Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Objective： To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods： Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen （PCNA） and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results： Amlodipine concentration of 8.25umol/L （1/2 of ICs0） affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion： The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

Proteomic and bioinformatic analyses of possible targetrelated proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells

Gambogic acid （GA） is an anticancer agent in phase IIb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early （3 h） and late stage （24 h） of treatment were searched using a proteomic technology, two-dimensional electro- phoresis （2-DE）. The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related pro- teins such as cytoskeleton-related proteins.

Inhibition Effect of shRNA on VEGF-C in Breast Cancer Cells

Objective： To investigate the effect of RNA interfering on VEGF-C in MDA-MB-231 cells. Methods： Three small interfering RNAs （siRNAa, siRNAb, siRNAc） were prepared. The most efficient one was screened and short hairpin （shRNA） was designed, the recombinant plasmid pGenesil-1/VEGF-C was constructed, and transfeeted into MDA-MB-231 cells by Lipofectamine TM 2000. RT-PCR, Western-blot an immunohistochemical methods were performed to detect the expression of VEGF-C. Results： RT-PCR results showed that siRNAa, siRNAb, siRNAc could inhibit the growth of MDA-MB-231 cells, among which, siRNAa was the most significant, with an inhibition rate of 72.1%. The recombinant plasmid pGenesil-1/VEGF-C was successfully constructed using shRNA and pGenesil-1. VEGF-C expression was significantly inhibited as determined by RT-PCR, immunocytochemistry staining and Western blot （P〈0.05）. Conclusion： shRNA RNAi technology could silence the expression of VEGF-C in MDA-MB-231 cells, which suggested that the technology may be one of the effective methods for inhibiting lymphangiogenesis in breast cancer.

Silencing Filamin A Inhibits the Invasion and Migration of Breast Cancer Cells by Up-regulating 14-3-3σ

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer. However, the exact relationship between them is still unknown. The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer. RNA interference technology was employed to silence filamin A in MDA-MB-231 cells. Real-time PCR and Westem blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels, respectively. Double immunofluorescence was applied to show their colocalization morphologically. Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells. The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ. In addition, double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells. Silencing filamin A led to the enhanced fluorescence of 14-3-3σ. Furthermore, cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro. In conclusion, silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Inhibition of γ-synuclein (SNCG) expression in breast cancer MDA-MB231 cell line

Objective:The aim of the study was to evaluate the inhibition of different chemotherapy drugs on γ-synuclein (SNCG) positive expression of breast cancer cell line MDA-MB231,and the effects on cell cycle and apoptosis,and to explore the related mechanism as well.Methods:We treated the breast cancer cell line MDA-MB231 for the inhibition of SNCG with chemotherapy drugs such as irinotecan,nedaplatin and 5-fluorouracil using RT-PCR and immunohistochemistry,and adopted flow cytometry to detect cell cycle distribution and apoptosis.Results:At the transcription and translation levels,the SNCG expression level in nedaplatin group and 5-fluorouracil group was lower than that of other groups and there was statistically significance (P < 0.01) compared with the control group,while there was not statistically significant between irinotecan group and the control group.After drugs action,cell cycle and distribution in each experiment group changed obviously,where the cells in G0G1 phase increased,especially the cells in the nedaplatin group and 5-fluorouracil group changed most significantly,as well as the obvious change in the cells of nedaplatin group and 5-fluorouracil group in the apoptosis period.Conclusion:There was a stronger inhibition of SNCG expression in nedaplatin and 5-fluorouracil groups,and can cause significant cell cycle and apoptosis changes.It may also be concluded that nedaplatin and 5-fluorouracil could make effects by the mechanisms of inhibiting cancer cell proliferation and inducing cell apoptosis.

Growth Inhibiton of Human Breast Cancer Cell Line MDA-MB-231 by Rosiglitazone through Activation of PPARγ

OBJECTIVE To investigate the anti-proliferative effect of rosiglitazone and its relationship to peroxisome proliferator-activated receptor γ （PPARγ） in human breast cancer cell line MDA-MB-231 and evaluate the potential application value of rosiglitazone for breast cancer therapy. METHODS The cytostatic effect of rosiglitazone on MDA- MB-231 cells was measured by the MTT assay. Cell-cycle kinetics was assessed by flow cytometry. Apoptotic cells were determined by the TUNEL assay. MDA-MB-231 cells were treated with rosiglitazone or in combination with the PPARy antagonist GW9662 to investigate the effect of rosiglitazone on cell proliferation and its relationship to PPARγ. RESULTS The results showed that rosiglitazone could inhibit growth of MDA-MB-231 cells in a dose- and time-dependent manner with an IC50 value of 5.2 μmol/L at 24 h after the drug was added into the culture. Cell cycle analysis showed that the percentage of G0/G1 phase cells increased, S phase cells decreased, and cells were arrested in G1 phase with increasing concentrations of rosiglitazone. Detectable signs of apoptotic cell death caused by rosiglitazone occurred at a concentration of 100 μmol/L and the apoptotic rate was （18 ± 3）%. PPARγ selective antagonist GW9662 could partially reverse the inhibitory effect of rosiglitazone on proliferation of MDA-MB-231 cells. CONCLUSION It was concluded that rosiglitazone can inhibit growth of MDA-MB-231 cells via PPARy activation and a high concentration of rosiglitazone can also induce MDA-MB-231 cell apoptosis. These results suggest that PPARy represents a putative molecular target for chemopreventive therapy and rosiglitazone may be effective in the treatment of breast cancer.

