Journal of Cancer Research and Therapeutics

: 2017  |  Volume : 13  |  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

Correspondence Address:
Shanmugam Manoharan
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Chidambaram - 608 002, Tamil Nadu


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.

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;13:471-476

How to cite this URL:
Buddhan R, Manoharan S. Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction. J Can Res Ther [serial online] 2017 [cited 2022 Aug 15 ];13:471-476
Available from:

Full Text


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.[1]

Skin cancer incidence increases every year and imposes a significant health problem throughout the world.[2] 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.[3] The incidence of skin cancer in Australia is 2–3 times higher than that of Canada, USA and UK.[4] It has been pointed out that 17,000 new cases and 5000 cancer-related deaths are reported to occur in European countries each year.[5] Skin cancer accounts for 2–4% of all neoplasms in Asian Indians and accounts for 1–2% of all neoplasms in South Asians.[6]

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.[7]

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.[8] Diosmin inhibited chemically induced cancer in rodents, including N-methyl-N-amyl nitrosamine-induced esophageal cancer,[9] 4-nitroquinoline 1-oxide-induced oral cancer and azoxymethane-induced colon cancer.[10] Diosmin augmented the effectiveness of interferon-alpha in the treatment of melanoma.[11] Diosmin protected lymphocytes against radiation exposure.[12] Diosmin is prescribed for the treatment of chronic venous insufficiency and hemorrhoids in Europe.[13],[14] Diosmin protected secondary restless leg syndrome caused by chronic venous insufficiency.[15] Diosmin has been used for the treatment of diabetes mellitus,[16] neurological problems like Alzheimer's diseases,[17] melanoma,[18] colon cancer and lymphedema.[19] Diosmin showed blood lipid lowering, antioxidant and anticarcinogenic activities.[20] Diosmin showed chemopreventive efficacy in urinary bladder carcinogenesis.[21] It also exhibited anti-inflammatory,[22] antimutagenic[23] and free radical scavenging activities. It has been reported that the anti-cell proliferative potential of diosmin is due to its metabolite diosmetin.[24] 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.

Drug exposure

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.[25]


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}

Reactive oxygen species generation

The intracellular reactive oxygen species (ROS) generation was measured according to the method of Pereira et al.,[26] using a nonfluorescent probe 2',7'-dichlorofluorescein diacetate.

Dual staining

The apoptotic induction potential of the diosmin was assessed using acridine orange/ethidium bromide dual staining.[27] 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).

DNA fragmentation

The DNA fragmentation assay was carried out according to the method of Herrmann et al.[28]

Western blotting

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.[29]

Statistical analysis

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.


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}

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}

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}

Western blotting

[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}

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}


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.[30] 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.[31] 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.[32] 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.[33] Apoptotic cells are characterized by cell shrinkage, nuclear damage and multiple DNA fragmentation.[34] 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.[35] MMPs also play a pivotal role in tumor angiogenesis.[36] 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.[37] Overexpression of MMP-2 and MMP-9 was associated with poor clinical outcome.[38] 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.[39] 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.[40] Lowered expression of p53 and increased expression of Bcl-2 proteins were well documented in various cancer cell lines.[41] 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 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.


