Indole-3-carbinol inhibits the proliferation of colorectal carcinoma LoVo cells through activation of the apoptotic signaling pathway

Abstract

Indole-3-carbinol (I3C) is a phytochemical that exhibits growth-inhibitory activity against various cancer cells. However, there are limited studies on the effects of I3C on colon cancer cells. In this study, the growth-inhibitory activity of I3C against the human colorectal carcinoma cell line (LoVo) was examined. The results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, colony formation, and cell counting assays revealed that I3C suppressed the proliferation of LoVo cells. Microscopy and wound-healing analyses revealed that I3C affected the morphology and inhibited the migration of LoVo cells, respectively. I3C induced apoptosis and DNA fragmentation as evidenced by the results of fluorescein isothiocyanate-conjugated annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay, respectively. Additionally, I3C arrested the cell cycle at the G0/G1 phase and enhanced the reactive oxygen species levels. Western blotting analysis revealed that treatment with I3C resulted in the activation of apoptotic proteins, such as poly(ADP-ribose) polymerase, caspase-3, caspase-7, caspase-9, Bax, Bim, and p53 in LoVo cells. These results indicate that I3C induces apoptosis in LoVo cells by upregulating p53, leading to the activation of Bax and caspases. Taken together, I3C exerts cytotoxic effects on LoVo cells by activating apoptosis.

Keywords

Indole-3-carbinol colon cancer LoVo cells apoptosis p53

Introduction

Globally, colon cancer is the fourth most common cancer with the fifth highest mortality rate. In 2018, more than half a million colon cancer-related deaths were reported.¹ The most common therapeutic strategies for nonmetastatic colorectal cancer are colonic resection and adjuvant chemotherapy.² The surgical treatment has been recognized as the most effective treatment of colorectal cancer. However, surgical treatment and chemotherapy are associated with several adverse effects, which decrease the quality of life of patients with cancer and may lead to cognitive and functional impairments, such as defecatory dysfunction.^3–5 Thus, there is a need to develop novel therapeutic strategies to improve the quality of life of patients and mitigate these side effects. Some natural products are reported to mitigate chemotherapy-induced toxicity and thus can serve as alternative therapeutics for cancer. However, further studies are needed to examine the therapeutic efficacy of natural compounds in cancer and elucidate the underlying mechanisms.⁴

Phytochemicals, which are naturally occurring compounds in plants, are key for the development of novel drugs. Some phytochemicals exhibit growth-inhibitory activities against various human cancers.^6,7 In addition to exhibiting anticancer properties, some phytochemicals mitigate the adverse effects of various chemotherapies.⁴ Indole-3-carbicol (I3C), a phytochemical, is found naturally in high doses in cruciferous vegetables, such as kale, cabbage, sprouts, and cauliflower.⁸ The structure of I3C is shown in Figure 1. I3C is reported to exert anticancer effects by disrupting the cell cycle regulation and the survival-related signals in the cancer cells.⁹ In particular, I3C induces apoptosis in various cancers, such as prostate, bone, and liver cancers, as well as leukemia. The mechanisms underlying the anticancer effects of I3C are mediated by multiple factors.^10–13 For example, I3C induced cell cycle arrest at the G1 phase and apoptosis in leukemia cell lines and suppressed the growth of prostate cancer cells by inactivating Akt.^12,14 In lung cancer, I3C induces apoptosis through p53 and caspase-8 pathway activation.¹⁵ However, there are limited studies on the growth-inhibitory effects of I3C against human colon cancer and the underlying molecular mechanisms.

Figure 1.

Chemical structure of indole-3-carbinol.

