Capsaicin induces autophagy through ROS generation and tumor-associated NADH oxidase (tNOX) -sirtuin1 (SIRT1) down-regulation in melanoma cancer cells

惡性黑色素瘤是一種罕見的皮膚癌，罹患的人口在全世界都有內穩定上升的趨勢，並成為癌症相關死亡的主要原因。 近年來，具有抗癌特性的天然化合物在癌症預防中受到更多關注。 辣椒素是辣椒的主要成分，具有抗癌活性。 眾所周知，辣椒素抑制癌症/轉化細胞中tNOX的活性。然而辣椒素在A375細胞中的抗癌活性和tNOX的表達之間的關係仍不清楚。 在目前的研究中，我們已經證明細胞自噬(autophagy)參與tNOX表達辣椒素對黑素瘤癌細胞的作用機制。 我們的數據表明，辣椒素會藉由以劑量和時間的不同下調tNOX的表達，並隨後在黑素瘤細胞中誘導自噬。 此外，我們發現辣椒素可以觸發A375細胞中活性氧(ROS)的產生，而ROS清除劑N-乙酰半胱氨酸(NAC)可以完全減弱ROS的產生。 NAC預處理A375細胞使辣椒素對自噬的作用無效，並且還逆轉了對Akt途徑磷酸化的抑制。重要的是，辣椒素直接與tNOX和SIRT1結合併增強其活性。 總之，我們的數據表明，辣椒素在A375中通過抑制腫瘤相關的NADH氧化酶(tNOX)和sirtuin 1(SIRT1)誘導產生ROS，而帶來作為癌症治療相關藥物的可能性。A rare form of skin cancer, malignant melanoma steadily risen world-wide and acts as a main cause to related deaths of cancer disease. In recent years, the natural compounds having anti-cancer properties gathered more attention in cancer prevention. Capsaicin is the major ingredients of hot chili peppers which show anticancer activities. It is well known that capsaicin inhibits the activity of tNOX in both cancer/transformed cells. However, the anticancer activity of capsaicin in A375 cells in terms of tNOX expression still unclear. In the current study, we have demonstrated the autophagy involvement in tNOX expression on mechanism of action of capsaicin on melanoma cancer cells. Our data indicated that the capsaicin downregulates expression of tNOX in a dose and time- dependent manner and subsequently induced autophagy in melanoma cells. Moreover, we found that the capsaicin triggers generation of reactive oxygen species (ROS) in A375 cells, while the ROS scavenger N-acetyl cysteine (NAC) totally attenuated the ROS generation. The NAC pretreatment A375 cells nullified the capsaicin effect on autophagy and also reversed inhibition of phosphorylation of Akt pathway. Importantly, the capsaicin directly binds with tNOX and SIRT1 and enhancing their activity. All together, our data suggest that generation of ROS by capsaicin induced autophagy in A375 by inhibiting tumor-associated NADH oxidase (tNOX) and sirtuin 1(SIRT1) promoting its possibility as a therapeutic agent for cancer.摘要 i ABSTRACT ii Table of Contents iii Table of Illustrations iv 1.INTRODUCTION 1 2.RESULTS 4 Effects of capsaicin on tumor-associated NADH oxidase (tNOX) in A375 cells 4 Capsaicin decreases stability and directly engages of tNOX in A375 cells 5 Capsaicin downregulates tNOX protein expression and induces autophagy in A375 cells 6 Generation of ROS triggers autophagy in A375 Cells 7 Capsaicin induces generation of reactive oxygen species (ROS) in A375 cells 7 Capsaicin inhibits Akt/mTOR signaling through ROS modulation 8 3.DISCUSSION 10 4.METHODS AND MATERIALS 13 1. Drugs and Reagents 13 2. Cell culture and transfection 13 3. Western blot analysis 14 4. Reverse transcription-polymerase chain reaction (RT-PCR) 14 5. The cellular thermal shift assay (CETSA) 15 6. Measurement of reactive oxygen species (ROS) 15 7. Apoptosis Determination 15 8. Determination of Autophagy 16 9. Statistical analysis 16 5.FIGURE LEGENDS 17 6.REFERENCES 25 Supplementary Data 3