Induction of Apoptosis of DK-5-62, a Novel (-)-Catechin Derivative Through MAPKs Signaling Pathway in HCT116 Cells

Abstract

The present study was designed to investigate the molecular mechanisms of DK-5-62, a novel (-)-catechin derivative on HCT116 human colorectal cancer cells. DK-5-62 inhibited the proliferation in dose- and time-dependent manner accompanied by the morphological changes. Effects of DK-5-62 appeared to be mediated by the induction of apoptosis, as manifested through DNA-binding dye Hoechst 33258 staining. Analysis of the mechanism of these events indicated that DK-5-62-treated cells exhibited an increased ratio of Bax/Bcl-2, resulting in the activation of caspase-9, caspase-3, and poly-ADP-ribose polymerase in a dose-dependent manner. Moreover, DK-5-62-induced apoptosis was accompanied by phosphorylation of the mitogen-activated protein kinase family, c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase. These results suggest that HCT116 cells are moderately sensitive to growth inhibition by DK-5-62 via apoptosis, as evidenced by activation of ERK/p38/Bcl-2 family signaling, as well as alteration in caspase-9 and caspase-3.

INTRODUCTION

Colorectal cancer is the third most common cause of cancer mortality in the United States, with 53,200 deaths projected in 2020. Among people diagnosed with metastatic colorectal cancer, approximately 70% to 75% of patients survive beyond 1 year, 30% to 35% beyond 3 years, and fewer than 20% beyond 5 years from diagnosis. The primary treatment for unresectable metastatic colorectal cancer is systemic therapy as cytotoxic chemotherapy, biologic therapy such as antibodies to cellular growth factors, immunotherapy, and their combinations.¹ However, cytotoxic drugs and chemotherapeutic agents have been used with limited success due to their severe side effects² and hence, more effective and less toxic therapeutic agents are needed.

Apoptosis is a self-destruction mechanism involved in developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Disruption of the regulation of apoptosis may result in various diseases, including cancer.³ Two major apoptosis pathways have been identified: (a) extrinsic or death receptor pathways and (b) intrinsic or mitochondrial-related pathways.⁴ The intrinsic pathway is regulated by the anti-apoptotic and pro-apoptotic members of Bcl-2 family members.⁵ There is growing evidence for a role of mitochondria in apoptosis induction, leading to the oligomerization of adapter proteins and pro-caspases, resulting in auto activation of initiator caspases.⁶ This leads to activation of the caspase cascade.⁷ The activated form of caspase-3 is one of the key executioner caspases of apoptosis because it cleaves many proteins including poly-ADP-ribose polymerase (PARP).⁸ Taken together, ROS induces the oxidation of mitochondrial pores which contribute to cytochrome c release by mitochondrial membrane potential disruption.⁹ It seems that mitochondria are both source and target of ROS.

Catechin, obtained from the plant Camellia sinensis L, is a significant constituent in tea polyphenols, has multiple favorable health-beneficial properties, such as the antiviral,¹⁰ antibacterial,¹¹ antifungal,¹² anticancer,^13,14 Parkinson’s diseases,¹⁵ anti-Alzheimer’s diseases,¹⁶ and diabetes.¹⁷ Therefore, it is essential to identify new catechin components with biological activity.^18,19 Among the catechin derivatives, DK-5-62 showed broad-spectrum antiproliferative activity with IC₅₀ value of 7.9, 6.6, 8.2 and 5.5 μM against the four cancer cell lines, human alveolar adenocarcinoma epithelial cell line (A549), human breast adenocarcinoma cell line (MDA-MB-231), human prostate cancer cell line (DU145), and human hepatocellular liver carcinoma cell line (HepG2), respectively.¹⁸

In the present study, we examined the basics of hypothesis testing, and investigated apoptosis in HCT116 human colorectal cancer cells, which were treated with various concentrations of DK-5-62, a novel (-)-catechin derivative. After incubation with various concentrations of DK-5-62, cell viability was significantly decreased in doseand time-dependent manners, which, in turn, led to caspase dependent signals.