Apoptosis, which is a natural cell death mechanism, is a critical therapeutic target for cancer because most cancers acquire chemoresistance and radioresistance mainly through the inhibition of apoptosis.^16,17 The cells undergoing apoptosis detach from other cells and the extracellular matrix and exhibit a round morphology.¹⁸ Phosphatidylserine on the cell surface and chromosomal DNA fragmentation, which leads to the activation of poly(ADP-ribose) polymerase (PARP) and consequently the cleavage of PARP, are major markers of apoptosis.^19,20 Various factors are involved in the induction of apoptosis. Apoptotic stimuli, such as genetic damage and enhanced oxidative stress promote mitochondrion-mediated apoptosis by increasing mitochondrial permeability, which is one of the typical apoptotic mechanisms. The mechanisms of apoptosis are tightly regulated and involve multiple factors, such as p53, caspase-3, caspase-7, caspase-9, and Bcl-2 family members.²¹

p53, a tumor suppressor protein, is involved in DNA damage response.²² The functions of p53 are dependent on its phosphorylation at several serine residues at the N-terminal and C-terminal domains.²³ Phosphorylation of p53 at Ser15 promotes both the upregulation and functional activation of p53 in response to cellular stresses, including DNA damage, which is essential for the induction of cell cycle arrest.^22,24 p53 phosphorylated at Ser15 or Ser20 undergoes a conformational change, which decreases its affinity to MDM2, a negative regulator of p53.^25–27 Severe and irreparable DNA damage induces the phosphorylation of p53 at Ser46, which promotes p53-mediated apoptosis and inhibits tumor growth by inducing the expression of pro-apoptotic genes.²⁸ The p53-mediated regulation of cell cycle and apoptosis, which are crucial processes involved in anticancer mechanisms, is critical for inhibiting tumor development.²⁹ p53 modulates the expression of various genes involved in DNA repair, cell cycle arrest, apoptosis, and inhibition of metastasis. Additionally, p53 suppresses cell growth by arresting the cell cycle and activating apoptosis.^30,31 For example, p53 can attenuate Bcl-2 function, which results in the activation of Bax (Bcl-2 associated X protein) and caspases and consequently the activation of apoptosis.¹⁶

Bcl-2 family proteins and caspases play a critical role in the regulation of apoptosis.^32,33 Most chemotherapeutic drugs exert cytotoxic effects against cancer cells by inducing apoptosis. Hence, the expression level and activation of Bcl-2 family members and caspases determine the therapeutic efficacy of anticancer drugs.³⁴ The Bcl-2 family comprises both pro-apoptotic and anti-apoptotic molecules. The balance between pro-apoptotic and anti-apoptotic factors determines the sensitivity of cells to death signals.^33,35 Apoptosis is mediated by BH3-only proteins (such as Bcl-2 interacting mediator of cell death (Bim)), which belong to the Bcl-2 family. BH3-only proteins promote apoptosis by activating pro-apoptotic proteins, such as Bax and Bak (Bcl-2 antagonist/killer), and suppressing anti-apoptotic proteins, such as B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-xl).³⁶ Activated Bax and Bak, which are pore-forming proteins, enhance mitochondrial outer membrane permeability and mediate the intrinsic apoptotic pathway through the activation of caspase-9. Activated caspase-9 functions as an effector of cell death by directly cleaving caspase-3 and caspase-7, which degrade several target proteins widely distributed throughout the cell.^37,38

This study aimed to examine the growth-inhibitory activity of I3C against the human colorectal carcinoma LoVo cells. In particular, this study focused on the I3C-mediated activation of apoptosis-related proteins, such as p53, caspase-3, caspase-7, caspase-9, and Bcl-2 family.

Materials and methods

Reagents

The stock solution (300 mM) of I3C (Sigma-Aldrich, St. Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored at −20°C until use. The anti-p27, anti-phospho-p53(S15), anti-caspase-3, anti-caspase-7, anti-cleaved caspase-7, and anti-Bim antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-PARP, anti-cleaved PARP, anti-caspase 9, anti-cleaved caspase-3, anti-phospho-p53(S20), anti-phospho-p53(S46), anti-Bax, anti-Bcl-xl, anti-Bcl-2, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-p35 and anti-cleaved caspase-9 antibodies were purchased from Youngin Frontier (Seoul, Korea). The anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology.

Cell culture

The human colon cancer cell line HCT-15 and HCT116 cells were purchased from the Korea Cell Bank (Seoul, Korea). The human colorectal carcinoma LoVo cells were purchased from the American Type Culture Collection (Manassas, Virginia). HCT-15, HCT116, and LoVo cells were cultured in Rosewell Park Memorial Institute (GIBCO, NY) medium supplemented with 0.25% glucose, 0.24% HEPES, 0.15% NaHCO₃, 1% penicillin/streptomycin (GIBCO), and 10% fetal bovine serum in a 100-mm plate under standard conditions (37°C, 5% CO₂, and 95% humidity).

