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

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

PKCδ-dependent Activation of the Ubiquitin Proteasome System is Responsible for High Glucose-induced Human Breast Cancer MCF-7 Cell Proliferation,Migration and Invasion

Type 2 diabetes mellitus (T2DM) has contributed to advanced breast cancer development over the past decades.However, the mechanism underlying this contribution is poorly understood. In this study, we determined thathigh glucose enhanced proteasome activity was accompanied by enhanced proliferation, migration and invasion,as well as suppressed apoptosis, in human breast cancer MCF-7 cells. Proteasome inhibitor bortezomib (BZM)pretreatment mitigated high glucose-induced MCF-7 cell growth and invasion. Furthermore, high glucoseincreased protein kinase C delta (PKCδ)-phosphorylation. Administration of the specific PKCδ inhibitorrottlerin attenuated high glucose-stimulated cancer cell growth and invasion. In addition, PKCδ inhibition byboth rottlerin and PKCδ shRNA significantly suppressed high glucose-induced proteasome activity. Our resultssuggest that PKCδ-dependent ubiquitin proteasome system activation plays an important role in high glucoseinducedbreast cancer cell growth and metastasis.     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.     

The Effect of 17 β-Estradiol on Invasion by the Ovarian Clear Cell Adenocarcinoma Cell Line ES-2 and the Molecular Mechanism Involved

OBJECTIVE To assess the effect of 17 β-estradiol（E2） on cell proliferation, cell invasiveness and its regulation of MTA3, Snail and matrix metalloproteinase 2 （MMP-2） expression in the ovarian clear cell adenocarcinoma cell line ES-2, and to further investigate the mechanism involved. METHODS We first investigated expression of ERα, ERβ, PR and E-cadherin of ES-2 cells by RT-PCR and Western blots. Before all experiments, the ES-2 cells were grown in medium depleted of steroid for more than 7 days. Following treatment with 10^-7,10^-8 and 10^-9 M E2, cell viability of the ES-2 cells was determined by the MTT method, and the cell cycle distribution and apoptosis were examined by flow cytometry （FCM）. Invasion and mobility assays were performed using modified Boyden chambers. MTA3, Snail and MMP-2 mRNA expression was measured by RT-PCR, and Snail, MMP-2 protein levels were determined by IHC. MMP-2 activity was assayed by zymography. RESULTS RT-PCR and Western Blots showed that theexpression of ERα and E-cadherin mRNA and protein in the ES-2 cells was negative, while ERβ and PR expression was positive. E2 at 10^-7,10^-8 or 10^-9M stimulated cell proliferation. A level of 10^-8M E2 reduced the proportion of G0-G1 phase cells and increased the proportion of cells in the S phase, but it had no effect on apoptosis. Invasiveness and mobility of the ES-2 cells was significantly increased by 10^-8M E2. Treatment with 10^-8M E2 led to reduced MTA3 mRNA expression, and elevated Snail and MMP-2 mRNA and protein levels. CONCLUSION E2 enhanced invasion by the ES-2 cells. The effects observed maybe mediated by down-regulation of MTA3 and up-reguation of Snail and MMP-2.     

Effect of 5-aza-2’-deoxycytidine on Cell Proliferation of Nonsmall Cell Lung Cancer Cell Line A549 Cells and Expression of the TFPI-2 Gene

Objective: The present study employed 5-aza-2’-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer(NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene. Methods:Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 μmol/L 5-Aza-CdR, a specificdemethylating agent, for 24 ,48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM)to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), realtime polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 genemethylation status, mRNA expression and protein expression. Results: MTT assay showed that the growth ofA549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group(0 μmol/L 5-Aza-CdR). After treatment with 0, 1, 5, 10 μmol/L 5-Aza-CdR for 72h, FCM showed their proportionin G0/G1 was 69.7±0.99%, 76.1±0.83%, 83.8±0.35%, 95.5±0.55% respectively (P<0.05), and the proportion in Swas 29.8±0.43%, 23.7±0.96%, 15.7±0.75%, 1.73±0.45%, respectively (P<0.05), suggesting 5-Aza-CdR treatmentinduced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 genewas detected in control group (0 μmol/L 5-Aza-CdR), and demethylation appeared after treatment with 1, 5,10 μmol/L 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were1±0, 1.49±0.14, 1.86±0.09 and 5.80±0.15 (P<0.05) respectively. Western blotting analysis showed the relativeexpression levels of TFPI-2 protein were 0.12±0.01, 0.23±0.02, 0.31±0.02, 0.62±0.03 (P<0.05). TFPI-2 proteinexpression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration.Conclusions: TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expressionin the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation ofTFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinicaltreatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be onemolecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.     