EXPERIMENTAL

Materials and Methods

Chemistry. Novel (-)-catechin derivatives, DK-5-29, DK-5-31, and DK-5-62, were provided by Professor Dong-Soo Shin of Changwon National University, Changwon, Korea.¹⁸ [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and streptomycin-penicillin were purchased from Gibco (Carlsbad, CA). The primary p38, p-p38, JNK, p-JNK, ERK1/2, p-ERK1/2, caspase-9, caspase-3 and PARP antibodies were purchased from Cell Signaling Technologies (Danvers, MA). All other chemicals and reagents were of the highest analytical grade.

Cell Culture and Viability Assay. The human colorectal cancer cells, HCT116, was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were maintained in Roswell Park Memorial Institute Media 1640 (RPMI 1640), supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin, and incubation was carried out at 37°C in a humidified incubator in a 5% CO₂ atmosphere. Cell counts were performed using a hemocytometer from Hausser Scientific (Horsham, PA). The cytotoxic effects of (-)-catechin derivatives against HCT116 cells were estimated colorimetric using the MTT method, which is based on the reduction of tetrazolium salt by mitochondrial dehydrogenase in viable cells.²⁰ Briefly, cells were seeded (2× 10⁶ cells/mL) in a 96-well plate and were then treated with (-)-catechin derivatives at final concentrations of 10, 25, 50, 75, and 100 μM. After 72 h incubation, MTT solution was added to each well at a final concentration of 0.4 mg/mL. After 2 h of incubation, the supernatants were aspirated and replaced with 150 μL of dimethyl sulfoxide (DMSO) to dissolve the formazan product. The absorbance at 540 nm was then read using a spectrophotometric plate reader. Results were calculated as percentages of the unexposed control.

Nuclear Staining with Hoechst 33258. The nuclear morphology of the cells was observed using the DNA-specific blue fluorescent dye Hoechst 33258. The viable cells were stained homogeneously, whereas apoptotic cells which had undergone chromatin condensation and/or nuclear fragmentation were not stained.^21,22 The HCT116 cells were treated with DK-5-62 at different concentrations. Cells were then fixed for 30 min in 100% methanol, washed with PBS, and stained with Hoechst 33258 (2 μg/mL). The cells were observed under a fluorescence microscope (Olympus Optical Co., Tokyo, Japan).

Measurement of Intracellular ROS. The Intracellular ROS generation was assessed using the stable nonpolar dye DCF-DA (Sigma-Aldrich; St. Louis, MO) followed by published method.²⁰ ROS production was measured by FACSCalibur flowcytometry (Becton Dickinson, San Jose, CA).

Apoptosis Analysis. Annexin V/PI double staining assay was then carried out in order to further differentiate between early apoptosis and late apoptosis stages. It was determined using an ApoScanTM Annexin V-FITC apoptosis detection Kit (BioBud, Seoul, Korea) in DK-5-62-treated HCT116 cells. The cells were trypsinized, harvested, washed with PBS. The cells were resuspended in 1× binding buffer (500 μL) and incubated with 1.25 μL of Annexin V-FITC (200 μg/mL) at room temperature for 15 min. The supernatant was then removed after centrifugation. The cells were resuspended in 500 μL of 1× binding buffer and cell suspensions were then stained with 10 μL of PI (30 μg/mL) at 4°C in the dark. Fluorescence was quantified using FACSCalibur flowcytometry. The amounts of early apoptosis and late apoptosis were determined as the percentage of Annexin V+/PI− or Annexin V+/PI+ cells, respectively.

Western Blot Analysis. Western blot analyses were performed as previously described.²³ The cells were cultured, harvested, and lysed on ice for 30 min in an appropriate lysis buffer (120 mM NaCl, 40 mM Tris (pH 8.0), and 0.1% NP 40) and were then centrifuged at 13,000×g for 15 min. Lysates from each sample were mixed with 5× sample buffer (0.375 M Tris-HCl, 5% SDS, 5% β-mercaptoethanol, 50% glycerol, 0.05% bromophenol blue, pH 6.8) and were then heated to 95°C for 5 min. Equal amounts of protein were separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto a nitrocellulose membrane. The membranes were then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.05% Tween-20 (TBST) and were then blocked in TBST containing 5% nonfat dried milk. The membranes were then incubated with their respective specific primary anti-bodies overnight at 4°C. After three washes in TBST, membranes were incubated with the appropriate secondary antibodies coupled to horseradish peroxidase (HRP) for 1 h at room temperature. The membranes were then washed again, and detection was carried out using an enhanced chemiluminescence Western blotting detection kit (Bio-Rad Laboratories, Inc., Hercules, CA). The values for the specific protein levels are presented as the fold-change relative to the control and densitometry was performed using Image J (National Institutes of Health, Bethesda, MD).