Cell proliferation and cell counting assays

The effect of I3C on the viability of colon cancer cells was examined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The LoVo, HCT-15, and HCT116 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultured for 24 h. Next, the cells were treated with various concentrations (0, 100, 200, 300, 400, 500, 600 µM, and 700 µM) of I3C prepared in DMSO for 24 or 48 h. The I3C-treated cells in each well were incubated with 20 µL of MTT (5 mg/mL) for 2 h at 37°C. The supernatant was removed and 150 µL of DMSO was added to each well to dissolve formazan. The samples were incubated on a shaker for 30 min. The absorbance of the samples at 570 nm was examined using a microplate reader. To perform the cell counting assay, LoVo cells were seeded in a six-well plate (10⁵ cells/well) and cultured for 24 h under standard conditions. The cells were then treated with different concentrations (0, 200, 400, and 600 µM) of I3C for 24 or 48 h. The number of cells was counted using a hemocytometer.

Colony formation assay

LoVo cells were seeded in six-well plates (1000 cells/well) and incubated for 24 h under standard conditions. The medium was replaced with a medium containing 200, 400, or 600 μM of I3C, and the cells were cultured for 24 h. Next, the medium was replaced with a fresh medium and the cells were cultured for 2 weeks. The cells were then washed twice with phosphate-buffered saline (PBS) (5 min per wash) and fixed using 4% formaldehyde for 20 min at 4°C. Further, the cells were washed twice with PBS (5 min per wash) and stained with a 1% crystal violet (Sigma-Aldrich) solution for 30 min at room temperature. The colonies were washed with distilled water and the number of colonies was counted.

Microscopy and wound-healing assay

LoVo cells treated with I3C at concentrations of 0, 200, and 400 μM were cultured until approximately 70% confluency. The cells were observed at 100× magnification using an inverted light microscope (Olympus CKX53, Tokyo, Japan) after 24 and 48 h. The wound-healing assay was performed to examine the effect of I3C on cell migration and invasion. LoVo cells were seeded in six-well plates (4 × 10⁵cells/well) and cultured overnight. The center of the monolayer was scraped using a pipette tip and the cells were washed twice with PBS. Next, the cells were incubated with the medium containing various concentrations of I3C (0, 200, and 400 μM) for 24 and 48 h. The wound area was observed at 40× magnification using an inverted microscope (Olympus CKX53, Tokyo, Japan).

Analysis of apoptosis

The effect of I3C on apoptosis was examined using the fluorescein isothiocyanate (FITC) annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). LoVo cells were seeded in a six-well plate (3 × 10⁵ cells/well) and cultured for 24 h. The cells were then treated with various concentrations of I3C (0, 200, 400, and 600 μM) for 24 h. Next, the cells were washed with PBS, resuspended in 1× binding buffer, and stained with FITC-labeled annexin V and propidium iodide (PI) for 15 min at room temperature in the dark. Further, the cells were analyzed using a flow cytometer (Beckman Coulter Gallios, Fullerton, California, USA).

DNA fragmentation analysis

The effect of I3C on DNA fragmentation was examined using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay with TdT enzyme and the fluorometric TUNEL system (Promega, Fitchburg, WI). LoVo cells were seeded in six-well plates (2 × 10⁵ cells/well), cultured for 24 h, and treated with 0, 200, 400, and 600 μM of I3C for 24 h. Next, the cells were fixed with 4% formaldehyde (pH 7.40) for 25 min at 4°C and washed twice with PBS for 5 min. The cells were then permeabilized using 0.2% Triton X-100 for 5 min at room temperature. Further, the cells were washed twice with PBS for 5 min and treated with 50 µL of TdT incubation buffer containing nucleotide mix and TdT enzyme for 1 h at 37°C in the dark. The cell nuclei were stained with a 10 mM Hoechst stain solution for 10 min and the labeled strand breaks were detected using a fluorescence microscope (CKX53; Olympus, Shinjuku, Tokyo, Japan).

