Chemotherapeutics are widely recognized for their adverse side-effects during anti-cancer regiments. One of the complementary approaches to circumvent this dilemma could be the exploitation of natural compounds, which could optimally counteract the cellular damages during chemotherapy. The present study ventures to evaluate the natural flavonoid, Chrysin (5, 7-dihydroxyflavone) for its therapeutic immunomodulatory properties along with the chemotherapeutic drug, Cyclophosphamide (CP). Male Wistar albino rats were utilized for this study. Assays were conducted for Acute Toxicity, Hemolysis, Phagocytosis, Natural Killer (NK) cell cytotoxicity, and oxidative stress. RT-PCR, ELISA and Western Blot were performed to assess the expression of inflammatory markers. Assay results such as Phagocytosis Index ( 0.009 ± 0.001), NK Cell cytotoxicity (61.10 ± 4.99 % ), expression of Perforin (0.45 ± 0.05 fold) and Granzyme ( 0.86 ± 0.01 fold ), hepatic antioxidative enzymes GSH (27.75 ± 1.54 mg/mg ), SOD ( 7.10 ± 0.35 U/mg ) and CAT (249.06 ± 31.30 mM/Min/mg ) and splenic hepatic antioxidative enzymes GSH (20.88 ± 0.74mg/mg), SOD (7.10 ± 0.35 U/mg) and CAT (249.06 ± 31.30mM/Min/mg) among the CP-treated groups were compared with those for the CP+Chrysin treated groups which were evaluated to be significantly increased with values of 0.016 ± 0.001, 82.73 ± 2.87 %, 0.77 ± 0.08 fold,1.11 ± 0.02 fold, 47.60 ± 3.02mg/mg, 08.97 ± 0.42 U/mg, 467.19 ± 15.92 mM/Min/mg, 29.02 ± 1.59 mg/mg, 5.17 ± 0.94 U/mg, 310.29 ± 9.1330 mM/Min/mg, respectively. Histopathological examination indicated that CP+Chrysin treated groups could recover from cellular damage triggered during the CP treatment. Results indicate the cytoprotective role of Chrysin, which in turn, could be reliably administered as a complementary therapy along with CP during chemotherapy.
Keywords: Chemotherapeutic agents, Cyclophosphamide, Chrysin, Inflammation,Toxicity
Full text and supplemented materials
References
Boothapandi, M. and Ramanibai, R., Immunomodulatory effect of natural flavonoid chrysin (5, 7–dihydroxyflavone) on LPS stimulated RAW 264.7 macrophages via inhibition of NF–κB activation, Process Biochem., 2019, vol. 84, pp. 186–195.
Boothapandi, M. and Ravichandran, R., Antiproliferative activity of chrysin (5, 7–dihydroxyflavone) from Indigofera tinctoria on human epidermoid carcinoma (A431) cells, Eur. J. Integr. Med., 2018, vol. 24, pp. 71–78.
Bose, B., Chung, E.Y., Hong, R., et al., Immunosuppression therapy for idiopathic membranous nephropathy: systematic review with network meta–analysis, J. Nephrol., 2022, vol. 35, no. 4, pp. 1159–1170. https://doi.org/10.1007/s40620-022-01268-2
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. B-iochem., 1976, vol. 72, nos. 1–2, pp. 248–254. https://doi.org/10.1006/abio.1976.9
Chang, Y., Guo, A., and Jing, Y., et al., Immunomodulatory activity of puerarin in RAW264.7 macrophages and cyclophosphamide–induced immunosuppression mice, Immunopharmacol. Immunotoxicol., 2021, vol. 43, no. 2, pp. 223–229. https://doi.org/10.1080/08923973.2021.1885043
Chen, X., Sun, W., and Xu, B., et al., Polysaccharides from the roots of Millettia speciosa champ modulate gut health and ameliorate cyclophosphamide–induced intestinal injury and immunosuppression, Front. Immunol., 2021, vol. 12, p. 766296. https://doi.org/10.3389/fimmu.2021.766296
Chen, X., Bi, M., and Yang, J., et al., Cadmium exposure triggers oxidative stress, necroptosis, Th1/Th2 imbalance and promotes inflammation through the TNF–α/NF–κB pathway in swine small intestine, J. Hazard. Mater., 2022, vol. 421, p. 126704. https://doi.org/10.1016/j.jhazmat.2021.126704
Dantas, A.C., Batista–Júnior, F.F., and Macedo, L.F., et al., Protective effect of simvastatin in the cyclophosphamide–induced hemohrragic cystitis in rats, Acta Cirúrgica Brasileira, 2010, vol. 25, pp. 43–46. https://doi.org/10.1590/s0102-86502010000100011
Dollery, C., Therapeutic Drugs, Edinburg: Churchill Livingstone.
