Growth Of Human Breast Cancer Cells Research Articles

Inhibitory effects of PAMAM nanoparticle-mediated 5-fluorouracil combined with microRNA-21 inhibitor on breast cancer cells growth in vitro

Objective To study the effect of PAMAM-mediated 5-fluorouracil combined with miR-21 inhibitor gene therapy to suppress MCF-7 human breast cancer cell growth in vitro. Methods 5-Fu/PAMAM complex was prepared by dialysis method and then incubated with miR-21 inhibitor at room temperature. Transmission electronic microscopy (TEM) was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy (UV). The transfection of PAMAM dendrimer was detected by flow cytometry. MTT assay was carried out to determine MCF-7 cell growth survival rate. Cell apoptosis was analyzed by flow-cytometry. Transwell assay was performed to detect invasion ability after MCF-7 cells treated with 5-Fu chemotherapy combined with miR-21 inhibitor gene therapy. Results The morphology of the complex was sphere observed under TEM. Encapsulation efficiency and loading efficiency of drug were (66. 21±4. 11)% and (31.77±0. 73)% , respectively. Flow cytometry revealed that 5-Fu/PAMAM transfection efficiency was (60.54 ±6. 97)%. 5-Fu combined with miR-21 inhibitor treatment significantly suppressed cell growth, and the survival rate was only (55. 85±3. 71)% on the 6th day of the observation period. The apoptosis rate in combined treatment group was (18. 32±2.42)% , dramatically higher than in control group (F=58. 326,P<0. 01). In combined treatment group, the number of invasion cells was only 18. 96 ±3. 14, suggesting the greatly decreased invasion ability of MCF-7 cells (F=16. 409,P < 0. 01). Conclusion PAMAM could effectively deliver miR-21 inhibitor and 5-Fu simultaneously, and combined therapy can suppress growth of MCF-7 cells effectively in vitro. Key words: Polyamide; 5-Fu; miR-21 inhibitor; Breast carcinoma

RNAi-mediated downregulation of UHRF1 results in decreased human breast cancer cell growth

The UHRF1 gene plays a fundamental role in cell proliferation through its NIRF_N domain, a PHD domain, a SRA domain and a RING domain. In addition, UHRF1 gene was involved in multidrug resistance in breast cancer treatment. In this study, we performed specific inhibition of UHRF1 using the short hairpin RNA (shRNA) lentiviral system in two human breast cancer cell lines (MDA-MB-231 and MCF-7). In vitro cell proliferation analysis by methylthiazoletetrazolium (MTT) and bromodeoxyuridine (BrdU) indicated a remarkable decrease in the proliferative potential of the UHRF1 siRNA treated cells. Moreover, dowregulation of UHRF1 in both breast cancer cell lines significantly inhibited the colony formation capacity. These data showed the involvement of UHRF1 in breast cancer growth and that inhibition of UHRF1 via RNA interference lentiviral system may provide an effective way for breast cancer therapy. RNAi-mediated downregulation of UHRF1 results in decreased human breast cancer cell growth Feng Yana* Xuyan Tana Yao Gengb Huangxian Jub Yanfang Gaoc Mingchen Zhua Xuelian Maoa aJiangsu Institute of Cancer Prevention and Cure, Nanjing 210009, PR China bDepartment of Chemistry, Nanjing University, Nanjing 210093, PR China cThe First Clinic Medical College of Nanjing Medical University, Nanjing 210029, PR China doi:10.4172/2155-9929.1000034

Calcitriol Derivatives with Two Different Side Chains at C-20. V. Potent Inhibitors of Mammary Carcinogenesis and Inducers of Leukemia Differentiation

Calcitriol is implicated in many cellular functions including cellular growth and differentiation, thus explaining its antitumor effects. It was shown that gemini, the calcitriol derivative containing two side chain at C20, is also active in gene transcription with enhanced antitumor activity. We have now further optimized both the A-ring and the two side chains. The chemical structures of the resulting 18 geminis were correlated with biological activities. Those containing the 1alpha-fluoro A-ring are the least active. Those featuring 23-yne and 23(E) side-chains are generally more active in human breast cancer cell growth inhibition and human leukemia cell differentiation induction than their 23(Z) counterparts. On the basis of these evaluations, we selected as lead compound a 20(R) gemini, related to calcitriol in terms of it is A-ring, where one side chain was modified by introduction of a 23-yne function and replacement of the geminal methyl groups with trifluoromethyl groups, the other created by extension of C21 with a 3-hydroxy-3-trideuteromethyl-4,4,4-trideutero-butyl moiety.