Cell cycle analysis

The cell cycle was analyzed using a flow cytometer (Becton Dickinson FACS). The cells were seeded at a density of 3 × 10⁵ cells in six-well plates and treated with different concentrations of I3C (0, 200, 400, and 600 μM) for 24 h. Next, the cells were fixed with cold 70% ethanol overnight at −20°C and incubated with cell cycle reagent containing PI (50 µg/mL) and RNase A (200 μg/mL) for 30 min at 37°C in the dark. Flow cytometric data were analyzed using the CXP software program (Beckman Coulter) and represented as the proportion of cells at different cell cycle phases.

Analysis of reactive oxygen species (ROS) level

To measure the intracellular accumulation of ROS in the I3C-treated LoVo cells, 5-(and-6)-carboxy-20,70–dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich), a stable nonpolar dye that readily diffuses into cells, was used. The cells were treated with 500 μM of I3C in the presence or absence of 10 mM N-acetylcysteine (NAC) for 24 h. Next, the cells were incubated with 10 mM DCF-DA for 30 min at 37°C in the dark and washed twice with PBS. The ROS levels were measured using a flow cytometer (Beckman Coulter).

WST-1 assay

The effect of I3C-induced ROS production on the proliferation of LoVo cells was examined using the WST-1 assay. The cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultured for 24 h. Next, the cells were treated with 500 μM of I3C in the presence or absence of 10 mM NAC for 24 h. To each well, 10 µL of EZ-cytox (DoGenBio, Korea) was added and the cells were incubated for 1 h at 37°C. The absorbance of the samples at 450 nm was quantified using a microplate reader.

Western blotting

LoVo cells were harvested and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) containing protease inhibitor (Sigma-Aldrich) and 10 mM phenylmethyl-sulfonyl fluoride (Sigma-Aldrich). The proteins were loaded onto a NuPAGE Bis-Tris Gel (Invitrogen) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 120 V for 2 h. The resolved proteins were electroblotted onto a nitrocellulose membrane at 55 V for 2 h. The membrane was blocked with 3% bovine serum albumin (Bovogen, 12 Williams Ave, Keilor East VIC 3033, Australia) and incubated with the following primary antibodies overnight at 4°C: anti-PARP, anti-cleaved PARP, anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7, anti-caspase-9, anti-cleaved caspase-9, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Bim, anti-p27, anti-p53, anti-phospho-p53, and anti-β-actin antibodies. Next, the membrane was washed thrice with Tris-buffered saline containing Tween-20 (TBS-T) and incubated with the secondary antibodies for 1 h at room temperature. The membrane was washed thrice with TBS-T and the protein signals were detected using an enhanced chemiluminescence reagent (iNtRON Biotechnology, South Korea) and the Chemi-Doc detection system (Bio-Rad, Hercules, CA, USA).

Statistical analysis

All data were obtained from at least three independent experiments. The data were analyzed using the One-way ANOVA test. The differences were considered significant at p < 0.05.

Results

I3C suppressed the proliferation of LoVo cells

The effect of I3C on the viability of LoVo, HCT-15, and HCT116 cells was examined using the MTT assay. There was no significant difference in viability of HCT-15 and HCT116 cells treated with I3C for 24 h. However, I3C concentration-dependently and time-dependently decreased the viability of LoVo cells (Figure 2(a)). Cell viability of LoVo cells treated with I3C for 48 h was much lower than that of HCT-15 and HCT116 cells treated with I3C for 48 h. The half-maximal inhibitory concentration value of I3C against LoVo cells after treatment for 48 h was 456 μM. The results of the cell counting assay also indicated the antiproliferative effect of I3C on LoVo cells (Figure 2(b)). The antiproliferative effects of I3C on LoVo cells were further examined using the colony formation assay. Treatment with I3C dose-dependently decreased the number of colonies (Figure 2(c) and (d)). These findings suggest that I3C exerts antiproliferative effects on LoVo cells.

Figure 2.