Ekeanyanwu, R.C. and Njoku, O.U., Acute and subacute oral toxicity study on the flavonoid rich fraction of Monodora tenuifolia seed in albino rats, Asian Pac. J. Trop. Biomed., 2014, vol. 4, no. 3, pp. 194–202. https://doi.org/10.1016/S2221-1691(14)60231-8
Ganeshpurkar, A. and Saluja, A.K., Protective effect of catechin on humoral and cell mediated immunity in rat model, Int. Immunopharmacol., 2018, vol. 54, pp. 261–266. https://doi.org/10.1016/j.intimp.2017.11.022
GLOBOCAN, New Global Cancer Data, 2020. https:// www.uicc.org/news/globocan–2020–new–global–cancer–data. Accessed July 20, 2023.
Gunawan, S., Broeke, C.T., Ven, P.V., et al., Parental experiences with chemotherapy–induced alopecia among childhood cancer patients in Indonesia, Asian Pac. J. Cancer Prev., 2016, vol. 17, no. 4, pp. 1717–1723. https://doi.org/10.7314/apjcp.2016.17.4.1717
Gutteridge, J.M., Lipid peroxidation and antioxidants as biomarkers of tissue damage, Clin. Chem., 1995, vol. 41, no. 12, pp. 1819–1828.
Hartner, L., Chemotherapy for oral cancer, Dental Clinics, 2018 vol. 62, no. 1, pp. 87–97.
Hassan, M.S.U., Ansari, J., Spooner, D., et al., Chemotherapy for breast cancer, Oncol. Rep., 2010, vol. 24, no. 5, pp. 1121–1131.
Hutter, D. and Greene, J.J., Influence of the cellular redox state on NF-κB-regulated gene expression, J. Cell. Physiol., 2000, vol. 183, no. 1, pp. 45–52. https://doi.org/10.1002/(SICI)1097-4652(200004)183:1<45::AID-JCP6>3.0.CO;2-P
Inoue, H. and Tani, K.J., Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments, Cell Death Differ., 2014, vol. 21, no. 1, pp. 39–49. https://doi.org/10.1038/cdd.2013.84
Ishwarya, M. and Narendhirakannan, R.T., 3, 4 Dihydroxycinnamic acid stimulates immune system function by modifying the humoral antibody response – An in vivo study, Cell. Immunol., 2017, vol. 314, pp. 10–17. https://doi.org/10.1016/j.cellimm.2017.01.006
Jiao, X., Zhang, X., Li, N., et al., Treg deficiency-mediated TH1 response causes human premature ovarian insufficiency through apoptosis and steroidogenesis dysfunction of granulosa cells, Clin. Transl. Med., 2021, vol. 11, no. 6, p. e448. https://doi.org/10.1002/ctm2.448
King, P.D. and Perry, M.C., Hepatotoxicity of chemotherapy, Oncologist, 2001, vol. 6, no. 2, pp. 162–176.
Kumar, V.P. and Venkatesh, Y.P., Alleviation of cyclophosphamide–induced immunosuppression in Wistar rats by Onion lectin (Allium cepa agglutinin), J. Ethnopharmacol., 2016, vol. 186, pp. 280–288. https://doi.org/10.1016/j.jep.2016.04.006
La Mantia, L., Milanese, C., Mascoli, N., et al., Cyclophosphamide for multiple sclerosis, Cochrane Database Syst. Rev., 2002, no. 3.
Lalla, R.V., Brennan, M.T., Gordon, S.M., et al., Oral mucositis due to high–dose chemotherapy and/or head and neck radiation therapy, J. Natl. Cancer Inst., 2019, no. 53, p. lgz011. https://doi.org/10.1093/jncimonographs/lgz011
Lee, J., Choi, J.W., Sohng, K., et al., The immunostimulating activity of quercetin 3–O–xyloside in murine macrophages via activation of the ASK1/MAPK/NF–κB signaling pathway, Int. Immunopharmacol., 2016, vol. 31, pp. 88–97. https://doi.org/10.1016/j.intimp.2015.12.008
Li, X., Huang, Q., Ong, C.N., et al., Chrysin sensitizes tumor necrosis factor–α–induced apoptosis in human tumor cells via suppression of nuclear factor–kappaB, Cancer Lett., 2010, vol. 293, no. 1, pp. 109–116. https://doi.org/10.1016/j.canlet.2010.01.002
Li, Y., Yu, P., Fu, W., et al., Ginseng–Astragalus–oxymatrine injection ameliorates cyclophosphamide–induced immunosuppression in mice and enhances the immune activity of RAW264.7 cells, J. Ethnopharmacol., 2021, vol. 279, p. 114387. https://doi.org/10.1016/j.jep.2021.114387
Liu, L., Li, H., Xu, R.H., et al., Exopolysaccharides from Bifidobacterium animalis RH activates RAW 264.7 macrophages through toll–like receptor 4, Food Agric. Immunol., 2017, vol. 28, no. 1, pp. 149–61.
