Pelargonidin induces apoptosis and cell cycle arrest via a mitochondria mediated intrinsic apoptotic pathway in HT29 cells

Abstract

Pelargonidin (PE) is an anticancer anthocyanidin that is abundant in berries. In this study, we found that PE irreversibly inhibits human colorectal adenocarcinoma cells (HT29) at micromolar concentrations in a dose- and time-dependent manner, which was determined using the MTT assay, and the GI₅₀ value was found to be 0.31 μM for a 24 h exposure. Inverted light microscopy observation and AO/EtBr staining of treated HT29 cells showed apoptotic morphological changes such as chromatin condensation and cell shrinkage. In addition, PE treatment showed a DNA ladder pattern, which is the hallmark of typical intrinsic apoptotic characterization by molecular DNA fragmentation analysis in HT29 cells. PE accumulates cells at G2/M of the cell cycle arrest in a dose-dependent manner as determined by flow cytometry analysis of propidium iodide staining. The G2/M phase cell cycle arrest peak of DNA, characteristic of apoptosis, was observed in the PE-treated cells. In addition, treatment with PE caused the release of cytochrome c from mitochondria into the cytosol. Western blotting analysis showed that PE significantly upregulated the activities of caspase-3 and -9 in comparison with a control group; subsequently, cleavage of PARP-1 protein is suggested, whereas PE significantly downregulated the expression of Bcl-2 and Bcl-xL and upregulated the expression of Bax and Bid. Furthermore, the downregulation of CDC25C, cyclin B1, and CDK1 expression resulted in the induction of G2/M cell cycle arrest in the HT29 cells. In contrast, PE induces the expression of p53 and p21^waf1 in HT29 cells. Collectively, these results show that PE has anticancer activities against HT29 cells through cell cycle arrest, DNA damage, and activation of the mitochondrial signalling pathway. This might provide a potential therapeutic option in the management of colon adenocarcinoma.

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1. Introduction

Cancer is a major public health problem with a high and increasing incidence and a high mortality. In recent decades, colon cancer is the third most common malignancy and the second most common cause of cancer-related deaths.¹ Cyclin-dependent kinases (CDKs) drive cell cycle progression from G1 to S phase and from G2 to M phase.² Among the four CDKs (CDK1, CDK2, CDK4, and CDK6), CDK4 and CDK6 are not required for the cell cycle of normal cells but are essential for driving cell cycle progression in various types of cancer.^3–5

Fruits, vegetables, and plants are a tremendous source of flavonoids. These flavonoids have received emerging attention in the area of pharmacology due to their various curative properties. Anthocyanins are water-soluble, glycosidic polyhydroxyl and polymethoxyl derivatives of flavylium salts, which are responsible for the red and blue colors of plant organs and are most abundant in berries, grapes, and red cabbage, among food stuffs. Chemically, anthocyanidin is a derivative of 2-phenylbenzopyrylium, having two benzoyl rings separated by a heterocyclic ring. Among several chemotherapeutic phytomedicines, flavonoids have an immense role in blocking cell proliferation and metastasis. Anthocyanins, one of the major subclasses of flavonoids, help in preventing the devastating conformational changes of colonic epithelial cells. The typical purple-colored pigment PE, commonly found in fruits, berries, and vegetables, exhibited exceptional anti-inflammatory effects^6–9 in human colon adenocarcinoma cells (HT29).

Both in vitro and in vivo experiments have shown the antioxidant properties of anthocyanins. Moreover, anthocyanins have an important role in the prevention against mutagenesis and carcinogenesis mediating some physiological functions related to cancer suppression. They show inhibitory effects on the growth of some cancer cells.¹⁰

It is well accepted that defective regulation of the cell cycle is one characteristic of cancer cell cycle regulation that is closely linked to cell proliferation, and one of the notable features of a tumor is abnormal cell cycle management.^11,12 Recently, many cell types have modulated expression of cell-cycle regulatory molecules responsible for cell-cycle arrest or apoptosis,^13–15 including cyclins and CDKs. The regulation of the cell-cycle process involves environmental stimuli that lead to the activation of cyclin-dependent serine/threonine kinases (CDKs), regulated by cyclins (CCCNs) and the inhibitors of cyclin-dependent kinases (CDKIs).¹⁶ Distinct pairs of cyclins and CDKs regulate progression through the different stages of the cell cycle. CDKs are modulated by their periodic phosphorylation and interaction with CDK inhibitors.^17,18 Recently, molecular markers were found to be involved in the regulation of the cell cycle as a target for prognosis and cancer treatment.^17–19 The activity of the CDKs play an important role for the discovery of new anticancer drugs specifically targeting the cell-cycle proteins. Previously, it was proven that CDK2 participates in the majority of cancer cases during the G1–S transition of the cell cycle when combined with cyclin E.²⁰ During the G1 phase, there is a progressive accumulation of cyclin E, and its expression above the threshold level leads the cell to enter the S phase.²¹ Cyclin B1 was reported to be an essential cell-cycle component required for the transition from G2 to M phases.^22–25 This study was conducted to determine the molecular mechanism of PE-induced G2M phase cell cycle arrest by investigating some major cell-cycle regulatory proteins, p53, p21, cyclin B1, cyclin D1, CDC25C, CDK1, CDK4, and CDK6 in HT29 cells.

