Anticarcinogenic effect of indole-3-carbinol (I3C) on human hepatocellular carcinoma SNU449 cells

Abstract

Many cruciferous vegetables, including cabbage, contain indole-3-carbinol (I3C), which is a known anticarcinogen. However, the anticarcinogenic effects of I3C on liver cancer have not been investigated. Therefore, this study was conducted to evaluate the anticarcinogenic effects of I3C in human hepatocellular carcinoma (HCC) SNU449 cells. The results of MTT and WST-1 assays indicated that treatment of SNU449 cells with I3C decreased viability in dose- and time-dependent manners, while colony formation assays indicated that I3C also inhibited proliferation of SNU449 cells. Moreover, fluorescence-activated cell sorter analysis showed that I3C induced apoptosis in SNU449 cells in dose- and time-dependent manners. Furthermore, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling revealed that I3C induced DNA fragmentation in SNU449 cells in a time-dependent manner, while Western blotting showed that apoptotic proteins such as p53, cleaved PARP, caspase-3, and caspase-7 were activated in SNU449 cells following treatment with I3C. Finally, reactive oxygen species-related protein peroxiredoxin-1 and thioredoxin-1 expression decreased in I3C-treated SNU449 cells. The aim of our study is to investigate the unknown mechanisms responsible for the apoptotic effects of I3C on human HCC SNU449 cells, and the results suggest that I3C may be useful for the prevention and treatment of liver cancer.

Introduction

In some regions of Asia and Africa, cancer of the liver is the most common cancer, where it accounts for 15% of all deaths. ¹ Moreover, the prevalence of hepatocellular carcinoma (HCC) is increasing rapidly in Western countries, ² especially in less developed countries, where HCC is a major cause of death among males. ³ Therefore, HCC is considered a medical issue that needs to be addressed globally. Although many treatments for HCC have been developed, and these therapies have several drawbacks, including detrimental effects of radiation therapy on normal liver tissue. ⁴ Accordingly, there is significant interest in developing novel therapeutic agents for the treatment of liver cancer with fewer side effects.

Biologically active compounds derived from various plants have played a crucial role in producing medicines to alleviate pain. ⁵ Indeed, plant-derived compounds play a crucial role in the treatment of various kinds of cancers. ⁶ Plants produce various phytochemicals that facilitate growth and act against pathogens, predators, or competitors. Phytochemicals can be divided into flavonoids and polyphenols, and many studies have indicated that phytochemicals have anticancer effects against various cancer cells.

3,3′-Diindolylmethane (DIM) is a typical phytochemical compound involved in cruciferous vegetables, including broccoli and cabbage. Many recent studies have shown that DIM exerts anticancer effects against many cancer cells through various intracellular mechanisms. Additionally, DIM is known to exert antiproliferation effects through the activation of reactive oxygen species (ROS) and endoplasmic reticulum stress pathways. ⁷ Moreover, DIM induces cell death through the activation of protective autophagy and the AMPK signaling pathway in prostate cancer cells. ⁸ DIM also exerts inhibitory effects against epithelial–mesenchymal transition and suppresses tumor growth in xenograft mouse models of breast cancer. ⁹

Ganoderma lucidum is an oriental fungus that has been widely used to promote health in many Asian countries, including China and Japan. ¹⁰ Recent studies have indicated that G. lucidum is an effective anticancer compound. ¹¹ Treatment of human breast adenocarcinoma MCF-7 cells with G. lucidum total triterpenes changes the expression levels of apoptotic proteins, including cyclin D1, Bcl-2, Bcl-xL, Bax, and Caspase-9, in a concentration-dependent manner. ¹²

Indole-3-carbinol (I3C) is a typical phytochemical found naturally in cruciferous vegetables such as kale, sprouts, and cabbage, ¹³ which is known to suppress the proliferation of many types of cancers, including human breast, colon, prostate, and endometrial cancers. ¹⁴ Indeed, many studies have demonstrated the anticancer effects of I3C in a variety of cancer cells. ^{15 –18} Moreover, I3C can act as an antioxidant in osteoblasts by suppressing cytotoxicity and can induce apoptosis in osteoblastic cells. ¹⁹ I3C also exerts significant suppressive effects on prostate cancer cells in vitro and in vivo. ²⁰ Moreover, I3C induces apoptosis in human lung epithelial carcinoma A549 cells by regulating the p53 and the caspase-8 pathway. ²¹ However, the anticancer effects of I3C in human liver cancer have not been investigated.

