Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E_2) on Breast Cancer MCF-7 Cells

Objective： To investigate whether dietary daidzein interact with endogenous 17β-Estradiol （E2） to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods： Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein （1, 5 μmol/L） and E2 （0.1-10 nmol/L） for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERI3） and ERβ-estrogen response element （ERE） dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53, bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results： Daidzein enhanced the antiapoptotic effect in an Ea dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression of p53 and bax, but had no effect on E2-upregulated expression of bcl-2. Conclusion： Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.

Study on Cisplatin Aggravating DNA Damage and Causing a High Apoptosis Rate on Breast Cancer MCF-7 Cells

[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.

Soy isoflavone extracts stimulate the growth of nude mouse xenografts bearing estrogen-dependent human breast cancer cells(MCF-7)

We explored the effects of different lifetime exposures to soy isoflavone extracts on the growth of estrogen- dependent human breast cancer cells （MCF-7） implanted into athymic mice of different ovarian statuses. The athymic mice, ovariectomized or not, were implanted with MCF-7 cells. Mice were fed with low, moderate and high doses of soy isoflavone extract, at dietary concentrations of 6.25, 12.5 and 25 g/kg, in different reproductive models, respectively. The expression of ki-67 was detected by immunohistochemistry, pS2 expression in tumors was analyzed by real-time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme im- munoassay. Total genistein and daidzein levels in serum and urine were determined by liquid chromatography- electrospray tandem mass spectrometry （LC-ES/MS/MS）. In Group A, on week 4, nude mice were exposed to different doses of soy iosflavone extracts. In Group B, the experimental diets were given to the nude mice follow- ing ovariectomy and tumor implantation. In both groups, 6.25 and 12.5 g/kg soy isoflavone extracts stimulated the growth of MCF-7 xenografts, increased pS2 expression, proliferation and estrogen level in serum. In both Group B （postmenopausal mouse model） and Group C （premenopausal mouse model）, soy isoflavone extracts at doses of 6.25 and 12.5 g/kg showed stimulatory effects on the growth of MCF-7 tumors. In conclusion, administration of soy isoflavone extracts at doses of 6.25 and 12.5 g/kg during adolescence or later in life stimulated tumor growth in both menopausal and postmenopausal mouse models.

QCM Detection of Adhesion, Spreading and Proliferation of Human Breast Cancer Cells (MCF-7) on a Gold Surface

The quartz crystal microbalance （QCM） was used to monitor the one-day incubation of human breast cancer cells （MCF-7） on the gold electrode. In combination with an optical microscope simulation experiment, the cell-population pictures at various stages, the QCM responses to the cells＇ adhesion, spreading and proliferation on the electrode surface were discussed. The △f0 and △R1 responses were found mainly from mixed effects of viscodensity and surface stress, and in proportion to the cell coverage, rather than to the number of cells at the electrode. The significant fore-and-aft changes in cyclic voltammetry and electrochemical impedance spectroscopy of the ferri-ferrocyanide redox couple also proved that the cells were adhesion to the gold surface.

Sonic Hedgehog stimulates migration of MCF-7 breast cancer cells through Rac1

As one of the most common tumors in women, breast cancer has drawn considerable interest from investigators and clinicians in recent years. Despite early diagnosis and best therapeutic regimens available, the prognosis of malignant or metastatic breast cancer patients is still not optimistic. Hedgehog signaling, a classical pathway indispensable to embryonic development, participates in the growth of a variety of tumors. In the present study,the effect of Sonic Hedgehog(Shh) on breast cancer cells was investigated. We identified that Shh signal stimulated the migration of MCF-7 breast cancer cells. Smo and Gli1 were involved in Shh-stimulated migration of MCF-7 cells. Activating Smo and Gli1 induced cell migration, which was blocked by their specific antagonists.The effect of Shh signaling on MCF-7 cells was independent of Wnt5 a, Dvl2 and Rab35, but directly dependent on Rac1. In conclusion, our study suggested that Shh promotes breast cancer cell migration via Rac1 independently of the non-canonical Wnt signaling pathway, which may represent a rational molecular target for combination medication in breast cancer.

