Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.     

Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells

Estrogen sensitizes the MCF-7 estrogen-responsive breast cancer cell line to the mitogenic effect of insulin and the insulin-like growth factors (IGFs). This sensitization is specific for estrogen and occurs at physiological concentrations of estradiol. Dose-response experiments with insulin, IGF-I, and IGF-II suggested that the sensitization is mediated through the type I IGF receptor. Binding experiments with 125I-IGF-I and hybridization of a type I IGF receptor probe to RNA showed that the levels of the type I IGF receptor and its mRNA are increased 7- and 6.5-fold, respectively, by estradiol. IGF-I and estradiol had similar synergistic effects on other estrogen-responsive breast cancer cell lines, but IGF-I alone increased the proliferation of the MDA MB-231 cell line which is not responsive to estrogens. These experiments suggest that an important mechanism by which estrogens stimulate the proliferation of hormone-dependent breast cancer cells involves sensitization to the proliferative effects of IGFs and that this may involve regulation of the type I IGF receptor.     

原位杂交检测microRNA-205在乳腺癌中的表达

目的探讨microRNA-205表达与乳腺恶性病变的关系。方法乳腺疾病及癌组织芯片原位杂交分析microRNA-205的表达;实时定量RT-PCR方法检测正常乳腺细胞株、恶性程度不同的乳腺癌细胞株中microRNA-205的表达。结果原位杂交分析显示,36例正常与良性乳腺病变中,33例(91.67%)表达阳性;36例乳腺癌中,23例(63.89%)表达阳性。microRNA-205的表达在乳腺正常与良性病变中的表达较恶性病变中高且有统计学差异(P=0.011),但与乳腺癌TNM分期、临床分期无关(P0.05)。实时定量RT-PCR结果显示,四个高度恶性乳腺癌细胞株(MDA-MB-231、HS578T、BT549和SUM159PT)中microRNA-205的表达较永生化正常乳腺上皮细胞株MCF10A和四个低度恶性细胞株(MDA-MB-468、T-47D、ZR-75-1和SKBR3)中为低(P0.05)。结论原位杂交适用于microRNA-205的表达分析;组织芯片标本原位杂交与乳腺细胞株实时定量RT-PCR分析结果提示,microRNA-205可能参与了乳腺癌的发生、发展,并随着乳腺癌的演进呈下调趋势。     

Comparative analysis of the cytotoxic activity of extracts from different parts of A. absinthium L. on breast cancer cell lines and correlation with active compounds concentration

The aim of the present study was to compare the cytotoxicity of different extracts of the plant Artemisia absinthium on breast cancer cell lines and to establish the correlation between the cytotoxicity and the active constituent’s level in these extracts. The cytotoxicity of the extracts was evaluated on the breast cancer cell lines, MCF-7 and MDA MB-231 by MTT assay and LDH release assay. An HPTLC method was developed for the simultaneous estimation of active constituents, that is, artemisinin, artemisinic acid, and alpha-thujone in different parts of A. absinthium. The whole extract was best among all the extracts tested with least IC₅₀ value and high LDH release that is, 491.19?µg/µL with 27.92% for MCF-7 and 459.97?µg/µL with 29.43% for MDA MB-231 cell lines respectively. Although, the concentration of all three quantified active compounds was higher in the extract from aerial part; however, the whole extract showed the best cytotoxicity among all extracts evaluated on the breast cancer cell lines. Surprisingly, our results demonstrate that the quantified active compounds were not solely responsible for the cytotoxic activity of the plant parts and further studies may be conducted to identify the compounds with synergistic, allosteric or antagonistic effects.     

Centchroman induces G0/G1 arrest and caspase-dependent apoptosis involving mitochondrial membrane depolarization in MCF-7 and MDA MB-231 human breast cancer cells

Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/-ve MCF-7 and MDA MB-231 HBCCs respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity, Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was detected by Annexin-PI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC50 of CC in MCF-7 and MDA MB-231 cells to be 10 and 20 microM (P < 0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G0/G1 arrest (P < 0.01) along with the presence of prominent sub-G0/G1 peak (P < 0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependent apoptosis even at a dose of 1 microM which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.     

Beta-lactam type molecular scaffolds for antiproliferative activity: synthesis and cytotoxic effects in breast cancer cells

A series of novel beta-lactam containing compounds are described as antiproliferative agents and potential selective modulators of the oestrogen receptor. The purpose of the study is to evaluate the antiproliferative effects of these compounds on human MCF-7 and MDA MB-231 breast cancer cells. The compounds are designed to contain three aryl ring substituents arranged on the heterocyclic azetidin-2-one (beta-lactam), thus providing conformationally restrained analogues of the triarylethylene arrangement exemplified in the tamoxifen type structure. The compounds demonstrated potency in antiproliferative assays against MCF-7 human breast cancer cell line at low micromolar to nanomolar concentrations with low cytotoxicity and moderate binding affinity to the oestrogen receptor. The effect of a number of aryl and amine functional group substitutions on the antiproliferative activity of the beta-lactam products was explored and a brief computational structure-activity relationship investigation with molecular simulation was investigated.     

