MAPK signaling mediates sinomenine hydrochloride-induced human breast cancer cell death via both reactive oxygen species-dependent and -independent pathways: an in vitro and in vivo study

Sinomenine, the main alkaloid extracted from the medicinal plant Sinomenium acutum, is known for its anti-inflammatory effects. Recent studies have suggested its anti-cancer effect in synovial sarcoma, lung cancer and hepatic cancer. However, the underlying molecular mechanism for its anti-cancer effect still remains unclear. This study investigated the anti-tumor activity of sinomenine hydrochloride (SH), a hydrochloride form of sinomenine, in human breast cancer cells in vitro and in vivo. We found that SH potently inhibited cell viability of a broad panel of breast cancer cell lines. Two representative breast cancer cell lines, namely ER(−)/PR(−) MDA-MB-231 and ER(+)/PR(+) MCF-7, were used for further investigation. The results showed that SH induced G1/S cell cycle arrest, caused apoptosis and induced ATM/Chk2- and ATR/Chk1-mediated DNA-damage response in MDA-MB-231 and MCF-7. The anti-cancer effect of SH was regulated by increased expression levels of p-ERK, p-JNK and p-38 MAPK. Further studies showed that SH resulted in an increase in reactive oxygen species (ROS) and inhibition of ROS by N-acetyl-L-cysteine (NAC) almost blocked SH-induced DNA damage but only mitigated SH-induced MAPK expression changes, suggesting that both ROS-dependent and -independent pathways were involved in MAPK-mediated SH-induced breast cancer cell death. The in vivo study demonstrated that SH effectively inhibited tumor growth without showing significant toxicity. In conclusion, SH induced breast cancer cell death through ROS-dependent and -independent pathways with an upregulation of MAPKs, indicating that SH may be a potential anti-tumor drug for breast cancer treatment.In recent decades, breast cancer is increasing in both developed and developing countries.^{1, 2, 3} Breast cancer has become the most common cancer and the leading cause of death in women all over the world.⁴ Although current strategies targeting breast cancer have improved markedly, breast cancer patients often develops metastasis⁵ and drug resistance.⁶ Therefore, it is necessary to search for new effective therapies for breast cancer treatment.Plants are one of the most important sources of compounds for chemoprevention and >60% of cancer therapeutics on the market or in preclinical trials are based on natural products.^{7, 8} The medicinal plant Sinomenium acutum Rehd. et Wils. (Fam. Menispermaceae) has been used to effectively treat rheumatoid arthritis for centuries in the Far East.⁹ Since its main effective component sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one, C19H23NO4, molecular weight: 329.38 Da, Figure 1a), a pure alkaloid, was extracted from the plant, numerous studies have been conducted on its underlying mechanisms for rheumatoid arthritis treatment^{10, 11} and other possible pharmacological effects, such as attenuation of ischemia/reperfusion injury,^{12, 13} treatment of neurodegenerative disorders¹⁴ and reduction of analgesic tolerance.¹⁵ Sinomenine hydrochloride (SH, Figure 1b), a hydrochloride chemical form of sinomenine, is widely used in clinical treatment of rheumatoid diseases for its anti-inflammatory and anti-immune effects.¹⁶ Recently, its anti-tumor activity has been found in synovial sarcoma, lung cancer and hepatic cancer;^{17, 18, 19} however, the molecular mechanisms and the signaling pathways of SH against cancer are still not clarified, and no studies have investigated whether SH could induce breast cancer cell death.Open in a separate windowFigure 1SH inhibited human breast cancer cell viability. Chemical structures of (a) sinomenine and (b) SH. (c) A panel of human breast cancer cell lines (MDA-MB-231, MCF-7, SK-BR-3, ZR-75-30, BT474 and T47D) were treated with SH (0, 0.1, 0.5 and 5.0 μmol/ml) for 48 h. Cell viability was measured by MTT assay. (d) Time-dependent inhibition of SH was evaluated by MTT assay. Data are represented as mean±S.D. of three independent experiments. *P <0.05, ^#P <0.01, SH-treated group compared with the untreated control group. (e) Cell colony formation was evaluated by clonogenic assay. (f) Morphology changes of breast cancer cells treated with SH. Representative data from three independent experiments are shownThere exist seven classes of mitogen-activated protein kinase (MAPK) intracellular signaling cascades, and four of them are implicated in breast diseases and function in mammary epithelial cells, including the extracellular-regulated kinase (ERK)1/2 pathway, the c-Jun N-terminal kinase (JNK) pathway, the p38 MAPK pathway and the ERK5 pathway.²⁰ In this study, we especially focused on three prominent MAPK pathways, namely ERK1/2, JNK and p38. Milde-Langosch et al.²¹ have pointed out in a clinical study that high phosphorylated ERK proteins are good prognostic indicators in breast cancer. Sustained phosphorylation of p38 and JNK in breast cancer cells are also involved in anti-cancer treatment.²² Considering the important roles of MAPKs in breast cancer progression and cell proliferation, we hypothesized that SH inhibited breast cancer growth via modulation of MAPK pathways.In this study, we first demonstrated the anti-proliferative effect of SH on breast cancer cells in vitro and in vivo. We found that SH induced G1/S cell cycle arrest, caused cell apoptosis and triggered oxidative DNA damage in breast cancer cells. The results also demonstrated that SH induced breast cancer cell death by upregulating MAPK pathways and increasing intracellular reactive oxygen species (ROS) generation. The ROS scavenger N-acetyl-L-cysteine (NAC) almost blocked SH-induced DNA damage but only mitigated SH-induced MAPK expression changes, indicating that both ROS-dependent and -independent pathways were involved in the MAPK-mediated anti-cancer effect of SH.