| Breast cancer is the most common type of malignancy in women.At present,surgery,radiotherapy,endocrine therapy,chemotherapy and immunot herapy are the main methods for the treatment of breast cancer.Mitogen-activated protein kinases(MAPKs)are expressed in many cell types a nd modulate a variety of physiological processes such as cell growth,metabolis m,differentiation and cell death.Up to date,three distinct groups of MAPKs have been characterized in mammals:extracellular signal-regulated kinases(ERK),Jun N-terminal kinases(JNK)and p38.It has been recently reported MAPKs play important roles in the occurrence and development of breast cancer.Ginkgo biloba(Ginkgo),mainly planted in Asia,has been used as a tradit ional herbal medicine for many years.In recent years,some active gradients fr om Ginkgo leaves has been extracted and identified as several kinds of glycosi des and terpenoids.Among these ingredients,ginkgetin(GK)has received exte nsive attentions because of its powerful biological activities in vivo and vitro,i ncluding anti-inflammatory,antioxidant as well as anti-cancer effects.In previo us studies,GK was demonstrated to possess anti-tumor activity against many h uman cancer cells,such as osteosarcoma,prostate cancer and ovarian adenocarc inoma cells.Moreover,GK regulates p-ERK1/2 expression induced by glutamat e insult.In the present study,we aim to characterize anti-tumor function of GK in breast cancer,and then assess whether MAPKs signaling decades involv ed in the action of GK.Objective:To observe Ginkgetin activity against human breast cancer cells and explore its effects on function and signal transduction of MAPKs.The current study will provide the experimental basics for developing and applicating of Ginkgetin as an anti human breast cancer.Methods:1 MTT assay detects the proliferation in human breast cancer cellsBriefly speaking,a total of 5.0×103 growing cells were seeded into each well of 96-well plates.The samples treated with GK,GK+MAPKs specific inhibitors was added to each well with final concentration varied from 5-300 μM.Two duplicates were created for each concentration with a total volume of 200μl per well.After 24h-96h incubation,the cells were treated with MTT in each well,followed by dissolving the formazan crystals with DMSO.The absorbance at 570 nm was determined and recorded by a microplate reader.The cells viability was assessed as the percent values compared with untreated control group,IC50(the concentration required to induce 50%cells growth inhibition)was determined by interpolation from dose response curves using Graphpad prism5.2 The effects of GK on apoptosis of human breast cancer cells were evaluatedThe terminal deoxyneucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)assay was used to evaluate MCF-7 cells apoptosis according to the manufacture’s instruction.Briefly,the MCF-7 cells were incubated with different concentrations of GK for 48 h.The cells were fixed in 10%buffered neutral formalin,dehydrated,and embedded in paraffin.Similarly,the tumor tissue samples were fixed in 10%formalin before embedded in paraffin and 4μm thick sections were prepared and stained for TUNEL analysis.Slides were then counterstained with hematoxylin solution.Finally,TUNEL-positive and DAPI-positive staining were viewed by a confocal laser scanning microscope.Percentage of apoptotic cells was determined from labeled nuclei with respect to the total number of nuclei counted at 10 randomly chosen microscopic fields at 400×magnifications.Moreover,GK,GK+MAPKs specific inhibitors induction of apoptosis was examined by FACS analysis with an annexin-V-FITC and propidium iodide(PI)double staining.The experiment was performed as previously described.3 The effects of GKon caspases activity of human breast cancer cells were evaluated by commercial kits.MCF-7 cells were treated with GK for 48h,and harvested and lysed in lysis buffer on ice for 10 min.After centrifugation,the supernatant was collected.Caspase-3,caspase-8 and caspase-9 activities were assessed using caspase colorimetric assay kits.Briefly,the supernatants were incubated with caspase-3,caspase-8 and caspase-9 substrates in reaction buffer,followed by detecting at 405 nm in a microplate reader.4 Western blot analysisThe protein concentration of cell lysates submitted for western blot analysis was estimated using a Bio-Rad protein assay.Protein samples were loaded onto 10%SDS-PAGE gels and electrotransferred to polyvinylidene difluoride membranes The membranes were then blocked with 5%BSA in buffer,and probed with primary antibodies of Bcl-2,Bax,survivin,p38,p-p38,JNK,p-JNK,ERK1/2,p-ERK1/2,cleaved-caspase-8,cleaved-caspase-9,cleaved-caspase-3 and cleaved-poly adenosine diphosphate ribose polymerase overnight at 4℃.After incubation with secondary antibody,the blots were visualized by an enhanced chemiluminescence detection kit.Quantitative analysis of blots was carried out using the Image-Pro Plus 6.P-actin was used as an internal control.5 MCF-7 xenograft nude mouse modelThe animals study was approved by the ethic committee of Anhui Medical University.Six-week-old female BALB/c nude mice(20±2g)were purchased from Model Animal Research Center of Nanjing University.The mice were maintained