|Year : 2017 | Volume
| Issue : 3 | Page : 471-476
Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction
Rajamanickam Buddhan, Shanmugam Manoharan
Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, Tamil Nadu, India
|Date of Web Publication||31-Aug-2017|
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Chidambaram - 608 002, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Objectives: Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells.
Materials and Methods: The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest).
Results: Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells.
Conclusion: The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.
Keywords: Apoptosis, diosmin, skin cancer
|How to cite this article:|
Buddhan R, Manoharan S. Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction. J Can Res Ther 2017;13:471-6
| > Introduction|| |
Cancer, a major public health burden worldwide, arises due to multiple genetic changes in a single cell, which further proliferate and forms a clone, termed as a tumor. Although various risk factors are documented to be involved in the pathogenesis of carcinogenesis, environmental factors are involved in 90% of cancers. Skin cancer is a cancer that arises from the three layers of the skin, epidermis, dermis and subcutaneous layers. Basal cell carcinoma and squamous cell carcinoma are the two most common types of skin cancer worldwide.
Skin cancer incidence increases every year and imposes a significant health problem throughout the world. The annual incidence rate of melanoma in African-Americans is 1/100,000. It has been estimated that one in five Americans will get skin cancer in their lifetime. The incidence of skin cancer in Australia is 2–3 times higher than that of Canada, USA and UK. It has been pointed out that 17,000 new cases and 5000 cancer-related deaths are reported to occur in European countries each year. Skin cancer accounts for 2–4% of all neoplasms in Asian Indians and accounts for 1–2% of all neoplasms in South Asians.
A431 (ATCC-CRC 1555) is a human cancer cell line established from an epidermoid carcinoma of the vulva of an 85-year-old female patient. Researchers utilized this cell line to study the molecular aspects of skin carcinogenesis as well as to investigate the cytotoxic/anti-cell proliferative potential of natural products and synthetic entities.
Diosmin, chemically named as 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-[(2R, 3R, 4R, 5R, 6S)-3, 4, 5-trihydroxy-6-methyl oxan-2-yl] oxymethyl] oxan-2-yl] oxychromen-4-one, is a glycosylated polyphenolic compound present in citrus species and olive leaves. Diosmin attenuated 7, 12-dimethylbenz (a) anthracene-induced cytotoxicity in a dose-dependent manner. Diosmin inhibited chemically induced cancer in rodents, including N-methyl-N-amyl nitrosamine-induced esophageal cancer, 4-nitroquinoline 1-oxide-induced oral cancer and azoxymethane-induced colon cancer. Diosmin augmented the effectiveness of interferon-alpha in the treatment of melanoma. Diosmin protected lymphocytes against radiation exposure. Diosmin is prescribed for the treatment of chronic venous insufficiency and hemorrhoids in Europe., Diosmin protected secondary restless leg syndrome caused by chronic venous insufficiency. Diosmin has been used for the treatment of diabetes mellitus, neurological problems like Alzheimer's diseases, melanoma, colon cancer and lymphedema. Diosmin showed blood lipid lowering, antioxidant and anticarcinogenic activities. Diosmin showed chemopreventive efficacy in urinary bladder carcinogenesis. It also exhibited anti-inflammatory, antimutagenic and free radical scavenging activities. It has been reported that the anti-cell proliferative potential of diosmin is due to its metabolite diosmetin. The present study explores the cytotoxic potential of diosmin in A431 skin cancer cells.
| > Materials and Methods|| |
The skin cancer cell line, A431 was purchased from the National Centre for Cell Sciences, Pune, India and cultured in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum, penicillin G, and streptomycin. The cells were placed in 5% CO2 incubator and the further experiments were started after the confluency stage was attained.
Log phase A431 cells were incubated at 37°C with the various doses of diosmin (10, 25, 50 μg/ml) in a CO2 incubator to find out the inhibitory concentration of (IC50) diosmin.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to assess the effect of the diosmin of A431 cell's viability.
The dose of the diosmin which inhibited the A431 cell proliferation by 50% (IC50) was calculated by plotting various concentrations of diosmin versus percentage of cell viability. The experimental protocol which was used to asses the cytotoxic efficacy of diosmin is given in [Figure 1].
|Figure 1: The experimental protocol that was used to asses the cytotoxic efficacy of diosmin|
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Reactive oxygen species generation
The intracellular reactive oxygen species (ROS) generation was measured according to the method of Pereira et al., using a nonfluorescent probe 2',7'-dichlorofluorescein diacetate.