A Variant of Human Estrogen Receptor-α,hER-α36 Weakens Docetaxel Drug Efficacy against Human Breast Cancer Cell Line MCF-7

Objective： hER-α36 is a variant of estrogen receptor-a, identified and cloned by a team of American. This research is to determine whether hER-α36 can enhance or weaken chemosensitivity to docetaxel in breast cancer cell line MCF-7（ERα66 positive）. Methods： RT-PCR was used to detect the expressions of ERα66 and ERa36 in the two human breast cancer cell lines MCF-7（MCF-7/ERα66） and MCF-7 transfected with ERa36（MCF-7/ERα36）. The two cell lines were treated with docetaxel（0-100umol/L）, and cell growth and apoptosis were evaluated using MTT （3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyl tetrazolium bromide） assay （using adriamycin （0-50umol/L） as the control） and flowcytometry. Western blot analysis was used to measure the effect of docetaxel on phosphor-ERKl/2 expression in the two cell lines. Results： The expressions of ERct36 and ERα66 were detectable in both MCF-7/ERα66 and MCF-7/ERα36 cell lines, while the expression of ERα36 in MCF-7/ER36 cells was higher. Both docetaxel and adriamycin inhibited the proliferation of both cells lines in a dose and time dependent manner. In comparison with MCF-7/ERα36 cell line, the MCF-7/ERα66 cells produced greater growth inhibition and apoptosis after treatment with docetaxel, but there was no significant difference in growth inhibition between the two cell lines treated with adriamycin; The MCF-7/ERα36 cell line resulted in a significant activation （phosphorylation） of ERK1/2 after treatment with docetaxel in a dose-dependent manner, but in the MCF-7/ERα66 cell line , a decrease in the level of phosphor- ERK1/2 expression was observed as the dose of docetaxel increased. Conclusion： ERa36 may be an agent that weakens chemosensitivity to docetaxel in breast cancer, probably by activating the expression of ERKI/2.

Inhibition of cell proliferation by siRNA targeting hPRLR in breast cancer MCF-7 cell line

Objective： To study the inhibition of proliferation of breast cancer by small interfering RNA（siRNA） targeting human prolactin （hPRLR） and the underlying mechanisms. Methods：The siRNA targeting hPRLR was chemically synthesized and transfected into MCF-7 cells, the expression of hPRLR was analyzed by real-time quantitive PCR, cell growth inhibition was measured with MTT assay, cell cycle of the transfected cells was examined by flow cytometry, meanwhile, expression of cyclin D1 was tested by semi-quantitative RT-PCR, Results：24 h after transfection with 100 nmol/L siRNA-PRLR, the expression of hPRLR mRNA was suppressed by 65%, cells in G1 phase increased, but cells in S phase decreased. Down regulated hPRLR expression exhibited significant inhibition in cell proliferation. And the expression of cyclin D 1 was down regulated. Conclusion：The results indicate that siRNA-hPRLR is a useful tool for silencing hPRLR expression and inhibiting cell proliferation in breast cancer MCF-7 cell line, and it may be a possible new approach for breast cancer gene therapy.

Doxorubicin Induces Apoptosis through down Regulation of miR-21 Expression and Increases miR-21 Target Gene Expression in MCF-7 Breast Cancer Cells

miRNAs play an important regulatory role in variety of cellular functions and several diseases, including cancer. MicroRNA-21 (miR-21) is overexpressed in almost all types of human cancers. Studies revealed that the knockdown of miR-21 results in reduced tumor cell growth, cell cycle arrest and cell apoptosis. In this study, we evaluated the effect of doxorubicin on miR-21 expression in mcf-7 breast cancer cells. miRNA was extracted from mcf-7 cells treated with doxorubicin and untreated cells using miRNeasy Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed using miScript II RT Kit (Qiagen) and Real Time-PCR was performed using Real Q Plus 2x Master Mix Green-(Ampliqon, Denmark). The relative expression of miR-16 and miR-21 was calculated using comparative Ct method. All tests were run in triplicate to minimize the experimental errors. Samples with a Ct > 37 were excluded from the analysis. Statistically, a significant decrease in cell proliferation of mcf-7 cells was found in doxorubicin group compared with control groups 24 hours after transfection, dose dependently (p value< 0.001). After 24 hours, Doxorubicin (100 μm) significantly decreased miR-21 expression in mcf-7 cells (p = 0.0001). Also, the expression of caspase 9 significantly increased after Doxorubicin (100 μm) treatment (p = 0.0003). Together, these findings indicate that miR-21 plays a key role in regulating cell apoptosis in mcf-7 cells and may serve as a target for effective therapies.

