Norcantharidin induces apoptosis through autophagosome accumulation in breast cancer MDA-MB-231 cells / 中国药科大学学报

@#The aim of this study was to investigate the effect of norcantharidin (NCTD) on the proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231.Western blot was used to detect the effect of NCTD on the expression levels of apoptosis-related proteins Bax/Bcl-2, cleaved-PARP/PARP/PARP, cleved-caspase-9, cleaved-caspase-3 and MCL-1 in MDA-MB-231 cells.Also, the expression levels of autophagy-related proteins LC3-II/LC3-I, Parkin and PINK1 in MDA-MB-231 cells were measured by Western blot.Flow cytometry was used to measure the effect of NCTD on the changes of mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS).The effect of NCTD on autophagy flow in cells expressing mCherry-EGFP-LC3 was detected by a confocal microscope.Moreover, the effects of NCTD combined with chloroquine (CQ) or 3-methyladenine (3-MA) on the apoptosis of MDA-MB-231 cells were detected by flow cytometry.The results showed that NCTD significantly increased the expression levels of Bax/Bcl-2, cleaved-PARP/PARP, cleaved-caspase-9, cleasved-caspase-3 and LC3-II/LC3-I proteins, and promoted the mitochondrial translocation of Parkin, and blocked the autophagic flow in MDA-MB-231 cells. Moreover, NCTD combined with CQ accelerated apoptosis, while NCTD combined with 3-MA decreased apoptosis.These results suggest that NCTD can induce autophagy accumulation and lead to apoptosis of MDA-MB-231 cells.

Effect of Curdione on MDA-MB-231 Cell Cycle and Apoptosis / 中国实验方剂学杂志

Objective:To investigate the effects of curdione on the proliferation， apoptosis and cell cycle of triple negative breast cancer cell line MDA-MB-231. Method:MDA-MB-231 cells were cultured<italic> in vitro</italic> with capecitabine （positive control） and curdione at different concentrations （125， 250， 500， 1 000， and 2 000 μmol·L^-1）， respectively， for detecting their viability using the cell counting kit-8 （CCK-8） at 24 and 48 h. Three effective inhibitory concentrations （250， 500， and 1 000 μmol·L^-1） against cell proliferation were selected for subsequent experiments. The effect of curdione on cell cycle was determined by flow cytometry combined with propidium iodide （PI） staining. After the set-up of high-concentration （2 000 μmol·L^-1） group， the effect of curdione on cell mitochondrial membrane potential was measured by JC-1（5，5，6，6-tetrachloro-1，1，3，3-tetraethylbenzimidazolylcarbocyanine iodide） staining， followed by the detection of cell apoptosis by flow cytometry combined with Annexin V-FITC/PI double staining. The changes in cell cycle status and apoptosis-related protein expression following curdione intervention were assayed by Western blot. Result:Compared with the blank control， curdione at 250， 500， 1 000， and 2 000 μmol·L^-1significantly inhibited the proliferation of MDA-MB-231 cells （<italic>P</italic><0.01）， exhibiting a concentration- and time-response relationship. The half maximal inhibitory concentration （IC₅₀） values at 24 and 48 h were 1 607 and 1 401 μmol·L^-1， respectively. Curdione at 250， 500， and 1 000 μmol·L^-1 arrested cells in G₁ phase. Curdione at 250 μmol·L^-1had no effect on cell mitochondrial membrane potential， which， however， declined significantly in the 500， 1 000， and 2 000 μmol·L^-1groups （<italic>P</italic><0.05， <italic>P</italic><0.01）. Curdione at 250， 500， and 1 000 μmol·L^-1obviously increased the proportion of apoptotic cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. Curdione at each concentration elevated the Bcl-2-associated X protein （Bax）/B-cell lymphoma 2 （Bcl-2） ratio （<italic>P</italic><0.05， <italic>P</italic><0.01）， but did not change the cysteinyl aspartate-specific protease-3 （Caspase-3） expression. The protein expression levels of Caspase-9， cleaved Caspase-9， cleaved Caspase-3， p53， and p21 were up-regulated （<italic>P</italic><0.05）. Conclusion:A certain concentration of curdione inhibits the proliferation of MDA-MB-231 cells， which may be related to its efficacy in arresting cell cycle and inducing apoptosis.