Lyman, G.H., Abella, E., and Pettengell, R., Risk factors for febrile neutropenia among patients with cancer receiving chemotherapy: a systematic review, Crit. Rev. Oncol., 2014, vol. 90, no. 3, pp. 190–199. https://doi.org/10.1016/j.critrevonc.2013.12.006
Masella, R., Di Benedetto, R., Varì, R., et al., Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione–related enzymes, J. Nutr. Biochem., 2005, vol. 16, no. 10, pp. 577–586. https://doi.org/10.1016/j.jnutbio.2005.05.013
Michael, A., Syrigos, K., and Pandha, H., Prostate cancer chemotherapy in the era of targeted therapy, Prostate Cancer Prostatic Dis., 2009, vol. 12, no. 1, pp. 13–16.
Mirzaei, S., Saghari, S., Bassiri, F., et al., NF-κB as a regulator of cancer metastasis and therapy response: A focus on epithelial–mesenchymal transition, J. Cell. Physiol., 2022, vol. 237, no. 7, pp. 2770–2795. https://doi.org/10.1002/jcp.30759
Misra, H.P. and Fridovich, I., The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase, J. Biol. Chem., 1972, vol. 247, no. 10, pp. 3170–3175.
Moraes, J.P., Pereira, D.S., Matos, A.S., et al., The ethanol extract of the inner bark of Caesalpinia pyramidalis (Tul.) reduces urinary bladder damage during cyclophosphamide–induced cystitis in rats, Sci. World J., 2013. https://doi.org/10.1155/2013/694010
Moron, M.S., Depierre, J.W., and Mannervik, B., Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver, Biochim. Biophys. Acta, 1979, vol. 582, no. 1, pp. 67–78. https://doi.org/10.1016/10.1016/0304-4165(79)90289-7
Nafees, S., Rashid, S., Ali, N., et al., Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway., Chem.-Biol. Interact., 2015, vol. 231, pp. 98–107. https://doi.org/10.1016/j.cbi.2015.02.021
Nathan, C. and Shiloh, M.U., Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens, Proc. Natl. Acad. Sci. U. S. A., 2000, vol. 97, no. 16, pp. 8841–8848. https://doi.org/10.1073/pnas.97.16.8841
Naval, J., Villacampa, M.J., Goguel, A.F. and Uriel, J., Cell–type–specific receptors for alpha–fetoprotein in a mouse T–lymphoma cell line, Proc. Natl. Acad. Sci. U. S. A., 1985, vol. 82, no. 10, pp. 3301–3305. https://doi.org/10.1073/pnas.82.10.3301
Niakani, A., Farrokhi, F., and Hasanzadeh, S., Decapeptyl ameliorates cyclophosphamide–induced reproductive toxicity in male Balb/C mice: histomorphometric, stereologic and hormonal evidences., Iran. J. Reprod. Med., 2013, vol. 11, no. 10, p. 791.
Novin, M.G., Alvani, M.S., Balani, M.M., et al., Therapeutic effects of edaravone on azoospermia: free radical scavenging and autophagy modulation in testicular tissue of mice, J. Reprod. Infertil., 2022, vol. 23, no. 2, p. 73. https://doi.org/10.18502/jri.v23i2.8990
Olayinka, E.T., Ore, A., Ola, O.S., et al., Ameliorative effect of Gallic acid on cyclophosphamide–induced oxidative injury and hepatic dysfunction in rats, Med. Sci., 2015, vol. 3, no. 3, pp. 78–92. https://doi.org/10.3390/medsci3030078
Prager, I. and Watzl, C., Mechanisms of natural killer cell–mediated cellular cytotoxicity, J. Leukocyte Biol., 2019, vol. 105, no. 6, pp. 1319–1329. https://doi.org/10.1002/JLB.MR0718-269R
Pratheeshkumar, P. and Kuttan, G., Cardiospermum halicacabum inhibits cyclophosphamide induced immunosupression and oxidative stress in mice and also regulates iNOS and COX–2 gene expression in LPS stimulated macrophages, Asian Pac. J. Cancer Prev., 2010, vol. 11, no. 5, pp. 1245–1252.
