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Biochanin A induces anticancer effects in SK‑Mel‑28 human malignant melanoma cells via induction of apoptosis, inhibition of cell invasion and modulation of NF‑κB and MAPK signaling pathways
Retraction in: /10.3892/ol.2021.12724
- Authors:
- Published online on: September 14, 2017 https://doi.org/10.3892/ol.2017.6945
- Pages: 5989-5993
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Copyright: © Xiao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Malignant melanoma accounts for approximately 75% of the skin cancer-related deaths in the world. Malignant melanoma is an aggressive form of cancer and and its incidence has increased significantly in the recent past (1). Transformation of melanoma into the metastatic stage is a very complex process involving various biochemical processes like cell cycle disruption, evasion of apoptosis, abnormalities in the adhesion and dilapidation of the extracellular matrix, cell migration and cell invasion (1–3). As for treatment of malignant melanoma is concerned, there are only few chemotherapeutic agents available and those too have serious side effects (4). Patients with metastatic melanoma and who are not suitable for surgical resection, 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC) has been reported to be very effective chemotherapeutic agent (4). However, in single-dose regimen, its overall response rate is very poor and the duration of response is very low. Further, it has been reported that combination of DTIC with other chemotherapeutic agents like cisplatin, carmustine, tamoxifen etc has led to higher response rates (4,5). But, unfortunately, this has also resulted in increased unwanted side-effects affecting the quality of life (5). In brief, the current treatment options for malignant melanoma especially metastatic melanoma are very limited and unsatisfactory. As a result there is a pressing need for development of new and effective chemotherapeutic agents for the treatment of malignant melanomas. Scientists are exploring the traditional and seemingly safe natural medicines for the anticancer therapeutics. Plant based drugs including taxol, vincristine, vinblastine, topotecan, irinotecan, camptothecin, podophyllotoxin etc have been successfully used for the treatment of a variety of human tumors. These compounds have been used as prototypes for the design and development of future anticancer drugs (6). Since, most of these drugs are associated with lot of side effects, there is need to explore less toxic and more effective drugs. Consistent with this, isoflavones are generally well tolerated and have not exhibited toxicity in humans (7) and may therefore prove potential candidates for anticancer drug development. Biochanin A is one such flavonoid and has been reported to exhibit anticancer activity against a range of cancer cells which include prostate cancer (8), pancreatic cancer (9) and breast cancer (7). However, the anticancer activity of Biochanin A has not been evaluated against human malignant melanoma cells. Therefore the aim of the present investigation was to evaluate the anticancer and apoptotic effects of Biochanin A in SK-Mel-28 human malignant melanoma cells along with investigating its effects on cell migration and invasion, cell cycle arrest and nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signalling pathway.
Materials and methods
Chemical and other biochemical reagents
Biochanin A (95% purity by HPLC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, Penicillin, streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (5% heat-inactivated), 3.5% trypsin, Dulbecco's modified Eagle's medium were purchased from Gibco (Carlsbad, CA, United States). Acridine orange (AO) and propidium iodide (PI) were purchased from Beyotime Institute of Biotechnology (Shanghai, China).
Cell line and cell culture conditions
Human malignant melanoma cell line (SK-Mel-28) was purchased from the Cell Institute, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium comprising of 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin with 5% CO2 at 37°C.
Cell cytotoxicity assay
In brief, human malignant melanoma cell line (SK-Mel-28) cells at a density of 2×106 cells/ml were cultured in a 96-well chamber slide for 24 h before using them in the experiment. SK-Mel-28 cells were then treated with 0, 10, 25, 50, 75 and 100 µM dose of Biochanin A dissolved in DMSO (0.1%) for 48 and 72 h. Subsequently, MTT was added into each well and then again cultured for 3 h before the supernatant was discarded. The formazan crystals formed were dissolved in DMSO and the absorbance values were examined on Automated Microplated Reader (BioTek Instruments, Inc., Winooski, VT, USA) at 570 nm. Cytotoxicity was represented as the concentration of Biochanin A inhibiting cell growth by 50% (IC50 value).
