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.

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.

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.

雷公藤内酯醇通过调控miR-142-3p/HSP70通路抑制人乳腺癌MCF-7细胞增殖、侵袭和迁移

目的:探究雷公藤内酯醇(TP)通过miR-142-3p/HSP70信号通路对人乳腺癌MCF-7细胞恶性生物学行为的影响。方法:常规培养MCF-7细胞,将其分为6组:对照组、TP组、miR-142-3p inhibitor组、TP+inhibitor组、miR-142-3p mimic组和TP+mimic组,用转染试剂将相应的核酸或质粒转染MCF-7细胞。qPCR法、EdU细胞增殖实验、Transwell小室实验、细胞划痕实验、WB法分别检测转染后各组MCF-7细胞中miR-142-3p和HSP70 mRNA的表达,MCF-7细胞的增殖、侵袭、迁移能力和HSP70蛋白表达水平。结果:TP或miR-142-3p过表达能显著促进MCF-7细胞中miR-142-3p和HSP70的表达,敲减miR-142-3p则可明显抑制MCF-7细胞中miR-142-3p和HSP70的表达,TP可逆转由敲减miR-142-3p对MCF-7细胞中miR-142-3p和HSP70表达的影响;TP、过表达miR-142-3p均可明显抑制MCF-7细胞的增殖、迁移和侵袭能力(均P<0.05),敲减miR-142-3p则均可促进MCF-7细胞的增殖、迁移和侵袭能力(均P<0.05),TP可逆转由敲减miR-142-3p对MCF-7细胞恶性生物学行为的影响(均P<0.05)。结论:TP可通过调控miR-142-3p/HSP70信号通路,进而抑制MCF-7细胞的增殖、侵袭和迁移能力。

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.

Triterpenoid of avocado (Persea americana) seed and its cytotoxic activity toward breast MCF-7 and liver HepG2 cancer cells

Objective:To determine the structure of triterpenoid isolated from avocado seeds and the cytotoxic effect on MCF-7 and Hep G2 cells.Methods:The powder sample was macerated with ethanol,followed with separation of the extract by column chromatography.The target compound was monitored on thin layer chromatography plate and reagent Lieberman–Buchard.The isolated compound was characterized by spectral analysis,mainly ultraviolet,infrared,and liquid chromatographymass spectroscopy and their spectroscopic data with those reported in literature were compared.In vitro cytotoxic activity was investigated against Vero,MCF-7,and Hep G2 cell lines using MTT assay.Results:A triterpenoid compound was isolated from ethanol extract.The extracts,fraction(F3),and the isolated compound showed a significant cytotoxic activity against all investigated cell lines.MTT assay showed that the triterpenoid isolate inhibited cell proliferation of MCF-7 and Hep G2 cell line with the IC50 values of 62 mg/m L and 12 mg/m L,respectively,and was safe to normal cells.Conclusions:The results of the present study reveal that triterpenoid from avocado seeds have the potential for further development as anticancer agents.

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.

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.

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.

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.

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.

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.

TNF-ɑ调控LRG1促进乳腺癌MCF-7细胞增殖、侵袭和迁移

目的:研究TNF-α对乳腺癌MCF-7细胞中LRG1蛋白表达的调控及其对乳腺癌MCF-7细胞增殖、侵袭和迁移能力的影响及相关分子机制。方法:MTT实验检测不同浓度TNF-α处理后MCF-7细胞活力;EdU实验、Transwell实验和划痕实验分别检测抑制LRG1表达后细胞增殖、侵袭以及迁移能力;Western blot检测细胞内MAPK信号通路中p-p38蛋白表达。结果:低浓度TNF-α处理乳腺癌MCF-7细胞,细胞活力增强;抑制LRG1表达后细胞增殖能力下降,侵袭细胞数、细胞迁移率以及p-p38蛋白表达均下降。结论:TNF-ɑ通过调控LRG1的表达促进乳腺癌MCF-7细胞增殖、侵袭和迁移,这一过程可能通过激活p38MAPK信号通路来实现。

