Naphthazarin suppresses cell proliferation and induces apoptosis in human colorectal cancer cells via the B-cell lymphoma 2/B-cell associated X protein signaling pathway
- Authors:
- Published online on: October 26, 2016 https://doi.org/10.3892/ol.2016.5319
- Pages: 5211-5216
Abstract
Introduction
Colorectal cancer is the third and second most common malignant tumor in males and females worldwide, respectively (1). According to the International Agency for Research on Cancer, 1.2 million novel cases of colorectal cancer were diagnosed worldwide in 2008, accounting for 8% of all cancer-related mortalities (2,3). The incidence of colorectal cancer is highest in developed countries and regions: Due to economic development and rapid urbanization in recent years in China, which has resulted in dietary and lifestyle changes within the population, the colorectal cancer morbidity and mortality rates in China are now higher than the average rates, worldwide (4,5).
In 2013, the incidence of colorectal cancer was 45.1 per 100,000 individuals, with >5,000 deaths per year and an average of 13.1 years of life lost (1). Colorectal cancer operation is an important method for the treatment of colorectal cancer (5). The clinical manifestations of colorectal cancer may appear in the following ways: Altered defecation habits, stomach ache, abdominal masses, gastrointestinal hemorrhage, jaundice and a change in character of the stool. All patients have postoperative complications, with have a great impact on the patient's quality of life, and even endanger the patient's life (6). Therefore, it is important that physicians investigate multiple potential treatments for colorectal cancer, with the aim of preventing these treatment complications (7).
Naphthazarin is a natural bioactive substance present in numerous plants that has been demonstrated to exhibit antitumor effects (8). Naphthazarin is an important active ingredient, which exhibits extensive pharmacological activities, including antitumor activity, and due to its low toxicity, it has gained considerable attention (9,10). However, to date the anticancer effects of naphthazarin on human colorectal cancer cells have not been reported. In the present study, the anticancer effects of naphthazarin, as well as its effect on the B-cell lymphoma 2 (Bcl-2)/B-cell associated X protein (Bax) signaling pathway were investigated in human SW480 colorectal cancer cells.
Materials and methods
Reagents
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), naphthazarin (Fig. 1) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution and a lactate dehydrogenase (LDH) assay were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis and caspase-3 activation kits were purchased from KeyGen Biotech Co., Ltd., (Nanjing, China).
Cell culture and cell viability assay
The human colorectal cancer SW480 cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM (Sigma-Aldrich; Merck Millipore) supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. A total of 1×104 cells/well were seeded in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1, 5, 10 and 20 µM naphthazarin for 24 h. Subsequently, 20 µl MTT solution (Sigma-Aldrich; Merck Millipore) was added to each well and incubated at 37°C in a 5% CO2 incubator for 4 h. Following incubation, the culture medium was replaced and 200 µl DMSO was added to each well and agitated for 20 min at room temperature. Cell viability was analyzed at a wavelength of 490 nm using an ELISA reader (Multiskan EX; Thermo Labsystems, Helsinki, Finland). .
LDH assay
SW480 cells (1×104 cells/well) were seeded in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, SW480 cells were treated with to 0, 0.5, 1, 5, 10 and 20 µM naphthazarin for 24 h. Subsequently, 100 µl LDH solution was added to each well and cultured for 30 min. The absorbance was read at a wavelength of 490 nm using an ELISA reader (Multiskan EX; Thermo Labsystems).
Annexin V-FITC/PI apoptosis assay
SW480 cells (1×106 cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were trypsinized (Sangon Biotech Co., Ltd., Shanghai, China), washed with phosphate-buffered saline (PBS) and fixed in precooling 75% ethanol at 4°C overnight. Next, Annexin-V FITC and PI were added and incubated for 10 min at room temperature in the dark. Flow cytometry analysis was performed on a FACScan flow cytometer (BD Biosciences, San Diego, CA, USA) using emission filters of 525 and 575 nm. Data were analyzed using CellQuest Pro software version 5.1 (BD Biosciences).
