Study On Anti-breast Cancer Activity And Mechanism Of Curcumol Derivatives

Objective:Breast cancer is the most common malignancy in the female population.Treating breast cancer can be accomplished through a variety of methods,such as surgery,chemotherapy,and endocrine therapy.Chemotherapy drugs are particularly important in the treatment of breast cancer.However,due to the rapid resistance and toxic side effects of chemotherapeutic drugs,it is urgently needed to develop new drugs for breast cancer treatment.Natural products are an important source of new drugs.Curcumol is derived from the traditional Chinese medicine Curumae Rhizoma,which has significant anti-tumor activity.However,because of its special structure,it has low solubility and low bioavailability.In order to further improve the anticancer activity and solubility,our research group previously synthetized a series of curcumol derivatives through structure modification.This study is to screen out compounds with anti-breast cancer activity from these curcumol derivatives,and study their anti-breast cancer effects and molecular mechanisms.To provide a preliminary material basis for the treatment of breast cancer and a theoretical basis for the comprehensive utilization and development of the traditional Chinese medicine Curcumae Rhizoma.Methods:（1）MTT assay was used to screen active compounds and detect their effects on the viability of breast cancer cells with different concentrations and time.Then the growth curve was drawn.The effect of active compounds on cell proliferation was detected by clone formation assay.（2）Wounding heal assay and Transwell assay were used to evaluate the migration and invasion of MDA-MB-231 cells.The effect of active compounds on cell adhesion was measured by adhesion assay.（3）RNA-seq and bioinformatics were used to analyze differentially expressed genes.（4）Flow cytometry was used to evaluate the effects of active compounds on cell cycle,apoptosis,ferroptosis-related ROS,lipid peroxidation and LIP levels.（5）The effects of active compounds on mitochondrial membrane potential was detected by JC-1 kit.（6）Western blot was applied to analyze apoptosis and cycle-related protein expression in breast cancer cells treated with active compounds.（7）The protein and m RNA expression of key genes associated with ferroptosis in treated breast cancer cells were detected by Western blot and q RT-PCR.（8）Mouse mammary cancer cells 4T1-luc（firefly luciferase labeled）were injected into the mammary fat pad of mice as an in vivo breast cancer model to evaluate the anti-breast cancer effect of active compounds in vivo.Results:（1）A Curcumol derivative,HCL-23,had an excellent inhibitory effect on breast cancer MDA-MB-231 cells with IC₅₀ value 7.18±0.41μM.After HCL-23 treatment,the cell morphology was changed from spindle to circular shape.HCL-23 caused an obvious cell damage at the concentration of 20μM.In addition,HCL-23 can inhibit the proliferation,migration,invasion and adhesion of MDA-MB-231 cells in a concentration dependent manner.（2）Through RNA-seq analysis,990 differentially expressed genes were identified,including 366 upregulated and 624 downregulated genes after HCL-23 treatment.functional enrichment analysis of differentially expressed genes showed that HCL-23might exert its anti-breast cancer effect by regulating several pathways,such as ECM-receptor interaction,ferroptosis and apoptosis.（3）HCL-23 can induce apoptosis and activate the Caspase cascade.the decrease of cell viability caused by HCL-23 can be partially blocked by Caspase pan inhibitor Z-VAD-FMK（Z-VAD）.（4）HCL-23 can increase the levels of lipid peroxidation,malondialdehyde（MDA）,LIP and ROS levels in MDA-MB-231 cells.In addition,the enhancement of lipid peroxidation and LIP induced by HCL-23 was partially alleviated with the treatment of ferroptosis inhibitor ferrostatin-1（Fer-1）and deferoxamine（DFO）.Meantime,Fer-1 and DFO could abate the decrease of MDA-MB-231 cell viability due to HCL-23 treatment.This suggested that HCL-23 triggers ferroptosis in MDA-MB-231 cells.RNA-seq analysis,q RT-PCR and Western blot comfirmed that the expression of heme oxygenase 1（HMOX1,HO-1）was significantly upregulated by HCL-23.Knockdown of HO-1 attenuated the upregulated level of lipid peroxidation and LIP caused by HCL-23,as well as the decrease of MDA-MB-231 cell viability.It is suggested that HCL-23-induced cellular ferroptosis is dependent on upregulation of HO-1.（5）HCL-23 induced cell cycle arrest at G2/M phase and downregulated the protein expression of CDK-1,Cyclin B1 and p-Cdc25C.（6）The results showed that HCL-23 could significantly attenuate bioluminescent signal of tumor sites,the tumor weight and volume.Results of Western blotting demonstrated a significant up-regulation of Cleaved-Caspase 3,Cleved-PARP,and HO-1 in the HCL-23 treatment group when compared to the control group.The expression of HO-1 in HCL-23 treated mouse breast tumors was also observed by immunohistochemistry（IHC）.HE staining showed nuclear division and the ratio of nucleus to cytoplasm were decreased after HCL-23 treatment.However,there was no obvious impact on body weight of HCL-23.Moreover,HE staining revealed that the liver and kidneys of mice in the HCL-23 treatment group showed no significant pathological changes compared to the control group.Conclusions:In this study,we screened HCL-23 from a series of curcumol derivatives,which significantly inhibited the proliferation and metastasis of MDA-MB-231 cells,and further study showed that this inhibitory effect was achieved by activating Caspase-mediated apoptosis and HO-1-dependent ferroptosis.