Rasheed, H.M., Rasheed, F., Qureshi, A.W., et al., Immunostimulant activities of the aqueous methanolic extract of Leptadenia pyrotechnica, a plant from Cholistan desert, J. Ethnopharmacol., 2016, vol. 186, pp. 244–250. https://doi.org/10.1016/j.jep.2016.03.039
Rego, A.C., Vesce, S., and Nicholls, D.G., The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1–7 neural cells, Cell Death Differ, 2001, vol. 8, no. 10, pp. 995–1003. https://doi.org/10.1038/sj.cdd.4400916
Reilly, C.A. and Aust, S.D., Measurement of lipid peroxidation, Curr. Protoc. Toxicol., 1999, no. 1, pp. 2–4. https://doi.org/10.1002/0471140856.tx0204s00
Ritter, A.T., Shtengel, G., Xu, C.S., et al., ESCRT–mediated membrane repair protects tumor–derived cells against T cell attack, Science, 2022, vol. 376, no. 6591, pp. 377–382. https://doi.org/10.1126/science.abl3855
Ruggiero, A., Rizzo, D., Catalano, M., et al., Acute chemotherapy–induced nausea and vomiting in children with cancer: Still waiting for a common consensus on treatment, Int. J. Med. Res., 2018, vol. 46, no. 6, pp. 2149–2156. https://doi.org/10.1177/0300060518765324
Sak, K., Chemotherapy and dietary phytochemical agents, Chemother. Res. Pract., 2012. https://doi.org/10.1155/2012/282570
Salem, M.L., Matsuzaki, G., Madkour, G.A., et al., Beta-estradiol-induced decrease in IL-12 and TNF-α expression suppresses macrophage functions in the course of Listeria monocytogenes infection in mice, Int. J. Immunopharmacol., 1999, vol. 21, no. 8, pp. 481–497. https://doi.org/10.1016/s0192-0561(99)00027-2
Salva, S., Marranzino, G., Villena, J., et al., Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide–treated mice, Int. Immunopharmacol., 2014, vol. 22, no. 1, pp. 209–221. https://doi.org/10.1016/j.intimp.2014.06.017
Sangeetha, P., Das, U.N., Koratkar, R., et al., Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer, Free Radical B-iol. Med., 1990, vol. 8, no. 1, pp. 15–19. https://doi.org/10.1016/0891-5849(90)90139-a
Saxena, M., Saxena, J., Nema, R., et al., Phytochemistry of medicinal plants, J. Pharmacogn. Phytochem., 2013, vol. 1, no. 6.
Schwab, C.E. and Tuschl, H., In vitro studies on the toxicity of isoniazid in different cell lines, Human Exp. Toxicol., 2003, vol. 22, no. 11, pp. 607–615. https://doi.org/10.1191/0960327103ht401oa
Senthilkumar, S., Devaki, T., Manohar, B.M., et al., Effect of squalene on cyclophosphamide-induced toxicity, Clin. Chim. Acta, 2006, no. 364, nos. 1–2, pp. 335–342. https://doi.org/10.1016/j.cca.2005.07.032
Shokrzadeh, M., Ahmadi, A., Chabra, A., et al., An ethanol extract of Origanum vulgare attenuates cyclophosphamide-induced pulmonary injury and oxidative lung damage in mice, Pharm. Biol., 2014, vol. 52, no. 10, pp. 1229–1236. https://doi.org/10.3109/13880209.2013.879908
Simpson, M.A. and Gozzo, J.J., Spectrophotometric determination of lymphocyte mediated sheep red blood cell hemolysis in vitro, J. Immunol. Methods, 1978, vol. 21, nos. 1–2, pp. 159–165. https://doi.org/10.1016/0022-1759(78)90232-6
Sinha, A.K., Colorimetric assay of catalase, Anal. Biochem., 1972, vol. 47, no. 2, pp. 389–394. https://doi.org/10.1016/0003-2697(72)90132-7
Sonenshein, G.E., Rel/NF–κB transcription factors and the control of apoptosis, Semin. Cancer Biol., 1997, vol. 8, no. 2, pp. 113–119. https://doi.org/10.1006/scbi.1997.0062
Souza, L.C., Antunes, M.S., Borges Filho C., et al., Flavonoid Chrysin prevents age–related cognitive decline via attenuation of oxidative stress and modulation of BDNF levels in aged mouse brain, Pharmacol. Biochem. Behav., 2015, vol. 134, pp. 22–30. https://doi.org/10.1016/j.pbb.2015.04.010
Suleyman, H., Gul, H.I., and Asoglu, M., Anti-inflammatory activity of 3-benzoyl-1-methyl-4-phenyl-4-piperidinol hydrochloride, Pharmacol. Res. Commun., 2003, vol. 47, no. 6, pp. 471–475. https://doi.org/10.1016/s1043-6618(03)00015-x
Susskind, B.M. and Chandrasekaran, J., Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine, J. Immunol., 1987, vol. 139, no. 3, pp. 905–912.