Effects of indole-3-carbinol (I3C) on the viability of LoVo cells. (a) The growth-inhibitory activity of I3C against LoVo, HCT-15, and HCT116 cells was examined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. These cells were treated with various concentrations of I3C for 24 and 48 h. I3C dose-dependently and time-dependently decreased the viability of LoVo cells. Data are presented as mean ± standard error of mean. *p < 0.05 compared with the control cells. (b) Cell counting was performed using a hemocytometer. LoVo cells were treated with 200, 400, and 600 μM of I3C for 24, 48, and 72 h. I3C dose-dependently decreased the number of LoVo cells. Data are presented as mean ± standard deviation. *p < 0.05 compared with the control cells. (c) Effects of I3C on the colony formation ability of LoVo cells. LoVo cells were treated with 50, 100, 150 μM of I3C for 24 h. I3C dose-dependently decreased the number of colonies. These results demonstrated that I3C exerts an antiproliferative effect on LoVo cells. (d) The number of colonies in the I3C-treated group was compared with the control (DMSO) group. Data are presented as mean ± standard deviation. The results were obtained from three independent experiments and the error bars indicate standard deviations. * p < 0.05 compared with the control cells.

I3C altered the morphology and suppressed the migration of LoVo cells

The effect of different concentrations (0, 200, and 400 μM) of I3C on the morphology of LoVo cells was examined using bright-field microscopy. Treatment with I3C affected the morphology of LoVo cells (Figure 3(a)), exhibited decreased growth and round morphology. Additionally, I3C dose-dependently and time-dependently inhibited the cell adherence. The effect of I3C on the migration of LoVo cells was examined using the wound-healing assay. LoVo cells were treated with various concentrations (0, 200, and 400 μM) of I3C for 24 h and 48 h. In the control group (DMSO-treated cells), the wound area decreased with time. However, treatment with 200 and 400 μM of I3C did not decrease the wound area with time (Figure 3(b)). This indicated that I3C inhibited the migration and invasion of LoVo cells.

Figure 3.

Morphological observation and wound-healing assay performed using a bright-field microscope. (a) The effect of indole-3-carbinol (I3C) treatment on the morphology of LoVo cells varied after 24 and 48 h of treatment. Magnification: 100×. (b) Wound-healing assay of the I3C-treated LoVo cells. I3C dose-dependently decreased the number of cells migrating to the wound. Magnification: 40×.

I3C induced apoptosis in LoVo cells

Annexin V-PI staining identifies cells with exteriorized phosphatidylserine from the inner side of the cell membrane to the cell surface, which is one of the hallmarks of early apoptosis. I3C dose-dependently increased the proportion of apoptotic cells. The proportions of viable cells after treatment with 0, 200, 400, and 600 μM of I3C for 24 h were 92.26%, 89.93%, 82.96%, and 42.19%, respectively, whereas those of apoptotic cells were 6.83%, 8.67%, 12.89%, and 47.48%, respectively (Figure 4(a) and (b)). These findings demonstrated that I3C induced apoptosis in LoVo cells.

Figure 4.

Flow cytometric analysis of LoVo cells double-stained with annexin V and propidium iodide (PI). (a) The fluorescence intensities of fluorescein isothiocyanate-labeled annexin V (X-axis) and PI (Y-axis) are represented in the logarithmic scale. LoVo cells were treated with different concentrations of indole-3-carbinol (I3C) (0, 200, 400, and 600 μM) for 24 h. I3C dose-dependently induced apoptosis in LoVo cells. Scatter plots represent the distribution of annexin V/PI staining in the control and I3C-treated LoVo cells. The cells were categorized as “viable” (lower left), “early apoptotic” (lower right), and “late apoptotic” (upper right). (b) Quantitative analysis of the proportion of apoptotic cells after treatment with I3C for 24 h. Data are represented as mean ± standard deviation. *p < 0.05 compared with control cells.

I3C induced DNA fragmentation in LoVo cells

The annexin V and PI staining results were further confirmed using the TUNEL assay, which measures DNA cleavage, a characteristic feature of apoptotic cells. LoVo cells were treated with various concentrations of I3C (0, 200, 400, and 600 μM) for 24 h and analyzed using a fluorescence microscope. The control group yielded negative results in the TUNEL assay. In contrast, I3C dose-dependently increased DNA fragmentation (observed as green fluorescence) in LoVo cells (Figure 5). Additionally, western blotting analysis revealed that treatment with I3C increased the expression levels of cleaved PARP, a characteristic event of apoptosis (Figure 8(a)). These findings indicated that I3C promoted DNA damage in LoVo cells.