2. Materials and methods

2.1. Reagents

Roswell Park Memorial Institute (RPMI-1640) medium, dimethyl sulfoxide (DMSO), penicillin, streptomycin, tryspin–EDTA, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and 5-fluorouracil (5-FU) were purchased from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Gibco-BRL (Gaithersburg, MD). Anti-Bcl-2, anti-Bax, anti-Bcl-xL, anti-Bid, anti-p21, anti-p53, anti-procaspase-3, anti-cleaved caspase-3, anti-procaspase-9, anti-cleaved caspase 9, anti-mitochondrial cytochrome c, and anti-cytosolic cytochrome c were purchased from Cell Signaling Technology (Beverly, MA). Anti-cyclinD1, anti-cyclin B1, anti-CDK1, anti-CDK2, anti-CDK4, anti-CDK6, anti-CDC25C, anti-CDC2, anti-poly (ADP-ribose) polymerase (anti-PARP), anti–anti-cleaved PARP, and anti-β-actin antibodies, and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of reagent or analytical grade.

2.2. Cell culture and drug preparations

The HT29 cell lines were purchased from National Center for Cell Sciences (NCCS), Pune, India. Cells were maintained in RPMI-1640 medium (Gibco-BRL), supplemented with heat-inactivated 10% FBS (Gibco-BRL) and 2 mM L-glutamine (Sigma Chemical), 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin, and maintained at 37 °C in an atmosphere of a 5% CO₂ incubator at 95% humidified air. The PE stock solution was prepared in DMSO and stored at −20 °C until use. The DMSO used for the study was at a final concentration of 0.1%.

2.3. Formazan-based viable cell mass assay (MTT assay)

HT29 cells were treated with PE and 5-FU (10–0.039 μM). Cell viability was determined at 24 h based on the MTT assay described earlier.²⁶ Briefly, the cells were seeded in a 96-well plate at a density of 4 × 10³ cells per well and allowed to adhere overnight. After removing the medium, 200 μL of fresh medium per well, containing 10 mmol L⁻¹ HEPES (pH 7.4), was then added. Then, 50 μL of MTT was added to the wells and the plate was incubated for 2–4 h at 37 °C in the dark. The medium was removed, and 200 μL of DMSO and 25 μL of Sorensen’s glycine buffer were added to the wells. Absorbance was measured using an ELISA plate reader at 570 nm.

2.4. Morphological observation

The HT29 cells were grown (4 × 10³ cells per coverslip) and incubated with PE at their IC₅₀ concentration (0.25, 0.31, and 0.5 μM) for 24 h, and then, they were dissolved in methanol/acetic acid (3 : 1, v/v). The morphometric studies were conducted using coverslips and gently mounted on glass slides. Morphological changes of HT29 cells were analyzed with a bright-field inverted light microscope (Nikon, Japan) at 400× magnification.²⁷

2.5. Acridine orange and ethidium bromide staining

Cells (1 × 10⁵ cells per mL) were seeded in a 96 well plate and treated with PE of different concentrations (0.25, 0.31, and 0.5 μM) for 24 h. After incubation, the plates were centrifuged. 200 μL of dye mixture [100 mg mL⁻¹ acridine orange (AO) and 100 mg mL⁻¹ ethidium bromide (EtBr)] was added to each well. After incubation for 2–3 min, the cells were visualized under a fluorescence microscope (Nikon Eclipse, Japan) at 40× magnification with an excitation filter at 510–590 nm.²⁸

2.6. Analysis of DNA fragmentation

HT29 cells treated with PE at different concentrations (0.25, 0.31, and 0.5 μM) were grown in 10 cm Petri dishes. Both the attached and unattached cells were harvested and washed thrice with ice-cold PBS. Cells were dissolved in 100 μL of DNA lysis buffer [50 mM Tris–HCL (pH 8.0), 10 mM EDTA, 0.5% N-lauroylsarcosine, and 2 mg mL⁻¹ proteinase K] and were incubated for 3 h at 55 °C, and RNAse A (Amresco, Solon, OH) was added for another 3 h. The DNA was extracted twice with equal volumes of phenol and once with chloroform–isoamylalcohol (24 : 1, v/v). DNA was then precipitated with 0.1 vol sodium acetate (pH 4.8) and 2.5 vol ethanol at 20 °C overnight and pelleted at 12 000g for 1 h. Samples were electrophoresed in a 1.5% (w/v) agarose gel, and DNA was visualized by EtBr staining.²⁶

2.7. Cell cycle analysis by flow cytometry

Investigation of apoptosis detection and analysis of cell-cycle distribution by flow cytometry were executed, as described earlier.²⁹ In brief, HT29 cells (1 × 10⁵ cells per mL) were incubated for 24 h in medium with less FBS to arrange the cell cycle. HT29 cells were then treated with medium containing 10% FBS and PE at different concentrations (0.25, 0.31, and 0.5 μM) for 48 h incubation, respectively. Cells were trypsinized, washed twice with PBS, incubated with 0.125% Triton X-100, and stained with propidium iodide in PBS-containing RNAse (0.1 mg mL⁻¹). The stained cells were analyzed with the flow cytometer FACS Calibur (Becton-Dickinson, San Jose, CA), and for each concentration, cells were counted until a 10 000 cell count was reached in a predefined G1 gate. The data of percentage of cells in sub-G1, G₀/G1, and G₂/M phases were consequently determined using BD Cell Quest Pro software (version 5.1).