Nitro-aspirin inhibits MCF-7 breast cancer cell growth: Effects on COX-2 expression and Wnt/β-catenin/TCF-4 signaling

There is current evidence implicating the Wnt/β-catenin/TCF pathway in breast cancer. We investigated the effect of para- and meta-positional isomers of nitric oxide-releasing aspirin (NO-ASA), and aspirin (ASA) on MCF-7 human breast cancer cell growth and β-catenin/TCF signaling. The p- and m-NO-ASA isomers strongly inhibited cell growth and β-catenin/TCF transcriptional activity compared to ASA; the IC 50s for growth inhibition were 57 ± 4, 193 ± 10 and >5000 μM, and for transcriptional inhibition they were 12 ± 1.8, 75 ± 6.5 and >5000 μM for p-, m-NO-ASA and ASA, respectively. p-NO-ASA reduced the expression of Wnt/β-catenin downstream target gene cyclin D1, and total cellular β-catenin levels. COX-2 expression was induced by p-NO-ASA, protein kinase C inhibitors reversed this induction. p-NO-ASA blocked the cell cycle transition at S to G 2/M phase. These studies suggest a targeted chemopreventive/chemotherapeutic potential for NO-ASA against breast cancer.

Effects of adiponectin and leptin co-treatment on human breast cancer cell growth

Obesity is a risk factor for postmenopausal breast cancer (BC), but the specific mechanisms for this relationship are not well understood. Studies on adipocyte-derived adiponectin and leptin reveal opposing effects on BC cell proliferation in vitro, suggesting they may play a role in BC pathogenesis. In the current study we examined effects on proliferation of five BC cell lines treated with varying adiponectin:leptin (A/L) ratios. A decrease in proliferation was noted for MCF-7 and T47-D cells with increasing ratios (2-500), while an increase was seen in similarly treated MDA-MB-231 and MDA-MB-361 cells. For SK-BR-3 cells, an increase was seen at a ratio of 8. We identified differential effects on some pro-mitogenic, survival and apoptosis-related proteins in MCF-7 and T47-D cells treated at an A/L ratio of 100. Specifically, the AKT and MAPK pathways were not activated in MCF-7 cells, but AKT activation occured within 30 min and MAPK activation was sustained at 48 h in T47-D cells. p53 and Bax were elevated in MCF-7, but were below basal in T47-D cells at 30 min. While co-treatment enhanced apoptosis in MCF-7, similar treatment had the opposite effect in T47-D cells. There were no differences in cell cycle distribution between treated or untreated MCF-7 or T47-D, although T47-D cells had a slightly higher proportion in the G1/G0 phase after co-treatment. The effects of A/L ratio on mediating proliferation may have some specificity since the cell lines exhibited different responses. This may explain previous inconsistencies for the relationship of serum leptin with BC. More studies are needed to better understand the complex interactions that exist between these two adipokines.

Stat3-siRNA induces Fas-mediated apoptosis in vitro and in vivo in breast cancer

Stat3, a member of the signal transducer and activator of transcription family, has the potential to mediate cell survival, growth and differentiation. Stat3 is constitutively activated in numerous cancers, including >50% of breast cancers. Previous studies demonstrated that constitutively activated Stat3 plays an important role in breast cancer development and progression by promoting cell proliferation and inhibiting apoptosis. The present study was designed to investigate the potential use of RNA interference (RNAi) to block Stat3 expression and activation, as well as the subsequent effect on human breast cancer cell growth. Our studies show that knockdown of STAT3 expression by siRNA reduced expression of Bcl-xL and survivin in MDA-MB-231 cells, and also led to Fas mediated intrinsic apoptotic pathway by activating caspases -8, -9, -3 and PARP1 cleavage. In nude mice, pRNAi-Stat3 significantly suppressed tumor growth compared with controls. It also suppressed Stat3 expression, and downregulated BcL-xL and upregulated Fas, Fas-L and cleaved caspase-3 expression within the tumor, which significantly induced apoptosis and led to tumor suppression. Thus, targeting Stat3 signaling using siRNA may serve as a novel therapeutic strategy for the treatment of breast cancers expressing constitutively activated Stat3.