4′,6-diamidino-2-phenylindole (DAPI) staining assay
SW480 cells (1×106 cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. Following incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were then incubated with 0.1% sodium citrate containing 0.1% Triton X-100 (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at 4°C. Cell nuclei were observed using a fluorescent microscope (Zeiss Axio Observer A1; Carl Zeiss, Inc., Oberkochen, Germany).
Western blot analysis
SW480 cells (1×106 cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were harvested with PBS and extracted in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the by Pierce BCA protein assay kit (BD Biosciences). Equal amounts of protein were resolved on 6–15% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Membranes were then incubated with antibodies against poly (ADP-ribose) polymerase (PARP; 1:1,000; D161071; Santa Cruz Biotechnology, Inc.), Bax (1:500; AF0057; Beyotime Institute of Biotechnology), Bcl-2 (1:500; AF0060; Beyotime Institute of Biotechnology) and β-actin (1:500; AA128; Beyotime Institute of Biotechnology) overnight at 4°C. Membranes were next washed with TBS containing Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; Sangon Biotech, Co., Ltd.) at 37°C for 2 h. The protein band was detected using ImageLab 3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Caspase-3 activation assay
SW480 cells (1×106 cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were harvested with PBS and extracted in cold RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the Pierce BCA protein assay kit (BD Biosciences). Equal amounts of the total protein were mixed with Ac-DEVD-pNA (Beyotime Institute of Biotechnology) for caspase-3 expression and incubated at 37°C for 2 h in the dark. Caspase-3 activation was analyzed at a wavelength of 405 nm using an ELISA reader (Multiskan EX; Thermo Labsystems).
Statistical analysis
Data are presented as the mean ± standard deviation of three independent experiments. Differences between groups were analyzed using the Student's t-test and the SPSS 17.0 program (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Naphthazarin decreases SW480 cell viability
The effect of naphthazarin on cell viability was investigated by MTT assay in SW480 cells. Following treatment with 5, 10 and 20 µM naphthazarin significantly decreased cell viability of SW480 cells in a dose-dependent manner (P<0.01; Fig. 2). These results indicate that naphthazarin exhibits an anticancer effect on human colorectal cancer cells.
Naphthazarin increases cytotoxicity of SW480 cells
A LDH assay was performed to investigate the effect of naphthazarin on SW480 cell cyotoxicity. Naphthazarin significantly increased cytotoxicity of SW480 cells in dose-dependent manner following treatment with 5, 10 and 20 µM naphthazarin for 24 h (Fig. 3).
Naphthazarin induces apoptosis of SW480 cells
The effect of naphthazarin on SW480 cell apoptosis was investigated. Upon treatment with 1 and 5 µM naphthazarin for 24 h, the rate of cell apoptosis was significantly increased in SW480 cells in a dose-dependent manner (Fig. 4).
Naphthazarin induces nuclear apoptosis in SW480 cells
SW480 cells were stained with DAPI and observed under a fluorescent microscope to investigate the effect of naphthazarinon nuclear apoptosis. Nuclear apoptosis was observed in SW480 cells following treatment with 0.5, 1 and 5 µM naphthazarin for 24 h (Fig. 5).
Effect of naphthazarin on PARP of SW480 cells
To determine whether PARP is associated with the effects of naphthazarin on cell viability and apoptosis in SW480 cells, PARP protein expression was evaluated by western blotting. As shown in Fig. 6, treatment with 0.5, 1 and 5 µM naphthazarin for 24 h decreased PARP protein expression, however, no significant differences were observed when compared with the control group.