毛酸浆内酯通过抑制STAT3诱导人乳腺癌MCF-7细胞凋亡

[目的]旨在探讨信号转导和转录激活因子3(STAT3)在毛酸浆内酯(PPB)诱导人乳腺癌MCF-7细胞凋亡中发挥的作用。[方法]采用荧光染色法分析PPB诱导MCF-7细胞凋亡;使用生物信息学方法预测PPB抗乳腺癌的潜在机制;采用噻唑蓝(MTT)法考察STAT3抑制剂S3I-201以及STAT3小干扰RNA(siRNA)对PPB抑制MCF-7细胞生长的作用;采用蛋白免疫印迹(Western Blot)法考察PPB单独处理或STAT3 siRNA预处理后对MCF-7细胞中STAT3、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶8(Caspase8)、半胱氨酸天冬氨酸蛋白酶9(Caspase9)、细胞色素c(Cytochrome c)以及多聚ADP核糖聚合酶(PARP)蛋白表达的影响。[结果]MCF-7细胞经PPB作用后凋亡形态特征明显,凋亡比例上升;生物信息学结果显示PPB与乳腺癌疾病的共同靶点STAT3在乳腺癌组织中高表达,单基因GSEA结果提示STAT3高表达与凋亡信号通路呈负相关;Western Blot法检测结果显示PPB能够抑制STAT3的磷酸化;S3I-201抑制剂或siRNA敲降STAT3均能进一步促进PPB抑制MCF-7细胞生长;此外,敲降STAT3进一步增加PPB对促凋亡蛋白Bax、Cytochrome c、裂解的Caspase8(Cleaved-Caspase8)、裂解的Caspase9(Cleaved-Caspase9)以及裂解的PARP(Cleaved-PARP)的促进作用,并增加PPB对抗凋亡蛋白Bcl-2的抑制作用。[结论]PPB通过抑制STAT3诱导人乳腺癌MCF-7细胞凋亡。

香豆酸诱导MCF-7细胞凋亡的代谢与氧化应激影响

目的 探讨香豆酸诱导乳腺癌细胞MCF-7凋亡的机制及氧化应激与代谢的影响。方法 采用氧化相关蛋白活性研究香豆酸对MCF-7氧化应激的影响;液相质谱仪结合代谢组学探索香豆酸对MCF-7细胞代谢的影响。结果 香豆酸呈浓度依赖式对MCF-7细胞凋亡具有一定的促进作用,并上调细胞的氧化应激性导致MCF-7细胞氧化系统失衡;同时香豆酸影响MCF-7的细胞代谢,主要是体现糖代谢,富集显著通路集糖酵解;并筛选到差异代谢物563个,其中上调463个,下调100个。结论 香豆酸诱导MCF-7细胞凋亡作用机制可能是通过影响细胞的氧化应激性以及调控细胞糖代谢来实现。

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.

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.

LMAN2在HR阳性乳腺癌组织中的表达与患者预后的关系及其对MCF-7细胞增殖和迁移的影响

目的:探究甘露糖结合凝集素2(LMAN2)在激素受体(HR)阳性乳腺癌组织中的表达水平与乳腺癌患者预后的关系及其对MCF-7细胞增殖和迁移的影响。方法:通过TCGA、Bc-GenExMiner、GEPIA和Kaplan-Meier Plotter数据库分析LMAN2在乳腺癌组织和正常乳腺组织中的差异性表达及其与患者预后的关系。采用小RNA干扰技术将si-LMAN2#1、si-LMAN2#2及si-NC转染至MCF-7细胞,将过表达LMAN载体(pc-LMAN)及空载体pcDNA3.1阴性对照(pc-NC)转染至MCF-7细胞,实验分为si-LMAN2#1、si-LMAN2#2、si-NC、pc-LMAN2和pc-NC组。通过qPCR和WB实验检测各组细胞中LMAN2 mRNA和蛋白的表达水平,CCK-8、克隆形成、Transwell迁移、WB等实验检测敲低和过表达LMAN 2对MCF-7细胞增殖、克隆形成、迁移及AKT信号通路相关蛋白表达的影响。结果:LMAN2在乳腺癌组织中的表达水平显著高于正常乳腺组织(P<0.001)。HR阳性乳腺癌组织中LMAN2表达水平显著高于HR阴性乳腺癌组织(P<0.001);LMAN2高表达与HR阳性乳腺癌患者不良预后有关联。敲低LMAN2可显著降低MCF-7细胞的增殖和迁移能力(P<0.01或P<0.001),过表达LMAN2可显著提高MCF-7细胞的增殖和迁移能力(均P<0.001)。敲低LMAN2组MCF-7细胞中PTEN和P21蛋白表达水平均显著升高,p-AKT蛋白表达水平显著降低(均P<0.01)。结论:LMAN2在乳腺癌组织和HR阳性乳腺癌组织中高表达,且与不良预后有关联。LMAN2高表达与MCF-7细胞增殖和迁移有关联,其作用机制可能涉及AKT信号通路。