Statistical Analysis. All measurements were made in triplicate, and all values are given as the mean±the standard deviation (SD). The results were subjected to analysis of variance (ANOVA) followed by the Tukey range test to analyze differences between conditions. In each case, a p-value of < 0.05 was determined statistically significant.

RESULTS AND DISCUSSION

In the present study, we examined the effects of novel (-)-catechin derivatives on the growth of HCT116 human colorectal cancer cells by the MTT assay. Chemical structure of compound is illustrated in Fig. 1.

Figure 1. Chemical structure of (-)-catechin derivatives.

Cells were exposed to various concentrations (0–100 μM) of (-)-catechin derivatives for 72 h and their viability was determined. Cytotoxicity was determined as the percentage of viable treated cells in comparison with viable cells of untreated control cells. As shown in Fig. 2A, 2B, DK-5-62 significantly inhibited the proliferation of HCT116 cells in dose-and time dependent manners. After 72 h of exposure, DK-5-62 induced 28.1% growth inhibition at 50 μM, 36.5% at 75 μM, and 52.9% at 100 μM, respectively. As shown in Fig. 2C, DK-5-62 did not affect the proliferation of FHC colorectal normal cells in a dose-dependent manner. Therefore, the concentrations of 50, 75, and 100 µM DK-5-62 were used for the experiments.

Figure 2. (A) Cytotoxic effects of (-)-catechin derivatives, DK-5-29, DK-5-31, and DK-5-62 on HCT116 human colorectal cancer cells for 72h. (B) Cytotoxic effects of DK-5-62 on HCT116 cells in a time-dependent manner. (C) Cytotoxic effects of DK-5-62 on FHC normal human colorectal cells for 72 h. *p<0.05, significantly different from control cells.

Apoptosis is characterized by a series of morphology changes and chromatin condensation of cellular nucleus by distinct mechanisms. Nuclear Hoechst 33342 staining was performed in order to determine whether the anti-proliferative effect of DK-5-62 was due to apoptosis. As shown in Fig. 3, HCT116 cells which were treated with DK-5-62 showed a number of morphological changes, including cell shrinkage, condensed chromatin, and a higher density of apoptotic bodies compared with the untreated control cells. At the higher dose 100 µM of DK-5-62, crescentshaped nuclei, which are one of the typical characteristics of apoptotic cells, were also found. These results suggest that DK-5-62 showed antiproliferative activities in a dose-dependent manner, and is consistent with MTT results. In case of mitochondrial-dependent apoptosis, it is largely regulated by direct or indirect intracellular ROS. The treatment of DK-5-62 and N-acetyl-L-cysteine (NAC) slightly attenuated relative to only DK-5-62 treatment, suggesting that NAC, antioxidant, might be ROS scavenger (Fig. 4). These results presented that ROS might be involved in DK-5-62-mediated apoptotic process.

Figure 3. Microscopy image of DK-5-62 treated HCT116 cells. After incubation with various concentrations of DK-5-62 for 72 h, the cells were observed by fluorescent microscopy using Hoechst 33258 staining (arrow indicates the formation of bodies).

Figure 4. Changes in ROS levels in DK-5-62 treated HCT116 cells. After incubation with NAC and DK-5-62 for 72 h, ROS levels were measured by flow cytometry. (A) Relative ROS levels in a dose-dependent manner. (B) Statistical analysis for ROS levels. *p<0.05, significantly different from control and DK-5-62 treated cells.