Canola oil inhibits human breast cancer cell growth by regulating caspase‐3 and p53

Epidemiological studies indicate that consuming a high‐fat diet may be correlated with an increased risk of breast cancer in women. However, increasing evidence suggests that it is not the quantity of fat but the type of fat intake that influences breast cancer risk. It is also suggested that the protective effect of omega‐3 fatty acids on breast cancer risk depends on the level of omega‐6 fatty acids, with a recommended omega‐6 to omega‐3 fatty acids ratio at 2:1 or lower. Canola oil has one of the lowest levels of saturated fat (7%, about half the level present in corn oil, olive oil and soybean oil). It has a relatively high level of oleic acid (monounsaturated fatty acid, 62%) and omega‐3 fatty acids (alpha‐linolenic, 10%) compared to most vegetable oils. It is thought that the low saturated fat, high oleic acid, and relatively high omega‐3 fatty acids content in canola oil give it particularly healthful properties. To investigate the effects of canola oil on breast cancer cell growth and apoptosis, two estrogen receptor‐positive breast cancer cell lines, T47D and MCF‐7, as well as a normal mammary cell line, MCF‐10A, were cultured and cell proliferation, caspase‐3 and p53 activities were measured. Canola oil supplementation significantly inhibited the growth of both T47D and MCF‐7 cell lines, but not MCF10A cell line. Canola oil also significantly increased the expression of p53, a tumor suppressor gene involved in cancer cell death, as well as the activity of caspase‐3 enzyme, one of the crucial enzymes regulating apoptosis pathway. Dietary strategies for cancer prevention and therapy are low‐risk and cost‐effective. Therefore, these results could be useful in the development of improved nutritional strategies for breast cancer prevention and therapy to reduce breast cancer risk. Supported by USDA‐CSREES (#2007‐38624‐18602).

Inhibition of Human Breast Cancer Cell Growth and Enzymatic Activity by a Fermented Nutraceutical

Human breast cancer cell lines MCF-7 (ER-positive) and Hs578T (ER-negative) were cultured and one lot incubated for 48 h with 5-50 mug/ml of a fermented phytocompound (MK: Manda-Koso, Innoshima, Japan). In vitro, it appeared a dose-dependent decrease of cell viability (5-57%) in MK group in both cell lines (P < 0.001, plateau: 30 microg/ml), decreased beta-galactosidase activity, enhanced apoptosis, and inversely increased Bax/Bcl2 ratio (P < 0.01) with an upregulation of p53 (P < 0.05). In the in vivo model, Balb-c mice were inoculated with tumor cells and the treatment group was fed with 20 mg of MK. Tumor weight in MK-fed group was time-course reduced by 22% to 51% at 2 and 4 weeks, respectively (P < 0.05) with increased survival (P < 0.05). Tumour tissue of MK-fed mice showed a downregulated Bcl-2 with increased Bax/Bcl-2 ratio, reduced PCNA, and activated caspase 3. Although more studies are ongoing to foster the clinical applicability of MK integrated within a rational chemopreventive and therapeutic strategy, a p53-mediated mechanism is likely to play a relevant role, besides its reported antioxidant capacity, NK cell activity enhancement, cancer-cytostatic activity properties.

Soy isoflavones and their bone protective effects

Several observational studies have suggested that populations with a high dietary soy intake have a lower incidence of osteoporosis-related fractures when compared to Western populations. However, there has not been consistent data to show that soy isoflavones protect against or lessen bone loss. Studies in our laboratory showed that genistein, the major soy isoflavone, could stimulate osteoblastic functions as well as human breast cancer cell growth. These studies raised the concern of whether it would be safe for women who have a prior history of breast cancer to consume soy isoflavone for management of postmenopausal osteoporosis. As increasing the purity of genistein is known to increase its ability to induce human breast cancer cell growth, current effort in our laboratory is to determine if the in vivo bone protective effects will be affected by the complexity of the soy isoflavones extract in ovariectomized mice.