Let-7 miRNA and CDK4 siRNA co-encapsulated in Herceptin-conjugated liposome for breast cancer stem cells

Recently,breast cancer stem cells(BCSCs)have rapidly emerged as a novel target for the therapy of breast cancer as they play critical roles in tumor growth,maintenance,metastasis,and recurrence.Let-7 miRNA is known to be downregulated in a variety of cancers,especially BCSCs,whereas CDK4 being overexpressed in human epidermal growth factor receptor 2(HER-2)overexpressing tumor cells.In this study,let-7 miRNA and CDK4-specific siRNA were chosen as therapeutic agents and co-encapsulated in Herceptinconjugated cationic liposomes for breast cancer therapy.Particle size,zeta potential,and encapsulation efficacy of mi/siRNA-loaded PEGylated liposome conjugated with Herceptin(Her-PEG-Lipo-mi/siRNA)were 176 nm,28.1 mV,and 99.7%±0.1%,respectively.Enhanced cellular uptake(86%)was observed by fluorescence microscopy when SK-BR-3 cells were treated with Her-PEG-Lipo-mi/siRNA.Also,the increased amount of let-7a mRNA and decreased amount of cellular CDK4 mRNA were observed by qRT-PCR when SK-BR-3 cells were treated with Her-PEG-Lipo-mi/siRNA,which was even more so when SK-BR-3 stem cells were used(197 vs 768 times increase for let-7a,62%vs 68%decrease for CDK4).Growth inhibition(65%)andmigration arrest(0.5%)of the cellswere achieved by the treatment of the cells with Her-PEG-Lipo-mi/siRNA,but not withmi/siRNA complex or other formulations.In conclusion,an efficient liposomal delivery system for the combination of miRNA and siRNA to target the BCSCs was developed and could be used as an efficacious therapeuticmodality for breast cancer.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

Studies on mechanism of cis9,trans11-CLA and trans10,cis12-CLA inducing apoptosis of human breast cancer cell line MCF-7

Objective： The aim of the study was to explore the activities of cis9, trans11-CLA （C9, t11-CLA） and transl0, cis12-CLA （t10, c12-CLA） inhibiting tumor, and investigate their relationships with PPARy and apoptotic proteins, and mechanism of anti-cancer. Methods： The inhibitory rate, cell growth curve and apoptotic morphological observation of MCF-7 cells were obtained by MTT assay, trypan blue staining and Hoechst33342 fluorescence staining. The apoptotic rate and cell cycle were detected with flow cytometry. Transcriptional level of genes was detected with RT-PCR semi-quantitative method, and Western blot was performed to detect proteins levels. Results： The two CLA isomers could reduce cell proliferation （P 〈 0.05）, increase apoptotic rate （P 〈 0.05）, and increase obviously the transcriptional and protein levels of PPARy （P 〈 0.01）. The synchronism and correlation between the effects of CLA to PPARy and apoptotic proteins Bax, Bcl-2, Caspase 3 changes were found with the dose- and time-dependent manners. There was cooperative relation between the levels of PPARy and the rates of Bax/Bcl-2, Caspase 3 （small fragment） by experiments of PPARy inhibitor GW9662 and ligand Rosiglitazone. Conclusion： The apoptotic pathway of PPARy-Bcl-2-Caspase 3 signaling was found. The C9, t11-CLA and tl0, c12-CLA could inhibit MCF-7 cell proliferation and promote apoptosis via activating PPARy-Bcl-2-Caspase 3 pathway. CLA may be a kind of activator of PPARv.

Green tea polyphenols induce cell death in breast cancer MCF-7 cells through induction of cell cycle arrest and mitochondrial-mediated apoptosis

In order to study the molecular mechanisms of green tea polyphenols（GTPs） in treatment or prevention of breast cancer, the cytotoxic effects of GTPs on five human cell lines（MCF-7, A549, Hela, PC3, and HepG2 cells） were determined and the antitumor mechanisms of GTPs in MCF-7 cells were analyzed. The results showed that GTPs exhibited a broad spectrum of inhibition against the detected cancer cell lines, particularly the MCF-7 cells. Studies on the mechanisms revealed that the main modes of cell death induced by GTPs were cell cycle arrest and mitochondrialmediated apoptosis. Flow cytometric analysis showed that GTPs mediated cell cycle arrest at both G1/M and G2/M transitions. GTP dose dependently led to apoptosis of MCF-7 cells via the mitochondrial pathways, as evidenced by induction of chromatin condensation, reduction of mitochondrial membrane potential（ΔΨ_m）, improvement in the generation of reactive oxygen species（ROS）, induction of DNA fragmentation, and activations of caspase-3 and caspase-9 in the present paper.