2.8. Immunoblot analyses

Western blotting was carried out as described earlier.³⁰ Immunoblotting, immunocomplexes, or total cell lysates were denatured with sample lysis buffer. Samples were subjected to SDS–PAGE on a 12% or 16% gel and separated proteins were transferred onto a membrane by Western blotting. Membranes were blocked with blocking buffer for 1 h at room temperature, and, as desired, the primary antibody, namely anti-Bax (1 : 250), anti-Bcl-2 (1 : 500), anti-Bcl-xL (1 : 500), anti-Bid (1 : 1000), anti-procaspase-3 (1 : 2000), anti-cleaved caspase-3 (1 : 2000), anti-procaspase-9 (1 : 1000), anti-cleaved caspase 9 (1 : 1000), anti-mitochondrial cytochrome c, anti-cytosolic cytochrome c, anti-cyclin B1 (1 : 1000), anti-cyclin D1 (1 : 1000), anti-CDK1 (1 : 500), anti-CDK4 (1 : 500), anti-CDK6 (1 : 500), anti-CDC25C (1 : 1000), anti-CDC2 (1 : 500), anti-PARP (1 : 1000), anti-p53 (1 : 500), anti-p-21 (1 : 1000) and anti-β-actin (1 : 5000), overnight at 4 °C, followed by HRP-conjugated goat anti-rabbit Ig (1 : 3000) polyclonal antibody and the Chemiluminescence ECL Plus detection reagents following the manufacturer’s procedure (AmershamBioscience).

2.9. Statistical analysis

All the data were analyzed using statistical software SPSS 16 and expressed as mean ± SD. Statistical analysis was conducted using the one way ANOVA and values of p < 0.0001, p < 0.01 and p < 0.05 were considered significant.

3. Results

3.1. Cytotoxicity assay

The control, PE, and 5-FUwere incubated individually with HT29 cells to study the inhibition of the cell growth rate using the MTT assay. The corresponding cell viability graph for different concentrations is shown in Fig. 1. The absorption percentage of treated cells compared with control cells was used to calculate the percentage of cell viability. A dose-dependent growth inhibition was observed at concentrations ranging from 10 to 0.039 μM (Table 1), and each compound exhibited different sensitivity to the cell line. In the case of PE, a cell viability was obtained at 74.41 ± 0.70% in a broad concentration range of 10–0.039 μM for 24 h when compared with control cells. Fifty percent cell death, which determines the Growth Inhibition (GI₅₀) value of PE against HT29 cells held at 0.3125 μM in 24 h, and for standard 5-FU, it was found to be 0.15625 μM in 24 h. For further studies, doses of 0.25, 0.312, and 0.5 μM PE and a treatment time of 24 h was selected. To evaluate the cytotoxic activity of PE on Normal Colon cell lines (FHC, human fetal normal colonic mucosa), the FHC cells were treated with various concentrations (10 to 0.039 μM) of PE for 24 h. Cell viability was determined by the MTT assay. The results of the cytotoxicity assay are presented in (Fig. 1B) which determines PE induced cell cytotoxicity in a concentration dependent manner. The GI₅₀ for PE on FHC cells was found to be 7 μM and the maximum cytotoxicity was found to be 40% at 10 μM. This data clearly suggest that PE potentially affects cancer cells (HT-29) compared to normal FHC cells.

Fig. 1 (A) & (B) Effect of PE toward HT29 & FHC cells as determined by the MTT assay. The cells were incubated with different concentrations of drugs (0.039–10 μM) for 24 h. (A) Cell morphological and biochemical changes induced by PE. Control received only 0.1% of DMSO. Cells were visualized under an inverted light microscope. Magnification, 20×. (B) Graphical representation of cell viability. Data presented are the means ± SD of results from three independent experiments. GI₅₀ concentration is 0.31 μM and 2.5 μM respectively.

Table 1 Cell cycle analysis of HT-29 cells after treatment with various concentrations of sample 1 and 5-FU

S. no.	Concentration (μM mL^−1)	PE	5 Flurouracil
1	10	3.52 ± 0.35	2.83 ± 0.10
2	5	8.67 ± 0.52	6.53 ± 0.67
3	2.5	15.47 ± 0.52	12.58 ± 0.33
4	1.25	23.43 ± 0.64	18.54 ± 0.63
5	1	28.33 ± 0.45	22.35 ± 0.59
6	0.625	32.17 ± 0.64	26.36 ± 1.33
7	0.5	43.28 ± 1.14	32.25 ± 0.58
8	0.3125	50.32 ± 0.40	35.23 ± 0.33
9	0.25	55.40 ± 0.61	42.20 ± 0.55
10	0.15625	57.57 ± 0.58	48.06 ± 0.46
11	0.078	64.31 ± 0.67	52.28 ± 0.59
12	0.039	74.41 ± 0.70	56.72 ± 0.65
13	Cell control	100	100

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3.2. Morphological observation

The effects of PE on morphological features in HT29 cells were examined using optical-inverted light microscopy. The day after seeding, cells were treated with (0, 0.25, 0.312, and 0.5 μM) PE incubated for the next 24 h. HT29 cells are polyhedral or irregular shaped and grow in a tightly connected manner. The untreated cells displayed normal, healthy shape with a distinct cytoskeleton (Fig. 2a and i). The PE-treated cells and the cellular morphology of HT29 cells were severely distorted, and some cells turned round in shape when compared with the control untreated cells in a dose-dependent manner (Fig. 2b–d and j–l). Significant cytotoxic apoptosis is revealed by detached cells from the culture dish and showed the characteristic membrane blebbing.