Cyclopamine inhibition of human breast cancer cell growth independent of Smoothened (Smo)

Altered hedgehog signaling is implicated in the development of approximately 20-25% of all cancers, especially those of soft tissues. Genetic evidence in mice as well as immunolocalization studies in human breast cancer specimens suggest that deregulated hedgehog signaling may contribute to breast cancer development. Indeed, two recent studies demonstrated that anchorage-dependent growth of some human breast cancer cell lines is impaired by cyclopamine, a potent hedgehog signaling antagonist targeting the Smoothened (SMO) protein. However, specificity of cyclopamine at the dosage required for growth inhibition (> or =10 microM) remained an open question. In this paper we demonstrate that hedgehog signaling antagonists, including cyclopamine, and a second compound, CUR0199691, can inhibit growth of estrogen receptor (ER)-positive and ER-negative tumorigenic breast cancer cells at elevated doses. However, our results indicate that, for most breast cancer cell lines, growth inhibition by these compounds can be independent of detectable Smo gene expression. Rather, our results suggest that cyclopamine and CUR0199691 have unique secondary molecular targets at the dosages required for growth inhibition that are unrelated to hedgehog signaling.

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase‐independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.

Cyclic Peptidyl Inhibitors of Grb2 and Tensin SH2 Domains Identified from Combinatorial Libraries

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. In this work, a novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 3.2 x 10(6)) were synthesized and screened against the SH2 domains of Grb2 and tensin. Potent, selective inhibitors were identified from the libraries and were generally more effective than the corresponding linear peptides. One of the inhibitors selected against the Grb2 SH2 domain inhibited human breast cancer cell growth and disrupted actin filaments. This method should be applicable to the development of cyclic peptidyl inhibitors against other protein domains, enzymes, and receptors.

APKC Inhibitors Might be the Sensitizer of Chemotherapy and Adoptive Immunotherapy in the Treatment of hASIPa-Overexpressed Breast Cancer

The atypical protein kinase C (aPKC) plays an important role in cell growth through the interaction with its substracts, including human ASIP (hASIP), which contains an aPKC phosphorylation site encoded by exon 17b of the gene. hASIP is expressed as numerous alternative splicing isoforms in the cells. Our results showed that hASIPa, an exon 17b-containing isoform of hASIP, is overexpressed in human breast cancer (HBC) MDA-MB-231, SK-BR-3, and MCF-7 cell lines and HBC specimens. The anticancer effects of 5-FU chemotherapy or adoptive immunotherapy and the synergic action of aPKC inhibitor against hASIPa-overexpressed HBC cells were tested. The results indicated that HBC MDA-MB-231 and SK-BR-3 cells were sensitive to 5-FU treatment in vitro. The combined treatment of aPKC inhibitor and 5-FU raised the anticancer activities against hASIPa-overexpressed HBC cells. The coculture of cytokine-induced killer (CIK) cells and autologous dendritic cells (DCs) with or without Her-2 peptide GP2 pulse created two new populations of effective immune-active T-cell populations called DC-modulated and cytokine-induced killer (DCIK) cells and peptide-DC-modulated and cytokine-induced killer (DCIK-P) cells. The DCIK cells showed cytotoxic activities on MDA-MB-231, SK-BR-3, and MCF-7 cells in MHC unrestricted manner. The DCIK-P cells possessed extra-enhanced cytotoxic activities against HLA-A2+/Her-2+ MDA-MB-231 cells in MHC restricted manner, but not for HLA-A2+/-/Her-2+ SK-BR-3 cells and HLA-A2+/Her-2+/- MCF-7 cells. The data suggested specific cytotoxic T-lymphocyte (CTL) activity of DCIK-P cells on MDA-MB-231 cells. The combined treatment of aPKC inhibitor with DCIK/DCIK-P cells further raised the anticancer activities against hASIPa-overexpressed HBC cells. The results demonstrated that the hASIPa/aPKC signaling pathway functions as an important regulator in the growth of HBC cells and aPKC inhibitor treatment showed the synergic activities on 5-FU or DCIK/DCIK-P cells adoptive immunotherapy against hASIPa-overexpressed HBC cells.