Inhibitory Effect and Mechanism of Jingulian Capsule on Human Breast Cancer MDA-MB-231 Cells / 中国实验方剂学杂志

Objective:To observe effect of Jingulian capsule on the proliferation of human breast cancer MDA-MB-231 cells and investigate its action mechanism against triple negative breast cancer （TNBC）. Method:The ingredients of Jingulian capsule were identified by ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）. The inhibitory effect of Jingulian capsule at different doses （0.125，0.25，0.5，1，and 2 g·L^-1） against the proliferation of MDA-MB-231 cells were detected by methyl thiazolyl tetrazolium （MTT） assay. After treatment for 24 h， the morphological changes in nuclear apoptosis of MDA-MB-231 cells were detected by Hoechst 33258 staining. The effect of different concentrations of Jingulian capsule on the apoptosis and cycle of MDA-MB-231 cells after different treatment time were determined by flow cytometry. The protein expression levels of Poly-ADP-ribose polymeras （PARP）， proto-oncogene c-Myc， cyclin B₁， and phosphorylated extracellular signal-regulated kinase （p-ERK） in each group were assayed by Western blot. Result:A total of 113 compounds in Jingulian capsule were identified by UPLC-MS/MS. As revealed by MTT assay，compared with blank group，Jingulian capsule （0.125，0.25，0.5，1，2 g·L^-1） significantly inhibited viability of MDA-MB-231 cells （<italic>P</italic><0.01）， with the half maximal inhibitory concentration （ IC₅₀） of（0.13±0.02）g·L^-1. According to flow cytometry，compared with the blank group，Jingulian capsule at 1 g·L^-1 significantly induced the apoptosis of MDA-MB-231 cells （<italic>P</italic><0.05）and Jingulian capsule at 0.5， 1 g·L^-1 obviously increased the number of MDA-MB-231 cells in S phase （<italic>P</italic><0.05，<italic>P</italic><0.01）. The results of Western blotting demonstrated that the protein expression levels of PARP，c-Myc，and cyclin B₁ in 0.5， 1 g·L^-1Jingulian capsule groups were remarkably down-regulated as compared with those in the blank group（<italic>P</italic><0.01）， and the protein expression level of p-ERK in 1 g·L^-1Jingulian capsule group was also down-regulated （<italic>P</italic><0.01）. Conclusion:Jingulian capsule is able to inhibit the proliferation of MDA-MB-231 cells，induce S phase cell cycle arrest， and promote their apoptosis， which may be related to the inactivation of the MAPK signaling pathway.

Effect and Its Mechanism of Plantago asiatica Polysaccharide on the Proliferation ，Migration and Invasion of Breast Cancer Cells / 中国药房

OBJECTIVE：To study the effects of Plantago asiatica polysaccharide on the proliferation ，migration and invasion of breast cancer cells ，and to investigate its mechanism preliminarily. METHODS ：Using human breast cancer cell MDA-MB- 231 as subjects ，MTT method was adopted to detect the effects of different concentrations of P. asiatica polysaccharide（8，16，32，64 mg/L）on the cell proliferation ability ，and survival rate of the cells was calculated. Scratch test and Transwell invasion test were used to detect the effects of different concentrations of P. asiatica polysaccharide（8，16 mg/L）on cell migration ability and invasion ability. Western blot assay was used to detect the expression of epithelial-mesenchymal transition （EMT）-related proteins [matrix metalloproteinase- 2（MMP-2），MMP-9，E-cadherin，N-cadherin，vimentin]. RESULTS ：Results of MTT assay showed that survival rate of the cells in 32，64 mg/L P. asiatica polysaccharide groups were significantly lower than control group （P＜0.05 or P＜0.01），so that 8，16 mg/L，which did not affect the cell survival rate ，were used as the follow-up drug concentrations. Compared with control group ，relative mobility （12，24 h），relative invasion rate and relative expression of MMP- 2，MMP-9， N-cadherin and vimentin protein were decreased significantly in 8，16 mg/L P. asiatica polysaccharide groups （P＜0.05 or P＜ 0.01），while relative expression of E-cadherin protein was increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. asiatica polysaccharide can inhibit the proliferation of breast cancer cells MDA-MB- 231，and inhibit the migration and invasion of the cells by regulating the expression of metastasis and EMT-related proteins.

