Molecular Mechanism Of Anti-Breast Cancer Activity Of C-phycocyanin And Targeting Nano-Drug C-PC/CMC-CD59sp

Breast cancer is one of the common malignant tumors in women worldwide.It is a great threat to women’s life and health.Its morbidity and mortality have been high and continue to rise.Triple-negative breast cancer is a common biological subtype of breast cancer,and its pathological features are progesterone receptor(PR)-negative,estrogen receptor(ER)-negative and HER2-negative.Patients with triple-negative breast cancer have a higher early recurrence rate,poor disease-specific survival,more prone to cancer spread,and a higher rate of visceral and central nervous system metastasis.At present,there is a lack of effective treatment strategies for triple-negative breast cancer.Therefore,the development of new drugs provides new hope for the treatment of triple-negative breast cancer.C-phycocyanin,a marine multi-subunit protein extracted from spirulina,has rich amino acid composition,high essential amino acid content,high nutritional value,and C-phycocyanin is safe,non-toxic and good water solubility.It is widely used in many industries such as food,health care products,cosmetics,scientific research and dyes.C-phycocyanin has many physiological activities such as anti-oxidation,anti-inflammatory,anti-cancer,enhance immunity,liver protection and nerve protection.A growing number of in vitro and in vivo studies have shown that phycocyanin exerted an anti-tumor effect in various cancer types such as breast cancer,liver cancer,lung cancer,colon cancer and leukemia.Therefore,C-phycocyanin has important development and research value as a potential anti-tumor drug.The combination of nanotechnology and medicine provides new ideas for the development of new drugs.Nanomedicine can reduce the side effects of traditional drug treatment,prolong the half-life of traditional drugs,change the metabolic pathway of drugs in the body,enhance the pharmacological activity of drugs,thereby develop into a new strategy for cancer treatment.Tumor-targeted nano-drugs have the characteristics of selective and targeted delivery of drugs,which can selectively target drugs to tumor sites,improve the targeting ability and accuracy of nano-drugs,and further improve the anti-tumor effect of nano-drugs.The field of cancer treatment has attracted widespread attention.Targeted nanomedicine can selectively focus on tumor sites to kill cancer cells,thereby reducing toxic side effects on normal sites.This study aims to investigate the anti-tumor effect of C-phycocyanin on triple-negative breast cancer MDA-MB-231 cells,and construct C-phycocyanin nano-drug using nanomedicine technology.C-phycocyanin nano-drug can overcome the shortcomings of C-phycocyanin itself,such as easy degradation and poor stability,which makes it possible to stably transport C-phycocyanin in vivo.This study provides a new direction for the development of C-phycocyanin and tumor-targeted therapy,and has important theoretical significance and research value.Objective:In vitro cell experiments were conducted to further investigate the effects of C-phycocyanin on proliferation,cell cycle,apoptosis and cell migration of triple-negative breast cancer MDA-MB-231 cells,and to explore the role of MAPK cascade in the anti-tumor effect of C-phycocyanin,further to reveal the molecular mechanism of C-phycocyanin anti-breast cancer activity.At the same time,using nanomedicine technology,carboxymethyl chitosan(CMC),C-phycocyanin(C-PC),CD59 ligand peptide(CD59sp)were organically combined to construct C-PC/CMC-CD59 sp Nano-drug to maximize the anti-tumor effect of C-PC.C-PC acted as a model drug against tumors,CMC was a nano drug carrier,CD59 ligand peptide was a nano drug targeting