Antitumor Mechanisms On Growth And Metastasis Of Jatrorrhizine Targeting TNIK In Mammary Carcinoma

Mammary carcinoma has become one of the most malignant tumor and major health burden for women.Postoperative metastasis is the main reason for mammary carcinoma difficult to be cured.Traf-2 and Nck interacting serine protein kinase(TNIK)is an attractive multifunctional tumour target protein and highly expressed in breast cancer tissues.TNIK could participate in activating the Wnt/?-catenin pathway,and is essential for the invasion and metastasis of breast cancer.Jatrorrhizine,a protoberberine isoquinoline alkaloids,shows a strong cytotoxic effect on various cancer cells.Molecular docking simulations-target structures shows that TNIK may be the target inhibitory protein for jatrorrhizine in cancer cells.Therefore,analyzing the molecular mechanism of TNIK regulating metastasis and exploring targeted drugs for inhibiting invasion and metastasis are of great significance for the treatment of mammary carcinoma.In this study,CRISPR/Cas9 gene editing technology was employed to knockout TNIK,and MDA-MB-231/TNIK-KO and MDA-MB-231/TNIK-Control were stably transfected and detected by immunofluorescence and Western blotting.Expression of TNIK,p-TNIK,and key proteins of Wnt/?-catenin pathway demonstrated that knockout of TNIK could obstruct and regulate pathway transportation and EMT expression,and the effects were potentiated with jatrorrhizine treatment.Jatrorrhizine could obviously inhibit the viability of MDA-MB-231/TNIK-KO cells,and induce cell apoptosis.Results of MTT,flow cytometry,immunofluorescence and western blotting showed that jatrorrhizine could inhibit the growth and proliferation of MDA-MB-231 and MCF-7 cells,block the cell cycle,and induce mitochondrial membrane potential dysfunction and early apoptosis features.Results of colony formation,wound healing,immunofluorescence and western bloting proved that jatrorrhizine could disrupt F-actin structure to regulating the actin cytoskeleton,regulate the expression of TNIK and subsequently inhibiting cell migration.Moreover,jatrorrhizine could down-regulate the expression of ?-catenin and up-regulate GSK-3?expression of Wnt pathway,and reverse the EMT by increasing the expression of EMT epithelial marker E-cadherin and decreasing the mesenchymal marker N-cadherin expression.In addition,jatrorrhizine could reverse the doxorubicin resistance in MCF-7/DOX cells and decrease transcription of the corresponding ATP-Binding Cassette transporters.The above results were further corroborated by the 4T1 tumour-bearing mice,in which jatrorrhizine showed significantly suppression on tumor growth and metastasis without obvious toxicity.In summary,jatrorrhizine can distinctly inhibit proliferation of the mammary carcinoma cells,induce early apoptosis involving mitochondrial apoptotic pathway,and regulate Wnt/?-catenin pathway by targeting TNIK.These findings could provide experimental and theoretical basis for studies on antitumor activity of jatrorrhizine and potential of metastatic inhibition.Meanwhile,the study could provide an overall perspective that jatrorrhizine is potentially used as a novel TNIK inhibitor for mammary cancer targeted therapy.