The Evaluation of the Effects of Temozolomide on MGMT Gene Expression in MCF-7 and SKBR3 Human Breast Cancer Cell Lines

Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter region. Methods: The MTT test was performed to determine the effective dose of TMZ. The time-dependent cell survival test was performed after the IC50 value was found. Western blotting was performed to determine MGMT gene expression levels. High Resolution Melting (HRM) technique was used to determine the methylation levels of MGMT gene promoter region. Results: TMZ has been shown to have a high cytotoxic effect on SKBR3 cell line and low cytotoxicity on MCF-7. When MGMT expression levels before and after TMZ treatment were observed by western blotting, the gene expression levels of TMZ treatment were shown to decrease in both cell lines. It was observed that MGMT gene promoter region was hypermethylated in two cell lines, and that the application of TMZ further increased the methylation levels in the promoter region. Conclusions: It was seen that TMZ could be used as a single agent in SKBR-3 cell line. With this study on breast cancer, it is expected that temozolomide treatment will lead future in vitro and in vivo studies for breast cancer.

Cytotoxic Activity of <i>Thelesperma megapotamicum</i>Organic Fractions against MCF-7 Human Breast Cancer Cell Line

Thelesperma megapotamicum (Asteraceae) is commonly used in Argentine to treat various diseases (renal, digestive affections, and as anaesthesia). The present study showed the mechanisms involved “in vitro” cytotoxicity of T. megapotamicum Fractions. Five Fractions (F1 - F5) were separated by column chromatography (Silica gel) using hexane:diethyl ether as eluents. Viability was evaluated in Human breast carcinoma cell line (MCF-7) by staining with crystal violet. With respect to F1 Fraction treatment, the cell survival was 49.14% ± 8.87%, while the F2 and F3 ones exhibited a strong reduction of cell viability to only 26.35% ± 1.63% and 23.3%1 ± 0.53% of the control cell at 50 μg/ml, respectively. Apoptotic effect of these Fractions was detected using FITC-labeled Annexin V and propidium iodide binding assays and was confirmed by a higher proportion of apoptotic cells due to F2 and F3 treatments. T. megapotamicum active Fractions could facilitate the tumoral cells death by decreasing the activity of the enzyme Gamma-glutamyltranspeptidase and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.

Triple Effect of Doxorubicin, 5-Fluorouracil, Propranolol on Cell Survival on MCF-7 Breast Cancer Cell Line

Purpose: Investigating the triple effect of doxorubicin, 5-fluorouracil, propranolol on MCF-7 (ER+, WTp53) breast cancer cell line with MTT test and survival analysis. Materials/Methods: In order to determine effective dosages of a combination of doxorubicin, 5-fluorouracil, propranolol on the MCF-7 cell line by using MTT and survival analysis technique. Result: IC50 values acquired by MTT tests are 0.01 mg/ml for doxorubicin, 6 mg/ml for 5-fluorouracil, 30 mg/ml for propranolol and 0.2/1/30 mg/ml (with previous respect) if all three agents are combined. It is found that the use of doxorubicin, 5-fluorouracil, and propranolol in combination is much effective than their single application. Discussion: Moderate concentrations of doxorubicin, 5-fluorouracil, and propranolol, if they are applied individually, showed high toxicity. When we used these drugs in combination;toxic effects lessened with respect to monotherapy. In the MCF-7 cell line, doxorubicin (IC50: 0.01 μM) increases cell death rates significantly and propranolol (IC50: 3 μM) has minimum effects in monotherapy in contrast to others. Propranolol is only superior to itself in combination therapy (IC50: 4 μM). However 5-fluorouracil (IC50: 30 μM) showed antagonistic effects with respect to other drugs. Additionally, having applied the three drugs in combination on the MCF-7 cell line for the first time in literature, it is highly possible to assess the application of doxorubicin, 5-fluorouracil and propranolol combination as a novel therapy option.