Naphthazarin decreases Bcl-2 and increases Bax expression in SW480 cells
To further investigate the anticancer effect of naphthazarin, the protein expression of Bcl-2 and Bax in human colorectal SW480 cancer cells was analyzed by western blotting following naphthazarin treatment (Fig. 7A). The results revealed that treatment with 5 µM naphthazarin for 24 h significantly decreased Bcl-2 expression in cells compared with the control (P<0.01; Fig. 7B). Furthermore, treatment with 1 and 5 µM naphthazarin for 24 h significantly increased Bax protein expression in a dose-dependent manner compared with the control (Fig. 7C).
Effect of naphthazarin on caspase-3 of SW480 cells
To confirm that potential mechanism of naphthazarin on cell apoptosis of human colorectal cancer cell, we also examined the activation of caspase-3 after naphthazarin treatment with for 24 h. Treatment with 5 µM naphthazarin resulted in significantly increased levels of caspase-3 activation in SW480 cells compared with the control (Fig. 8).
Discussion
Globally, colorectal cancer is the third most common type of malignant tumor, after lung and breast cancer (11). The incidence of colorectal cancer exhibits differences in regional distribution: Incidence is highest in developed countries and regions, such as Australia, New Zealand, Europe and North America, and lower in Asia and Africa (12–14). The highest mortality rates occur in Central and Eastern European countries, and the lowest mortality rates are observed in central African regions (15). In China in 2009, colorectal cancer incidence and mortality rates were higher than the world averages: Rates were than lower that observed in Japan, Singapore and South Korea, but higher than that of countries such as Iran, Laos and India (16,17). In the present study, it was demonstrated that naphthazarin significantly decreased cell viability, increased cytotoxicity and induced cellular and nuclear apoptosis of SW480 cells in a dose-dependent manner. Recent studies have demonstrated that naphthazarin induces apoptosis of human breast cancer (18) and gastric cancer cells (19).
PARP is involved in DNA damage recognition and signal transduction: PARP inhibitors can selectively prevent defects in the DNA of tumor cells (20). A previous clinical trial revealed that PARP inhibitors used for the treatment of ovarian cancer patients harboring mutations in breast cancer susceptibility genes achieved a good response rate, with few side effects (21). However, in the present study, treatment with naphthazarin did not affect PARP protein expression levels in SW480 cells. These results indicate that the PARP signaling pathway may not be involved with the anticancer effects of naphthazarin on human colorectal cancer cells.
Colorectal cancer is one of the most common malignant tumors worldwide (22). It has been demonstrated that the occurrence of tumor development depends on the dynamic balance between cell proliferation and apoptosis (23). Caspase-3 is a important apoptotic protein for various cells, and caspase-3 activation can induce apoptosis in cancer cells (24). The Bcl-2 family of apoptosis-related proteins includes important regulatory factors: Bcl-2 inhibits apoptosis, whereas Bax and Bak promote apoptosis. Therefore, changes in expression of these proteins affects the apoptosis of both normal cells and tumor cells (25,26). The results of the present study revealed that naphthazarin promoted Bax expression and inhibited Bcl-2 protein expression, and increased caspase-3 activation in SW480 cells. These results are in accordance with those of Acharya et al (9) who reported that naphthazarin increases the Bax/Bcl-2 protein ratio in A549 lung cancer cells.
In conclusion, the present study demonstrated that naphthazarin suppressed cell proliferation and induced apoptosis in human colorectal cancer cells via the Bcl-2/Bax signaling pathway. Thus, we hypothesize that naphthazarin may present a potential chemotherapeutic agent for colorectal cancer. However, further studies are required to investigate the mechanisms underlying the anticancer effects of naphthazarin on human colorectal cancers.
