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. 2011;6(11):e27444.
doi: 10.1371/journal.pone.0027444. Epub 2011 Nov 8.

Grape Proanthocyanidins Induce Apoptosis by Loss of Mitochondrial Membrane Potential of Human Non-Small Cell Lung Cancer Cells in Vitro and in Vivo

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Free PMC article

Grape Proanthocyanidins Induce Apoptosis by Loss of Mitochondrial Membrane Potential of Human Non-Small Cell Lung Cancer Cells in Vitro and in Vivo

Tripti Singh et al. PLoS One. .
Free PMC article

Erratum in

  • PLoS One. 2012;7(6): doi/10.1371/annotation/23a8e553-4fce-4c73-9946-7a2a6a5729e9

Abstract

Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GSPs induce apoptosis in non-small cell lung cancer cells.
(A) In vitro treatment of A549 and H1299 cells with GSPs induces apoptosis in a dose-dependent manner. Apoptotic cell death was analyzed using FACS analysis, and total percentage of apoptotic cells in A549 and H1299 cells are summarized as mean ± SD, n = 3. Significant difference versus non-GSPs-treated controls: * P<0.05; P<0.01; P<0.001. (B) Treatment of A549 and H1299 cells with GSPs results in a dose-dependent reduction in the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xl while increasing the expression of the pro-apoptotic protein, Bax, as determined by western blot analysis. Data are representative of three independent experiments with similar results.
Figure 2
Figure 2. GSPs disrupt mitochondrial membrane potential in non-small cell lung cancer cells
(A & B) Immunoblotting for cytochrome c and Smac/DIABLO using cytosolic and mitochondrial fractions prepared from A549 and H1299 cells following treatment with indicated concentrations of GSPs for 48 h. The blots were stripped and re-probed with anti-COX IV antibody to ensure equal mitochondrial protein loading as well as to rule out cross-contamination of mitochondrial and cytosolic fractions. (C) Treatment of A549 and H1299 cells with GSPs induces loss of mitochondrial membrane potential. Cells were treated with indicated doses of GSPs for 48 h. JC-1 dye-stained cell were analyzed by flow cytometry, as described in Materials and Methods. Data are representative of two separate experiments with identical observations.
Figure 3
Figure 3. Induction of apoptosis in lung cancer cells by GSPs is mediated through caspase-3 activation
(A) Treatment of A549 and H1299 cells with GSPs reduces the level of caspase-9 while increases the activation/cleavage of caspase-9, caspase-3 and PARP in a dose-dependent manner. Cells were treated with varying concentrations of GSPs (0, 20, 40, 60 and 80 µg/mL) for 48 h, thereafter cells were harvested, cell lysates prepared and subjected to western blot analysis to detect the levels of caspase-9, cleaved caspase-9, caspase-3 and PARP. A representative blot is shown from three independent experiments with similar results. (B) The caspase-3 activity in cell lysates from the samples of Panel A was measured using a colorimetric protease assay (ApoTarget Kit). GSPs treatment to A549 and H1299 cells increases the activity of caspase-3 dose-dependently. Significant difference versus non-GSPs treatment group, P<0.01 and * P<0.001. (C) The effect of GSPs (60 and 80 µg/mL) on apoptosis of A549 and H1299 cells was determined after 48 h in the absence or presence of 60 µmol/L of the caspase-3 inhibitor (z-DEVD-fmk). The content of apoptotic cells was determined using cell death detection ELISA kit, as detailed in Materials and Methods. The cells treated with z-DEVD-fmk blocked the GSPs-induced apoptosis in A549 and H1299 cells. The percentage of apoptotic cells in different treatment groups was summarized and data are presented as mean ± SD from two repeated experiments. Significant inhibition by caspase-3 inhibitor versus GSPs alone treated cells, * P <0.001. CI = caspase-3 inhibitor, C = control, without any treatment.
Figure 4
Figure 4. Effect of GSPs on cell cycle progression of A549 and H1299 cells
