Antitumor Effects Of Digoxin And Gemcitabine Nanomaterials On MDA-MB-231 Cells Of Triple Negative Breast Cancer In Vitro And In Vivo

Breast cancer is the most common menacing health issue affecting women worldwide as well as the second leading cause of cancer-related deaths to date.According to Global Cancer Statistics 2018,It accounts for a quarter of all cancer cases.Breast cancer occurs mainly in the high age group of women over 55 with ratio of 1 to 10.Triple negative breast cancer(TNBC)is a subtype of breast cancer that lacks the special types of estrogen receptor(ER),receptor(PR)and human epidermal growth factor receptor-2(HER-2).It has the characteristics of invasion,metastasis and recurrence;The result is poor prognosis.At present,chemotherapy is the main treatment for TNBC after clinical surgery,but its cure rate is low,easy to develop drug resistance.Gem is often used as a first-line treatment,and resistance to it can develop in treating tumors,limiting its better clinical use to some extent,But in combination with other anti-tumor drugs;It has been clinically identified as one of the treatment options for patients with recurrent or metastatic breast cancer.TNBC subtypes of breast cancer are most commonly associated with hypoxia.Many studies have shown that HIF-1? a transcription factor,is an important mediator of anoxic response and promotes tumor metastasis and development.HIF-1? can activate the transcription of a variety of hypoxic response genes,among which the most effective angiogenic growth factor is the generation of vascular endothelial growth factor(VEGF),which mediates hypoxic-driven angiogenesis by HIF-1?.Therefore,hypoxic cancer cells are invasive and metastatic,as well as resistant to cancer treatment,and the combination of HIF-1? and VEGF constitutes a lethal cancer phenotype.Considering these factors,we should look for inhibitors that can inhibit HIF-1? and achieve anti-cancer effects.Studies have confirmed that digoxin is a new inhibitor of hif-1,which can inhibit the proliferation of tumor cells by inhibiting the synthesis of HIF-1? protein and the target gene of HIF-1? in cancer cells,thus causing the growth stagnation of subcutaneous transplanted tumors in nude mice.It was found that digoxin is a new class of tumor targeted drugs,which can inhibit the proliferation of tumor cells,induce apoptosis,block the migration and metastasis of tumor cells,and reverse the multi-drug resistanceChemotherapy single drug makes the human body has a greater chance of developing resistance to specific drugs,but the efficacy is not ideal combined with nano-drug preparation to pack the chemotherapy drugs in a nano-capsule,to construct a two-drug combination of nano-drug preparation,improve the bioavailability and therapeutic effect,reduce the drug dose,and reduce the toxic and side effects on normal cells.The combined application of a variety of anti-tumor drugs can reverse the drug resistance of tumor cells,induce the apoptosis of tumor cells,improve the treatment effect and reduce the recurrence rate.Taking the tumor microenvironment as the starting point,the effects of digoxin combined with gemcitabine nanometer preparation on the biological characteristics of MDA-MB-231 of triple negative breast cancer cells were discussed from the perspective of overall animal experiments and cell level,and the anti-tumor effects of digoxin combined with gemcitabine nanometer preparation on MDA-MB-231 cells were further studied.Objective: To investigate the effect of Digoxin combined with Gemcitabine nanometeron apoptosis of MDA-MB-231 cells of triple negative breast cancer in vitro and vivo,an3 possible mechanism.Method: 1.Effects of digoxin and gemcitabine nanomaterials on MDA-MB-231 in triple negative breast cancer cells: Breast cancer MDA-MB-231 cells were cultured in vitro and divided into control group and drug administration group.CCK-8 method was used to detect the effects of at different drug administration group on the activity of MDA-MB-231 cells.Flow cytometry was used to quantitatively investigate the effects of different drug groups on apoptosis,cell cycle and mitochondrial membrane potential.Transmission electron microscopy and Hoechst staining were used to observe the effects of different drug groups on the morphology of MDA-MB-231 cells.The effect of CD31 and VEGF on angiogenesis was detected by immunofluorescence.Expression of Bax,Bcl-2,Caspase3,Cyt-C and HIF-1? in MDA-MB-231 cells was detected by Western Blot.2.Effect of digoxin and gemcitabine nanomaterials on transplanted tumor of triple negative breast cancer MDA-MB-231 in nude mice: Female BALB/c nude mice were selected,weighing 20-22 g.After 5 days of acclimatization,MDA-MB-231 cells were inoculated subcutaneously in the right lower limb.To construct the xenograft model of triple negative breast cancer in nude mice,After 7 days,a mass was palpable in the right lower extremity.Randomly divided into 7 groups,control group,PLGA group,PLGA-Dig group(2mg/kg),PLGA-Gem group(5mg/kg)and PLGA-Gem-Dgroup(2mg/kg +5mg/kg).Six animals in each group were injected through the tail vein for 28 consecutive days.The drug was administered once a day from Monday to Friday in the treatment group,and the model group and the nanocyst group were given the same volume.The body weight and tumor size of each group were weighed once every 2-3 days,and the growth curve of transplanted tumor was