Fullerenol: A new nanopharmaceutic?

Chemical modification of fullerenes to hydrosoluble cluster molecules made fullerenes interesting for biological investigation. Among them, polycarbonated and polyhydroxylated fullerene C60 derivatives showed the most interesting biological activities. In this paper, we present the most important recent results of in vitro and in vivo biological studies with fullerenol C60(OH)24. Fullerenol C60(OH)24 was strong antioxidant: it reacted with superoxide anion radical, hydroxy radical and nitrous oxide radical in chemical and biological systems. Fullerenol C60(OH)24 did not inhibit human breast cancer cell growth at concentrations from 0.8 to 3.45 ?M, but strongly modulated cytotoxic effects of doxorubicin and cis-platinum after 24 and 48 hours of treatment. Radioprotective effects of fullerenol C60(OH)24 were shown in different in vitro and in vivo models. Fullerenol C60(OH)24 (100 mg/kg) protected rat heart from doxorubicin toxicity. Biodistribution studies of fulelrenol were also investigated. Accumulating data from the literature and from our studies suggest that fullerenol, as a nanoparticle might be a new promising pharmaceutical in the near future.

RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion

Metastasis is a leading cause of mortality and morbidity in breast cancer. Recently, dramatic overexpression of ecto-5'-nucleotidase (CD73), a glycosylphosphatidylinositol-anchored cell surface protein has been found in estrogen receptor-negative [ER (-)] breast cancer cell lines and in clinical samples. In this study, CD73 small interfering RNA (siRNA) plasmid was constructed and stably transfected into breast cancer cell MB-MDA-231 to determine the role of CD73 in breast cancer metastasis and the possible mechanism. Our study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression at mRNA and protein level in MB-MDA-231 cells, leading to in vivo and in vitro growth suppression, prevention of adhesion to extracellular matrix (ECM), and inhibition of invasion and migration. These properties correlate with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity as well as reduction of epidermal growth factor receptor (EGFR) expression. Demonstration of the role of CD73 in breast cancer may lead to new targeted therapies for breast cancer.

Effects of ecto-5'-nucleotidase on human breast cancer cell growth in vitro and in vivo

Ecto-5'-nucleotidase (CD73) is an essential enzyme that generates adenosine, an essential molecule for cell growth. CD73 increases significantly in many breast cancers. In this study, alpha,beta-methylene adenosine-5'-diphosphate (APCP), a specific CD73 inhibitor was used to block the hydrolase's activity. Effects of CD73 were examined on human breast cancer cells MDA-MB-231 in culture for proliferation, cell cycle progression, and apoptosis before and after APCP treatment. The in vivo effect of CD73 was examined on MDA-MB-231 tumor xenograft growth in nude mice. Cell growth curve, cell cycle and apoptosis were observed with MTT assays and flow cytometry, respectively. Microvessel density (MVD) and lymph vessel density (LVD) of implanted tumor tissues was analyzed by immunohistochemistry for CD31 and VEGFR-3 staining respectively. Our results showed that APCP inhibited MDA-MB-231 viability in a dose-dependent manner. APCP (12 microM) increased the percentage of G0/G1 phase cells from 49.75 to 59.16% while it decreased S phase and G2/M cells from 24.85 and 18.65% to 21.65 and 12.55%, respectively. The percentages of early and late apoptotic cells were also decreased after APCP treatment. However, APCP treatment did not affect the percentage of normal cells. Xenograft of MDA-MB-231 cells in the APCP treatment group had lower volume and weight than those of control group (2.70+/-1.14 vs 1.41+/-0.39 cm(3) and 2.7+/-0.5 vs 1.3+/-0.2 g), accompanied with less vessel formation with a MVD of 5+/-1 compared to the control group's 10+/-2 and an LVD of 4+1 vs 7+2. Our results suggest that CD73 may promote tumor growth and serve as a marker of breast cancer progression.

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER + MCF-7 and ER − MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G 2/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G 2 exit and initiation of mitosis, namely the p34 CDC2/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21 WAF, proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (ΔΨ) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.