Figure 5.

Effects of indole-3-carbinol (I3C) on DNA fragmentation examined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay. LoVo cells were treated with 0, 200, 400, and 600 μM of I3C for 24 h. DNA strand breaks were visualized using fluorescence microscopy (Magnification: 100×). Blue fluorescence represents nuclei, while green fluorescence represents DNA fragmentation. The merged images show blue-stained nuclei with green-stained nicked DNA. I3C induced DNA fragmentation in the nuclei of LoVo cells.

I3C arrested the cell cycle of LoVo cells

The effect of I3C on the cell cycle of LoVo cells was examined using flow cytometry with PI staining. LoVo cells were treated with various concentrations of I3C (0, 200, 400, and 600 μM) for 24 h. Treatment with I3C dose-dependently increased the proportion of cells at the sub-G1 phase, which represents the late apoptotic stage. The proportions of cells at the sub-G1 phase after treatment with 0, 200, 400, and 600 μM were 0.87%, 0.98%, 1.32%, and 2.32%, respectively (Figure 6(a)). Additionally, treatment with I3C increased the proportion of cells at the G0/G1 phase. The proportions of cells at the G0/G1 phase after treatment with 0, 200, 400, and 600 μM of I3C were 20.21%, 23.16%, 28.7%, and 29.47%, respectively (Figure 6(a)). These results suggest that I3C induced apoptosis and G0/G1 arrest in LoVo cells.

Figure 6.

Effects of indole-3-carbinol (I3C) treatment for 24 h on the LoVo cell cycle. (a) Analysis of the cell cycle. I3C dose-dependently increased the proportion of cells at the sub-G1 and G0/G1 phases. (b) Quantitative analysis of fluorescence intensity of the cells at the sub-G1 phase. Data are represented as mean ± standard deviation. *p < 0.05 compared with the control cells. (c) Quantitative analysis of fluorescence intensity of the cells at the G0/G1 phase. Data are represented as mean ± standard deviation. *p < 0.05 compared with the control cells.

I3C enhanced ROS production and inhibited the proliferation of LoVo cells

I3C-mediated intracellular ROS generation in LoVo cells was measured using the DCF-DA assay. The cells were treated with 500 μM of I3C and NAC for 6 h. Flow cytometric analysis revealed that the mean fluorescence intensity (MFI) values of control, NAC-treated, I3C-treated, and I3C and NAC-treated cells were 74, 65.5, 113, and 92.7, respectively (Figure 7(a)). The MFI values in the I3C-treated cells were higher than those in the control cells and I3C and NAC-treated cells. Interestingly, the viability of 500 μM I3C-treated group was 58%, whereas that of I3C/NAC-treated group was similar to control group (Figure 7(c)). These results indicated that I3C promotes intracellular ROS generation, which results in the suppression of LoVo cell proliferation.

Figure 7.

Effects of indole-3-carbinol (I3C) on the production of reactive oxygen species (ROS) in LoVo cells. (a) The cells were treated with 500 μM I3C and/or 10 mM N-acetylcysteine (NAC) for 6 h. The ROS levels were measured using 5-(and-6)-carboxy-20,70–dichlorofluorescein diacetate staining. (b) Quantitative analysis of fluorescence intensity. Data are represented as mean ± standard deviation. *p < 0.05 and #p < 0.05 compared with the control and I3C-treated cells, respectively. (c) The viability of LoVo cells treated with 500 μM I3C and/or 10 mM NAC for 48 h was examined using the WST-1 assay. Data are represented as mean ± standard deviation. *p < 0.05 and #p < 0.05 compared with the control and I3C-treated cells, respectively.