Fig. 2 Light microscopy and fluorescent microscopy images of cellular morphology, AO/EtBr staining of (A and E) control and (B and F) 0.25 μM, (C and G) 0.31 μM, and (D and H) 0.50 μM of PE treated HT29 cells. AO/EtBr staining of (I and M) control and (J and N) 0.039 μM, (K and O) 0.078 μM, and (L and P) 0.156 μM of FU treated HT29 cells.

3.3. Acridine orange and ethidium bromide staining

Staining cells with fluorescent dyes, including AO and EtBr, is used to evaluate the nuclear morphology of apoptotic cells. To corroborate that apoptosis has been induced by PE and the positive control 5FU, HT29 cells were analyzed following AO and EtBr (AO/EB) staining. AO is a vital dye that will stain both live and dead cells, whereas EtBr will stain only those cells that have lost their membrane integrity. Three concentrations (0.25, 0.312, and 0.5 μM) for PE and (0.039, 0.078 μM and 0.156 μM) for 5FU were chosen based on the GI₅₀ values determined by the MTT assay. As a control, HT29 cells were cultured in complete media and stained with AO/EB (Fig. 2e–h & m–p). The figure shows PE induced apoptosis after a 24 h incubation at all the concentrations of PE tested. Cells-stained green represent viable cells whereas those stained yellow represent early apoptotic cells, and those stained reddish or orange represent late apoptotic cells. As shown in Fig. 2f, HT29 cells treated with 0.25 and 0.31 μM PE show changes in cellular morphology, including chromatin condensation, membrane blebbing, and fragmented nuclei (Fig. 2g and h) whereas following treatment with 5FU cells showed the same morphological and staining properties as the lower dose as mention above. Therefore, using the AO/EB staining procedure, the morphological features of an HT29 cell line undergoing apoptosis were shown to be dose dependent, that is, a stronger apoptotic signal was induced with higher concentrations of PE, which was proven by the positive control drug 5FU (Fig. 2i and p).

3.4. DNA fragmentation assay

To explain the apoptotic induction by PE in cancer cells, a DNA fragmentation assay was performed in HT29 cells. DNA fragmentation is one of the archetypal biochemical features of apoptosis. During the late stage of apoptosis, nuclear DNA is cleaved at intervals of 180–200 base pairs (bp) by endonucleases, and due to this, DNA bands appear like a ladder on an agarose gel. HT29 cells were treated with three concentrations of PE (0.25, 0.31, and 0.5 μM) based on the IC₅₀ concentration that was predetermined by the MTT assay, resulting in degradation of chromosomal DNA into small oligonucleosomal fragments, which results in a ladder-like pattern of DNA that might be due to the presence of early apoptotic cells (membrane blebbing and late stage of apoptosis) with clear nuclear fragmentation (Fig. 3), when compared with control. Treatment with different concentrations of PE (Lanes 3–5) for 24 h showed typical features of DNA laddering on an agarose gel, whereas untreated cells produced intact genomes (Lane 2), which can also be inferred from the result of the AO/EB fluorescence staining. Therefore, we can conclude that, with short incubation time, PE brings about early apoptosis, but when incubated for longer duration, a later stage of apoptotic cells with more nuclear fragmentation was observed.

Fig. 3 Analysis of DNA fragmentation induced by PE in HT29 cell line. Cells were treated for 24 h with PE, and DNA fragmentation was assessed by 1.5% agarose gel electrophoresis and ethidium bromide staining. PE concentrations used to treat HT29 were 0.25, 0.31, and 0.50 μM, respectively. The data are representative of three independent experiments carried out under the same conditions.

3.5. Cell-cycle analysis

To clarify whether PE produces an antitumor effect only by inducing apoptosis or they also induce cell-cycle arrest, we assessed cell-cycle distribution by FACS analysis in HT29 cells. The effect of PE on the HT29 cell cycle is represented in Fig. 4. Our results showed that PE induced arrest of the cell cycle at the G2/M phase. Treatment with PE at 0.25, 0.5, and 0.5 μM shifted the population of HT29 cells into the G2/M phase from the G1 phase. At 0.25 μM, 28.31% cells were in the G2/M phase, whereas, at 0.31 μM, cells in the G2/M phase increased to 40.42% with a reduced number of cells at the G1 phase, and on treatment with 0.5 μM of PE, 58.54% cells were in the G2/M phase as shown in Fig. 4B–D, when compared with control Fig. 4A. Thus, with flow cytometry observation, we could say that PE induces arrest of the cell cycle at the G2/M phase, but the role of different proteins involved in the induction of G2/M arrest in HT29 cells still needs further investigation.

Fig. 4 Effects of PE treatment on cell cycle phase distribution. HT29 cells were treated with different concentrations of PE and analyzed at 24 h by DNA flow cytometry. Histogram representing the PI staining (FL2-A) of vehicle-treated cells. Cellular DNA was stained with PI and the distribution of the cells in the G0/G1, S, and G2/M phase were analyzed. Histograms showed number of cells per channel (vertical axis) vs. DNA content (horizontal axis). The values indicated the percentage of cells in the indicated phases of the cell cycle. The data shown are representative of three independent experiments with similar findings. Significant differences from control were indicated by *p < 0.01, **p < 0.05, and ***p < 0.0001 (considered as statistically significant).