Inhibitory effect of disulfiram combined with cisplatin on triple negative breast cancer MDA-MB-231 cells and its mechanism / 吉林大学学报(医学版)

Objective: To observe the inhibitory effect of combination of disulfiram (DSF) and cisplatin (CDDP) on the proliferation of triple-negative breast cancer (TNBC) MDA-MB-231 cells, and to explore its possible mechanism. Methods: The MDA-MB-231 cells in the logarithmic phase were divided into blank control group. DSF group. CDDP group and combination groups (60/xmol • L 1 CDDP+1. 2 and 4/imol • L ' DSF). MTT method was used to detect the survival rates of the MDA-MB-231 cells in various groups and flow cytometry was used to detect the apoptotic rates of the MDA-MB-231 cells in various groups, flow cytometry was used to detect the level of active oxygen ( ROS ) in the MDA-MB-231 cells in various groups, and Inductively Coupled Plasma-MassSpectrometry (ICP-MS) was used to detect the levels of Pt in the MDA-MB-231 cells in various groups. Results: The MTT method and flow cytometry results showed that after treated for 72 h. compared with 60/xmol • L ' CDDPgroup, the survival rates of the MDA-MB-231 cells in combination 1 group (60/xmol • L CDDP+1/xmol • L 1 DSF), combination 2 group (60/xmol • L 'CDDP+ 2/xmol • L 1 DSF) and combination 3 group (60/xmol • L 'CDDP+4/xmol • L 'DSF) were decreased ( P<0. 05). After treated for 24 h. compared with 60/x mol • L ' CDDP group and 2/x mol • L DSF group, the apoptotic rate of the MDA-MB-231 cells in combination group (60/xmol • L 'CDDP+2/xmol • L ' DSF) was increased ( P<0. 05). Compared with CDDP group, the level of ROS in the MDA-MB-231 cells in combination group (60/xmol • L 'CDDP+2/xmol • L 1 DSF) was increased (P<0. 05). The 1CP-MS results showed that compared with CDDP group, the level of Pt in the MDA-MB-231 cells in combination group (20/xmol • L 1CDDP+ 0.625/xmol • L ' DSF) was increased (P< 0.05). Conclusion: The combination of DSF and CDDP can significantly inhibit the proliferation of MDA-MB-231 cells, and its possible mechanism may be related to inhibiting the growth of tumor cells by increasing the ROS level and Pt content in the MDA-MB-231 cells.

Effect of Yanghetang on Apoptosis of Triple Negative Breast Cancer Cell Line MDA-MB-231 Based on Egr1/p21 Signaling Pathway / 中国实验方剂学杂志

Objective::To investigate the effect of ancient recipe Yanghetang and its drug-contained serum on apoptosis of triple negative breast cancer cell line MDA-MB-231 based on the signal pathway of early growth response gene 1 (Egr1)/cyclin dependent inhibitor (p21). Method::The drug-containing serum of Yanghetang was prepared from rats, and MDA-MB-231 cells were cultured in vitro. The blank control group was set, and MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were intervened for 24, 48, 72 h, respectively. After that, the cell counting kit-8(CCK-8) method was used to detect the cell proliferation of each group. The blank control group was set, while MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were treated for 48 h, and then flow cytometry was used to detect the apoptosis of each group and the distribution of cell cycle. The expression of Egr1 and p21 mRNA in each group was detected by quantificational Real-time polymerase chain reaction (Real-time PCR), while the expression of Egr1 and p21 protein in each group was detected by Western blot. Result::After MDA-MB-231 cells were intervened by Yanghetang for 24, 48, 72 h, MDA-MB-231 cell proliferation was significantly inhibited in Yanghetang high and middle dose groups as compared with the blank control group (P<0.01). After MDA-MB-231 cells were intervened by Yanghetang for 48 h, the apoptosis was significantly increased in Yanghetang high and middle dose groups as compared with the blank control group (P<0.05, P<0.01). In the Yanghetang high, middle dose groups, the proportion of cell cycle G0/G1 phase decreased, and the proportion of G2/M phase increased (P<0.05, P<0.01). The mRNA and protein expressions of Egr1, p21 were increased in Yanghetang high and middle groups (P<0.05, P<0.01). Conclusion::Yanghetang can activate Egr1/p21 signaling pathway in MDA-MB-231 cells, increase the expression of Egr1 and p21, and cause G2/M cell cycle arrest, thereby inducing apoptosis of MDA-MB-231 cells.

