptotic nucleus morphology, including chromatin condensation and apoptotic bodies, in all cell types after PAC treatment (** p ≤ 0.01, Fig. 6).

Flow cytometric analysis of apoptosis in HT-29, MCF-7, and PC-3 cancer cells

An annexin V-FITC and PI was used to detect apoptotic cells following PACs treatment in HT-29, MCF-7, and PC-3 cancer cells. The results in Fig. 7 show that the control group (untreated cells) in all three cancer cell lines showed approximately 100% cell viability, while HT-29 cells treated with PACs showed 21.9% viable cells, 0.50% necrotic cells, 22.1% early apoptotic cells, and 64.5% late apoptotic cells (Fig. 7B). MCF- cells treated with PACs showed 31.2% viable cells, 0.14 necrotic cells, 11.5% early apoptotic cells, and 52.2% late apoptotic cells (Fig. 7E), and PC-3 cells treated with PACs showed 40.0% viable cells, 1.81 necrotic cells, 11.5% early apoptotic cells, and 46.8% late apoptotic cells (Fig. 7H). Results shown in Figs. 7C, 7F, 7I demonstrate that in all three cancer cell lines treated with PACs, a significant cell loss (P < 0.001) was observed.

BCL-2 and BAX mRNA expression in HT-29, MCF-7, and PC-3 cancer cells

To determine the ability of PACs to induce apoptosis, we examined the expression of pro-apoptotic genes, such as BAX, and anti-apoptotic genes, such as Bcl-2, after 24 h exposure to PACs at their respective IC₅₀ concentration. BAX expression significantly increased (^∗∗p < 0.01 in HT-29 and PC-3 cells and ^∗p < 0.05 for MCF-7 cells), while BCL-2 expression significantly decreased (^∗p < 0.05, Fig. 8) after treatment with PACs compared to control cells.

Caspases 3, 8, and 9 enzyme activity assays in HT-29, MCF-7, and PC-3 cancer cell lines

It has been shown that the cells undergo apoptosis after caspases activation (Soung et al., 2008). Therefore, in this study the enzymatic activities of caspases 3, 8, and 9 were investigated in all cancer cell lines following PAC exposed for 24 h at their respective IC₅₀ concentration. Caspases 3, 8, and 9 enzyme activities were significantly increased. Treated HT-29 cells showed significant increase in caspases activities compared to control (p < 0.05, caspases 3 and 8, and p < 0.0001, caspase 9; Figs. 9A–9C), while treated MCF-7 cells showed significant increase in caspases 3, 8 and 9 activities compared to control (p < 0.01, Figs. 9D–9F); PC-3 cells also showed similar results (p < 0.05, Figs. 9G–9I).