molecule,which achieved targeted delivery of C-PC.The safety,targeting and high efficiency of the nano drug on MDA-MB-231 cells were studied.The in vitro and in vivo experiments were conducted to explore the targeting ability and growth inhibition effects of C-PC/CMC-CD59 sp nanomedicine on breast cancer cells,and to analyze the advantages and disadvantages of C-PC/CMC-CD59 sp nanomedicine compared with C-PC and C-PC/CMC nano-drug,.Methods:1.To investigate the effect of C-phycocyanin on the proliferation of MDA-MB-231 cells by CCK8 assay and colony formation assay;to detect the MDA-MB-231 cell cycle checkpoint after C-phycocyanin treatment by flow cytometry,and detect the expression of Cyclins,CDKs and CDKIs by Western Blot;to detect the apoptosis of MDA-MB-231 cells treated with C-phycocyanin by flow cytometry and TUNEL,and detect the expression of death receptor signaling pathway;to detect the migration ability of MDA-MB-231 cells after C-phycocyanin treatment by scratch assay and Transwell transfer assay,and observe cell morphology changes by immunofluorescence assay;to detect the expression of COX-2 by Western Blot and qPCR methods.2.To detect the activation of PI3K/AKT and MAPK signaling pathway in MDA-MB-231 cells treated with C-phycocyanin by Western Blot.3.Nano-targeted C-PC/CMC-CD59 sp nano-drugs were prepared by ionic crosslinking method,and the particle size and Zeta potential of the nano-drugs were detected by Malvern laser particle size analyzer.4.The experiment was divided into six groups: Control,CMC,CD59 sp,C-PC,C-PC/CMC and C-PC/CMC-CD59 sp treatment group.The cytotoxicities of five drugs on breast cancer MDA-MB-231 cells were detected by CCK8 experiment and colony formation assay;the MDA-MB-231 cell cycle detection points of five drugs were detected by flow cytometry,the expressions of Cyclins,CDKs and CDKIs were detected by Western Blot;flow cytometry and TUNEL were used to detect the apoptosis of MDA-MB-231 cells treated with five drugs,Western Blot was used to detect the death receptor signaling pathways;the migration ability of MDA-MB-231 cells treated by five drugs was detected by scratch test and Transwell assay;the expressions of COX-2 and MMP-2 were detected by Western Blot and qPCR.5.Flow cytometry was used to detect the C-phycocyanin uptake rate of MDA-MB-231 cells in different treatment groups;HCCLM3 CD59 overexpression stable cell line,HepG2 and MDA-MB-231 CD59 knockdown stable cells were constructed.The C-PC/CMC-CD59 sp uptake rates of the three stable cell lines were detected by flow cytometry.6.NU/NU nude mice were injected subcutaneously with MDA-MB-231 cells to construct a tumor-bearing mouse model,which were divided into Control,CMC,CD59 sp,C-PC,C-PC/CMC and C-PC/CMC-CD59 sp treatment group;after treatment,the volume and weight changes of the mice,the volume and weight of the tumor mass,the volume and weight of the spleen were observed,and the anti-tumor effects of the nanospheres in vivo were studied.Results:1.C-phycocyanin could effectively inhibit the proliferation and clonal formation of triple-negative breast cancer MDA-MB-231 cells;C-phycocyanin inhibited the expressions of p21 and p27 proteins,inhibited the expressions of Cyclin D1 and CDK2,induced G0/G1 phase cell cycle arrest in MDA-MB-231 cells;C-phycocyanin induced apoptosis by activating membrane death receptor signaling pathway;C-phycocyanin upregulated COX-2 expression at the protein and mRNA levels and inhibited MDA-MB-231 cell migration.2.C-phycocyanin inhibited the proliferation of breast cancer MDB-MA-231 cells by up-regulating the expression of phosphorylated JNK and p38,and down-regulating the expression of phosphorylated ERK1/2 protein;therefore,C-phycocyanin could regulate MAPK signaling pathway to play