Effect of glaucocalyxin A on proliferation and cell cycle of triple-negative breast cancer MDA-MB-231 cells / 中草药

Objective: To investigate the effect of glaucocalyxin A on proliferation and cell cycle of triple-negative breast cancer MDA-MB-231 cells and its mechanism. Methods The proliferation inhibition rates of MDA-MB-231 cells were measured by MTT assay. The cell cycle was analyzed by flow cytometry, and the expression of the protein cyclin B1, cyclin D1, CDK2, CDK4, p53, p21, p27, LSD1, H3K4me2, and H3K9me2 was detected by Western blotting. Results Growth of MDA-MB-231 cells was significantly inhibited by glaucocalyxin A in a dose-dependent and time-dependent manner. Flow cytometric analysis indicated that the percentage of MDA-MB-231 cells at G2/M phase was increased significantly. As the results of Western blotting, the protein expression levels of p53, p21, p27, H3K4me2, and H3K9me2 in MDA-MB-231 cells were increased, while that of cyclin B1, cyclin D1, CDK2, CDK4, and LSD1 were decreased after treated with glaucocalyxin A. Conclusion Glaucocalyxin A could inhibit the proliferation of MDA-MB-231 cells and induce cell cycle arrest at the G2/M phase, and the mechanism may be related to the activation of p53 protein expression and the regulation of histone methylation.

Induced effects of resveratrol on apoptosis and autophagy in human breast cancer MDA-MB231 cells / 中国药理学通报

Aim: To explore the induced effects of resveratrol (Res) on apoptosis and autophagy protein expression in human breast cancer MDA-MB231 cells and the mechanisms. Methods: MDA-MB231 cells were cultured in vitro. MTT was adopted to detect cell proliferation of MDA-MB231 cells inhibited by Res(0, l, 5, 10, 20, 50, 100, 200 mg · L-1). MDA-MB231 cells were treated by Res with different concentrations (0, 5, 20, 60 mg · L-1). The migration ability of cells was detected by scratch test. The early apoptotic rate was detected by Annexin V/PI double staining. The expression of cell LC3 I/II was detected by immunofluorescence method. MDA-MB231 cells were treated by Res with different concentrations (0, 20, 60 mg · L-1). And the autophagy related to p62 protein and Beclin 1 expression was detected by Western blot. Results: Res had significantly inhibitory effects on MDA-MB231 cell proliferation in a concentration-dependent manner. With the increase of Res concentration in MDA-MB231 cells, compared with control group, cell migration ability decreased (8.7 ± 1.1)%, (16. 5 ±2. 9)% and (32.2 ±3.6)%. The early apoptotic cells increased according to the results of flow cytometry and the laser confocal examination showed that LC3 I/II green fluorescence increased. Western blot detected that p62 protein expression decreased and Beclin 1 protein expression increased in the cytoplasm of MDA-MB231. Conclusions: Res can inhibit the proliferation of human breast cancer MDA-MB231 cells in vitro and its mechanism may be related to cell apoptosis and autophagy induced by Res.

