Biochemical and Biophysical Research Communications
Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells
Introduction
Nitric oxide (NO) is an endogenous, short-lived, pleiotropic signaling molecule, which is synthesized from l-arginine and oxygen by nitric oxide synthase (NOS). There are mainly three isoforms of NO synthase: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) [1], [2]. NO level and NOS activity have been found increased in multiple kinds of cancers, including breast cancer, lung cancer, gastric cancer, bladder cancer and oral cavity cancer [3], [4], [5]. NO promotes the hepatitis B virus X protein (HBx)-induced hepatocellular carcinoma by up-regulating HBx gene expression and JNK signaling pathway [6]. NO and its product peroxynitrite promote inflammation-related carcinogenesis by inducing DNA damage, facilitating formation of DNA lesion 8-nitroguanine and restraining DNA repair system. NO promotes cell proliferation by up-regulating endogenous basic fibroblast growth factor (bFGF), mitogen-activated protein kinase (MAPK) pathway and induction of heat-shock protein 90, 70 and 32. NO inhibits cell apoptosis with decreasing release of cytochrome C, suppressing caspase activation and stabilizing Bcl-2 by inducing cysteine S-nitrosylation [2], [5], [7], [8], [9], [10].
Nitric oxide regulates a lot of cellular responses by S-nitrosylation. Protein S-nitrosylation is a reversible post-translational modification which occurs on protein cysteine residues, by attaching an NO moiety with a reactive cysteine thiol group to form an S-nitrosothiol (SNO). Experimental evidence has indicated that the dysregulation of S-nitrosylation involves in numerous cancer-related pathological events, such as tumor origination, development, metastasis and treatment resistance [11], [12], [13], [14]. S-nitrosylation on the catalytic-site cysteines of caspase-3 inhibits its activity, and then suppresses apoptosis [8], [15]. For the antiapoptotic protein Bcl-2, S-nitrosylation on the cysteine residues (Cys158 and Cys229) inhibits ubiquitination and subsequent proteasomal degradation, thus enhances Bcl-2 stabilization [7], [16]. The activation of tumor suppressor gene PTEN is inhibited by S-nitrosylation, which could be prevented by S-sulfhydration [17].
Flavonoids are widespread in nature, regarded as safe and easily obtainable, because they can be extracted from natural plants, and many of them are part of the daily human diet [18], [19]. Flavonoids have been demonstrated to regulate NO-related cellular processes. Quercetin inhibits NO accumulation and activity of iNOS in SAS human oral cancer cells [20], cholangiocarcinoma (CCA) cells [21], and RINm5F rat insulinoma cells [22]. Acacetin suppresses transcriptional activities of Stat-1 and Stat-3 by inhibiting Tyr phosphorylations, and then reduces expressions of downstream targets iNOS and eNOS in human umbilical vein endothelial cells as well as human lung cancer and prostate cancer cells [23]. Epigallocatechin-3-gallate (EGCG) suppresses NO production and iNOS activity in cholangiocarcinoma (CCA) cells and murine mammary carcinoma cells, and reduces the LPS-induced S-nitrosylation of superoxide dismutase 2 (SOD2), seroxiredoxin (PRDX) and ubiquitin carboxyl-terminal hydrolase 14 (USP14) in murine BV-2 microglial cells [21], [24], [25]. Puerarin inhibits oxidative stress and protein S-nitrosylation in the rat diabetic kidneys [26].
Flavone, 2-phenyl-4H-1-benzopyran-4-one, is the core structure of flavonoids, which has been shown to possess antitumor function in human colon and breast cancer cells [27], [28], [29]. Some results implied that, maybe flavone's growth inhibitory effects are related to NO. In HT-29 human colon cancer cells, flavone induces cell apoptosis, activation of caspase-3-like and release of cytochrome C, which are all repressed by NO donor SNP, whereas the same concentration SNP treatment only do not cause any change in caspase-3-like activity [30]. In murine J774 macrophages and rat hepatocyte, flavone inhibits the LPS-induced NO production, iNOS expression and activation of nuclear factor-κB (NF-κB), which is an important transcription factor for iNOS [31], [32]. But, in cancer cells, there is still no definite experimental evidence about flavone's effect on NO.
In this study, we explored the flavone-induced anticancer effect and regulations on NO-related mechanism in MCF-7 and MDA-MB-231 breast cancer cells. Flavone inhibited cell proliferation and caused apoptosis by concentration- and time-dependent manner. We found flavone decreased NO production by inhibiting NOS activity, and then down-regulated the level of protein S-nitrosylation, which is a post-translational modification mediated by NO. In our knowledge, this is the first specific report focused on flavone-induced regulations on NO-related cellular processes and S-nitrosylation in cancer cells.
Section snippets
Cell culture
The human breast cancer cell lines MCF-7 and MDA-MB-231 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2–95% air.
MTT assay
MTT assay is based on the conversion of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Amresco, US) to formazan crystals by mitochondrial dehydrogenases. Briefly, MCF-7 cells were seeded in 96-well plates at a density of 2 × 104 cells per well for 18 h. Cells
Flavone inhibited cell viability and induced apoptosis
After treated with 100, 150, 200 and 250 μM flavone for 24 h, the inhibition rates on cell viability were 3.26 ± 8.20%, 11.04 ± 10.45%, 19.64 ± 3.52% and 20.41 ± 4.15%. Cell viability was significantly inhibited by 200 and 250 μM flavone (P < 0.01), and without significant effects by 100 and 150 μM flavone (Fig. 1A). After treated with 200 μM flavone for 48, 72 and 96 h, the inhibitory effects on cell viability increased with the extension of treatment time (Fig. 1B). Hoechst and propidium
Discussion
As the basic structure of flavonoids, flavone has been shown to inhibit cell growth by inducing DNA fragmentation, cell cycle arrests and caspase-3 activation in human colon and breast cancer cell lines [27], [29], [30]. Furthermore, flavone has selectivity to cause cell death in cancer cells but not in nontumorigenic epithelial cells [27], [28], [34]. In this study, our results indicated flavone inhibited cell proliferation and induced apoptosis with a concentration- and time-dependent manner
Conflict of interest
All authors declare no conflict of interest.
Acknowledgments
This work was supported by the Special Funds of the National Natural Science Foundation of China (Grant No. 31340035), the Science and Technology Development Program of Shandong Province (2012CSF12112), the Key Program of Shandong Provincial Natural Science Foundation of China (ZR2013CZ002),the Research Fund for the Doctoral Program of Higher Education of China (20113704110004), Science and Technology Program of Jinan (201202033).
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