I3C affected the expression of proteins involved in apoptosis and cell cycle

The expression levels of proteins in the I3C-treated LoVo cells were examined using western blotting to examine the molecular mechanisms of I3C. The expression levels of PARP, cleaved PARP, caspase-3, caspase-7, caspase-9, cleaved caspases, Bcl-2, Bcl-xl, Bax, Bim, p27, p53, and phospho-p53 were examined after treating LoVo cells with DMSO and 500 µM I3C for 48 h (Figure 8(a)). I3C upregulated the expression levels of pro-apoptotic proteins, such as cleaved PARP, cleaved caspases, Bax, and Bim, and downregulated the expression levels of anti-apoptotic proteins, such as Bcl-xl and Bcl-2. Furthermore, the expression levels of proteins that inhibit the cell cycle, such as p53 and p27 were upregulated upon treatment with I3C (Figure 8(b)). These findings suggest that I3C treatment promotes apoptosis in LoVo cells through caspases and regulates the expression of proteins related to the cell cycle.

Figure 8.

Effects of indole-3-carbinol (I3C) on the expression of proteins involved in different apoptotic pathways examined using western blotting. The cells were treated with dimethyl sulfoxide and 500 µM of I3C for 48 h. (a) The expression levels of poly(ADP-ribose) polymerase (PARP), cleaved PARP, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-9, cleaved caspase-9, Bcl-2, Bcl-xl, Bax, and Bim. β-actin was used as a loading control. (b) The expression levels of p27, p53, and p-p53. β-actin was used as a loading control. I3C altered the expression level of intracellular proteins associated with apoptosis and cell cycle in LoVo cells. Quantitative analysis of expression levels of proteins involved in apoptosis (c) and cell cycle regulation (d). The expression levels of target proteins were normalized to those of β-actin. Data are represented as mean ± standard deviation. *p < 0.05 compared with the control cells.

Discussion

Globally, colorectal cancer is one of the most commonly diagnosed cancers.³⁹ The current therapeutic strategies for colorectal cancer involve surgical resection and chemotherapy. However, patients with colorectal cancer exhibit poor prognosis. Additionally, drug toxicity and drug resistance are the major obstacles for treating patients with advanced-stage colorectal cancer.⁴⁰ Furthermore, conventional chemotherapy is associated with a low response and high toxicity.⁴¹ Thus, natural compounds with limited side effects are potential alternative therapeutic agents for cancer.⁴²

I3C is a phytochemical found in cruciferous vegetables.⁴³ Various studies have reported the therapeutic effects of I3C, such as cancer-preventive, anti-inflammatory, and anti-oxidative effects.^13,43,44 Additionally, I3C is reported to exhibit growth-inhibitory activity against various cancers, such as lung and liver cancers and glioblastoma. I3C targets the signaling pathways involved in cell proliferation and survival, which are upregulated in the cancer cells as a strategy to acquire resistance to chemotherapy.⁹ For example, I3C exhibits growth-inhibitory activity against cancer cells through the activation of caspases, Akt-NFκB signaling, cyclin-dependent kinase activities, and endoplasmic reticulum stress.^8,9,45,46 Additionally, I3C induces apoptosis in the liver cancer cells through the upregulation of Hsp27. In the osteosarcoma cells, I3C induces the FOXO3-mediated apoptotic signaling pathways.^10,11 The broad-spectrum anticancer activities and low toxicity of I3C indicate the therapeutic potential of I3C in cancer.⁹ However, there are limited studies on the cytotoxic effects of I3C on colon cancer cells and the underlying mechanisms. This study examined the growth-inhibitory activities of I3C against the human colorectal cancer cell line (LoVo) and examined the underlying mechanisms in colon cancer cell.

The effect of I3C on the proliferation of LoVo cells was examined using the MTT, cell counting, and colony formation assays. I3C exerted a cytotoxic effect on LoVo cells in dose- and time-dependent manners. Additionally, I3C altered the morphology and inhibited the migration of LoVo cells, which were examined using microscopy and wound-healing assay, respectively.

Next, annexin V/PI staining was performed to examine the effect of I3C on apoptosis. I3C dose-dependently increased the proportion of apoptotic cells. The effects of I3C on DNA fragmentation and cell cycle were examined using TUNEL assay and PI staining, respectively.⁴⁷ The I3C-treated LoVo cells exhibited enhanced DNA fragmentation. Additionally, I3C dose-dependently increased the proportion of cells at the sub-G1 phase. Furthermore, the proportion of cells at the G0/G1 phase increased upon treatment with I3C, which indicated that I3C arrested the cell cycle at the G0/G1 phase. The effect of I3C on ROS production in LoVo cells was examined using the DCF-DA assay.