Isolation of homogeneous polysaccharide from Poria cocos and effect of its sulfated derivatives on migration of human breast cancer MDA-MB-231 cells / 中国中药杂志

SATB1 plays a crucial role in the invasion and metastasis of breast cancer,and inhibition of SATB1 expression can effectively control breast cancer metastasis. In this study,homogeneous polysaccharides were isolated from Poria cocos and their sulfated derivatives were prepared to screen out the polysaccharide compositions with inhibitory effects on SATB1 expression. Smal-molecule components were removed from P. cocos by ethanol extraction,and P. cocos crude polysaccharide PPS was obtained by water extraction and ethanol precipitation. Then PPS was successively separated by DEAE Sepharose fast flow anion-exchange and Superdex-75 gel permeation chromatographic steps to give PPSW-1. The structure of PPSW-1 was identified and its sulfated derivatives were prepared. Then their inhibitory effects on human breast cancer MDA-MB-231 cells were investigated. A kind of polysaccharide,PPSW-1 with inhibitory effect on human breast cancer MDA-MB-231 cells,was obtained from P. cocos,with a relative molecular weight of 3. 06×104,and structure of 1,6-branched 1,3-α-D-galactan. PPSW-1 and its sulfated derivative Sul-W-1 showed good inhibitory effect on cells migration,and the water solubility of Sul-W-1 was better than that of PPSW-1. In addition,it was found that polysaccharide of P. cocos and its sulfated derivative can inhibit expression of SATB1. In this study,a kind of homogeneous polysaccharide with inhibitory effect on human breast cancer MDA-MB-231 cells was isolated from P. cocos,and its sulfated derivative with similar efficacy but better solubility was prepared,laying the foundation for the substance basis study of P. cocos.

In vitro study of genistein inducing apoptosis in MDA-MB-231 breast cancer cells via NF-κB and MAPK signaling pathways / 中国药理学通报

Aim To study the effect of genistein on apoptosis in human breast cancer MDA-MB-231 cells and the underlying mechanisms. Methods MTT as-say was used to observe the inhibitory rate on human breast cancer MDA-MB-231 cells treated with genistein. Colony assay was used to determine the cell colony formation rate on human breast cancer MDA-MB-231 cells treated with genistein. Western blot was used to detect the expression of Bcl-2, Bax, caspase-3,NF-κB, ERK, p-ERK, JNK and p-JNK in human breast cancer MDA-MB-231 cells treated with genistein. Results The results of MTT assay showed that genistein inhibited the viability of breast cancer MDA-MB-231 cells in a time- and concentration-de-pendent manner. Colony assay suggested that genistein had an antiproliferative effect on MDA-MB-231 cells. The expression levels of Bcl-2, NF-κB and p-ERK were significantly down-regulated compared with con-trol(P < 0.01). However, the expression of Bax, caspase-3 and p-JNK was significantly up-regulated(P<0.01). Conclusions Genistein could inhibit the growth of human breast cancer MDA-MB-231 cells and induce apoptosis,and the mechanism may be related to the inhibition of NF-κB, ERK/MAPK signaling path-ways and the activation of JNK/MAPK signaling path-way.

Assessment Of Cytotoxicity Potency Of Paclitaxel In Combination With Clinacanthus Nutans Extracts On Human Mda-Mb-231 Breast Cancer Cells / Jurnal Sains Kesihatan Malaysia

Clinacanthus nutans (C. nutans) leaf extracts have been widely used by cancer patients in Malaysia and local practiceclaims a cure to cancer. There were several studies done to determine the cytotoxicity potency of C. nutans extracts onvarious types of cells. However, there is still lacking on the knowledge regarding the combination effect of C. nutanswith anticancer drugs. Thus, the study was carried out to determine the cytotoxicity potency of C. nutans extracts andpaclitaxel (PTX) alone and, in combination on MDA-MB-231 cells. The cells were treated with 100% ethanol extract ofC. nutans (CNE) and water extract of C. nutans (CNA), PTX and combination of both extracts and PTX for 72 hours andthe cytotoxic activity was determined using SRB assay. Result showed that CNE had little cytotoxic activity, whereas CNAshowed no cytotoxic activity on MDA-MB-231 cells. For combination treatment of C. nutans extracts and PTX, only CNEshowed significant enhanced PTX-induced cytotoxicity (p < 0.05), meanwhile CNA inhibited PTX-induced cytotoxicitysignificantly (p < 0.05). As a conclusion, CNE was able to increase PTX potency to inhibit the viability of MDA-MB-231cells.