To quantify the extent of apoptotic cells, flowcytometry analysis was performed using double staining with Annexin V and PI. The Annexin V−/PI−population was considered to represent unaffected cells, Annexin V+/PI− as early apoptosis, Annexin V+/PI+ as late apoptosis, and Annexin V−/PI+ as necrosis. The results showed that DK-5-62-treated cells significantly increased apoptotic cells populations, compared to untreated control cells (Fig. 5A). DK-5-62-treated HCT116 cells showed that early apoptotic cell populations were increased 22.1% at 100 µM of DK-5-62, compared with 1.9% for the control. The late apoptotic cells were increased 18.9% at 75 µM, compared with 5.7% for the control. The total apoptotic cell populations were ncreased 21.1%, 31.8%, and 39.4% at 50, 75, and 100 µM, respectively, compared with 7.6% for the control. The quantitative data are presented in Fig. 5B. These results suggest that DK-5-62 can induce apoptosis in HCT116 cells. Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the Annexin V binding assay to detect apoptotic cell death. In the early stages of apoptosis, there are alterations of PS, which translocate the outer layer from the inner side of the plasma membrane. This allows early recognition of dead cells by which PS exposes at the external surface of the cell.²⁴ This is followed by an enabling characteristic paraben can cause DNA damage in the short term,²⁵ but more study is needed to figure out long-term and low-dose mixtures.

Figure 5. Effects of DK-5-62 on apoptosis in HCT116 cells. (A) Flow cytometric analysis of HCT116 cells incubated with DK-5-62 for 72 h. The right bottom quadrant represents Annexin V-stained cells (early-phase apoptotic cells). The top right quadrant represents PI- and Annexin V-stained cells (late-phase apoptotic cells). (B) Statistical analysis of apoptosis. *p<0.05, significantly different from control cells in intact cells; early apoptotic; late apoptotic; and total apoptotic.

To study the apoptotic effects of DK-5-62 in HCT116 cells, we examined the expression levels of a number of apoptosis regulatory proteins, including Bcl-2, Bax, caspase-9, caspase-3, and PARP. The mitochondrial pathway is an important apoptosis pathway as it regulates the apoptotic cascade via a convergence of signaling at the mitochondria. Bcl-2 interacts with the mitochondrial plasma membrane and prevents mitochondrial membrane pores from opening during apoptosis, blocking the signals of apoptotic factors, such as Bax.²⁶ As a result, DK-5-62 increased Bax expression but decreased the expression of Bcl-2, each in a dose-dependent manner. The mitochondrial plasma membrane disruption by DK-5-62 was followed by the activation of caspase-9, caspase-3, and its target, PARP. Also, a densitometric analysis of the bands showed that DK-5-62 caused a dose-dependent increased the Bax/Bcl-2 ratio (Figs. 6A, 6B, 6C).

Figure 6. Effects of DK-5-62 on expression of apoptosis-related proteins in HCT116 cells. Cells were treated with DK-5-62 (0, 50, 75, and 100 µM) for 72 h. (A) The cell lysates were electrophoresed, and Western blotting with Bax, Bcl-2, caspase-9, caspase-3, and cleaved PARP antibodies. (B) Statistical analysis in Bax/Bcl-2 ratio. (C) Statistical analysis in caspases/β-actin ratio. *p<0.05, significantly different from control cells.