Suvarna, S.K., Layton, C., and Bancroft, J.D., Theory and Practice of Histological Techniques, London: Churchill Livingstone, Elsiver, 2013, pp. 173–187.
Tanaka, T., Narazaki, M., Kishimoto, T., IL–6 in inflammation, immunity, and disease, Cold Spring Harbor Perspect. Biol., 2014, vol. 6, no. 10, p. a016295. https://doi.org/10.1101/cshperspect.a016295
Tekin, S. and Seven, E., Assessment of serum catalase, reduced glutathione, and superoxide dismutase activities and malondialdehyde levels in keratoconus patients, Eye, 2022, vol. 36, no. 10, pp. 2062–2066. https://doi.org/10.1038/s41433-021-01753-1
Teles, K.A., Medeiros–Souza, P., Lima, F.A., et al., Cyclophosphamide administration routine in autoimmune rheumatic diseases: a review, Rev. Bras. Reumatol., 2017, vol. 57, pp. 596–604. https://doi.org/10.1016/j.rbre.2016.09.008
Van Horssen, R., Ten Hagen, T.L., and Eggermont, A.M., TNF–α in cancer treatment: molecular insights, antitumor effects, and clinical utility, Oncologist, 2006, vol. 11, no. 4, pp. 397–408. https://doi.org/10.1634/theoncologist.11-4-397
Villar, I.C., Jiménez, R., Galisteo, M., et al., Effects of chronic chrysin treatment in spontaneously hypertensive rats, Planta Med., 2002, vol. 68, no. 09, pp. 847–850. https://doi.org/10.1055/s-2002-34400
Voiculescu, C.L., Rosu, L., and Rogoz, S., Modulation of mouse spleen natural killer (NK) cell activity by beta–interferon, interleukin–1, and prostaglandins, Lymphology, 1988, vol. 21, no. 3, pp. 144–151.
Wang, C.C., Huang, Y.J., Chen, L.G., et al., Inducible nitric oxide synthase inhibitors of Chinese herbs III. Rheum palmatum, Planta Med., 2002, vol. 68, no. 10, pp. 869–874. https://doi.org/10.1055/s-2002-34918
Won, J.H., Im, H.T., Kim, Y.H., et al., Anti–inflammatory effect of buddlejasaponin IV through the inhibition of iNOS and COX–2 expression in RAW 264.7 macrophages via the NF–κB inactivation, Br. J. Pharmacol., 2006, vol. 148, no. 2, p. 216. https://doi.org/10.1111/cas.14933
Yasuda, H., Yasuda, M., and Komatsu, N., Chemotherapy for non-Hodgkin lymphoma in the hemodialysis patient: A comprehensive review, Cancer Sci., 2021, vol. 112, no. 7, pp. 2607–2624. https://doi.org/10.1111/cas.14933
Yu, Q., Nie, S.P., Wang, J.Q., et al., Chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide–induced mice, Int. J. Biol. Macromol., 2014, vol. 64, pp. 395–401. https://doi.org/10.1016/j.ijbiomac.2013.12.029
Yuan, W., Dasgupta, A., and Cresswell, P., Herpes simplex virus evades natural killer T cell recognition by suppressing CD1d recycling, Nat. Immunol., 2006, vol. 7, no. 8, pp. 835–842. https://doi.org/10.1038/ni1364
Zhai, J., Zhang, F., Gao, S., et al., Schisandra chinensis extract decreases chloroacetaldehyde production in rats and attenuates cyclophosphamide toxicity in liver, kidney and brain, J. Ethnopharmacol., 2018, vol. 210, pp. 223–231. https://doi.org/10.1016/j.jep.2017.08.020
Zhao, G., Kan, J., Li, Z., et al., Characterization and immunostimulatory activity of an (1→ 6)–a-D–glucan from the root of Ipomoea batatas, Int. Immunopharmacol., 2005, vol. 5, no. 9, pp. 1436–1445. https://doi.org/10.1016/j.intimp.2005.03.012