Effect of Guizhi Fuling capsule and its extracts on human breast cancer cells proliferation

OBJECTIVE To evaluate the effect of Guizhi Fuling Capsule active pharmaceutical ingredient(API)and its fractions on human breast cancer cells proliferation by high-throughput screening assay.METHODS The crude fractions were obtained from the extraction and elution of the API of Guizhi Fuling Capsule,and 929 standard fractions were obtained by the optimal separation conditions.Sulforhodamine B(SRB)method was used to evaluate the effects of the Guizhi Fuling capsule API and929 kinds of fractions on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.RESULTS The Guizhi Fuling capsule API had a strong ability to inhibit the proliferation of MCF-7 cells at high concentration and the ability to inhibit MDA-MB-231 cells' proliferate at low concentration following 72 h treatment;some samples of 929 fractions(5μg·mL^(-1))was found to have a breast cancer cell growth inhibition rate above 50%,without toxicity on HUVECs proliferation.CONCLUSION The API of Guizhi Fuling capsule had significant cytotoxicity effects on these two human breast cancer cells,with significant concentration-and time-dependent manner.

Investigating the effects of Pentoxifylline on human breast cancer cells using Raman spectroscopy

Breast cancer is one of the leading causes of cancer-related deaths in a global scenario.In the present study,biochemical changes exerted upon Pentoxifylline(PTX)treatment had been ap-praised in human breast cancer cells using Raman spectrosecopy.There are no clinically approved methods to monitor such therapeutic responses available.The spectral profiling is suggestive of changes in DNA,protein and lipid contents showing a linear relationship with drug dosage.Further,multivariate analysis using principal component based linear-discriminant-analysis(PC-LDA)was employed for dlassifying the control and the PTX treated groups.These findings support the feasibility of Raman spectroscopy as an alternate/adjunct label-free,objective method for monitoring drug-induced modifications against breast cancer cells.

A 3D biophysical model for cancer spheroid cell-enhanced invasion in collagen-oriented fiber microenvironment

The process of in situ tumors developing into malignant tumors and exhibiting invasive behavior is extremely complicated.From a biophysical point of view,it is a phase change process affected by many factors,including cell-to-cell,cell-to-chemical material,cell-to-environment interaction,etc.In this study,we constructed spheroids based on green fluorescence metastatic breast cancer cells MDA-MB-231 to simulate malignant tumors in vitro,while constructed a three-dimensional(3D)biochip to simulate a micro-environment for the growth and invasion of spheroids.In the experiment,the 3D spheroid was implanted into the chip,and the oriented collagen fibers controlled by collagen concentration and injection rate could guide the MDA-MB-231 cells in the spheroid to undergo directional invasion.The experiment showed that the oriented fibers greatly accelerated the invasion speed of MDA-MB-231 cells compared with the traditional uniform tumor micro-environment,namely obvious invasive branches appeared on the spheroids within 24 hours.In order to analyze this interesting phenomenon,we have developed a quantitative analyzing approach to explore strong angle correlation between the orientation of collagen fibers and invasive direction of cancer cell.The results showed that the oriented collagen fibers produced by the chip can greatly stimulate the invasion potential of cancer cells.This biochip is not only conducive to modeling cancer cell metastasis and studying cell invasion mechanisms,but also has the potential to build a quantitative evaluation platform that can be used in future chemical drug treatments.

Microfluidic chip enables single-cell measurement for multidrug resistance in triple-negative breast cancer cells

Aims:Triple-negative breast cancer patients are commonly treated with combination chemotherapy.Nonetheless,outcomes remain substandard with relapses being of a frequent occurrence.Among the several mechanisms that result in treatment failure,multidrug resistance,which is mediated by ATP-binding cassette proteins,is the most common.Regardless of the substantial studies conducted on the heterogeneity of cancer types,only a few assays can distinguish the variability in multidrug resistance activity between individual cells.We aim to develop a single-cell assay to study this.Methods:This experiment utilized a microfluidic chip to measure the drug accumulation in single breast cancer cells in order to understand the inhibition of drug efflux properties.Results:Selection of single cells,loading of drugs,and fluorescence measurement for intracellular drug accumulation were all conducted on a microfluidic chip.As a result,measurements of the accumulation of chemotherapeutic drugs(e.g.,daunorubicin and paclitaxel)in single cells in the presence and absence of cyclosporine A were conducted.Parameters such as initial drug accumulation,signal saturation time,and fold-increase of drug with and without the presence cyclosporine A were also tested.Conclusion:The results display that drug accumulation in a single-cell greatly enhanced over its same-cell control because of inhibition by cyclosporine A.Furthermore,this experiment may provide a platform for future liquid biopsy studies to characterize the multidrug resistance activity at a single-cell level.