Investigation of Methylation Profiles of TP53, Caspase 9, Caspase 8, Caspase 3 Genes Treated with DNA Methyl Transferase Inhibitor (DNMTi) Zebularine (ZEB) and Caffeic Acid Phenethyl Ester (CAPE) on MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.

Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression

Objective:To investigate the effect of piperine on human breast cancer cells.Methods:The effect of piperine on proliferation and migration of human breast cancer cells,MCF-7 and MDA-MB-231,was investigated using colony formation assays,wound healing assays,Matrigel migration assays,flow cytometry,RT-qPCR,and Western blotting assays.Results:Piperine inhibited the growth of MCF-7 and MDA-MB-231 cells and suppressed colony formation.Cell reduction at the G_(0)/G_(1) phase and cell arrest at the G_(2)/M phase were observed in breast cancer cells.However,the significant effect was only demonstrated in MDA-MB-231 cells.Moreover,cancer cell migration was suppressed by piperine at low concentration.RT-qPCR and Western blotting assays showed that piperine downregulated Rac1 gene and protein expression.Conclusions:Piperine could inhibit growth and migration of breast cancer cells by reducing Rac1 gene and protein expression.

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.

The effects of various chemotherapy regimens on the expression of PCNA and human breast cancer xenograft (MCF-7) transplanted in nude mice

Objective: To investigate the antitumor activity of different combination regimens to human breast cancer xeno-graft (MCF-7) transplanted in nude mice and the effects on the expression of PCNA, and to evaluate the value of PCNA as predictive factor for the response of chemotherapy and individualized treatment. Methods: (1) 88 nude mice models of human breast cancer xenograft (MCF-7) were established, and then were randomly divided into control group and 10 chemotherapy groups (each group, n = 8). Among them, the mice of 5 chemotherapy groups were treated intraperitoneally/orally by 5 com-bination chemotherapy regimens (CMF, CAF, NP, TP, Xeloda) respectively at 1/3 LD10 dosage schedule (dose lethal to 10% of the mice), and that in another 5 chemotherapy groups were treated at 2/3 LD10 dosage schedule. Control animals were administered intraperitoneally with normal saline. (2) The body weight of nude mice and transplanted tumor growth were ob-served and recorded, then inhibition rate of tumor growth was calculated. (3) The pathological features of transplanted tumor were studied under microscope. The expression of proliferating cell nuclear antigen (PCNA) was comparatively studied in chemotherapy group and control group by SP immunohistochemical method and flow cytometry analysis. Results: (1) Body weight, tumor weight and inhibition rate of tumor growth of athymic mice bearing cancer: Body weights and tumor weights of nude mice in every 2/3 LD10 chemotherapy group were significantly lower than those of the control group (P < 0.05), and the inhibition rates of tumor growth were 83.1%, 75.5%, 84.6%, 87.9% and 91.0%, respectively. Body weights of athymic mice in every 1/3 LD10 chemotherapy group were lower than that of the control (P < 0.05). The results showed that the 2/3 LD10 chemotherapy groups could reflect the effect of combination chemotherapy on the nude mice and the clinical dependability was better. So the data of 2/3 LD10 chemotherapy groups were appropriated for successive study. (2) Immunohistochemical studies: The expressions of PCNA in every chemotherapy group were significantly lower than that of the control (P < 0.05). Moreover, the expression of PCNA in NP group was significantly lower than those of CMF, CAF, TP and Xeloda groups (P < 0.05), while the expressions of TP and Xeloda groups were significantly lower than those of CMF and CAF groups (P < 0.05). (3) FCM analysis: FI values of PCNA in every chemotherapy group were significantly lower than that of the control (P < 0.05). FI values of PCNA in TP and Xeloda groups were significantly lower than those of CMF and CAF groups (P < 0.05), while the value of NP group was significantly lower than that of CMF group (P < 0.05). (4) Relationship between PCNA expression and pathologic response: The expression of PCNA was significantly correlated with pathological therapeutic response of transplanted breast carcinoma (P = 0.001). Conclusion: In vivo chemosensitivity testing with 2/3 LD10 dosage combinations in nude mice bearing cancer can reflect the effects of chemotherapeutics and affects of organism exactly. Various chemotherapy regimens all can decrease the expression of PCNA in breast cancer. The PCNA can be regarded as the factor to judge the response to chemotherapy, and it become possibly one of the prospective factors in the selection of chemotherapy regimen and play a rule in individualized therapy in the clinic.