3.6. Western blot analysis

3.6.1. PE modulates Bcl-2 family proteins in HT29 cells. Several cytoplasmic proteins, particularly, members of the Bcl-2 family, are critical in apoptosis regulation. Of them, the pro-apoptotic subgroup, including Bax, promotes cell death, while members of the Bcl-2 family inhibit apoptosis. Most anti-apoptotic proteins, such as Bcl-xL and Bcl-w, share four regions of homology with Bcl-2. We determined the effect of PE treatment of HT29 cells on the protein levels of the Bcl-2 family members. As shown in Fig. 5A and B, after being treated with PE, immunoblot analysis data showed that the expression of Bax (pro-apoptotic protein) was significantly upregulated dose-dependently in HT29 cells (*p < 0.05 and **p < 0.001, respectively). Meanwhile, the BH3 interacting-domain (BID) death agonist, a pro-apoptotic member of the Bcl-2 protein family, was upregulated on treatment with PE (*p < 0.05 and **p < 0.001, respectively), whereas the expression of Bcl-2 and Bcl-xL (anti-apoptotic protein) was significantly down-regulated in a dose-dependent manner after a 24 h treatment with 0.25, 0.312, and 0.5 μM PE (*p < 0.05 and **p < 0.001, respectively). Furthermore, to investigate the effect of cytosolic Bcl-2 and Bax levels, cytosolic extracts were prepared after treatment with PE and the expression level of Bax and Bcl-2 was analysed by immunoblotting techniques, as shown in Fig. 6A and B. Treatment with PE significantly increased the expression of Bax in cytosolic extracts in a concentration dependent manner compared with control (p < 0.05) whereas there a down-regulation of Bcl-2 was observed after treatment with PE in a dose dependent manner. These results indicate that PE induced the translocation of Bax from mitochondria to the cytosol to induce apoptosis in HT-29 cell lines. Our results showed that PE significantly increases the Bcl-2/Bax ratio, thereby leading to apoptosis of HT29 cells. β-Actin was used as a loading control, which showed equal intensity bands, confirming equal protein concentration in all samples. These experimental findings suggest that PE induced apoptosis of HT29 cells via the mitochondrial pathway.

Fig. 5 Expression of apoptosis-related proteins in HT29 cells after PE treatment for 24 h as assessed by Western blot analysis: PE-mediated upregulation of Bax and Bid and downregulation of Bcl-2 and Bcl-XL. HT29 cells were treated with PE (0, 0.25, 0.31, 0.5, and 1 μM) for 24 h, respectively. Then, 50 μg of total cell protein lysates were used for Western blot analyses. The cells were lysed, and then cellular proteins were separated by SDS–polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. β-Actin was used as an internal control. β-actin is shown as the loading control. A representative experiment of three independent experiments is shown in the figure. Quantitative expression of proteins after normalization to β-Actin. Data presented are the mean ± SD of results from three independent experiments (**p < 0.05, *p < 0.001 vs. control).

Fig. 6 Effects of PE on cytosolic apoptotic and anti-apoptotic proteins in colon cancer cell lines (HT29). Cells were pretreated in the presence or absence of PE for 24 h. Data represent the results of three independent experiments. The expression level of Bax and Bcl-2 was analysed by Western blot assay. Dose-dependent effects of PE on the release of apoptotic protein was upregulated in cytosol. β-Actin expression was used as an internal control. Data presented are mean ± SD of results from three independent experiments (**p < 0.05, ***p < 0.001 vs. control).

3.6.2. PE-induced cytochrome c release-mediated apoptosis. Release of the vital mitochondrial respiratory-chain protein cytochrome c into the cytosol from mitochondria is an upstream event in caspase activation, which seems to be the hallmark of apoptotic cells treated with inducers. We, therefore, assessed the expression of cytosolic cytochrome c in 0.25, 0.312, and 0.5 μM PE-treated HT29 cells by Western blotting. The results showed that mitochondrial cytochrome c protein expression was significantly downregulated in the mitochondrial fraction of PE-treated cells. In contrast, the expression of cytosolic cytochrome c was significantly upregulated in treated cells when compared with untreated cells (*p < 0.05 and **p < 0.001, versus control), as shown in Fig. 7A and B. Furthermore, to validate, the expression of cellular apoptotic proteins caspase-3 and caspase-9 was elucidated by immunoblot analysis. Aspartate-specific cysteine proteases, that is, caspase-9 and caspase-3, play a central role in the execution of the apoptotic program. We found a marked upregulation of caspase-9 and caspase-3 activity after treatment with PE in a concentration-dependent manner. At a concentration of 0.25 and 0.31 μM, the expression of cleaved caspase-9 and caspase-3 was found to be increased gradually. Meanwhile, at a higher GI₅₀ concentration (i.e., 0.5 μM), the expression was significantly upregulated when compared with the control (*p < 0.05 and **p < 0.001, versus control). Concomitant decrease was observed in the pro form of caspase-9 and caspase-3 in PE-treated HT29 cells. The cleavage of PARP, one of the several known cellular substrates of caspases, is the hallmark of apoptosis. At the final stage of apoptosis, proteolytic cleavage of PARP was activated. Exposure of HT29 cells to PE treatment for 24 h showed that the expression of PARP and cleaved PARP was found to be significantly upregulated in a dose-dependent manner (*p < 0.05 and **p < 0.001) when compared with control. The obtained results revealed that PE activates caspase-9 and caspase-3 induced by cytochrome c release, which trigger PARP cleavage.

Fig. 7 Effects of PE on proteins of the caspase-dependent apoptotic pathway in colon cancer cell lines (HT29). Activation of caspase-dependent apoptotic pathways by PE. Cells were pretreated in the presence or absence of PE for 24 h. Data represent the results of three independent experiments. Proteolytic cleavage of PARP, caspase-3, and caspase-9 in a dose-dependent manner under treatment of PE, and apoptosis was analyzed by a Western blot assay. Dose-dependent effects of PE on the release of cytochrome c into the cytosol was observed. β-Actin expression was used as an internal control. Quantitative expression of caspase-3, -9, PARP, and cytochrome-C after normalization to β-actin. Data presented are mean ± SD of results from three independent experiments (**p < 0.05, ***p < 0.001 vs. control).