The apoptotic induction potential of the diosmin was assessed using acridine orange/ethidium bromide dual staining. The cells were categorized as living cells (normal green nucleus), early apoptotic cells (bright green nucleus with condensed or fragmented chromatin), late apoptotic cells (orange-stained nuclei with chromatin condensation or fragmentation) and necrotic cells (uniformly orange-stained cell nuclei).
The DNA fragmentation assay was carried out according to the method of Herrmann et al.
Western blotting was utilized to assess the expression pattern of p53, Bcl-2, matrix metalloproteinases (MMP)-2, MMP-9, caspase-3, and caspase-9.
Cell cycle analysis
Cell cycle analysis in control and diosmin treated A431 cells was performed in flow cytometry according to the procedure of Nicoletti et al.
Values are expressed as mean ± standard deviation. Statistical comparisons were performed by one-way analysis of variance followed by Duncan's multiple range test. The results were considered statistically significant if the P values were <0.05.
| > Results|| |
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
[Figure 2] represents the diosmin efficacy on cell viability and morphology of A431 skin cancer cells respectively. Diosmin significantly inhibited the proliferation of A431 cells and the IC50 was attained at 45 μg/ml.
|Figure 2: Effect of morphological changes in control and diosmin treated A431 cells for 24 h. (a) Untreated A431 cells; (b) 10 μg/ml of diosmin; (c) 25 μg/ml of diosmin; (d) 50 μg/ml of diosmin|
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Reactive oxygen species generation and apoptotic induction
[Figure 3] depicts the effect of diosmin on ROS generation and apoptotic induction potential in A431 cells. Diosmin at a concentration of 45 μg/ml excessively generated ROS in A431 cells. Fluorescence microscopic analysis showed bright green fluorescence in diosmin-treated A431 cells as compared to untreated cells. Diosmin also induced apoptosis in A431 cells as evidenced by orange to red nuclei with condensed chromatin.
|Figure 3: Reactive oxygen species generation and apoptotic induction in control and diosmin treated A431 cells for 24 h. Reactive oxygen species generation: (a) Untreated A431 cells; (b) diosmin treated A431 cells arrows (→) represents reactive oxygen species generation. Apoptotic induction: (c) Untreated A431 cells, (d) diosmin treated A431 cells arrows (→) indicate apoptosis|
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Apoptotic DNA fragmentation
DNA fragmentation in untreated cells and diosmin treated A431 cells is shown in [Figure 4]. DNA fragmentation was higher in A431 cells treated with diosmin (45 μg/ml) for 24 h as compared to untreated cells.
|Figure 4: DNA fragmentation in control and diosmin treated A431 cells. L1-25 bp DNA ladder; L2-DNA isolated from untreated A431 cells; L3-DNA isolated from diosmin treated A431 cells|
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[Figure 5] depicts the expression pattern of p53, Bcl-2, caspases-3, caspases-9, MMP-2 and MMP-9 in diosmin treated A431 cells and untreated cells. Overexpression of p53, caspase-3, caspase-9 and decreased expression of Bcl-2, MMP-2 and MMP-9 was noticed in diosmin treated A431 cells as compared to untreated A431 cells.
|Figure 5: Expression pattern of p53, Bcl-2, caspase-3, caspase-9, MMP-2 and MMP-9 in control and diosmin treated A431 cells. β-actin served as an internal control. L1:Untreated A431 cells; L2: Diosmin treated A431 cells|
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Cell cycle analysis by flow cytometry
[Figure 6] shows the effect of diosmin on cell cycle arrest in A431 cells. We noticed a very few apoptotic cells in untreated A431 cells. However, we noticed an accumulation of apoptotic cells in G2/M phase (late apoptosis) in diosmin treated A431 cells.
|Figure 6: Effect of diosmin on cell cycle arrest in A431 cells. Untreated A431 cells; B. A431 cells treated with 45 μg/ml of diosmin|
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| > Discussion|| |
The present study explores the anti-cell proliferative potential of the diosmin in A431 skin cancer cell line. MTT assay, the most commonly employed procedure to assess the cell viability, is utilized to test the anti-cell proliferative efficacy of the natural products and their synthetic compounds. The viable cells convert MTT into a purple colored product, which can be determined colorimetrically. The cytotoxic potential of the test compound is proportional to the intensity of the color. The present study observed significant reduction in the cell viability in diosmin treated A431 cells as compared to control cells. Our results thus focus the cytotoxic potential of the diosmin in A431 cells.