1Lomas A, Leonardi-Bee J, Bath-Hextall F. A systematic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol 2012;166:1069-80.
2Kolk A, Wolff KD, Smeets R, Kesting M, Hein R, Eckert AW. Melanotic and non-melanotic malignancies of the face and external ear – A review of current treatment concepts and future options. Cancer Treat Rev 2014;40:819-37.
3Siegel RL, Fedewa SA, Miller KD, Goding-Sauer A, Pinheiro PS, Martinez-Tyson D, et al. Cancer statistics for Hispanics/Latinos, 2015. CA Cancer J Clin 2015;65:457-80.
4Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
5Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. Eur J Cancer 2013;49:1374-403.
6Kumar S, Mahajan BB, Kaur S, Yadav A, Singh N, Singh A. A study of Basal cell carcinoma in South Asians for risk factor and clinicopathological characterization: A hospital based study. J Skin Cancer 2014;2014:173582.
7Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, et al. In vitro cultivation of human tumors: Establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 1973;51:1417-23.
8Suresh K, Rajasekar M, Arun Kumar R, Sivakumar K. Dose tolerance study of diosmin against 7, 12-dimethylbenz (a) anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Int J Pharm Biol Arch 2014;5:82-9.
9Tanaka T, Makita H, Kawabata K, Mori H, Kakumoto M, Satoh K, et al. Modulation of N-methyl-N-amylnitrosamine-induced rat oesophageal tumourigenesis by dietary feeding of diosmin and hesperidin, both alone and in combination. Carcinogenesis 1997;18:761-9.
10Tanaka T, Makita H, Ohnishi M, Mori H, Satoh K, Hara A, et al. Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats by flavonoids diosmin and hesperidin, each alone and in combination. Cancer Res 1997;57:246-52.
11Alvarez N, Vicente V, Martínez C. Synergistic effect of diosmin and interferon-alpha on metastatic pulmonary melanoma. Cancer Biother Radiopharm 2009;24:347-52.
12Hosseinimehr SJ, Ahmadi A, Mahmoudzadeh A, Mohamadifar S. Radioprotective effects of Daflon against genotoxicity induced by gamma irradiation in human cultured lymphocytes. Environ Mol Mutagen 2009;50:749-52.
13Maksimovic ZV, Maksimovic M, Jadranin D, Kuzmanovic I, Andonovic O. Medicamentous treatment of chronic venous insufficiency using semisynthetic diosmin – A prospective study. Acta Chir Iugosl 2008;55:53-9.
14Diana G, Catanzaro M, Ferrara A, Ferrari P. Activity of purified diosmin in the treatment of hemorrhoids. Clin Ter 2000;151:341-4.
15Carpentier PH, Mathieu M. Evaluation of clinical efficacy of a venotonic drug: Lessons of a therapeutic trial with hemisynthesis diosmin in “heavy legs syndrome”. J Mal Vasc 1998;23:106-12.
16Jain D, Bansal MK, Dalvi R, Upganlawar A, Somani R. Protective effect of diosmin against diabetic neuropathy in experimental rats. J Integr Med 2014;12:35-41.
17Rezai-Zadeh K, Douglas Shytle R, Bai Y, Tian J, Hou H, Mori T, et al. Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease beta-amyloid production. J Cell Mol Med 2009;13:574-88.
18Martínez Conesa C, Vicente Ortega V, Yáñez Gascón MJ, Alcaraz Baños M, Canteras Jordana M, Benavente-García O, et al. Treatment of metastatic melanoma B16F10 by the flavonoids tangeretin, rutin, and diosmin. J Agric Food Chem 2005;53:6791-7.
19Hnátek L. Therapeutic potential of micronized purified flavonoid fraction (MPFF) of diosmin and hesperidin in treatment chronic venous disorder. Vnitr Lek 2015;61:807-14.
20Mohamed D, Tawakkol SM. Fluorimetric determination of diosmin and hesperidin in combined dosage forms and in plasma through complex formation with terbium. Bull Fac Pharm Cairo Univ 2013;51:81-8.
21Yang M, Tanaka T, Hirose Y, Deguchi T, Mori H, Kawada Y. Chemopreventive effects of diosmin and hesperidin on N-butyl-N-(4-hydroxybutyl) nitrosamine-induced urinary-bladder carcinogenesis in male ICR mice. Int J Cancer 1997;73:719-24.
22Crespo ME, Gálvez J, Cruz T, Ocete MA, Zarzuelo A. Anti-inflammatory activity of diosmin and hesperidin in rat colitis induced by TNBS. Planta Med 1999;65:651-3.
23Kuntz S, Wenzel U, Daniel H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur J Nutr 1999;38:133-42.
24Androutsopoulos VP, Mahale S, Arroo RR, Potter G. Anticancer effects of the flavonoid diosmetin on cell cycle progression and proliferation of MDA-MB 468 breast cancer cells due to CYP1 activation. Oncol Rep 2009;21:1525-8.
25Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
26Pereira C, Santos MS, Oliveira C. Involvement of oxidative stress on the impairment of energy metabolism induced by A beta peptides on PC12 cells: Protection by antioxidants. Neurobiol Dis 1999;6:209-19.
27Baskic D, Popovic S, Ristic P, Arsenijevic NN. Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int 2006;30:924-32.
28Herrmann M, Lorenz HM, Voll R, Grünke M, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 1994;22:5506-7.
29Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139:271-9.
30Nayak D, Pradhan S, Ashe S, Rauta PR, Nayak B. Biologically synthesised silver nanoparticles from three diverse family of plant extracts and their anticancer activity against epidermoid A431 carcinoma. J Colloid Interface Sci 2015;457:329-38.
31Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012;48:158-67.
32Alarifi S, Ali D, Alakhtani S, Al Suhaibani ES, Al-Qahtani AA. Reactive oxygen species-mediated DNA damage and apoptosis in human skin epidermal cells after exposure to nickel nanoparticles. Biol Trace Elem Res 2014;157:84-93.
33Smina TP, Mohan A, Ayyappa KA, Sethuraman S, Krishnan UM. Hesperetin exerts apoptotic effect on A431 skin carcinoma cells by regulating mitogen activated protein kinases and cyclins. Cell Mol Biol (Noisy-le-grand) 2015;61:92-9.
34Tang HL, Tang HM, Mak KH, Hu S, Wang SS, Wong KM, et al. Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response. Mol Biol Cell 2012;23:2240-52.
35Cathcart J, Pulkoski-Gross A, Cao J. Targeting matrix metalloproteinases in cancer: Bringing new life to old ideas. Genes Dis 2015;2:26-34.
36Tang Y, Nakada MT, Kesavan P, McCabe F, Millar H, Rafferty P, et al. Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases. Cancer Res 2005;65:3193-9.
37Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: Regulators of the tumor microenvironment. Cell 2010;141:52-67.
38Akgül B, Pfefferle R, Marcuzzi GP, Zigrino P, Krieg T, Pfister H, et al. Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice. Exp Dermatol 2006;15:35-42.
39Kumar S. Caspase function in programmed cell death. Cell Death Differ 2007;14:32-43.
40Jänicke RU, Sohn D, Schulze-Osthoff K. The dark side of a tumor suppressor: Anti-apoptotic p53. Cell Death Differ 2008;15:959-76.
41Eberle J, Hossini AM. Expression and function of bcl-2 proteins in melanoma. Curr Genomics 2008;9:409-19.