The mitochondrial-related pathway is regulated by anti-apoptotic (Bcl-2, Bcl-x, and Bcl-XL) and pro-apoptotic members (Bax, Bak, Bid, Bad and Bim) of the Bcl-2 family. The anti-apoptotic proteins on the outer membranes of mitochondria maintain the integrity of the mitochondria, through inhibiting apoptosis in the presence of various apoptotic stimuli.²⁷ The ratio of Bax and Bcl-2 protein determines the susceptibility of cells to cell survival as well as death.²⁸ These results suggested that DK-5-62 can induce apoptosis through the regulation of apoptosis-related protein expression in HCT116 cells. Caspases are cysteinyl aspartate proteinases (cysteine proteases that cleave their substrates following an Asp residue), which is essential phase in apoptosis. While the upstream caspases for the intrinsic pathway is caspase-9, of the extrinsic pathway is caspase-8. The intrinsic and extrinsic pathways converge to caspase-3. Caspase-3, then cleaves the inhibitor of the caspase-activated deoxyribonuclease, which is responsible for nuclear apoptosis. In this pathway, cell death signals lead to cytochrome c release from the mitochondria, which binds and facilitates the formation of the apoptosome that recruits and activates caspase-9.^29.30 The apoptosome-bound caspase-9 cleaves and activates caspase-3. Caspase-3 is one of the key protagonists of apoptosis, because it is either partially or completely responsible for the proteolytic cleavage of many key proteins, such as PARP. PARP is important for cell viability, but its cleavage facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis.^31,32 Therefore, the Western blotting experiments indicated that caspase-9 and caspase-3 appear to be activated in DK-5-62-induced HCT116 cells. These results suggest that HCT116 cells are highly sensitive to growth inhibition by DK-5-62 via the activation of apoptosis, as evidenced by activation of Bcl-2-mediated signaling, as well as alteration in caspase-9 and caspse-3.

Mitogen-activated protein kinases (MAPKs) signaling cascades, including c-Jun amino-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and p38 kinase, are found in all eukaryotes and play a central role in regulating cell proliferation, differentiation and apoptosis.³³ To further determine whether MAPKs are involved in the DK-5-62-induced HCT116 cytotoxicity, we examined the phosphorylation expression levels of MAPKs. The expressions of non-phosphorylated ERK, JNK and p38 MAPKs were not changed upon treatment with DK-5-62. By contrast, the accumulation of phosphorylated ERK, JNK, and p38 MAPKs were slightly elevated for 24 h (Fig. 7A). These results indicated that 100 µM of DK-5-62 induces apoptosis through ERK, JNK and p38 MAPKs in HCT116 cells. To further determine whether p38 are involved in 100 µM of DK-5-62-induced HCT116 cytotoxicity, the kinase-specific inhibitors, SB203580, was incubated. Co-treatment with DK-5-62 and SB203580 effectively blocked DK-5-62 mediated P-p38 up-regulation (Fig. 7B). The cumulative results suggested that p38 MAPK might involve in DK-5-62-induced mitochondrial apoptosis. As a group, MAPKs, which are found in all eukaryotes, regulate in a series of physiological processes, including cell growth, differentiation, and apoptosis.³⁴ Many evidences suggest that antitumor agents can regulate the activities of MAPK members in most cancer cell lines. Additionally, several chemotherapeutic agents, such as taxol, etoposide, and ceramide, also have been shown to induce MAPK activation in human cancer cells.³⁵

Figure 7. Regulations of MAPKs in treated with DK-5-62 in HCT116 cells. (A) Equal amounts of cell lysates were electrophoresed and JNK, ERK and p38 and their phosphorylated expression form were detected by western blotting analysis with corresponding antibodies. (B) Treatment with or without MAPK inhibitor, SB203580.

CONCLUSION

Colorectal Cancer is the third most common cancer diagnosed in the United States in 2020. While the incidence and the mortality rate of colorectal cancer has decreased due to effective cancer screening measures, there has been an increase in number of young patients diagnosed in colon cancer due to unclear reasons at this point of time.³⁶ It is thought to arise as a result of the transformation of normal colonic epithelial cells into a colorectal carcinoma as an adenomatous polyp. Many factors play a crucial role in colorectal cancer progression, such as mutation in tumor suppressor genes, cell proliferation, angiogenesis, and chronic inflammation.³⁷ Antitumor agents are developed with the aim of targeting the inhibition of cancer cells, but not normal cells; however, many are toxic to normal cells also. Therefore, novel natural or synthetic agents, which specifically target colorectal cancer but have lower toxicity for normal colonic epithelial cells, hold enormous potentials. In conclusion, we examined the mechanism involvement in DK-5-62-induced apoptosis in HCT116 cells. HCT116 cells are highly sensitive to growth inhibition by DK-5-62 via the activation of apoptosis, as evidenced by activation of MAPK-mediated signaling as well as alteration in Bcl-2 family protein expression and activation of caspase-9 and caspase-3.