3.6.3. Modulation of the expression of cell cycle-regulatory proteins. Dysregulation of the cell cycle is a hallmark of tumor cells. Activated CDKs regulate the progression of cell cycle activity, which is modulated by the number of regulatory subunits, named cyclins, and with a group of CDKI proteins. CDK complexes were immunoprecipitated using specific anti-CDK antibodies, and the levels of CDK-associated kinase activity were measured. Therefore, to investigate the mechanism by which PE mediated G2/M arrest, we examined the dose-dependent effects on various cell cycle regulators in HT29 cells (Fig. 8A). As PE was observed to cause an arrest of cells in the G2/M phase, we next assessed its effect on G2/M cell cycle regulators, including CDK1, cyclin B1, CDC25C, and CDC2. As shown by immunoblot analysis in Fig. 8A and B, PE treatment of cells caused a significant dose-dependent decrease in the levels of CDK1, cyclin B1, CDC25C, and CDC2 proteins when compared with control cells. Next, we had also assessed the effect of PE on the expression of CDK4, CDK6, and cyclin D1 (Fig. 8A and B). Together, these protein expression data correlated with the dose-dependent effect of PE on G2/M cell cycle arrest.

Fig. 8 Effect of PE on the expression of cyclin B1, CDC2, and CDC25C in HT29 cells. HT29 cells were treated with the indicated concentration of drugs for 24 h. The control group was treated with 0.1% DMSO for 24 h. Cell lysate was prepared and the protein level of cyclin B1, CDC2, CDC25C was determined by Western blotting analysis. The expression of cyclin and CDK after extract induction at different doses. β-Actin was used for normalization and verification of protein loading. Quantitative expression of proteins after normalization to β-actin. Data presented are the mean ± SD of results from three independent experiments (**p < 0.05, *p < 0.001 vs. control).

3.6.4. PE-induced G2/M arrest in HT29 cells is regulated by p21. In addition to cyclins and CDKs, CDKIs play an essential role in the tightly regulated activities of the cyclin/CDK complex. Among several CDKIs, p21 has been widely documented to be involved in the G2/M checkpoint by inhibiting the activity of the CDC2/cyclin B1 complex. To determine whether p21 had a role in PE-induced G2/M arrest, the effect of PE on the expression of p21 was investigated by Western blot analysis. As shown in Fig. 9A and B, treatment with PE significantly increased the protein levels of p21 in a time-dependent manner.

Fig. 9 Expression of cell cycle inhibitors in HT29 cells following PE treatment. Cell lysates were prepared from HT29 cells at the times indicated following treatment with PE. The expression of p21 and p53 was analyzed by Western blotting. The quantitative data are presented as mean ± SD of three repeats of three independent studies; *p < 0.05; **p < 0.005, compared with control at the respective times.

3.6.5. p21^waf1 was induced by the p53-dependent pathway. p21^waf1 is a universal inhibitor of CDKs. The expression of p21^waf1 is primarily regulated by p53 tumor suppressor protein, and its expression is upregulated by antiproliferative signals. According to our results, PE profoundly increased the level of p53 and p21^waf1 (Fig. 9A and B, respectively). The induction of p21^waf1 expression was clearly consistent with p53 expression and was followed by the inhibition of CDK expression. These results indicate that the increased level of p21^waf1 was induced by the p53-dependent pathway and may have been responsible for the reduced level of CDK1 and the subsequent G2/M cell cycle arrest.

4. Discussion

Donaldson³¹ reported that lifestyle and dietary management would prevent 30–40% of all cancers in the world. In recent decades, the anticancer drugs derived from natural products are used in clinical trials.³² Thus, the search for natural plant products with various bioactivities represents an interesting area due to its diversity and unique way of action.

Our study focused on the antiproliferative and cytotoxicity effect of PE, an anthocyanin, which is profoundly rich in strawberries, raspberries, pomegranates, and blackberries,^6,7 on human colon adenocarcinoma cells (HT29). The biological properties of the novel bioactive anthocyanin PE have not yet been fully evaluated; so, information on their biological activity is limited. Considering this, our study evaluated the anticancer activity of PE against human colon adenocarcinoma cell lines (HT29). In the first part of this study, the antiproliferative properties of PE were predetermined using the MTT assay. The principle of this assay is based on the reduction of a soluble tetrazolium salt, by mitochondrial dehydrogenase activity of viable tumor cells, into a soluble colored formazan product that can be measured spectrophotometrically after dissolution.³³ The results of the study demonstrated that PE exhibited antiproliferative activity against HT29, confirming its cytotoxic sensitivity. Despite the treatment with 50% growth, GI₅₀ of PE against HT29 after 24 h treatment was found to be 0.31 μM (Fig. 1). The effect of PE, a strong inducer of apoptosis, on HT29 cancer cell lines was shown by the presence of significant DNA nuclear fragmentation along with activation of the intrinsic caspase cascade pathway following treatment with the GI₅₀ concentration on HT29 cells.