Most studies indicated the antitumor effects of I3C in vivo or in vitro, while a few showed the evidence of carcinogenic effects of I3C depending on the exposure protocol and species. ²² In rats, I3C induced tumor progression through ROS-mediated Akt activation mechanism that inhibits the TGF-β/Smad signaling. Research showed that I3C promoted hepatocarcinogenesis in animal model. ²³

Apoptosis is the process of programmed cell death in multicellular organisms. Several morphological and physiological changes occur during apoptosis, including cell shrinkage, exposure to phosphatidylserine, and chromosomal DNA fragmentation. ²⁴ It is well-known that the mechanisms of apoptosis are complicated, involving many signaling pathways. ²⁵ A typical mechanism of apoptosis is mitochondria-mediated apoptosis. Bcl-xL is a transmembrane protein located in the mitochondria that plays an essential role in the regulation of apoptosis by suppressing the release of cytochrome c, which induces caspase activation. ²⁶ Other regulatory steps of mitochondria-mediated apoptosis include the formation of apoptosome. Cytochrome c is released from mitochondria to the cytoplasm, where it binds to apoptotic protease activating factor-1 (Apaf-1). ²⁷ When activated Apaf-1 forms apoptosome, it activates initiator procaspase-9, leading to apoptosis. ²⁷ During the apoptotic process, cleavage of poly (ADP-ribose) polymerase (PARP) is a hallmark event because DNA damage leads to activation of PARP. ²⁸

In this study, we investigated the antiproliferation effects of I3C in human HCC SNU449 cells and the mechanisms related to I3C-induced apoptosis in SNU449 cells. Specifically, we investigated the induction of apoptosis as well as the molecular basis for the induction of apoptosis in I3C-treated human HCC SNU449 cells. The results of our study indicated that I3C inhibited cell growth, induced DNA fragmentation, and activated apoptosis-related proteins, including caspase-3, caspase-7, PARP, thioredoxin-1, and peroxiredoxin-1.

Materials and methods

Reagents

I3C and crystal violet were purchased from Sigma Aldrich (St Louis, Missouri, USA). I3C was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. ²⁹ Crystal violet was dissolved in distilled water (1 g/100 ml). ³⁰

Cell culture

The human HCC cell line, SNU449, was purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured under standard conditions (5% carbon dioxide (CO₂), 37°C, and 95% humidity) in Roswell Park Memorial Institute medium 1640 (GIBCO, Grand Island, New York, USA) containing 10% heat-inactivated fetal bovine serum (GIBCO) and 1% penicillin/streptomycin (GIBCO). The cells were harvested at 85% confluence using 0.25% (w/v) trypsin–ethylenediaminetetraacetic acid (Sigma-Aldrich) and then seeded in culture plates and incubated for 24 h. After 24 h, the cells were treated with either DMSO (Sigma-Aldrich) or various concentrations of I3C.

MTT assay

SNU449 cells were seeded in 96-well plates (5 × 10³ cells/well) and incubated for 24 h under standard conditions (5% CO₂, 37°C, and 95% humidity), after which they were treated with various concentrations of I3C (DMSO, 200, 400, and 600 µM). After 24 and 48 h of treatment, 20 µL of MTT solution (2.5 mg/mL in phosphate-buffered saline (PBS)) was added to each well, and the cells were incubated for 2 h under standard conditions. ²⁴ After 2 h of incubation, the medium was removed and 200 µL of DMSO was added to each well to dissolve the formazan. The plates were then placed on a shaker for 15 min at 80 r/min, after which the optical density (OD) by spectrophotometry (PerkinElmer, Waltham, MA, USA) at 570 nm.

WST-1 assay

The WST-1 assay was conducted according to the manufacturer’s protocol. Briefly, SNU449 cells were seeded in 96-well plates (5 × 10³ cells/well) and incubated for 24 h under standard conditions (5% CO₂, 37°C, and 95% humidity). The cells were then treated with various concentrations of I3C (DMSO, 200, 400, and 600 µM). After 24 and 48 h of treatment, 10 µL of EZ-cytox (DoGenBio, Korea) was added to each well, and the samples were incubated for 1 h under standard conditions. Following incubation, the OD was measured by spectrophotometry at 450 nm.

