Enzyme HO1 that’s involved in heme catabolism and cleaves heme to kind biliverdin, carbon monoxide, and ferrous iron. HO1 is expressed in many sorts of cancers and is positively correlated with poor prognosis in individuals with cancers [23]. HO1 overexpression in cancer cells promotes proliferation, invasion, and survival [246]. On the other hand, current studies have indicated the antitumor effects of HO1. Gandini et al. found that HO1 activation induced apoptosis and inhibited migration and invasion by modulating epithelial esenchymal transition (EMT) in breast cancer cells [27]. Additionally, Yanagawa et al. reported that low HO1 expression is correlated with an increased danger of lymph node metastasis in OSCC [28]. These findings indicate the dual part of HO1 in cancer progression. On the other hand, the function of HO1 in OSCC still needs additional investigation. Artemisia annua L. is a Chinese traditional medicine which is extensively used to treat fever. Artemisinin, a sesquiterpene lactone isolated from A. annua L., exerts antiparasitic and anticancer effects [29,30]. Additionally, A. annua L. contains several phenolic compoundsCancers 2021, 13,3 ofincluding phenolic acids, flavonols, and flavones [31]. Handful of research have reported the anticancer and antiinflammatory properties of chrysosplenol D, a flavonol isolated from A. annua L. [32,33]. Moreover, flavonols which include casticin, quercetin, and kaempferol have been reported to market cancer cell apoptosis [346]. On the basis of those findings, we hypothesized that chrysosplenol D would inhibit cancer cell proliferation in OSCC. Thus, in this study, we investigated the effects of chrysosplenol D on OSCC and elucidated its mechanism underlying cell apoptosis. In addition, we evaluated the effect of HO1 on chrysosplenol Dtreated OSCC. 2. Components and Techniques two.1. Chemical substances and Reagents Chrysosplenol D (purity 98 ) was bought from ChemFaces (Wuhan Chem Faces Biochemical Co., Ltd., Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) to get a solution of 100 mM. Additionally, three(four, 5imethylthiazol2yl)2, 5diphenyltetrazolium bromide (MTT), 4,6diamidino2phenylindole (DAPI), protease inhibitor cocktail, and phosphatase inhibitor cocktail had been bought from Sigma Aldrich (St Louis, MO, USA). Main antibodies have been bought from Cell Signaling Technologies (Danvers, MA, USA). Secondary antibodies have been purchased from Jackson ImmunoResearch (West Grove, PA, USA). The final concentration of DMSO utilised in all treatment options was 0.1 . two.two. Cell Lines and Cell Culture Human OSCC cell lines, namely SCC9 and HSC3, were purchased from the American Form Culture Collection (Manassas, VA, USA). The OECM1 cell line was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). The HSC3M3 cell line, which features a higher metastatic possible for lymph nodes in human OSCC, is derived from the HSC3 cell line. The HSC3M3 cell line was bought from the Japanese Collection of Research Bioresources Cell Bank (Japan). For culturing, SCC9 cells have been grown in Dulbecco’s modified Eagle’s medium (DMEM): nutrient mixture F12 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10 fetal bovine serum (FBS), 1 mM Methoxyfenozide Autophagy Lglutamine, 1 penicillin/streptomycin, 1.5 g/L sodium bicarbonate, and 1 mM sodium pyruvate (Sigma Aldrich). HSC3 cells have been grown in DMEM medium (Gibco BRL) supplemented with 10 FBS, 1 mM glutamine, 1 penicillin/streptomycin, and 1.5 g/L sodium bicarbonate. OECM1 cells have been grown in RPMI 1640 medium (Gibco BRL).