In vitro wound healing assay
Human malignant melanoma cell line (SK-Mel-28) (1×106 cells/ml) were seeded in a 6-well plate and incubated at 37°C till 100% confluent monolayer of cells was attained. A straight cell-free wound was made using a 100 ml pipette tip. To each well, different doses (0, 10, 50 and 100 µM) of Biochanin A were added and the cells were washed with PBS to eliminate any cellular debris. After that the cells were stained using 0.5% crystal violet powder for 15 min. The arbitrarily selected fields were chosen and photographed using an inverted light microscope (IX71; Olympus, Tokyo, Japan). The percentage of cells that migrated into the scratched area were calculated and length of wounds was determined by Image J (version 1.46) software.
Invasion assay
After the SK-Mel-28 cells were washed with PBS, the cells were again suspended in serum free medium. Then 400 µl of cell suspension (2×106 cells/ml) was added to the upper chamber coated with Matrigel membrane (Millipore, Billerica, MA, USA) while as lower chamber was filled with medium containing 5% FBS. SK-Mel-28 cells were incubated with various doses (0, 10, 50 and 100 µM) of Biochanin A for 1 h at 25°C. After 48 h, the cells which had still remained in the upper face of the filters were removed, and the cells which had travelled to the lower face of the filters were fixed with 90% methanol and then stained with 0.35% crystal violet and then counted using light microscope.
Fluorescence microscopy using AO/PI
Human malignant melanoma cells (SK-Mel-28) were plated on a chamber slide at a density of 2×106 cells per chamber and then treated with varying doses (0, 10, 50 and 100 µM) of Biochanin A and incubated for 48 h before 10 µg/ml of AO and 10 µg/ml of PI were added to each chamber. The images of the treated and untreated cells were captured by a UV fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan). The images were taken at ×200 magnification so that the evidence of apoptosis in clear.
Western blot analysis
A western blot assay was carried out to examine the expression of proteins responsible for the anticancer effect. SK-Mel-28 cells were harvested and lysed with RIPA buffer and the collected protein samples were examined by bichinconinic acid protein assay kit for protein quantification. Approximately 200 µg of cellular protein from each sample was applied to 8–10% SDS-polyacrylamide gels and probed with specific antibodies followed by exposure to horseradish peroxidase-conjugated goat anti-mouse antibodies. Blots were then developed using the West Pico Chemiluminescent substrate (Pierce, Woburn, MA, USA). The antibodies against β-actin, p-MEK, MEK, NF-κB were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Statistical analysis
All data were expressed as the mean ± standard deviation. The means of the different groups were compared using one-way of variance (ANOVA) followed by the Student's t-test. Each experiment was performed at least three times. P<0.05 was considered to indicate a statistically significant difference.
Results
Cytotoxic effects of Biochanin A in SK-Mel-28 human malignant melanoma cells
The cytotoxic effects of Biochanin A in SK-Mel-28 cells are depicted in Fig. 1. The cytotoxicity of the compound was evaluated by MTT assay using a range of concentrations including 0, 10, 25, 50, 75 and 100 µM. The results point out that Biochanin A is a potent cytotoxic agent showing dose-dependent growth inhibition of these cancer cells. The cytotoxic effects of the compound were tested at 48 and 72 h incubation times and results revealed that the drug exerted time and concentration dependent anticancer effects. In addition, it should be noted that the cytotoxic activity of Biochanin A was not significant at 10 µM and the dose dependent only above 10 µM.
Biochanin A suppressed cell migration and invasion in SK-Mel-28 human malignant melanoma cells
The effect of Biochanin A on the cancer cell migration and invasion in SK-Mel-28 human malignant melanoma cells was evaluated by in vitro wound healing assay and invasion assay respectively. The results which are depicted in Fig. 2 revealed that a wound scratch in vehicle-treated control cells was virtually fully closed after 48 h of incubation. However, treatment with 0, 10, 50 and 100 µM dose of Biochanin A (Fig. 2B-E) led to suppression of cell migration in a dose-dependent manner. As can be seen from Fig. 3A-D, Biochanin A also inhibited cancer cell invasion in a dose-dependent manner. Thus these results indicate that Biochanin A might show anticancer effects by inhibiting cancer cell migration and invasion. Unlike MTT assay, the inhibition of cell migration and invasion were observed even at the lowest concentration of 10 µM Chemotherapeutic agents which suppress cancer cell migration and invasion are believed to be promising antitumor drugs because cancer cell migration and invasion have a direct relationship with cancer metastases.
