Therapeutic Effect And Mechanism Of Elemene On Triple-negative Breast Cancer Mice

Breast cancer has the highest incidence and mortality rate among female cancer patients.At present,clinical treatments for the breast cancer mainly include chemotherapy,hormone therapy,targeted therapy and immunotherapy,according to the classification of breast cancer in clinic.Among the classifications,triple-negative breast cancer(TNBC)is the one that is unable to benefit from hormone therapy and targeted therapy because the expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor-2(Her-2)are all negative.Moreover,the triple negative breast cancer is more likely to metastasize than the receptor-positive breast cancers.Currently,chemotherapies are applied to TNBC in a combination of taxanes,anthracyclines,cyclophosphamide and platinum,and the survival rate has been relatively low.At the same time,due to the lack of targeting,the traditional chemotherapeutics usually bring severe side effects to the patients,and those who are elderly,infirm,or have other severe diseases are hardly to tolerate the side effects,which leads to the difficult situation that no optional therapeutics for using.Traditional Chinese medicine has received increasing attentions as a supplementary medicine for tumor therapy,Rhizoma Zedoariae is a commonly used medicinal material for promoting blood circulation and removing blood stasis.Previous clinical trials and related basic research have shown that its volatile oil extract elemene has anti-tumor activity.In the clinical applications,elemene emulsion has shown a certain effect in the treatment of cancerous pleural and ascites effusion,and gastrointestinal tumors.At the same time,the toxic and side effects of the elemene emulsion were reported relatively low and could be tolerated by the elderly and infirm patients.Nevertheless,the pharmacological research on elemene is not adequate.So far most of the existing investigations have focused on the cytotoxic mechanisms of elemene,which are insufficient for the guidance of clinical application.On the basis of the clinical pharmacological characteristics of Rhizoma Zedoariae,we proposed to systematically investigate the effects and underlying mechanisms of elemene on TNBC at the level of molecule,cell,tissue and animal model,from the view of the modulation of tumor microenvironment by elemene,aiming to provide theoretical and experimental basis for formulating more effective treatment regimes of elemene in the clinical application.In the experiments,the cell viability was examined by using CCK8(Cell Counting Kit-8)on mouse breast cancer cell line 4T1 cells,mouse macrophage cell line RAW264.7 cells,human umbilical vein endothelial cell line HUVEC cells,mouse fibroblast cell line NIH3T3 cells,rat cardiomyocyte cell line H9C2 cells.The Annexin V-FITC/PI cell apoptosis detection kit was used to measure the apoptotic cells.To establish the TNBC orthotopic xenograft mouse model,4T1 cells were injected into the mammary fat pad of 6-8 weeks old Balb/c mice.After 7 days of the inoculation,when the solid tumor was developed,the animals were grouped as 1mg/kg,5mg/kg,10mg/kg and 20mg/kg,as well as non-treatment control,and the elemene was intraperitoneally injected once a day to the treatment group.A control group of weekly intraperitoneal injection of paclitaxel(PTX;20mg/kg)was also set for comparison.After three-week treatment.the mice were sacrificed,and organs were collected for histopathological studies.The metastasis of tumor cells in the liver and lung were observed by H&E staining,the expression of HIF-la,CD31,NLRP3,caspase-1 and IL-1? in the tissue were detected by immunohistochemical staining,and dihydroethidium(DHE)was used as a fluorescent probe to observe the level of reactive oxygen species(ROS)in tumor tissues.Experimental results indicated that elemene showed no obvious cytotoxicity to tumor cells and normal cells when the concentration was lower than 20 ?g/mL.When the concentration was increased to 30 ?g/mL,elemene showed significant cytotoxic effect and induced tumor cells apoptosis.According to these results,low and medium doses were used in the animal experiments.The results of animal survival experiment showed that the survival time of elemene treatment group(1mg/kg,5mg/kg,10mg/kg and 20mg/kg)and the low-dose paclitaxel group(20mg/kg)was similar,and all were significantly longer than that of the non-treatment control group.Histopathological studies showed that after three weeks' treatment,the low and medium doses of elemene(1mg/kg,5mg/kg,10mg/kg and 20mg/kg)could significantly inhibit the metastasis of tumor cells to the lung and liver,and alleviated the splenomegaly in mice bearing tumor at the same time,though did not inhibit the size of the tumor in situ in mice.In the mechanistic study,we found out that elemene could inhibit angiogenesis in the tumor tissues by down-regulating the expression of HIF-1? and its downstream proteins CD31 in tumor tissues of breast cancer mice,thereby reducing the metastasis of tumor cells.Meanwhile,elemene could inhibit the expression of inflammasome NLRP3 in the tumor tissues of the breast cancer mice,thereby downregulating the expression of IL-1? by inhibiting the activation of caspase-1,clearly suggested that elemene has anti-inflammatory effect.The results of electron spin resonance spectroscopy showed that elemene could eliminate reactive oxygen species(ROS)effectively,including DPPH,superoxide anion and hydroxyl free radicals.Cell experiment results showed that elemene significantly reduced the intracellular ROS as well as the transcription levels of inflammatory factors Il-1? and Tnf-? induced by lipopolysaccharide(LPS)in the RAW264.7 cells,which proved that elemene played an anti-inflammatory role by eleminating the increased ROS.At the same time,ROS levels in the tumor tissues of elemene treated mice were significantly down-regulated,evidenced by dihydroethidine staining,indicating that elemene could inhibit the expression of HIF-1? and NLRP3 by eliminating ROS.In summary,the elemene has the feature of relatively low cytotoxicity at low and medium doses while eleminating ROS effectively.We demonstrated that elemene was able to effectively improve the hypoxia and inflammatory microenvironment of the tumor tissue by scavenging ROS in the tumor tissue with the low and medium doses,which inhibited tumor neovascularization and led to the inhibition of the metastasis of tumor cells to the lung and liver.At the same time,the splenomegaly of tumor-bearing mice was alleviated significantly.These therapeutic effects in turn prolonged the survival of mice.The results of this study indicated that the advantage of elemene lies in its regulatory effect on the tumor microenvironment rather than cytotoxic effect.Therefore,low and medium doses of elemene are suggested to use in the clinical application to avoid systemic toxic effects while to take the advantage of the regulatory effects on the tumor microenvironmet.Meanwhile,it is suggested that elemene might be more suitable for tumor cells with high oxidative status;therefore it would be better to select the tumor type when elemene is going to be applied.