Light microscopy observation of the PE-treated HT29 cell line after 24 h of exposure showed typical morphological features of apoptosis. The level of cell shrinkage and cell blebbing was observed under inverted light microscopy after incubation of HT29 with GI₅₀ and sub-GI₅₀ concentrations of PE (Fig. 2A–D) after 24 h. HT29 cells are polyhedral or irregular shaped and grow in a tightly connected manner with a distinct cytoskeleton. The apoptotic characterization of observed morphological changes included reduction in cell volume, cell shrinkage, reduction in chromatin condensation, and formation of cytoplasmic blebs.³⁴ When the HT29 cells were treated with PE at GI₅₀ (Fig. 2B–D), the cellular morphology was severely distorted and round in shape when compared with control untreated cells (Fig. 2A). Thus, our drug showed significant cytotoxic apoptosis, which is revealed by detachment of cells from the culture dish and the characteristic membrane blebbing and/or bubble formation.

The morphological hallmark for apoptotic cells is staining with fluorescent dyes, including AO and EtBr. The vital dye AO stains both live and dead cells, whereas the cells that had lost their membrane integrity will be stained with EtBr.³⁵ AO permeates all cells, and the nuclei become green, whereas EB is only taken up by cells that lost their cytoplasmic membrane integrity, and their nuclei are stained red. As shown in Fig. 2E–H, HT29 cells were treated with PE at three concentrations based on the GI₅₀ values determined by the MTT assay, which were 0.25, 0.31, and 0.50 μM. Fig. 2G and H show early and late apoptotic cells respectively, that were observed in the 24 h treatment of PE. Moreover, morphological changes of apoptosis, including cell shrinkage and chromatin condensation, were observed in the experimental group when compared with control cells. On PE treatment, the percentage of apoptotic cells was found to be statistically significant with the proportional increase in concentration of PE.

The apoptotic hallmark, a DNA fragmentation assay, was performed to show that PE induces intrinsic apoptotic cell death. On treatment with PE with corresponding GI₅₀ concentrations, it showed a significant DNA ladder-like pattern. DNA fragmentation was used to determine whether the antiproliferative effect of Piper sarmentosum ethanolic extract on cells acts through the respective apoptosis pathway.³⁶ Our data are also in corroboration with the findings of Mishra et al., who reported a DNA ladder-like pattern with aqueous ethanol seed extract of Ziziphus mauritiana on HL60 cells in a concentration-dependent manner, and a time-dependent study showed typical ladder-like pattern owing to the induction of apoptosis.³⁷ The results obtained from our experiment finally confirm cell death via apoptosis by PE. Thus, this illustrates that the anticancer effect of PE may act through apoptotic signaling.

The pro-apoptotic Bcl-2 family members functionally incapacitate mitochondria, which leads to an intrinsic apoptotic pathway that is mediated by the loss of mitochondrial potential and activation of caspase-9 by release of cytochrome c that indeed activates executor caspases (3 and 7), which, in turn, causes the destruction of cellular proteins.³⁸ Adams and Cory³⁹ reported that Bcl-2 family proteins comprise both pro- and anti-apoptotic molecules. The membrane protein Bcl-2, which is mainly located on the outer membrane of mitochondria, on overexpression, inhibits apoptosis of the cells and the release of cytochrome c from mitochondria to the cytosol. On the other hand, it includes Bax and some other pro-apoptotic proteins which, upon overexpression, promote the release of cytochrome c by enhancing apoptosis.⁴⁰ According to the above-mentioned review, the results clearly state that treatment with PE on HT29 cells upregulated the expression of pro-apoptotic protein. Our results clearly suggest the expression of Bax and Bid was upregulated, whereas the anti-apoptotic proteins Bcl-2 and Bcl-xL were downregulated. It confirms that modulation of the Bcl-2 family proteins should be potential mechanism of PE against human colon cancer cells.

Apoptosis is a well-controlled process of cell deletion and has a basic role in the regulation of physiological growth control and homeostasis. Insufficient apoptosis plays a pivotal role in tumor progression and therapy resistance.⁴¹ In this study, apoptosis induction in the PE-treated HT29 cell line was also confirmed with DNA fragmentation, expression of cell cyclin-regulatory proteins, and cell cycle arrest. Agents that inhibit cell cycle progression, such as mifepristone and histone deacetylase inhibitors, were reported as potential therapeutics for endometrial cancers.^42,43 The findings of FACS calibur analysis of HT29 cells on PE treatment showed a decrease in the cell population in the G1 and S phase leading to a significant increase in the number of cells in the G2/M phase, stoutly indicative of a G2/M arrest. In this study, we have demonstrated that flavonoid PE is associated with cell growth inhibition in human colon cancer cells through the induction of G2/M cell cycle arrest. No significant changes in the population of cells in the G1 phase or in the levels of proteins related to the G1 phase (Fig. 4), such as CDK6 and CDK4, were observed (Fig. 8). To the best of our knowledge, this is the first study to demonstrate that PE induces G2/M cell cycle arrest in cancer cells.