Colony formation assay

Colony formation assay was conducted as previously described. ³⁰ Briefly, cells were seeded in 6 cm dishes (5 × 10³) and incubated for 24 h under standard conditions, after which they were treated with I3C (DMSO and100 µM) and incubated for an additional 24 h. The medium was then exchanged for new media containing 10% heat-inactivated fetal bovine serum (GIBCO) and 1% penicillin/streptomycin (GIBCO), and the samples were incubated for 2 weeks. Next, colonies were washed twice with PBS for 5 min each and fixed with 4% formaldehyde. Following fixation, the colonies were washed twice with PBS for 5 min each and stained with 1% crystal violet (Sigma Aldrich) solution for 30 min. After staining, the colonies were washed with distilled water, and the number of colonies was counted.

Annexin V/PI staining

We conducted Annexin V/PI staining according to our previous study ³¹ using a FITC Annexin V apoptosis detection kit (BD Bio-Sciences, Franklin Lakes, New Jersey, USA) to measure I3C-induced apoptosis in SNU449 cells. Briefly, cells were seeded in a six-well plate (3 × 10⁵ cells/well) and incubated for 24 h, after which they were treated with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 and 48 h. The cells were then washed with PBS and resuspended with 1× binding buffer containing FITC Annexin V for 15 min at room temperature (25°C) in the dark. Finally, cells were analyzed by flow cytometry (Beckman Coulter Gallios, Fullerton, California, USA).

TUNEL assay

We conducted a terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay according to the manufacturer’s protocol in conjunction with the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) to detect DNA fragmentation induced by I3C in SNU449 cells. Briefly, SNU449 cells were seeded in six-well plates (3 × 10⁵ cells/well) and then incubated for 24 h. Next, cells were washed twice with PBS for 5 min each and then fixed with 4% formaldehyde for 25 min at 4°C. Following fixation, the cells were washed twice with PBS for 5 min, after which they were permeabilized using Triton X-100 (0.2%, PBS) for 10 min. Following permeabilization, the cells were treated with a 50-µL TdT enzyme buffer for 1 h under standard conditions. Next, 10 µM Hoechst solution (in PBS) was added for 10 min, and DNA fragmentation was finally visualized by fluorescence microscopy (Nikon Eclipse TE 2000-U, Tokyo, Japan).

Western blotting

Western blotting was conducted to compare the protein expression between the control (DMSO) group and the I3C-treated (100, 200, and 300) groups. Briefly, SNU449 cells were seeded in 10 cm dishes (1.5 × 10⁶) and then incubated for 24 h under standard conditions. The cells were subsequently treated with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 h, after which they were harvested and lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA) containing protease inhibitor (Sigma-Aldrich) and phenylmethylsulfonyl fluoride (Sigma-Aldrich). The concentration of pro-tein was subsequently measured using the Qubit™ Fluorocytometer (Invitrogen, Carlsbad, CA, USA), after which proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 V for 2 h and then transferred to an Immobilon-P PVDF membrane (Sigma-Aldrich) at 55 V for 2 h. The membranes were subsequently blocked with 2% bovine serum albumin (BOVOGEN Biologicals, Australia) for 1 h, ³² after which they were incubated with pri-mary antibodies against various proteins (Akt, pAkt, p53, pJNK, phospho-(Ser) 14-3-3 binding motif, Bcl-xL, c-Jun, PARP, cPARP, caspase-3, caspase-7, thioredoxin-1, peroxiredoxin-1, Cell Signaling Technology) at 4C overnight. Finally, the membranes were washed with TBS-Tween (TBS-T), and fluorescence was measured using a Western blot detection system (YMS, Korea) and a Chemi-doc detection system (BioRad, USA).

Statistical analysis

The values shown are representative of three experiments. One-way analysis of variance followed by Bonferroni’s post hoc test was used for statistical analysis, and a p < 0.05 was considered significant statistically.

Results

I3C inhibited proliferation of SNU449 cells

To examine the antiproliferative effects of I3C against liver cancer, we treated human HCC SNU449 cells with various concentrations (0−600 µM) of I3C for 24 and 48 h and measured their survival rate through MTT, WST-1, and colony formation assays. The MTT assay results indicated that the IC50 (concentration that inhibits 50% of cell survival) value of I3C was 488 µM after 24 h. Additionally, the MTT and WST-1 results showed that I3C had antiproliferative effects against the SNU449 cells in dose- and time-dependent manners (Figure 1(a) and (b)). We also found that the number of colonies decreased in 100 µM I3C-treated SNU449 cells relative to the control (DMSO) group (Figure 1(c)). Taken together, these results suggest that I3C exerted anticancer effects in human HCC SNU449 cells.