Expression of miR-103a-3p in breast cancer tissues and its suppression on glycolysis and proliferation of breast cancer cells via down-regulating PDK4 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective： ： To explore miR-103a-3p expression in the tumor tissues and the serum of breast cancer patients, and its role and mechanism in breast cancer development. Methods: Pathologically confirmed 31 cases of tumor tissues and 21 cases of para-cancerous tissues resected at Department of Oncological Surgery of the Second Affiliated Hospital of Hainan Medical University (Haikou, China) from March 1, 2017 to August 31，2017 were collected for this study; in addition, serum samples from 38 breast cancer patients and 22 healthy subjects as well as the breast cancer cell lines MCF-7 and MDA-MB-231 were used in this study. pHBLV-U6-Luc-T2A-Puro and PLL3.7 lentivirus were applied to knock down miR-103a-3p and PDK4 in MCF-7 and MDA-MB-231 cells, respectively. qPCR and Western blotting were performed to examine the mRNA and protein expressions of miR-103a-3p and PDK4 in tissues and serums of breast cancer patients as well as the in cell lines, respectively; CCK-8 assay was applied to detect the proliferation of MCF-7 and MDAMB-231 cells; Olympus AU5400 was applied to detect the glucose consumption and lactate production in indicated cell line. Results： ： miR-103a-3p was significantly decreased in tumor tissues compared with the paracancerous tissues (P<0.01). miR-103a-3p knockdown activated the glucos consumption and lactate production (all P<0.01), increased the PKD4 expression (P<0.01) in MCF-7 and MDAMD-231 cells, and promoted the proliferation of MCF-7 and MDA-MB-231 cells (P<0.01). Furthermore, knockdown of PDK4 suppressed the glucose consumption, lactate production and proliferation in MCF-7 and MDA-MB-231 cells with miR-103a-3p silencing (all P<0.01). Conclusion： ：In the breast cancer, miR-103a-3p inhibited the proliferation of breast cancer cells through down-regulation of PDK4 and PDK4-mediated aerobic glycolysis.

Dipyridyl pyrrolidine inhibits MDA-MB231 breast cancer cell cycle by inhibiting AKT-mTOR / 中国药科大学学报

@#To investigate the induction of cell cycle arrest of human breast cancer MDA-MB231 cells by Di-indolyl pyrrolidine(DIPRD), a pyrrolidine-derived spirooxindoles compounds. The cytotoxic effect of DIPRD on MDA-MB231 cells was detected by CCK-8 method. The cell cycle arrest of MDA-MB231 cells was detected by DAPI/EdU double-staining. Phosphorylation levels of AKT, mTOR, apoptosis-related proteins p53, MDM2, and DNA repair enzyme PARP levels were detected by Western blot. DIPRD inhibited the viability of MDA-MB231 cells by downregulating the number of EdU-positive cells, increase G1 phase and reduce cell number in S/G2 phase, down-regulated the p-AKT(Ser473), p-mTOR, p-p53, cyclin D1, CDK4, and the upregulated the p-AKT(Thr308), p-MDM2 and Cleaved-PARP levels were detected in a dose-dependent manner at 12. 5, 25, and 50 mg/mL. DIPRD may play a role in cell cycle arrest through AKT signaling pathway and induce cell apoptosis.

Effects of Active Constituents of Sinopodophylli Fructus on Cell Proliferation,Cell Cycle and Mitochon-drial Membrane Potential of Human Breast Cancer Cell / 中国药房