References
Sakurai J, Matsui Y, Hiraki T, Iguchi T, Fujiwara H, Gobara H, Mitsuhashi T, Nagasaka T and Kanazawa S: Single center prospective phase II trial of CT-guided radiofrequency ablation for pulmonary metastases from colorectal cancer (SCIRO-1401). Acta Med Okayama. 70:317–321. 2016.PubMed/NCBI | |
Huang L, Xu Y, Cai G, Guan Z and Cai S: Downregulation of S100A4 expression by RNA interference suppresses cell growth and invasion in human colorectal cancer cells. Oncol Rep. 27:917–922. 2012.PubMed/NCBI | |
Kanefendt F, Lindauer A, Kinzig M, Strumberg D, Scheulen ME, Mross K, Fischer R, Moritz B, Sörgel F and Jaehde U: Biomarker response on exposure to sunitinib and its primary metabolite (SU12662) in metastatic colorectal cancer patients. Int J Clin Pharmacol Ther. 49:88–90. 2011.PubMed/NCBI | |
Xu F, Xu L, Wang M, An G and Feng G: The accuracy of circulating microRNA-21 in the diagnosis of colorectal cancer: A systematic review and meta-analysis. Colorectal Dis. 17:O100–O107. 2015. View Article : Google Scholar : PubMed/NCBI | |
Su B, Xu B and Wan J: Correlation between long-term aspirin use and F-fluorodeoxyglucose uptake in colorectal cancer measured by PET/CT. PLoS One. 9:e1094592014. View Article : Google Scholar : PubMed/NCBI | |
Cai Q, Lin J, Wei L, Zhang L, Wang L, Zhan Y, Zeng J, Xu W, Shen A, Hong Z and Peng J: Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of STAT3 signaling pathway. Int J Mol Sci. 13:6117–6128. 2012. View Article : Google Scholar : PubMed/NCBI | |
Josa V, Krzystanek M, Eklund AC, Salamon F, Zarand A, Szallasi Z and Baranyai Z: Relationship of postoperative thrombocytosis and survival of patients with colorectal cancer. Int J Surg. 18:1–6. 2015. View Article : Google Scholar : PubMed/NCBI | |
Kim MY, Park SJ, Shim JW, Yang K, Kang HS and Heo K: Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells. Int J Oncol. 46:1659–1666. 2015.PubMed/NCBI | |
Acharya BR, Bhattacharyya S, Choudhury D and Chakrabarti G: The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells. Apoptosis. 16:924–939. 2011. View Article : Google Scholar : PubMed/NCBI | |
Choi SY, Son TG, Park HR, Jang YJ, Oh SB, Jin B and Lee J: Naphthazarin has a protective effect on the 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine-induced Parkinson's disease model. J Neurosci Res. 90:1842–1849. 2012. View Article : Google Scholar : PubMed/NCBI | |
Kim SJ, Kim HJ, Kim HR, Lee SH, Cho SD, Choi CS, Nam JS and Jung JY: Antitumor actions of baicalein and wogonin in HT-29 human colorectal cancer cells. Mol Med Rep. 6:1443–1449. 2012.PubMed/NCBI | |
Tatsumi S, Matsuoka H, Hashimoto Y, Hatta K, Maeda K and Kamoshida S: Organic cation transporter 2 and tumor budding as independent prognostic factors in metastatic colorectal cancer patients treated with oxaliplatin-based chemotherapy. Int J Clin Exp Pathol. 7:204–212. 2013.PubMed/NCBI | |
Liu Z, Huang Q, Liu G, Dang L, Chu D, Tao K and Wang W: Presence of FOXP3(+)Treg cells is correlated with colorectal cancer progression. Int J Clin Exp Med. 7:1781–1785. 2014.PubMed/NCBI | |
Shimizu D, Ishikawa T, Ichikawa Y, Togo S, Hayasizaki Y, Okazaki Y, Danenberg PV and Shimada H: Prediction of chemosensitivity of colorectal cancer to 5-fluorouracil by gene expression profiling with cDNA microarrays. Int J Oncol. 27:371–376. 2005.PubMed/NCBI | |