The Cannabinoid CB1 Receptor Antagonist Rimonabant (SR141716) Inhibits Human Breast Cancer Cell Proliferation through a Lipid Raft-Mediated Mechanism

The endocannabinoid system has been shown to modulate key cell-signaling pathways involved in cancer cell growth. In this study, we show that cannabinoid receptor type 1 (CB1) antagonist Rimonabant (SR141716) inhibited human breast cancer cell proliferation, being more effective in highly invasive metastatic MDA-MB-231 cells than in less-invasive T47D and MCF-7 cells. The SR141716 antiproliferative effect was not accompanied by apoptosis or necrosis and was characterized by a G1/S-phase cell cycle arrest, decreased expression of cyclin D and E, and increased levels of cyclin-dependent kinase inhibitor p27KIP1. We have also shown that SR141716 exerted a significant antiproliferative action, in vivo, by reducing the volume of xenograft tumors induced by MDA-MB-231 injection in mice. On the other hand, at the concentration range in which we observed the antiproliferative effect in tumor cells, we did not observe evidence of any genotoxic effect on normal cells. Our data also indicate that the SR141716 antiproliferative effect requires lipid raft/caveolae integrity to occur. Indeed, we found that CB1 receptor (CB1R) is completely displaced from lipid rafts in SR141716-treated MDA-MB-231 cells, and cholesterol depletion by methyl-beta-cyclodextrin strongly prevented SR141716-mediated antiproliferative effect. Taken together, our results suggest that SR141716 inhibits human breast cancer cell growth via a CB1R lipid raft/caveolae-mediated mechanism.

Inhibitors of nm23 Suppress MDA‐MB‐435 Human Breast Carcinoma Mediated Angiogenesis In Vitro

We have previously discovered that human breast cancer cells secrete a factor that may support tumor angiogeneis. Inhibitors of nucleoside diphosphate kinase (NDPK) are represented by epigallocatechin gallate (EGCG) and ellagic acid (EA). We hypothesize that NDPK-B secreted by human breast cancer cells contributes to tumor angiogenesis and subsequent metastasis by modulating ATP levels, thereby activating endothelial P2Y purinergic receptors. Supporting this notion, extracellular purine nucleotides (e.g. ATP) have recently emerged as a novel class of proliferative agents with putative roles in cancer and angiogenesis. In proliferation assays, 10μM EGCG but only 3μM EA is required to significantly inhibit MDA-MB-435S human breast cancer cell growth while 3μM of either EGCG or EA significantly inhibited CD31+human endothelial cell growth. CD31+ cell tubule formation on Matrigel™ demonstrated that 30μM EGCG compared to only 1μM EA is sufficient to significantly inhibit angiogenesis. Using Matrigel™, we also determined that MDA-MB-435S cell conditioned media significantly induced angiogenesis in a cell number and time dependent manner. Furthermore, 1μM EA but not equamolar EGCG significantly attenuated this cell number induced angiogenesis. In a collagen based in vitro angiogenesis assay, we observed that P2Y1R activation by 100μM ATP or 10μM 2-methyl-thio-ATP significantly promoted angiogenesis. Generally, EA was a more potent inhibitor than EGCG for all experiments. We believe this is related to EA > EGCG potency on inhibiting NDPK activity. Further investigation and delineation of NDPK’s hypothesized role in human breast cancer induced angiogenesis may reveal unique therapeutic targets in its treatment and lethal metastatic spread.

Progesterone inhibits human breast cancer cell growth through transcriptional upregulation of the cyclin-dependent kinase inhibitor p27 Kip1 gene

The effects of progesterone derivatives on breast cancer development are still controversial, probably accounting for their biphasic, opposed effects on mammary cell-cycle regulation. Here, we demonstrate in vitro that the growth-inhibitory effects of progesterone on breast cancer T-47D cells require the transcriptional upregulation of the cyclin-dependent kinase inhibitor p27 Kip1 (p27) gene. A statistical analysis of human tumor biopsies further indicates that p27 mRNA levels correlate to progesterone receptor (PR) levels. Moreover, p27 gene expression is inversely associated with tumor aggressiveness, and is a prognostic factor of favorable disease outcome. Thus, progesterone derivatives selectively activating the p27 gene promoter could be promising drugs against breast cancer progression.