OBJECTIVE:To explore the effects and mechanism of extracts,active constituents and constituent combination of Sinopodophylli Fructus on cell proliferation of human breast cancer. METHODS:Acid phosphatase method was conducted to deter-mine the effects of 4 extracts [ethanol extract (Xc),petroleum ether extract from ethanol extract (Xp),ethyl acetate extract from ethanol extract (Xe),n-butanol extract from ethanol extract (Xz)],5 active constituents [podophyllotoxin (S1),deoxypodophyllo-toxin (S2),4-desmethyl deoxypodophyllotoxin (S3),8-isopentenyl kaempferol (S4),8,2′-diisoprenyl quercetin-3-methyl ether (S5)] and 3 active constituent combination [combination 1,S1-S2-S3-S4-S5 (2:4:1:4:32),Z1;combination 2,S2-S4 (1:1),Z2;combination 3,S3-S4(1:4),Z3] on the MDA-MB-231,MCF-7 cell proliferation;flow cytometry was adopted to detect the effects of above-mentioned samples on MDA-MB-231,MCF-7(T47D)cell cycle and mitochondrial membrane potential. RESULTS:The active constituent combination Z1 showed significant inhibitory effects on MDA-MB-231,MCF-7 cells,the half inhibitory concen-trations(IC50)were(0.27±0.2),(0.11±0.1)μg/mL;extracts Xc,Xp,Xe,active constituents S2,S4 and active constituent combi-nation Z2,Z3 showed relatively strong inhibitory effects on MDA-MB-231,MCF-7 (T47D) cell proliferation (IC50<15 μg/mL). Both extracts and active constituents can block MDA-MB-231,MCF-7 cell cycle in G2/M phase;all active constituents can block MDA-MB-231,T47D cell cycle in G0/G1 phase,and can reduce MDA-MB-231,T47D cell mitochondrial membrane potential. CONCLUSIONS:The active constituents and constituent combination of Sinopodophylli Fructus can inhibit cell proliferation of breast cancer by affecting cell cycle and mitochondrial mem-brane potential.

Inhibitory effect of Euphorbia helioscopia on human triple-negative breast cancer MDA-MB-231 cells and its possible mechanism of apoptosis / 药学实践杂志

Objective To investigate the effect of Euphorbia helioscopia on MDA-MB-231 cells and its mechanism.Methods The cell viability was detected by MTT assay.The production of ROS in MDA-MB-231 cells was measured by fluorescence microscopy.The apoptotic rate was detected by flow cytometry.Apoptosis DNA fragments were detected by TUNEL assay.Western blot was used to assess the expression of caspase-9, caspase-3 and PARP.Results MTT assay showed that the extract significantly inhibited the viability of MDA-MB-231 cells, which can be diminished by the ROS inhibitor NAC and the caspase inhibitor Z-VAD-FMK.The marked increase in the production of ROS induced by the extract was observed with fluorescence microscopy.Flow cytometry showed that the PI positive staining cells increased significantly after the treatment of the extract, but was diminished by NAC.Caspase-9 and caspase-3 were activated after the treatment of the extract while the PARP was cleaved.TUNEL showed that a significant increase in apoptotic DNA fragmentation induced by the extract, which can be diminished by NAC and Z-VAD-FMK.Conclusion Ethyl acetate extract inhibited the MDA-MB-231 cells and induced apoptosis.The mechanism may involve with the mitochondrial damage due to the excessive ROS.

Effects of ropivacaine on proliferation,apoptosis and cell cycle of MDA-MB-231 cells / 实用医学杂志

Objective To investigate the effects of ropivacaine on proliferation,apoptosis and cell cycle of MDA-MB-231 Cells. Methods The cultured MDA-MB-231 cells were treated with different concentrations of ropivacaine. The proliferation of MDA-MB-231 cells was detected by MTT method. Cell apoptosis and cell cycle were detected by Annexin V-FITC/PI and flow cytometry. Results After ropivacaine administration,MDA-MB-231 cells proliferation inhibition ratio increased significantly.Annexin V-FITC/PI and flow cytometry showed ropiva-caine had no significant effects on cell apoptosis and cell cycle.Conclusion Ropivacaine can inhibit the prolifera-tion of MDA-MB-231 cells,but can′t induce apoptosis and block cell cycle.