Krützfeldt J, Rösch N, Hausser J, Manoharan M, Zavolan M and Stoffel M: MicroRNA-194 is a target of transcription factor 1 (Tcf1, HNF1α) in adult liver and controls expression of frizzled-6. Hepatology. 55:98–107. 2012. View Article : Google Scholar : PubMed/NCBI | |
Dimou A, Syrigos KN and Saif MW: Disparities in colorectal cancer in African-Americans vs Whites: Before and after diagnosis. World J Gastroenterol. 15:3734–3743. 2009. View Article : Google Scholar : PubMed/NCBI | |
Mehrabani D, Shamsdin SA, Dehghan A and Safarpour A: Clinical significance of serum vascular endothelial growth factor and complement 3a levels in patients with colorectal cancer in southern Iran. Asian Pac J Cancer Prev. 15:9713–9717. 2014. View Article : Google Scholar : PubMed/NCBI | |
Kim MY, Park SJ, Shim JW, Yang K, Kang HS and Heo K: Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells. Int J Oncol. 46:1659–1666. 2015.PubMed/NCBI | |
Kim JA, Lee EK, Park SJ, Kim ND, Hyun DH, Lee CG, Lee JH, Yang KM, Heo K and Son TG: Novel anti-cancer role of naphthazarin in human gastric cancer cells. Int J Oncol. 40:157–162. 2012.PubMed/NCBI | |
Sung B, Kang YJ, Kim DH, Hwang SY, Lee Y, Kim M, Yoon JH, Kim CM, Chung HY and Kim ND: Corosolic acid induces apoptotic cell death in HCT116 human colon cancer cells through a caspase-dependent pathway. Int J Mol Med. 33:943–949. 2014.PubMed/NCBI | |
Hochster H, Wadler S, Runowicz C, Liebes L, Cohen H, Wallach R, Sorich J, Taubes B and Speyer J: Activity and pharmacodynamics of 21-Day topotecan infusion in patients with ovarian cancer previously treated with platinum-based chemotherapy. New York Gynecologic Oncology Group. J Clin Oncol. 17:2553–2561. 1999.PubMed/NCBI | |
Eng C, Bessudo A, Hart LL, Severtsev A, Gladkov O, Müller L, Kopp MV, Vladimirov V, Langdon R, Kotiv B, et al: A randomized, placebo-controlled, phase 1/2 study of tivantinib (ARQ 197) in combination with irinotecan and cetuximab in patients with metastatic colorectal cancer with wild-type KRAS who have received first-line systemic therapy. Int J Cancer. 139:177–186. 2016. View Article : Google Scholar : PubMed/NCBI | |
Prasad S, Yadav VR, Sung B, Reuter S, Kannappan R, Deorukhkar A, Diagaradjane P, Wei C, Baladandayuthapani V, Krishnan S, et al: Ursolic acid inhibits growth and metastasis of human colorectal cancer in an orthotopic nude mouse model by targeting multiple cell signaling pathways: Chemosensitization with capecitabine. Clin Cancer Res. 18:4942–4953. 2012. View Article : Google Scholar : PubMed/NCBI | |
Dastjerdi MN, Rarani MZ, Valiani A and Mahmoudieh M: The effect of adenosine A1 receptor agonist and antagonist on p53 and caspase 3, 8, and 9 expression and apoptosis rate in MCF-7 breast cancer cell line. Res Pharm Sci. 11:303–310. 2016. View Article : Google Scholar : PubMed/NCBI | |
Song J, Peng XL, Ji MY, Ai MH, Zhang JX and Dong WG: Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo. World J Gastroenterol. 19:4127–4136. 2013. View Article : Google Scholar : PubMed/NCBI | |
Chudecka-Głaz AM, Cymbaluk-Płoska AA, Menkiszak JL, Sompolska-Rzechuła AM, Toloczko-Grabarek AI and Rzepka-Górska IA: Serum HE4, CA125, YKL-40, bcl-2, cathepsin-L and prediction optimal debulking surgery, response to chemotherapy in ovarian cancer. J Ovarian Res. 7:622014. View Article : Google Scholar : PubMed/NCBI |