drawn..After 28 days,the nude mice were sacrificed for cervical dislocation,the tumor body was separated and weighed,and the heart,liver,spleen,lung and kidney were taken out and weighed in 5% formaldehyde fixation solution.Tumor volume and tumor control rate were calculated in each group.Paraffin sections of nude mouse xenograft with MDA-MB-231 cells were prepared,and HE staining was used to observe the differences between the tumor bodies,heart,liver,spleen,lung and kidney in each group.Apoptosis of transplanted tumor was detected by TUNEL staining,and protein expression levels of Ki-67,HIF-1?,CD31 and VEGF protein were detected by immunohistochemistry.1.Effects of digoxin and gemcitabine nanomaterials on triple negative breast cancer cells MDA-MB-231 CCK-8 results indicated that,compared with the control group,the activity of MDA-MB-231 cells decreased in the drug administration group at different time,with statistically significant difference(P<0.05 or P<0.01).Annexin V/PI double staining results indicated that compared with the control group,the apoptosis rate of the treated group increased with a statistically significant difference(P<0.05 or P<0.01).Cell cycle results showed that compared with the control group,MDA-MB-231 cells were subject to G2/M phase cycle arrest,significantly inducing apoptosis,with statistically significant differences.The results of JC-1 staining indicated that compared with the control group,the mitochondrial membrane potential of MDA-MB-231 cells in the drug administration group decreased,and the proportion of JC-1 decreased,with statistically significant difference(P < 0.05 or P < 0.01).The results of fluoroscopy Hoechst indicated that,compared with the control group,the cell morphology of the drug administration group underwent apoptotic changes,Meanwhile apoptotic corpuscles appear.Immunofluorescence test results indicated that compared with the control group,the fluorescence intensity of the drug administration group was significantly reduced,indicating that the drug administration group could inhibit tumor angiogenesis.Western-blot results indicated that,compared with the control group,the administration group up-regulated the expression of pro-apoptotic factor Bax,Caspase-3,Cyt-C,down-regulated the expression of anti-apoptotic factor Bcl-2,with statistically significant difference(P<0.05 or P<0.01).2.Effect of digoxin and gemcitabine nanometer preparation on xenograft of triple negative breast cancer MDA-MB-231 in nude mice 2.1 In vivo toxicity evaluation of digoxin and gemcitabine nanometer preparations on nude mice transplanted with MDA-MB-231 tumor Morphological changes in heart,liver,spleen,lung and kidney were detected by HE staining.The results showed that the cells in the control group and the nanocyst group Result:had complete,full,dense,regular arrangement and even distribution of blood vessels.In the solution group,the heart,liver,spleen,lung and kidney were significantly damaged,and the number of living cells was significantly reduced and the arrangement was irregular.The number of living cells was significantly reduced and the arrangement was irregular.The PLGA-Dig group,PLGA-Gem group and PLGA-Gem-Dig group had no obvious toxic effect on all organs,and the good cell growth state was almost non-toxic to all organs of nude mice,Compared with the solution group has a significant advantage.2.2 Effects of digoxin and gemcitabine nanomaterials on nude mice transplanted with MDA-MB-231 tumor The growth curve and tumor size of the transplanted tumor could be obtained.Compared with the control group,the tumor volume of the drug administration group gradually decreased and the growth rate slowed down.Dig and Gem nanometer preparations could inhibit the growth of xenograft tumors in nude mice.HE staining showed that,compared with the control group,the mass volume of tumor cells was significantly reduced,with partial necrosis and sparse arrangement of tumor cells Ki-67 expression declined,The results showed that digoxin and gemcitabine nanomaterials inhibited the proliferation of xenograft tumor in nude mice with MDA-MB-231 cells.The TUNEL test showed that the number of positive cells in the tumor cells increased in the treatment group compared to the control group.Immunohistochemical examination showed that the expression of HIF-1? protein and VEGF protein could be decreased,and the microvascular density of CD31 could be decreased.The results showed that digoxin and gemcitabine nanometer preparations promoted the apoptosis of xenograft tumor in nude mice MDA-MB-231Conclusion: 1.Digoxin and gemcitabine can enhance the inhibition of proliferation and apoptosis of MDA-MB-231 cells in breast cancer.Blocking the G2/M phase of the cycle,inhibiting the growth of xenograft tumor in nude mice and inducing its tumor tissue apoptosis,the combined effect of the two drugs is more significant 2.The mechanism of the effect of digoxin and gemcitabine nanomaterials on the malignant behavior of breast cancer may be related to the inhibition of tumor microangiogenesis and the reduction of tumor angiogenesis by inhibiting the expression of HIF-1? and reducing the expression of VEGF and CD31.At the same time,apoptotic proteins are regulated,and mitochondrial membrane potential is destroyed,leading to cell apoptosis,which may be one of the molecular mechanisms of apoptosis induced by digoxin and gemcitabine nanomaterials.