Figure 1.

Viability of I3C-treated SNU449 cells was investigated. SNU449 cells were treated with different concentrations of I3C (DMSO, 200, 400, and 600 µM) for 24 and 48 h. (a) MTT assay: Viability of SNU449 cells decreased in dose- and time-dependent manners. (b) WST-1 assay: Viability of SNU449 cells decreased in dose- and time-dependent manners. (c) Colony formation assay: The number of colonies in the 100 µM I3C-treated group was compared with the control (DMSO) group. The results indicated that I3C exerts antiproliferation effect on SNU449 cells. (d) Each bar represents the mean ± SEM. The results are based on triplicate analyses and error bars show the standard deviations (*p < 0.05). I3C: indole-3-carbinol; DMSO: dimethyl sulfoxide; SEM: standard error of mean.

I3C induced apoptosis in SNU449 cells

Exposure of phosphatidylserine on the cell surface is a typical feature of apoptotic cells ³³ that serves as an “eat me” signal, causing phagocytes to engulf the cells. ³⁴ The majority of classical apoptotic hallmarks can be examined by flow cytometry. ³⁵ We conducted an Annexin V/PI double-staining assay to examine I3C-induced apoptosis in SNU449 cells. Treatment with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 h decreased the proportions of intact cells from 92.49% to 85.00%, 78.80%, and 70.30%, respectively (Figure 2(a)). Treatment with different concentrations of I3C for 48 h also decreased the respective proportions of intact cells from 91.21% to 89.41%, 49.07%, and 37.80% (Figure 2(b)). different concentrations of I3C for 24 h increased the proportions of total apoptotic cells from 6.67% to 9.54%, 12.87%, and 19.28%, respectively (Figure 2(a)). Moreover, treatment with the test concentrations of I3C for 48 h increased the proportions of total apoptotic cells from 7.85% to 10.01%, 46.06%, and 50.11%, respectively (Figure 2(b)). Taken together, our results showed that the proportion of total apoptotic cells increased in response to I3C treatment in dose- and time-dependent manners.

Figure 2.

Annexin V/PI double staining was conducted by flow cytometry. FITC fluorescence (X axis) and PI fluorescence (Y axis) are represented as logarithmic fluorescence intensity. SNU449 cells were treated with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 and 48 h, which induced apoptosis in dose- and time-dependent manners. Upper left panel represents necrotic cells, the lower left live cells, the lower right early phase apoptotic cells, and the upper right late phase apoptotic cells. The rates of apoptotic cells (B2 and B4 section) are indicated. The results indicated that I3C induces apoptosis in SNU449 cells. DMSO: dimethyl sulfoxide; PI: propidium iodide; FITC: fluorescein isothiocyanate; DMSO: dimethyl sulfoxide; I3C: indole-3-carbinol.

I3C induced DNA fragmentation in SNU449 cells

Apoptosis is characterized by morphological and physiological features, such as cell shrinkage, exposure of phosphatidylserine on the cell surface, and fragmentation into membrane-bound apoptotic bodies. ³³ DNA cleavage is also typical of apoptotic cells. ³⁶ A TUNEL assay was conducted to examine I3C-induced DNA fragmentation in SNU449 cells. We found that I3C induced an increase of green fluorescence in dose- and time-dependent manners (Figure 3), indicating that DNA damage in the nucleus had occurred. Evaluation of the merged images showed DNA-damaged nuclei in SNU449 cells. During the apoptotic process, PARP is activated following DNA damage, ²⁸ and PARP cleavage is a hallmark event of apoptosis. Western blot analysis indicated an increase in the levels of cleavage of PARP in I3C-treated SNU449 cells (Figure 4(c)), which is consistent with the TUNEL assay results. Taken together, our results suggest that I3C induced DNA fragmentation and DNA damage in SNU449 cells.

Figure 3.

DNA damage caused by I3C in SNU449 cells. DNA strand breaks were visualized by TUNEL assay. We treated SNU449 cells with DMSO or 200 µM of I3C for 24 and 48 h. Blue fluorescence represents nuclei and green fluorescence represents DNA strand breaks. The merged image indicates the DNA-damaged nuclei. The results indicate that DNA in nuclei was fragmented by I3C in SNU449 cells. I3C: indole-3-carbinol. TUNEL: terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling; DMSO: dimethyl sulfoxide.