Amphiregulin Antisense RNA Delivered by Adnovirus Suppresses Growth of Breast Cancer Cells In Vitro and In Vivo

OBJECTIVE To investigate the therapeutic potential of amphiregulin antisense RNA delivered by adenoviral vector in a human breast cancer model.METHODS Human amphiregulin cDNA was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into an E1/E3-deleted type 5 adenoviral vector to obtain an AdA4 construct which expresses amphiregulin antisense mRNA. Both in vitro and in vivo antiproliferative effects of the antisense RNA were studied by infecting transformed human breast epithelial NS2T2A1 cells and tumors.RESULTS Amphiregulin protein expression was inhibited dramatically in the NS2T2A1 cells after infection with AdA4. The in vitro cell growth was inhibited significantly at day 4 post-AdA4 infection compared with control empty virus AdC1 at a MOl of 200 and 400 pfu/cell to 69.3% and 49.8%, respectively (P<0.02, P<0.005). After 3 intra-tumoral injections of 109 pfu AdA4, tumor volumes were reduced to 40.6% of that of the control group at day 35 (P<0.005).CONCLUSION The transfer of amphiregulin RNA antisense by adenoviral vector is effective for amphiregulin targeting strategy, leading to an inhibition of in vitro cell proliferation and in vivo tumor growth in this breast cancer model.     

Influence of 17β-Estradiol on 15-Deoxy-Δ12,14 Prostaglandin J2 -Induced Apoptosis in MCF-7 and MDA-MB-231 Cells

The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), is expressed in variouscancer cells including breast, prostate, colorectal and cervical examples. An endogenous ligand of PPARγ,15-deoxy-Δ12,14 prostaglandin J2 (PGJ2), is emerging as a potent anticancer agent but the exact mechanismhas not been fully elucidated, especially in breast cancer. The present study compared the anticancer effects ofPGJ2 on estrogen receptor alpha (ERα)-positive (MCF-7) and ERα-negative (MDA-MB-231) human breastcancer cells. Based on the reported signalling cross-talk between PPARγ and ERα, the effect of the ERα ligand,17β-estradiol (E2) on the anticancer activities of PGJ2 in both types of cells was also explored. Here we reportthat PGJ2 inhibited proliferation of both MCF-7 and MDA-MB-231 cells by inducing apoptotic cell death withactive involvement of mitochondria. The presence of E2 potentiated PGJ2-induced apoptosis in MCF-7, but notin MDA-MB-231 cells. The PPARγ antagonist, GW9662, failed to block PGJ2-induced activities but potentiatedits effects in MCF-7 cells, instead. Interestingly, GW9662 also proved capable of inducing apoptotic cell death.It can be concluded that E2 enhances PPARγ-independent anticancer effects of PGJ2 in the presence of itsreceptor.     

Cell cycle synchronization induced by tamoxifen and 17β-estradiol on MCF-7 cells using flow cytometry and a monoclonal antibody against bromodeoxyuridine

Summary Cell cycle synchronization of MCF-7 hormone-sensitive human breast cancer cells has been evaluated after sequential treatment with tamoxifen and 17-estradiol. The analysis was performed by flow cytometry. Two methods were used, one for single-parameter DNA content analysis, and one for bivariate analysis of DNA content and amount of incorporated bromodeoxyuridine (BrdUrd) into DNA using a specific monoclonal antibody. According to the BrdUrd method, tamoxifen was found (over a 30h period) to decrease (with respect to cells grown in control medium) the fraction of cells in S phase from 45% to 20%, to increase cells in G0 + G1 from 47% to 68%, and to induce a slight build-up of cells in G2 + M. Subsequent addition of estradiol resulted in partial synchronous recruitment of the cells from G0+G1 to progress through the S phase; after 6–8 h delay time, the percentage of cells in G0+G1 decreased by 50% and cells in S increased by 175%. The bivariate BrdUrd technique offered more reliable and detailed information than the single-parameter DNA analysis for differentiating and measuring the time course of estrogen-recruited cells as they progressed through early and late S phase, and has the potential for a very detailed cell kinetic analysis of bothin vitro andin vivo hormone-sensitive cells.     

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.     

