Capsaicin Induces Ca²⁺ Homeostasis Imbalance And Autophagy Of Anaplastic Thyroid Cancer Cells And Its Inhibition On Stemness

CAP（Capsaicin）,an alkaloid found in peppers,is the main active ingredient of red peppers,a kind of foodstuff flavoring.In recent years,CAP has shown its excellent anti-cancer ability in various types of cancers,and its potential pharmacological activity is paid more and more attention by numerous researchers.Based on the statistical data from World Health Organization,cancer incidence worldwide rised rapidly in 2020,and cancer mortality has also reached highest level in history.ATC（Anaplastic thyroid cancer）,the most rare and malignant thyroid tumor,is resistant to current therapies with its undifferentiated（stemness）status,resulting in a extremely low survival rate of ATC patients.Hence,this paper aimed to explore the inhibitory effect of CAP on the stemness of ATC cells and to find out the underlying mechanism.This study will provide insight of the physiological activity of CAP,and further supply theoretical support for CAP as an adjuvant drug in ATC treatment.The results from cell viability assays showed that 150-200μM of CAP could selectively kill8505C and FRO cells without affecting the growth of Nthy-ori-3.1 cells,indicating that CAP has a good toxic effect on the thyroid cancer cells.By using fluorescence spectrophotometer and flow cytometry methods,we further found that CAP could significantly increase the concentration of Ca²⁺in cytoplasm and mitochondrion in 8505C and FRO cells,followed by inducing the imbalance of cytoplasmic and mitochondrial Ca²⁺homeostasis(intracellular Ca²⁺homeostasis imbalance).Intracellular Ca²⁺homeostasis imbalance of8505C and FRO cells further promoted the continuous opening of mitochondrial permeability transition pore,which finally resulted in the cell death of the two cells.BAPTA-AM,a Ca²⁺chelator,could buffer the excessively elevated Ca²⁺and capsazepine,an antagonist against TRPV1 channel,could competitively bind to TRPV1 channel.After the combined treatment of BAPTA-AM（or capsazepine）and CAP,CAP could not significantly induce the imbalance of cytoplasmic and mitochondrial Ca²⁺homeostasis as well as the opening of m PTP in 8505C and FRO cells,indicating that CAP triggered the imbalance of intracellular Ca²⁺homeostasis in ATC cells by activating TRPV1channel.On the base of the above results,we explored the downstream biological effects caused by capsaicin-induced Ca²⁺homeostasis imbalance.By performing Western Blot method,it was found that CAP could markedly upregulate the expression level of autophagy marker protein LC3-II,and downregulate p62 expression level.Compared with CAP treatment alone,the co-treatment of CAP and the autophagy inhibitor 3-MA（3-methyladenine）,could siginificantly inhibit LC3-II expression level while the co-treatment of CAP and CQ（chloroquine）,another autophagy inhibitor,further significantly increased LC3-II expression level in 8505C and FRO cells,showing that CAP induced autophagy in ATC cells.CAP inhibited the phosphorylation of S6 and 4E-BP1 which are the key proteins of m TORC1 signaling pathway in 8505C and FRO cells,leading to the decreased expression of p-S6 and p-4E-BP1,indicating that CAP activated autophagy of ATC cells by inhibiting m TORC1signaling pathway.Moreover,compared with CAP treatment alone,the combined treatment of BAPTA-AM（or capsazepine）and CAP could significantly upregulate p-S6 expression level and inhibit LC3-II expression level at the same time.These results showed that CAP induced intracellular Ca²⁺homeostasis imbalance to inhibit the activity of m TORC1 signaling pathway and finally activated autophagy of ATC cells.The strong stemness of ATC cells is the main reason to limit the therapeutic effect of existing therapies.Using sphere formation assay and stemness-related proteins detection,we found that CAP could inhibit the stemness of 8505C and FRO cells.In addition,when 8505C and FRO cells were co-treated with 3-MA（or CQ）and CAP,CAP could not significantly inhibit the sphere formation ability and expression levels of stemness-related proteins in the two cells,declaring CAP depended on autophagy activation to suppress ATC stemness.Among the six stemness-related proteins（ALDH1A1、CD44、CD133、SOX2、OCT4A and NANOG）detected in our study,only the expression level of OCT4A was significantly inhibited both in 8505C and FRO cells treated with CAP.And after the co-treatment of 3-MA（or CQ）and CAP,only the expression of OCT4A could recover,indicating that CAP specifically promoted autophagy-lysosome degradation of OCT4A.The result that the m RNA expression of OCT4A was not affected by CAP further supported the above conclusion.Moreover,compared with CAP treatment alone,OCT4A protein level recovered in 8505C and FRO cells co-treated with BAPTA-AM（or capsazepine）and CAP,indicating that CAP promoted autophagy-lysosome degradation of OCT4A through Ca²⁺-dependent autophagy in both cells.In conclusion,CAP induced the Ca²⁺homeostasis imbalance in ATC cells by activating TRPV1channel,and finally killed ATC cells.Moreover,Ca²⁺homeostasis imbalance induced by CAP activated autophagy of ATC cells by inhibiting m TORC1 signaling pathway,enhanced the autophagy lysosomal degradation of stem cell biomarker OCT4A,and finally the stemness of ATC cells was suppressed by CAP.Therefore,CAP is expected to become a potential drug targeting cancer stem cells to improve the survival rate of ATC patients.