Figure 4.

Western blotting was performed to investigate different pathways related to apoptosis. SNU449 cells were treated with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 h and analyzed by Western blotting. (a) Akt, pAkt, and p53 expression. (b) pJNK, p14-3-3, Bcl-xL, and c-Jun expression. (c) PARP, cPARP, caspase-3, and caspase-7 expression. (d) Thioredoxin-1 and peroxiredoxin-1 expression. These results indicated that I3C altered intracellular protein expression level in SNU449 cells. I3C: indole-3-carbinol; DMSO: dimethyl sulfoxide.

I3C activated the apoptotic pathway in SNU449 cells

Western blot analysis was conducted to examine the expression of apoptotic proteins in I3C-treated SNU449 cells. Treatment of cells with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 h revealed that Akt, pAkt, and p53 expression decreased in a dose-dependent manner (Figure 4(a)). We also analyzed the Jun N-terminal kinase (JNK) signaling pathway (Figure 4(b)). pJNK expression increased in I3C-treated SNU449 cells. Moreover, the levels of phospho-(Ser) 14-3-3 binding motif expression and antiapoptotic protein Bcl-xL expression decreased in I3C-treated SNU449 cells. We also observed significant upregulation of c-Jun. Furthermore, PARP, caspase-3, and caspase-7 expression decreased, whereas cleaved PARP (cPARP) expression increased in response to I3C treatment (Figure 4(c)). Finally, thioredoxin-1 and peroxiredoxin-1 expression decreased in response to I3C treatment in a dose-dependent manner (Figure 4(d)).

Discussion

Despite rapid progress in diagnosis and therapy, drug resistance is a major clinical problem during the treatment of human hepatocellular cancer. Therefore, it is essential to identify new agents for the effective treatment of cancer. We focused on I3C, a phytochemical found in cruciferous vegetables such as broccoli, cabbage, and sprouts. Specifically, we examined the anticancer effects of I3C in human HCC SNU449 cells.

First, we conducted MTT, WST-1, and colony formation assays to investigate the antiproliferative effects of I3C in SNU449 cells. Our results showed that I3C decreased viability in SNU449 cells in dose- and time-dependent manners (Figure 1(a) and (b)) and that the number of colonies was significantly decreased in the 100 µM I3C-treated group when compared with the control (DMSO) group (Figure 1(c)). These results indicate that I3C exerts antiproliferative effects in SNU449 cells.

We next conducted Annexin/PI staining to evaluate the percentages of apoptotic cells after treatment with different concentrations of I3C (DMSO, 100, 200, and 300 µM) for 24 and 48 h. Our results showed that the percentages of apoptotic cells increased in dose- and time-dependent manners, demonstrating that I3C induces apoptosis in SNU449 cells.

Many physiological changes occur in apoptotic cells, one of the most prominent being DNA fragmentation. ³⁷ In this study, a TUNEL assay was conducted to visualize DNA strand breaks in I3C-induced apoptotic SNU449 cells. Briefly, we treated SNU449 cells with DMSO and 200 µM I3C for 24 and 48 h. When compared with the control (DMSO) group, more green fluorescence was observed in the 200 µM I3C-treated group, demonstrating that I3C induces DNA fragmentation in SNU449 cells.

It is well-known that there are many factors involved in apoptosis signaling pathways in apoptotic cells. ³⁸ Therefore, we examined the level of protein expression related to apoptosis signaling pathways. The Akt pathway plays a crucial role in cell survival and proliferation and promotes cell survival through phosphorylation and inhibition of Forkhead transcription factor. ³⁹ Additionally, the Akt pathway is a target for cancer therapy, because it regulates the cell cycle and apoptosis. ⁴⁰ Our results indicated that Akt and pAKT expression decreased in I3C-treated SNU449 cells. We also examined the expression of p53, which is a well-known tumor suppressor protein regulated by Akt signaling pathways. ^{41 –43} Akt activates MDM2, which subsequently induces degradation of p53. ^43,44 However, our results indicated that even though the level of Akt and pAKT decreased, p53 expression level also decreased in I3C-treated SNU449 cells. In short, the level of p53 expression decreased in SNU449 cells induced to undergo apoptosis by I3C treatment. Further investigation is needed to elucidate the mechanism of p53 expression in I3C-treated SNU449 cells.