In past decades research was aimed to trap DNA replication by administration of DNA alkylating agents, anti-metabolites, mitotic spindle assembly inhibitors or topoisomerase inhibitors targeting cancer cells. Meanwhile recent strategies focused on kinase inhibitor development which is essential and directly responsible for cellular aberrations.⁴⁴ The cell cycle is regulated by cyclin and CDKs in all eukaryotes. Apoptotic signals are activated by cell cycle checkpoints that in turn also enable cellular repair the damaged cells.^45,46 The alternative inhibition of cell proliferation can be done by targeting CDK1/cyclin B, which has a unique ability and essential mitotic functions to compensate for all other cell cycle cyclin-dependent kinases. The proliferation of cancer cells and tumours effectively blocked by targeting CDK1 and cyclin B were proposed and shown by researchers.^47–56 Specifically, the regulation of progression of the G2/M phase in the cell cycle was controlled by cyclin B and CDK1 proteins.⁵⁷ During DNA damage the cells were blocked in the G2/M phase and the cells were triggered to cell death by radiotherapy in the G2/M phase and the significantly increased induction of G2/M arrest causes cell death by apoptosis which would be the novel approach in cancer therapeutics.^58,59

The two CDKIs exert key roles in averting G2 exit both by inhibiting cyclin B1–CDK and cyclin A–CDK complexes, which control G2-M progression. Because many therapeutic agents used for the management of cancer treatment ultimately target damaged DNA,⁶⁰ the G2-M checkpoint blockage can be seen as a form of cellular resistance to chemotherapeutic regimens, thus allowing DNA repair mechanisms to be activated to promote continued cell viability.⁶¹ In eukaryotic cells, cell cycle progression is partly controlled by a family of protein kinase complexes, including CDKs and their activating partners, cyclins.² As the arrest of S and G2/M phases in CNE cells was observed after treatment with ESD, the levels of the regulators involved in the cell cycle were evaluated by western blotting. The CDC2/cyclin B1 is a critical complex regulating the G2/M phase of the cell cycle. The activation of CDC2 is responsible for cell entry into mitosis, which is controlled by phosphorylation of CDC25C.⁶² The expression levels of both CDC2 and CDC25C were reduced in a dose-dependent manner when treated with PE, although that of cyclin B1 was reduced (Fig. 9). These results suggested that alterations in the levels of various cell cycle-regulatory proteins were responsible for cell cycle arrest in PE-induced HT29 cell death.

On the other hand, we also assessed the effect of PE on the regulation of p21, which is known to control the entry of cells at the G2/M phase transition checkpoint. Our results demonstrated that treatment of HT29 cells with PE caused significant time-dependent induction of p21. Correspondingly, reductions in mitotic-CDKCDC2 and cyclin B1 expression were also observed (Fig. 9). These findings imply that PE promotes cell death through inducing G2/M arrest and apoptosis as well.

In principle, apoptosis is triggered by two alternative mechanisms, including the ligation of plasma membrane death receptors, which stimulate the “extrinsic” pathway, and the perturbation of intracellular homeostasis involving the “intrinsic” pathway.⁶³ In the execution of both apoptotic pathways, caspases, a family of cysteine aspartyl-specific proteases, are known to play a central role and are responsible for the characteristic biochemical and morphological changes in apoptosis.⁶⁴ We observed that PE induced cell death with characteristics of apoptosis (Fig. 8). Mitochondria have been demonstrated to perform a crucial function in the events resulting in caspase activation in many cell types undergoing apoptosis. In particular, the release of cytochrome c from mitochondria promotes procaspase-9 activation.^65,66 The activated caspase-9 cleaves the downstream effecter caspases, including caspases-3, -6, and -7, which lead to the hallmarks of apoptosis.^67,68 We observed an increased level of cytochrome c followed by cleavage of PARP to its substrate by PE in HT29 cells. These findings suggested that PE induced cell death via a mitochondria-mediated pathway. Swamy et al.⁶⁹ reported that PE increase nuclear localization of active p53. Treatment of HT29 cells might involve inhibition of function of mutated p53. Our results are similar to the study done by Peng et al.⁷⁰

In our work, we detected a potent effect of PE on the G2/M phase cell cycle arrest in HT29. There was an upregulation of p53 and p21 and a decrease in expression levels of cyclin B1 and CDC2 which were observed as well. Collectively, these results indicate that PE induced G2/M cell cycle arrest may be attributed to its inhibitory effect on colon cancer cells.

The growth, development and homeostasis in metazoans of cells were maintained by a distinct genetic and biochemical pathway called the apoptotic pathway, either the intrinsic or extrinsic apoptotic pathway.^71,72 In this present study on treatment with PE of HT29 cells, cytochrome c was released from mitochondria to the cytosol, which was induced by the over expression of pro-apoptotic protein Bax and decreased expression levels of anti-apoptotic protein like Bcl-2 and Bcl-xl. The released cytosolic cytochrome c in turn activates a caspase cascade i.e. cleaved caspases 9 was elevated to activate cleaved caspases 3 and PARP, subsequently inducing apoptosis.

5. Conclusion

In summary, we have shown that PE inhibits HT29 cell proliferation, and AO/EtBr staining revealed the morphological features of PE-induced cell apoptosis with chromatin condensation and membrane blebbing in HT29 cells. Furthermore, apoptosis was confirmed by a fragmented DNA ladder-like pattern using the DNA fragmentation assay, which is a hallmark of apoptosis. Indeed, PE-induced accumulation of cells in G2/M cell cycle arrest in a concentration-dependent manner was confirmed by cell cycle analysis. Investigated cell cycle check point results indicate that the increased level of p21^waf1 was induced by a p53-dependent pathway and may have been responsible for the reduced level of CDK1 and cyclin B1. The subsequent G2/M cell cycle arrest decreased following PE treatment which indicates that PE can selectively inhibit G2/M CDKs. The overall evaluated results strongly suggest that PE possess an anticancer property. Our findings suggest that PE promotes apoptosis via a mitochondria mediated intrinsic apoptotic pathway. The study on the CDK and cyclin complex was performed to provide strong evidence of its anticancer property. Moreover, these data suggest the possibility of using PE as a potential new chemotherapeutic drug for colon carcinoma.

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