ROS play an important role in divergent physiological, biological and molecular pathways at normal physiological concentrations. However, they produce deleterious effects in cells and tissues at higher concentrations, including DNA fragmentation and nuclear damage. Most of the researchers utilized the status of ROS generation as one of the characteristic features to assess the apoptotic efficacy of the test compound. An increase in ROS generation was observed in A431 cells exposed to diosmin for 24 h, as evidenced by intense green fluorescence level (dichloro-dihydro-fluorescein diacetate assay). The results of the present study thus indicate that diosmin might have inhibited cell proliferation through ROS-mediated apoptosis. The apoptotic ability of the diosmin was also tested by carrying out DNA fragmentation assay. The number of DNA fragments, in the form of ladder formation, observed in agarose gel electrophoresis could indicate the apoptotic efficacy of the test compound. DNA fragmentation was higher in the diosmin treated A431 cells as compared to the control cells, which further confirm the apoptotic/cytotoxic potential of the diosmin.
Apoptosis is an essential phenomenon to maintain the cellular balance between cell differentiation and cell death. Apoptotic pathway, thus, performs a critical role in the removal of genetically damaged cells from the body. Apoptotic cells are characterized by cell shrinkage, nuclear damage and multiple DNA fragmentation. Dual staining (acridine orange/ethidium bromide) is utilized to assess the efficacy of the test compound on apoptotic pathways. Apoptotic cells, in this method, are characterized by the red-orange fluorescence, while viable cells and early apoptotic cells exhibit bright green and green fluorescence respectively. A431 cells treated with diosmin revealed red orange fluorescence, which confirmed the apoptotic potential of the diosmin. We observed that exposure of A431 cells to the diosmin for 24 h caused an increase in the population of apoptotic cells gradually. Also, we noticed an increase in late apoptotic cells accompanied by decrease in early apoptotic cells after 24 h treatment with diosmin. Flow cytometry analysis also confirmed the accumulation of late apoptotic cells in diosmin treated A431 cells, which indicates that the diosmin can arrest the cell cycle in the G2/M phase.
MMPs play an important role in cancer invasion and were found to be overexpressed in almost all types of cancers. MMPs also play a pivotal role in tumor angiogenesis. Since MMP-2 and MMP-9 are involved in the regulation of tumor microenvironment, researchers utilized these proteins as a molecular target to test the anticell proliferative or anti-invasive potential of the natural products or synthetic agents. Overexpression of MMP-2 and MMP-9 was associated with poor clinical outcome. In the present study, Western blot analysis revealed over-expression of MMP-2 and MMP-9 in A431 cells, which lend credence to the observations of the other researchers. Diosmin treated A431 cells showed decreased MMP-2 and MMP-9 expression, which indicates the anti-invasive potential of the diosmin.
Caspases exists as inactive proenzymes in the cells and their activation triggers the process of apoptosis. Caspases 3 and 9 play a critical role in the programmed cell death and in particular caspase 3 is involved in the terminal phase of apoptosis. Activation of caspase cascade has been reported in various cancer cells treated with cytotoxic agents. Diosmin treated A431 cells showed increased expression of caspases 3 and 9, which confirmed the apoptotic induction potential of the diosmin.
p53, the molecular policemen, plays a critical and crucial role in DNA repair, cell cycle arrest, and apoptotic induction. Bcl-2, the anti-apoptotic protein in association with p53 stimulated apoptotic induction in several cancer cell lines. Lowered expression of p53 and increased expression of Bcl-2 proteins were well documented in various cancer cell lines. Our results corroborate these observations. Diosmin treated A431 cells exhibited downregulation of Bcl-2 accompanied by p53 upregulation, which explores diosmin as a prominent apoptotic agent. The present study thus explores the anticancer/cytotoxic potential of the diosmin in A431 skin cancer cell lines. The anticancer potential of the diosmin is due to its apoptotic induction as well as anti-tumor invasive potential in A431 cancer cell lines. The cytotoxic potential of diosmin is summarized in the flow chart [Figure 7].
|Figure 7: The flow chart that summarizes the cytotoxic potential of diosmin|
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The present study will be extended to in vivo model to further prove and confirm the anticancer potential of diosmin, which could explore diosmin as a promising candidature for skin cancer treatment.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]