JNK also plays an essential role in apoptotic pathways. ⁴⁵ When compared with the control (DMSO) group, pJNK is increased in I3C-treated (100, 200, and 300 µM) SNU449 cells. However, there appears to be no dose dependence in the expression of pJNK in I3Ctreated groups. The phospho-(Ser) 14-3-3 binding motif is a site that interacts with other phosphorylated proteins, such as phosphorylated Bad and FOXO3. ⁴⁶ 14-3-3 protein binds to phosphorylated Bad and is then sequestered in the cytosol, ⁴⁷ where it plays a crucial role in protecting cells against stress-induced apoptosis. ⁴⁸ Our results showed that phospho-(Ser) 14-3-3 binding motif expression level was decreased in I3C-treated SNU449 cells in a dose-dependent manner.

Bcl-xL is a member of the Bcl-2 family that plays an essential role in the regulation of apoptosis. ⁴⁹ The antiapoptotic protein Bcl-xL suppresses the apoptosis signaling pathway by inhibiting the release of cytochrome c from mitochondria into the cytoplasm. ⁵⁰ We found that the level of Bcl-xL expression was decreased in I3C-treated SNU449 cells.

C-Jun is a transcription factor and a member of the AP-1 family that is normally involved in cell cycle and differentiation. ⁵¹ c-Jun induces apoptosis by activating death signaling that has been inhibited by Bcl-2. ⁵¹ We observed that I3C induced activation of c-Jun in SNU449 cells.

Caspases are protease enzymes that play significant roles in apoptosis signaling pathways. ⁵² To activate procaspases, these proteins require proteolytic activation. ⁵³ Once cleaved, these caspases induce apoptosis, ⁵⁴ with caspase-3 and caspase-7 being key mediators of apoptosis. ⁵⁵ We found that I3C decreased procaspase-3 and procaspase-7 in I3C-treated SNU449 cells, suggesting that caspase-3 and caspase-7 were cleaved in I3C-treated SNU449 cells.

We also investigated activation of PARP protein in I3C-treated SNU449 cells. PARP plays a crucial role in genome repair, ⁵⁶ and its cleavage indicates that cells lose the ability to repair DNA damage originating from either apoptotic signaling pathways or external genotoxic substances. ⁵⁷ We found that the level of PARP expression decreased and cPARP increased in I3C-treated SNU449 cells, suggesting that I3C induced PARP cleavage in SNU449 cells.

Thioredoxin-1 is a peroxidase that plays important roles in many biological processes, including redox signaling. ⁵⁸ In addition, thioredoxin-1 is an ASK1-binding protein known to be antiapoptotic. ⁵⁹ Peroxiredoxin is an antioxidant enzyme that is recharged by thioredoxin after reducing hydrogen peroxide. ⁶⁰ Peroxiredoxin-1 also plays a crucial role in the regulation of apoptosis, ⁶¹ while peroxiredoxin-1 knockdown leads to apoptosis, indicating that it has antiapoptotic functions, ⁶² Our results indicated that I3C decreased the level of thioredoxin-1 and peroxiredoxin-1 expression as apoptosis progressed.

There is a controversy when it comes to anticarcinogenic effects of I3C on cancer. A few studies showed that I3C can also promote carcinogenesis depending on specific conditions. ²² However, most studies provided clear evidence of anticarcinogenic effects of I3C on various cancers in vitro and in vivo. Our results also support the idea that I3C is a promising chemotherapeutic candidate for the prevention and treatment of liver cancer.

Conclusion

In this study, we investigated mechanistic insight into the molecular basis of I3C-induced apoptosis in human HCC SNU449 cells. Our results indicated that I3C has an antiproliferative effect on SNU449 cells. We found that I3C induces apoptosis in dose- and time-dependent manners in SNU449 cells as well as DNA cleavage. We also found that PARP, caspase-3, caspase-7, and c-Jun are activated and that the level of Bcl-xL, thioredoxin-1, and peroxiredoxin-1 decreased in I3C-treated SNU449 cells. However, to fully investigate the potential of I3C for use as an anticancer agent, further in-depth studies including animal experiments are required to facilitate development of therapeutic methods for the treatment of human HCC.

Supplementary material

HET785235_Supplementary_material - Anticarcinogenic effect of indole-3-carbinol (I3C) on human hepatocellular carcinoma SNU449 cells

HET785235_Supplementary_material for Anticarcinogenic effect of indole-3-carbinol (I3C) on human hepatocellular carcinoma SNU449 cells by CM Lee, S-H Park, and MJ Nam in Human & Experimental Toxicology