Biochanin A induces apoptosis in SK-Mel-28 human malignant melanoma cells
AO/PI staining was also done to examine the apoptosis-inducing effects of Biochanin A in SK-Mel-28 human malignant melanoma cells. As compared to the untreated control cells which indicated homogenous green fluorescence, the Biochanin A-treated cells with 10, 50 and 100 µM dose showed orange and red fluorescence indicating onset of apoptosis in these cells (Fig. 4A-D). The population of these apoptotic cells (red fluorescence) increased with increasing doses of Biochanin A. Unlike the results of MTT assays, the results in apoptosis assay also showed apoptosis at 10 µM of Biochanin A.
Biochanin A leads to upregulation of NF-κB and MAPK signalling pathways
Western blot assay was used to study the expression levels of various key proteins involved in the anticancer action of Biochanin A in SK-Mel-28 human malignant melanoma cells. Biochanin A treatment to SK-Mel-28 cells led to activation of NF-κB in a dose-dependent manner (Fig. 5). Biochanin A also resulted in activation (upregulation) of the MAPK signalling pathways. Biochanin A significantly increased the phosphorylation of MAPK proteins. The upregulation of MAPK was seen to follow dose dependent pattern.
Discussion
Malignant melanoma accounts for about 75% of the skin cancer-related deaths in the world and there are limited treatment options available for its treatment. While as a number of drugs have been isolated from plants, many of them exibit toxicity. However, isoflavonoids have been reported to exhibit little or no cytotoxicity in human (7) and therefore may prove important targets for development of cancer chemotherapy. Biochanin A is a one such plant flavonoid and is mostly found in red clover, soy, peanuts and chickpea (10). Flavonoids are one of the common constituents in the human diet. Flavonoids have been reported to show chemopreventive action against different cancers. They are reported to show anticancer action by various mechanisms including cell cycle arrest, induction of apoptosis, carcinogen inactivation, and suppression of angiogenesis, antioxidative capacity and reversal of multidrug resistance (11–16).
In the present study, our main objective was to evaluate the antitumor activity of Biochanin A in SK-Mel-28 human malignant melanoma cells. Its effects on apoptosis, cell migration and invasion were also examined along with studying its effects on NF-κB and MAPK signalling pathway. The results suggest that Biochanin A inhibits cancer cell growth in SK-Mel-28 melanoma cells in a concentration dependent manner as well as time-dependent manner. Biochanin A also led to onset of orange/red fluorescence in these cells and the intensity of this red fluorescence increased with increasing dose of the drug indicating that Biochanin A induces apoptosis in SK-Mel-28 melanoma cells. These preliminary results will form basis for future exploration of the apoptosis inducing mechanism of Biochanin A in presence of zVAD with appropriate controls and in different cell lines. Further results revealed that Biochanin A also led to the inhibition of cell migration and cell invasion in these cells. Biochanin A might display antitumor effects by impeding cancer cell migration and invasion. Chemotherapeutic agents which suppress cancer cell migration and invasion are believed to be promising antitumor drugs because cancer cell migration and invasion have a direct relationship with cancer metastases(17,18). The migration and invasion of cancer cells are significant events in metastasis (17). Consequently, suppression of the invasion and metastasis of cancer cells has been thought to be a reasonable approach for the malignancy treatment (17). Western blot assay showed that Biochanin A led to upregulation of NF-κB and MAPK signalling pathways. While as biochanin did enhance the expression of NF-κB and MAPK, further investigations would confirm the involvement of these signalling pathways in biochanin induced anticancer activity. Moreover, the evaluation of the effect Biochanin A on MAPK downstream targets may aslo prove useful in and needs to be thoroughly studied in future. Taken together, the results indicate that Biochanin A may prove an important lead molecule.
In brief, the current study reports the antitumor and apoptotic effects of Biochanin A in SK-Mel-28 human malignant melanoma cells. The current study also reports that the anticancer effects of the compound were mediated via inhibition of cell migration and invasion Moreover the molecule also exerts its effects on NF-κB and MAPK signalling pathways.