the role of anti-breast cancer.3.C-PC/CMC nanospheres were successfully constructed;the morphology of nanospheres was uniform;the particle size of C-PC/CMC nanospheres was approximately 149.6 ± 52.5 nm;the Zeta potential of C-PC/CMC nanospheres was-19.8 ± 4.19 mV.4.C-PC/CMC-CD59 sp nanospheres inhibited the proliferation of breast cancer MDA-MB-231 cells,and the inhibition effect of CPC/CMC-CD59 sp nanospheres was concentration-dependent and time-dependent;C-PC/CMC-CD59 sp nanospheres inhibited the expression of Cyclin D1/CDK4 and Cyclin E/CDK2,and up-regulated the expression of p21 and p27 proteins,thereby induced cell cycle arrest in G0/G1 phase of MDA-MB-231 cells;C-PC/CMC-CD59 sp nanospheres activated Fas/FasL-mediated death receptor pathway and induced apoptosis of MDA-MB-231 cells;C-PC/CMC-CD59 sp nanospheres significantly down-regulated the expression of COX-2 and MMP-2 proteins and inhibited cell migration in a dose-dependent manner.5.The fluorescence intensity of C-PC/CMC-CD59 sp treatment group was the strongest;the fluorescence intensity of HCCLM3-CD59 cells was significantly stronger than that of HCCLM3 and HCCLM3-NC cells;the fluorescence intensity of MDA-MB-231-CD59-sgRNA cells was significantly lower than that of MDA-MB-231 and MDA-MB-231-NC-sgRNA;the fluorescence intensity of HepG2-CD59-sgRNA cells was significantly lower than that of HepG2 and HepG2-NC-sgRNA.6.Six groups of tumor-bearing mice showed no weight loss during the administration;in the C-PC,C-PC/CMC and C-PC/CMC-CD59 sp treatment groups,tumor growth of the tumor-bearing mice was inhibited.However,the inhibitory effect of C-PC and C-PC/CMC was significantly lower than that of C-PC/CMC-CD59 sp nanosphere group.The C-PC/CMC-CD59 sp nanosphere group had the smallest volume and the lightest weight.The tumor mass of C-PC/CMC-CD59 sp nanosphere group had the smallest volume and the lightest weight.Conclusions:1.C-phycocyanin up-regulated the expression of phosphorylated JNK and p38,and down-regulated the expression of phosphorylated ERK1/2 protein,thereby inhibited the proliferation of breast cancer MDB-MA-231 cells,induced cell cycle G0/G1 arrest,and induces cells apoptosis.Therefore C-phycocyanin could play a role in anti-breast cancer by regulating the MAPK signaling pathway.2.C-PC/CMC-CD59 sp nanospheres had a targeted anti-tumor effect.The C-PC/CMC-CD59 sp nanospheres could be targeted to the tumor site by binding the CD59 ligand peptide to the CD59 protein on the surface of the tumor cells.3.The anti-tumor effect of C-PC/CMC-CD59 sp nanospheres was mainly achieved by inhibiting the proliferation of breast cancer cells,promoting apoptosis,impeding cell cycle progression and preventing tumor cell metastasis.4.C-PC,C-PC/CMC and C-PC/CMC-CD59 sp nanospheres could inhibit tumor growth in animal models.The anti-tumor effects of C-PC/CMC-CD59 sp were most effective,while C-PC,C-PC/CMC and C-PC/CMC-CD59 sp nanospheres had no obvious toxicity;C-PC could further enhance its anti-tumor effects by nanotechnology,and the anti-tumor effects of C-PC/CMC-CD59 sp were more significant under the action of tumor targeting.This study successfully used the nanotechnology to contruct the targeted nano-drug C-PC/CMC-CD59 sp,and confirmed that the nano-drug had safety,targeting and high efficiency.The targeted anti-tumor effects of the targeted nano-drug were mainly achieved by inhibiting the proliferation,blocking cell cycle progression,inducing apoptosis,and inhibiting cell migration of the triple-negative breast cancer MDA-MB-231 cells.This study provided new ideas for the development of marine drugs and the clinical application of C-phycocyanin,and provided new directions and theoretical support for tumor targeted therapy and nanomedicine research.