Synthesis of benzo[d]imidazo[2,1-b]thiazole-chalcone conjugates as microtubule targeting and apoptosis inducing agents

A series of benzo[d]imidazo[2,1-b]thiazole-chalcone conjugates (5a-aa) were designed, synthesized and evaluated for their cytotoxic potency against a panel of human cancer cell lines like lung (A-549), breast (MDA MB-231), prostrate (DU-145) and colon cancer (HT-29). Preliminary results revealed that some of these conjugates like 5d and 5u exhibited significant antiproliferative effect against human breast cancer (MDA MB-231) with IC₅₀ values of 1.3 and 1.2 µM respectively. To investigate the mechanistic aspects underlying the activity, the detailed biological studies of these promising conjugates (5d and 5u) were carried out on the MDA MB-231 cancer cells. Flow cytometric analysis revealed that these conjugates induce cell-cycle arrest in the G2/M phase. The tubulin polymerization assay suggests that these conjugates effectively inhibit microtubule assembly. In addition, morphological changes, reactive oxygen species (ROS) detection by 2′, 7′–dichlorofluorescin diacetate (DCFDA) and annexin V–FITC/PI assays indicate that 5d and 5u induces apoptosis. Furthermore, in silico computational studies, including molecular docking studies have been carried out to rationalise the binding modes of these conjugates with the tubulin protein.     

Human umbilical cord mesenchymal stem cells suppress breast cancer tumourigenesis through direct cell-cell contact and internalization

The purpose of this study was to investigate how human umbilical cord mesenchymal stem cells (HUMSCs) affect breast cancer tumourigenesis. To observe the influence of HUMSCs on tumourigenesis in vitro, we performed a co-culture of MDA MB-231 breast cancer cells with HUMSCs, and a result of HUMSCs on tumourigenesis in vivo was achieved by injection of HUMSCs into nonobese diabetic/severe combined immunodeficient mice following tumour establishment with MDA-MB231. During the co-culture, apoptosis of MDA-MB231 was noted, which was driven either by binding with HUMSC through direct cell-cell contact or by formation of a novel cell-in-cell phenomenon after internalization of HUMSC. Also, treatment with HUMSC injection was efficacious in both in situ and metastatic breast cancers in the animal models. Since HUMSCs were proved to efficaciously suppress breast cancer tumourigenesis both in vitro and in vivo, it is our expectation that treatment with HUMSCs can be a viable therapy for breast cancer in the near future. In addition, we share a new point of view on the role of HUMSCs in foetal development during pregnancy.     

Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.     

CAPER,a novel regulator of human breast cancer progression

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.     

Gallic acid based steroidal phenstatin analogues for selective targeting of breast cancer cells through inhibiting tubulin polymerization

Phenstatin analogues were synthesized on steroidal framework, for selective targeting of breast cancer cells. These analogues were evaluated for anticancer efficacy against breast cancer cell lines. Analogues 12 and 19 exhibited significant anticancer activity against MCF-7, hormone dependent breast cancer cell line. While analogues 10-14 exhibited significant anticancer activity against MDA-MB-231, hormone independent breast cancer cell line. Compound 10 showed significant oestrogen antagonistic activities with low agonistic activity in in vivo rat model. These analogues also retain tubulin polymerization inhibition activity. The most active analogue 10 was found to be non-toxic in Swiss albino mice up to 300 mg/kg dose. Gallic acid based phenstatin analogues may further be optimized as selective anti-breast cancer agents.     

Long non‐coding RNA ZEB2‐AS1 promotes the proliferation,metastasis and epithelial mesenchymal transition in triple‐negative breast cancer by epigenetically activating ZEB2

The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.     

Altering bioelectricity on inhibition of human breast cancer cells

Background

Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer.

Methods

In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive).

Results

Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10–20 μM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 μM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil.

Conclusions

Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

     

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells

BackgroundCoumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed.MethodsAntiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot.ResultsThe inhibition concentration (IC₅₀) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC₅₀) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84 μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process.ConclusionStyrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer.     

Synthesis and anti breast cancer activity of biphenyl based chalcones

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33) were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer) cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 μM. The two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell cycle arrest and loss of mitochondrial membrane potential.     

Apoptosis‐inducing effect of garcinol is mediated by NF‐κB signaling in breast cancer cells

Garcinol, obtained from Garcinia indica in tropical regions, is used for its numerous biological effects. Its anti‐cancer activity has been suggested but the mechanism of action has not been studied in‐detail, especially there is no report on its action against breast cancer cells. Here we tested our hypothesis that garcinol may act as an anti‐proliferative and apoptosis‐inducing agent against breast cancer cell lines. Using multiple techniques such as MTT, Histone‐DNA ELISA, Annexin V‐PI staining, Western blot for activated caspases and cleaved PARP, homogenous caspase‐3/7 fluorometric assay and EMSA, we investigated the mechanism of apoptosis‐inducing effect of garcinol in ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. We found that garcinol exhibits dose‐dependent cancer cell‐specific growth inhibition in both the cell lines with a concomitant induction of apoptosis, and has no effect on non‐tumorigenic MCF‐10A cells. Our results suggested induction of caspase‐mediated apoptosis in highly metastatic MDA‐MB‐231 cells by garcinol. Down‐regulation of NF‐κB signaling pathway was observed to be the mechanism of apoptosis‐induction. Garcinol inhibited constitutive NF‐κB activity, which was consistent with down‐regulation of NF‐κB‐regulated genes. This is the first report on anti‐proliferative and apoptosis‐inducing action of garcinol against human breast cancer cells and the results suggest that this natural compound merits investigation as a potential chemo‐preventive/‐therapeutic agent, especially against breast cancer. J. Cell. Biochem. 109: 1134–1141, 2010. © 2010 Wiley‐Liss, Inc.