References
Hussein MR, Haemel AK and Wood GS: Apoptosis and melanoma: Molecular mechanisms. J Pathol. 199:275–288. 2003. View Article : Google Scholar : PubMed/NCBI | |
Soengas MS and Lowe SW: Apoptosis and melanoma chemoresistance. Oncogene. 22:3138–3151. 2003. View Article : Google Scholar : PubMed/NCBI | |
Senderowicz AM: Novel small molecule cyclin-dependent kinases modulators in human clinical trials. Cancer Biol Ther. 2 4 Suppl 1:S84–S95. 2003. View Article : Google Scholar : PubMed/NCBI | |
Comis RL: DTIC (NSC-45388) in malignant melanoma: A perspective. Cancer Treat Rep. 60:165–176. 1976.PubMed/NCBI | |
McClay EF and McClay ME: Tamoxifen: Is it useful in the treatment of patients with metastatic melanoma? J Clin Oncol. 12:617–626. 1994. View Article : Google Scholar : PubMed/NCBI | |
Cragg GM, Boyd MR, Cardellina JH, Grever MR, Schepartz SA, Snader KM and Matthew S: Role of Plants in the National cancer Institute drug discovery and development program. ACS Symposium Series. 534:80–95. 2009. | |
Sehdev V, Lai JC and Bhushan A: Biochanin A modulates cell viability, invasion, and growth promoting signaling pathways in HER-2-positive breast cancer cells. J Oncol. 2009:1214582009. View Article : Google Scholar : PubMed/NCBI | |
Seo YJ, Kim BS, Chun SY, Park YK, Kang KS and Kwon TG: Apoptotic effects of genistein, biochanin-A and apigenin on LNCaP and PC-3 cells by p21 through transcriptional inhibition of polo-like kinase-1. J Korean Med Sci. 26:1489–1494. 2011. View Article : Google Scholar : PubMed/NCBI | |
Bhardwaj V, Tadinada SM, Jain A, Sehdev V, Daniels CK, Lai JC and Bhushan A: Biochanin A reduces pancreatic cancer survival and progression. Anticancer Drugs. 25:296–302. 2014. View Article : Google Scholar : PubMed/NCBI | |
Medjakovic S and Jungbauer A: Red clover isoflavones Biochanin A and formononetin are potent ligands of the human aryl hydrocarbon receptor. J Steroid Biochem Mol Biol. 108:171–177. 2008. View Article : Google Scholar : PubMed/NCBI | |
Hodek P, Trefil P and Stiborová M: Flavonoids-Potent and versatile biologically active compounds interacting with cytochromes P450. Chem Biol Interact. 139:1–21. 2002. View Article : Google Scholar : PubMed/NCBI | |
Manthey JA, Guthrie N and Grohmann K: Biological properties of Citrus flavonoids Pertaining to cancer and inflammation. Curr Med Chem. 8:135–153. 2001. View Article : Google Scholar : PubMed/NCBI | |
Surh YJ: Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer. 3:768–780. 2003. View Article : Google Scholar : PubMed/NCBI | |
Galati G, Teng S, Moridani MY, Chan TS and Brien OPJ: Cancer chemoprevention and apoptosis mechanisms induced by dietary polyphenolics. Drug Metabol Drug Interact. 17:311–349. 2000. View Article : Google Scholar : PubMed/NCBI | |
Yang CS, Landau JM, Huang MT and Newmark HL: Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr. 21:381–406. 2001. View Article : Google Scholar : PubMed/NCBI | |
Banday JA, Shah SA, Kanth AH, Farozi A and Wani H: In Vitro screening for anticancer activity of petroleum ether and ethyl acetate extracts of Conyza canedensis growing in Kashmir region. Adv Biomed Pharma. 2:82–85. 2015. View Article : Google Scholar | |
Park GB, Kim DJ, Kim YS, Lee HK, Kim CW and Hur DY: Silencing of galectin-3 represses osteosarcoma cell migration and invasion through inhibition of FAK/Src/Lyn activation and β-catenin expression and increases susceptibility to chemotherapeutic agents. Int J Oncol. 46:185–194. 2015. View Article : Google Scholar : PubMed/NCBI | |
Li X, Yang G, Li X, Zhang Y, Yang J, Chang J, Sun X, Zhou X, Guo Y, Xu Y, et al: Traditional Chinese medicine in cancer care: A review of controlled clinical studies published in Chinese. PLoS One. 8:e603382013. View Article : Google Scholar : PubMed/NCBI |