Significance of the Expression of Rho-GDP Dissociation Inhibitorβ,γin Lung Squamous Cell Carcinoma and Adenocarcinoma

Objective:To study the expression of Rho-GDP dissociation inhibitor β,γ(Rho-GDIβ,Rho-GDIγ)in lung squamous cell carcinoma and adenocarcinoma and its relationship with the expression of RhoC(Ras homologus oncogenes C)and clinicopathologic parameters.Methods:Western blot assay was employed for Rho-GDIβ,Rho-GDIγ and RhoC in lung squamous cell carcinoma and adenocarcinoma and non-neoplastic lung tissues of 37 cases with fresh specimens.Results:The study showed that Rho-GDIβ,Rho-GDIγ and RhoC were expressed in lung cancer and non-neoplastic lung tissues,the level in lung cancer tissue was much higher than that in non-neoplastic tissues(P<0.001).In lung cancer,the expression of Rho-GDIβ was much higher in patients with lymph node metastasis(P=0.021),and the expression of Rho-GDIγ was much higher in poorly differentiated tumor than in well-differentiated and moderately differentiated tumor,but both of them were not correlated with other clinicopathologic parameters.The expressions of Rho-GDIβ and Rho-GDIγ were not correlated with the expression of RhoC.Conclusion:In lung cancer,Rho-GDIβ and Rho-GDIγ may play a role in the tumorigenesis,Rho-GDIβ may promote metastasis,and Rho-GDIγ may have some relationship with differentiation.     

Ellagic Acid Exerts Anti-proliferation Effects via Modulation of Tgf-Β/Smad3 Signaling in MCF-7 Breast Cancer Cells

Ellagic acid has been shown to inhibit tumor cell growth. However, the underlying molecular mechanismsremain elusive. In this study, our aim was to investigate whether ellagic acid inhibits the proliferation of MCF-7 human breast cancer cells via regulation of the TGF-β/Smad3 signaling pathway. MCF-7 breast cancer cellswere transfected with pEGFP-C3 or pEGFP-C3/Smad3 plasmids, and treated with ellagic acid alone or incombination with SIS3, a specific inhibitor of Smad3 phosphorylation. Cell proliferation was assessed by MTTassay and the cell cycle was detected by flow cytometry. Moreover, gene expression was detected by RT-PCR,real-time PCR and Western blot analysis. The MTT assay showed that SIS3 attenuated the inhibitory activityof ellagic acid on the proliferation of MCF-7 cells. Flow cytometry revealed that ellagic acid induced G0/G1 cellcycle arrest which was mitigated by SIS3. Moreover, SIS3 reversed the effects of ellagic acid on the expression ofdownstream targets of the TGF-β/Smad3 pathway. In conclusion, ellagic acid leads to decreased phosphorylationof RB proteins mainly through modulation of the TGF-β/Smad3 pathway, and thereby inhibits the proliferationof MCF-7 breast cancer cells.     

Inhibitory Effects of β-Cyclodextrin-Helenalin Complexes on H-TERT Gene Expression in the T47D Breast Cancer Cell Line - Results of Real Time Quantitative PCR

Background: Nowadays, the encapsulation of cytotoxic chemotherapeutic agents is attracting interest as amethod for drug delivery. We hypothesized that the efficiency of helenalin might be maximized by encapsulationin β-cyclodextrin nanoparticles. Helenalin, with a hydrophobic structure obtained from flowers of Arnicachamissonis and Arnica Montana, has anti-cancer and anti-inflammatory activity but low water solubility andbioavailability. β-Cyclodextrin (β-CD) is a cyclic oligosaccharide comprising seven D-glucopyranoside units,linked through 1,4-glycosidic bonds. Materials and Methods: To test our hypothesis, we prepared β-cyclodextrinhelenalincomplexes to determine their inhibitory effects on telomerase gene expression by real-time polymerasechain reaction (q-PCR) and cytotoxic effects by colorimetric cell viability (MTT) assay. Results: MTT assayshowed that not only β-cyclodextrin has no cytotoxic effect on its own but also it demonstrated that β-cyclodextrinhelenalincomplexes inhibited the growth of the T47D breast cancer cell line in a time and dose-dependent manner.Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentrationof β-cyclodextrin-helenalin complexes increased. Conclusions: β-Cyclodextrin-helenalin complexes exertedcytotoxic effects on T47D cells through down-regulation of telomerase expression and by enhancing Helenalinuptake by cells. Therefore, β-cyclodextrin could be superior carrier for this kind of hydrophobic agent.