in a climate controlled room(12:12 dark-light cycle with a constant room temperature of21 ± 1 ℃)and fed standard rodent chow and water ad libitum.To establish the nude mouse xenograft model,a toatal of MCF-7 cells(1×107)were injected subcutaneously into the right flank area of each mouse.As tumors reached an average size beyond 200 mm3,the mice were randomly divided into four groups with five in each.Animals were grouped as following:control group,GK(25,50,10Omg/kg)groups.All mice were orally administrated for consecutive eight weeks.During therapy,body weight and tumor volume were monitored and recorded every week.6 Immunohistochemical staining assayTumors tissue were removed from sacrificed mice,fixed in formalin,embedded in paraffin wax,and serially sectioned(4μm thick).The sections were deparaffinized in xylene and rehydrated through graded decreasing concentrations of alcohol,and incubated with antibodies of p-p38,p-JNK,and p-ERK1/2.Vectastain ABC kit was used to bind the antibodies and the binding sites were visualized using DAB kit.Each section was observed in 10 visual fields by an experienced pathologist,who is unaware of the grouped details.7 Statistical analysisData are presented as mean ± standard deviation(S.D.).A P value less than 0.05 was accepted as statistically significant.Statistical analyses were determined by SPSS software.The data were analyzed by SPSS 20.0 software.RESULTS:1 GK inhibited proliferation of breast cancer cells in vitroCytotoxic effects of GK on MDA-MB-231,BT-474 and MCF-7 cells were examined by MTT assay.The GK inhibited all tumor cells proliferation in a dose dependent manner.Moreover,we noted GK repressed growth of all breast cancer lines in a time dependent manner.However,response of the cells exposed to GK varied among cell lines.Based on IC50 values,the sensitivity of tumor cells to GK was as following:MCF-7>MDA-MB-231>BT-474.Moreover,GK showed almost no toxicity to MCF-10A cells even when its concentration was 150μM,which suggested a high selective cytotoxicity for cancer cells.2 GK induced MCF-7 cells apoptosis in vitroTUNEL staining was performed to analyze apoptosis of GK treated MCF-7 cells.The results demonstrated that GK treatment significantly induced apoptosis in MCF-7 cells.Compared with the control,GK with a concentration of 20-80 μM induced the apoptosis from 8.9%to 28.7%in MCF-7 cells.Moreover,we have also evaluated the apoptosis of MCF-7 cells by Annexin V/PI double positive staining.The data listed in clearly showed that GK treatment increased apoptotic rates of MCF-7 cells from 8.5%to 33.5%.3 GK regulated expression of apoptotic associated proteins and induced caspases activation in MCF-7 cellsWestern blot analysis indicated GK down-regulated expression of Bcl-2&survivin,and up-regulated Bax compared with control.We explored whether caspase-3,caspase-8 and caspase-9 were activated by GK treatment.Our results showed caspase-3,caspase-8 and caspase-9 activities of MCF-7 cells were all increased after GK actions.Further,we also confirmed expression of cleaved-caspase-8,cleaved-caspase-9,cleaved-caspase-3 and cleaved-PARP increased in MCF-7 cells by GK treatment.4 GK increased the expression of MAPKs in MCF-7 cellsGK treatment resulted in increased levels of p-p38,p-JNK as well as p-ERKi/2,but had no effects on the total expression of p38,JNK and ERK1/2.MCF-7 cells were co-incubated with GK and SB203580(p38 specific inhibitor),SP600125(JNK specific inhibitor)or U0126(MEK specific inhibitor)for 48 h.As shown in,all inhibitors obviously abrogated the apoptosis and growth inhibition induced by GK.These results indicate that activation of MAPKs in MCF-7 cells might play a partial but significant role in GK-induced cell growth inhibition.5 GK suppressed breast tumor growth in vivo.In MCF-7 xenograft nude mouse model.The results showed that GK didn’t alter body weight of tumor mice.However,reductions in tumor volume and weight were observed after treatment of GK.Consistent with results of the vitro experiment,we found GK treated mice showed increased rates of apoptotic cells compared with that of control mice.Therefore,the present finding indicates GK displays a well anti-tumor activity in MCF-7 xenograft nude mouse model.6 GK increased p-p38,p-JNK and p-ERK1/2 positive cells in the tumor tissue of nude miceTo explore effects of GK on MAPKs in vivo,we measured expression of p-p38,p-JNK and p-ERKi/2 in the MCF-7 xenografts nude mice by immumohistochemical staining.The results showed that treatment with GK significantly increased numbers of p-p38,p-JNK and p-ERK1/2 positive cells in the tumor tissue compared with that of control,which was similar with the vitro result.Conclusions:1 Ginkgetin has proliferation inhibitory effect on breast cancer cells in vitro and in vivo.2 The mechanism of Ginkgetin antitumor in vivo and in vitro may be related to Ginkgetin induced apoptosis,mediating the expression of apoptosis related proteins in Caspases and Bcl-2 family members,and regulating the MAPKs signaling pathway in breast cancer cells.3 This study provides a good experimental basis for the application of Ginkgetin anti-tumor effect.We show the characteristics and molecular mechanism of Ginkgetin in human breast cancer cells. |