Treatment with I3C increased the ROS levels in LoVo cells. The viability of I3C-treated LoVo cells was lower than that of I3C and NAC-treated LoVo cells. These results indicate that I3C decreased cancer cell viability by promoting the production of ROS. Several studies have demonstrated that I3C and its derivatives induce apoptosis through the induction of intracellular ROS production.^48–50 Therefore, the growth-inhibitory activity of I3C against LoVo cells may be attributed to the enhanced production of intracellular ROS, which are potent mediators of apoptosis.

Chemotherapeutic drugs for colorectal cancer induce apoptosis through the cleavage of PARP, downregulation of anti-apoptotic Bcl-2 protein, and activation of p53, Bax, caspase-3, caspase-7, and caspase-9 in the cancer cells.^51–53 For example, pinocembrin, a phytochemical, triggers Bax-dependent mitochondrial apoptosis in the colon cancer cells.⁵⁴ Therefore, the effect of I3C on the expression levels of proteins associated with the apoptotic signaling pathway was examined using western blotting.

PARP, which plays a crucial role in DNA repair, is activated immediately after DNA strand breaks.⁵⁵ The cleavage of PARP is critical for apoptosis as it inhibits the cellular response to DNA damage caused by external genotoxic substances or apoptotic signaling pathways.⁴⁶ Treatment with I3C downregulated the expression level of PARP and upregulated the expression level of cleaved PARP, which suggested that PARP was cleaved in the I3C-treated LoVo cells.

The expression levels of Bcl-2 and caspase family members were also examined in this study. Bcl-2 family members, which are central regulators of apoptosis comprising both pro-apoptotic and anti-apoptotic proteins, mediate the activation of intrinsic apoptotic pathways that results in the cleavage of caspases.⁵⁶ Caspases are cleaved by an upstream caspase or another protease.⁵⁷ The expression levels of Bcl-2 and Bcl-xl were downregulated, whereas those of Bax and Bim were upregulated in the I3C-treated LoVo cells. Additionally, treatment with I3C downregulated the expression levels of procaspase-3, procaspase-7, and procaspase-9 and upregulated the expression levels of cleaved caspase-3, caspase-7, and caspase-9. This suggested that I3C activates caspase-3, caspase-7, and caspase-9 in LoVo cells.

In this study, the expression levels of cell cycle regulatory proteins, such as p53 and p27 were also examined. p53, a tumor suppressor, is activated by various cellular stresses, including DNA damage and hypoxia. In response to these stresses, p53 regulates the transcription of genes involved in DNA damage repair, cell cycle arrest, and apoptosis, such as p27 and Bax.⁵⁸ The functions of p53 are dependent on the phosphorylation of several serine residues.⁵⁹ The phosphorylation of p53 at Ser15 promotes cell cycle arrest, while that at Ser20 inhibits the degradation of p53, which results in the accumulation of p53 and upregulation of the cell cycle inhibitor p27.^60,61 Similarly, the phosphorylation of p53 at Ser46 regulates apoptosis by enhancing the expression of several apoptosis-inducing genes.⁶² The expression levels of phosphorylated p53 (Ser15, Ser20, or Ser46) and p53 were upregulated in the I3C-treated LoVo cells.

In summary, this study demonstrated that I3C suppressed the growth and migration, induced apoptosis, DNA fragmentation, and cell cycle arrest, and elevated ROS levels in LoVo cells. Furthermore, I3C activated proteins involved in the apoptotic signaling pathways, such as PARP, Bcl-2 family, caspase-3, caspase-7, caspase-9, and p53. These findings indicated that the mechanism underlying I3C-induced apoptosis involves p53 activation, which leads to the upregulation of Bax and the activation of the caspase cascade in LoVo cells. The findings of this study provide experimental evidence for the chemotherapeutic potential of I3C in human colorectal carcinoma.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Gachon University (Grant Number: GCU-2019-0823).

ORCID iD

S-H Park

MJ Nam

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