Anti-Melanogenesis Effect In B16F10 Cells Regulated By CNSL Extract And Its Mechanism On Inducing Human Malignant Melanoma Apoptosis

Melanin is crucial in protecting the skin from UV damage and other environmental factors,but the abnormal accumulation of melanin can lead to serious skin diseases such as freckles and chloasma,and even cancer.It’s important to search for specific inhibitors to regulate melanin production.And melanoma is a malignant solid tumor originating from skin tissue,is the most aggressive skin cancer,with a high degree of malignancy and increasing harm to human health.Hence,it has great practical significance to develop new candidate adjuvant drugs for the treatment of melanoma.Cashew nut is a common food and cashew nut shell is the by-product contains a large amount of cashew nut shell liquid(CNSL).Cardols/Cardanols are 5 kinds of active phenols obtained from natural CNSL,which have a broad application prospect in biomedical and cosmetic research.At present,there is no research report for the interference of CNSL extract to melanogenesis and anti-malignant melanoma.In this paper,mouse B16F10 cells and M14 human malignant melanoma cells were used as research objects to explore the mechanism of Cardols/Cardanols in regulating melanogenesis and anti-malignant melanoma.Above all,the extraction and purification of high purity CNSL extract was optimized and the preparation process was established.Cardols/Cardanols were obtained by HPLC.Using B16F10 melanoma cells as the melanogenic model,it was found that Cardols/Cardanols could interfere with the melanogenic production of B16F10 cells to regulate the depth of cell color,inhibit pigmentation,reduce melanogenic capacity,inhibit TYR activity,and significantly down-regulate melanogenesis induced by α-MSH and Forskolin.Molecular mechanism studies found that the inhibition of key products of cAMP by Cardols/Cardanols lead to blocked signal transduction.Cardols/Cardanols activated the PI3K/Akt signaling pathway,synergistically inhibited the α-MSH/cAMP/PKA and MAPK signaling pathways,thereby regulating MITF,affected expression levels of downstream key proteins TYR,TYRP-1 and TYRP-2.Ultimately Cardols/Cardanols inhibited the production of melanin.On the other hand,Cardanol Monoene(CM),the main component of CNSL extract,could inhibit the proliferation of M14 human malignant melanoma cells in vitro significantly,and inhibited colony formation,cell migration and invasion ability.Meanwhile,CM induced cell cycle S phase block to inhibit proliferation and growth.We confirmed the CM-induced apoptosis of M4 cells through qualitative and quantitative analysis.Apoptotic rate 43.46%,and showing the classic morphological characteristics of apoptosis.Moreover,CM-induced M14 apoptosis was different from necrosis and autophagy.Neither 3-MA nor Necrostatin-1 could inhibit CM-induced M14 cells death,but Z-VAD-FMK inhibited CM-induced M14 cell apoptosis.Further studies found that intracellular ROS content increased,P53 activated,Bax/Bcl-2 ratio was up-regulated,mitochondrial structure changed,ΔΨm decreased,and Cyto c released from mitochondria to bind to Apaf-1,activating Caspase cascade,PARP was cleaved,finally leading to cell death.CM induced M14 cell apoptosis as the dose-dependent manner through an endogenous mitochondrial mediated pathway triggered by ROS.On the animal level,BALB/c nude mouse of human malignant melanoma xenograft model was established.It was found that CNSL extract inhibited the growth of melanoma solid tumors without causing pathological changes significantly.The national standard acute oral toxicity test also showed that CNSL extract was safe and had no side effects.CNSL extract relied on the endogenous mitochondrial related signaling pathway to induce the apoptosis of cells in tumor tissues,and down-regulates the expression of VEGF and PCNA to achieve the anti-tumor effect.In summary,the anti-melanogenesis effect in B16F10 cells regulated by CNSL extract and its biological effect against malignant melanoma were systematically studied,which gave a theoretical foundation for the development and application of CNSL extract,and provides new ideas and theoretical basis for the treatment of pigmentation related disorders and malignant melanoma.