Cells were treated either with vehicle (0.1% DMSO in medium) or 20, 40 and 60 µg/mL doses of GSPs in complete medium. After 48 h of treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and flow cytometric analysis was performed to analyze the cell cycle distribution, as detailed in the Materials and Methods. (A–D) Cell cycle distribution in A549 (left panels) and H1299 (right panels) cells with the treatment of various concentrations of GSPs. (E) Data from the cell cycle distribution were summarized and presented as the mean ± SD of three independent experiments. Statistically significant versus non-GSPs treated control group, P <0.05 and * P <0.01.
Figure 5
Figure 5. Effect of GSPs on G1 phase cell cycle regulatory proteins in A549 and H1299 cells.
The cells were treated with either vehicle (0.1% DMSO in medium) or GSPs (20, 40, 60 and 80 µg/mL) for 48 h and thereafter harvested, cell lysates prepared and then subjected to SDS-PAGE followed by Western blot analysis, as described in Materials and methods. Effect of GSPs was determined on the following: (A) the expression of cyclin D1, cyclin D2 and cyclin E, and the expression levels of Cdk2, Cdk4 and Cdk6; and (B) the expression of Kip1/p27 and Cip1/p21. β-actin was used to verify equal loading of the samples. Representative blots are shown from three independent experiments with almost identical results. (C) In binding assay, Cip1/p21 and Kip1/p27 were immunoprecipitated using specific antibody from total protein lysates followed by SDS-PAGE and western blot analysis for Cdk2, Cdk4 and Ckd6 as detailed in Materials and methods. IP, immunoprecipitation; IB, immunoblotting.
Figure 6
Figure 6. Administration of GSPs by gavage inhibits the growth of lung tumor xenografts in mice.
Each mouse was s.c. implanted with either 2×106 A549 or H1299 cells mixed with Matrigel on the right flank. Twenty-four h later, mice were given either PBS (100 µL) or GSPs (50, 100 or 200 mg/kg body weight in 100 µL PBS) by gavage 5 days/week. (A) Change in body weight of mice during the 8 weeks study was monitored. The body weights of the control and GSPs-fed mice did not differ significantly throughout the experiment protocol. (B) Average tumor volume ±SD/mouse (mm3) in each group. (C) Tumors were harvested at the termination of the experiment, and the wet weight of the tumor/mouse in grams in each group is reported as mean ± SD. Statistical significance of difference between control and GSPs-fed groups was analyzed by one-way ANOVA followed by Bonferroni t test. n = 10. Statistical significance vs non-GSPs-fed controls, P<0.05; P<0.01; * P<0.005.
Figure 7
Figure 7. Administration of GSPs by gavage induces apoptosis in tumor xenograft cells.
(A) The data on terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive cells in tumor xenograft tissues from GSPs-treated and GSPs-untreated samples are summarized. (B) GSPs inhibit the expression of anti-apoptotic proteins while increases the expression of pro-apoptotic protein in A549 and H1299 xenograft tissues. (C) GSPs enhance the activation of caspase-9, caspase-3 and PARP proteins in tumor xenografts. Tumor lysates were prepared from the tumors collected at the termination of the experiment and subjected to western blot analysis, as described in Materials and methods. Representative blots are presented from the independent experiments from at least six tumors from six different mice per group with identical observations. (D) Analysis of PCNA-positive cells for proliferation index. Immunohistochemical data in terms of percentage of positive cells (TUNEL-positive or PCNA-positive) are summarized and presented as mean ±SD of 6–7 tumor samples from each group. Statistical significance versus non-GSPs-fed controls, P<0.01; * P<0.001.
Figure 8
Figure 8. Effect of dietary GSPs on G1 phase cell cycle regulatory proteins in xenograft tissues.
The A549 tumor xenograft tissues were harvested at the termination of the experiment, and tumor lysates were subjected to the analyses of cell cycle proteins of G1 phase using Western blot analysis, as described in Materials and Methods. Effect of GSPs was determined on the following: (A) the expression of cyclin D1, cyclin D2 and cyclin E, and the expression levels of Cdk2, Cdk4 and Cdk6; and (B) the expression of Cdk inhibitory proteins, Kip1/p27 and Cip1/p21. β-actin was used to verify equal loading of the samples. Representative blots are presented from the independent experiments from at least six tumors from six different mice per group with identical expression levels of proteins.

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