Loquat (Eriobotrya japonica) leaf extract inhibits the growth of MDA-MB-231 tumors in nude mouse xenografts and invasion of MDA-MB-231 cells

BACKGROUND/OBJECTIVES: The present study was conducted to examine the inhibitory effect of loquat leaves on MDA-MB-231 cell proliferation and invasion. MATERIALS/METHODS: Female athymic nude mice were given a subcutaneous (s.c.) inoculation of MDA-MB-231 cells and randomly grouped to receive a s.c. injection of either 500 mg/kg ethanol, water extract or vehicle five times a week. Tumor growth, mitotic rate and necrosis were examined. MDA-MB-231 cells were cultured with DMSO or with various concentrations of loquat water or ethanol extract. Proliferation, adhesion, migration, invasion and matrix metalloproteinase (MMP) activity were examined. RESULTS: Tumor growth of xenograft nude mouse was significantly reduced by loquat extracts. The results of mitotic examination revealed that loquat extracts reduced tumor cell division. Both ethanol and water extracts significantly inhibited MDA-MB-231 cell proliferation. The protein expression of ErbB3 was significantly down-regulated by loquat leaf extracts. Loquat leaf extracts increased apoptosis of MDA-MB-231 cells following 24 hour incubation and the ethanol extract was more potent in inducing apoptosis than the water extract. Furthermore, loquat extracts inhibited adhesion, migration and invasion of MDA-MB-231 cells. MMP activity was significantly inhibited by loquat extracts. CONCLUSION: Our results show that extracts of loquat inhibit the growth of tumor in MDA-MB-231 xenograft nude mice and the invasion of human breast cancer cells, indicating the inhibition of tumor cell proliferation and invasion.

Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells / 中国天然药物

Gambogic acid (GA) is an anticancer agent in phase ‖b 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 electrophoresis (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 proteins such as cytoskeleton-related proteins.

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer / 中国病理生理杂志

AIM:To investigate the effect of microRNA ( miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS:miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls.MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells.Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b.Mcl-1 expression vector was constructed , and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay .RESULTS:Down-regulation of miR-193b was found in breast cancer patients .The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells.The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection.The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was sig-nificantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer .

Effects of anti-heparanase monoclonal antibody in combination with paclitaxel on proliferation, migration and invasion of breast cancer cell lines / 肿瘤

Objective: To investigate the effects of anti-heparanase monoclonal antibody (anti-HPA mAb) in combination with paclitaxel (PTX) on proliferation, migration and invasion of breast cancer MCF-7, MCF-7/F5 and MDA-MB-231 cells. Methods: The expression levels of HPAmRNA in MCF-7, MCF-7/Fs and MDA-MB-231 cells after treatment with anti-HPA mAb were detected by reverse transcription-PCR (RT-PCR). The proliferation of the three breast cancer cell lines after treatment with different concentrations of anti-HPA mAb or PTX alone or in combination was detected by MTT assay. The cell cycle distribution and the abilities of migration and invasion of these breast cancer cells after different treatment modalities were examined by flow cytometry (FCM), wound healing assay and Transwell assay, respectively. Results: The expression levels of HPA mRNA in MCF-7/F5 and MDA-MB-231 cells after treatment with 10 and 20 nmol/L anti-HPA mAb were lower than those of murine immunoglobulin C (IgG) treatment group (all P < 0.05). The proliferation of MCF-7, MCF-7/F5 and MDA-MB-231 cells after treatment with anti-HPA mAb or PTX alone was inhibited in a concentration-dependent manner (all P < 0.05). The proliferative inhibition rates of the three breast cancer cell lines after treatment with combination of anti-HPA mAb and PTX were higher than those of any single drug treatment groups (all P < 0.05). The ratios of G0/G1 phase of the three breast cancer cell lines after treatment with combination of anti-HPA mAb and PTX were lower than those of the control groups (three breast cancer cell lines without any treatment), and the ratios of G2/M phase were opposite (P < 0.05, P < 0.01), with a typical sub-diploid peak which appeared before C0/G1 phase. The abilities of migration and invasion of MCF-7/F5 and MDA-MB-231 cells after treatment with combination of anti-HPA mAb and PTX were lower than those of the control, murine IgG and single drug treatment groups (all P < 0.05). Conclusion: Anti-HPA mAb in combination with PTX have synergistic inhibitory effect on the abilities of proliferation, migration and invasion of breast cancer cells, and this mechanism may be associated with the arrest in G2/M phase.