Agents In Breast Cancer Cells Research Articles

Role of Coccinia indica in the prevention and management of breast cancer: A review

Cucurbitacin B is isolated from the fruit of Coccinia indica, have reported for treatment of several types of cancer due to its anticancer and antioxidant effect. Cucurbitacin B, inducing cell cycle arrest at G2/M as well as apoptosis. It can act as anti-proliferative agent for breast cancer cells in vitro and in vivo and reduces the ferrocynaide to ferrous. Cucurbitacin B inhibits telomerase activity in several breast cancer cell lines through down regulation of both the hTERT and c -Myc expression. The expression of the hTERT gene directly depends on the telomerase activity as the hTERT protein, is the catalytic rate-limiting determinant subunit of telomerase. Estrogen receptor activates a number of cellular signal transduction and also ERK/MAPK pathway. It leads to the translocation of activated MAP kinase to the nucleus where it regulates the expression of a number of transcription factors. Including pertinently, c -Myc but when cell lines treated with cucurbitacin B, directly modulates either the estrogen receptor or subsequent signalling pathway and the down regulate JAK/STAT pathway. Whereas some different biological pathway exist in estrogen negative cells, where c -Myc is primarily controlled at the level of RNA stability. However, cucurbitacin B exerts anticancer activity and inhibit the telomerase and induced apoptosis.

Small hydrophobe substitution on polyethylenimine for plasmid DNA delivery: Optimal substitution is critical for effective delivery

Cationic polymers have been turned into effective gene delivery agents by functionalizing with long-chain aliphatic lipids, but little information exists if small hydrophobic moieties can serve as effective substituents for this purpose. To explore this issue, we modified small molecular weight (1.2kDa) polyethylenimine (1.2PEI) by a small hydrophobe, propionic acid (PrA), through N-acylation and investigated the efficacy of resultant polymers to deliver plasmid DNA (pDNA) to breast cancer cells MDA-231 and MCF-7. A significant impact of PrA grafting was observed on physicochemical features of polymers and resultant pDNA complexes. pDNA binding capacity, as measured by BC50 (weight ratio for 50% binding), was decreased from 0.25 to 0.64 with PrA substitution. Hydrodynamic size of polymer/pDNA complexes was not altered, but the surface charge (ξ-potential) was increased with low PrA substitution and decreased at higher PrA substitutions. Similarly, in vitro pDNA transfection efficacy in MDA-231 and MCF-7 cells was significantly increased with PrA grafting and optimum efficacy was observed in polymers with modest substitution, 0.25–1.0 PrAs/PEI (mol/mol), but higher substitutions was detrimental to transfection. The transfection efficiency of PEI-PrAs was higher than aliphatic lipid (linoleic acid) substituted PEI and more stable than 25kDa branched PEI. However, unlike studies reported elsewhere, siRNA had no effect on transfection efficacy of pDNA/PEI-PrA complexes when used as an additive. We conclude that small hydrophobe substitution on low MW PEI converts it into effective pDNA delivery agent in breast cancer cells up to an optimal ratio, indicating that balancing hydrophobicity of polymer is critical for pDNA transfection. Statement of SignificanceThis manuscript investigated the influence of small hydrophobe (propionic acid, PrA, 3 carbon) grafted onto small molecular weight polyethylenimine (1.2PEI) in pDNA delivery. We have explored this approach as an alternative of common strategies to graft long chain and/or bulky lipids [linoleic acid (18 carbon), cholesterol]. At optimal substitution, transfection efficiency of these polymers was significantly higher than long chain lipid substituted 1.2PEI, emphasizing a proper hydrophobic/hydrophilic balance for optimum gene delivery. The overall results establish the feasibility of using small hydrophobes to create functional carriers, as long as the polymers are engineered with optimal ratio of substituent. The reported studies should facilitate the efforts of biomaterials scientists and engineers to design new carriers for gene therapy.

Intracellular delivery and ultrasonic activation of folate receptor-targeted phase-change contrast agents in breast cancer cells in vitro

Intracellular delivery and ultrasonic activation of folate receptor-targeted phase-change contrast agents in breast cancer cells in vitro

2-Amino-2-deoxy-glucose conjugated cobalt ferrite magnetic nanoparticle (2DG-MNP) as a targeting agent for breast cancer cells.

In this study, 2-amino-2-deoxy-glucose (2DG) was conjugated to COOH modified cobalt ferrite magnetic nanoparticles (COOH-MNPs), which were designed to target tumor cells as a potential targetable drug/gene delivery agent for cancer treatment. According to our results, it is apparent that, 2DG labeled MNPs were internalized more efficiently than COOH-MNPs under the same conditions in all cell types (MDA-MB-231 and MCF-7 cancer and MCF-10A normal breast cells) (p<0.001). Moreover, the highest amount of uptake was observed in MDA-MB-231, followed by MCF-7 and normal MCF-10A cells for both MNPs. The apoptotic effects of 2DG-MNPs were further evaluated, and it was found that apoptosis was not induced at low concentrations of 2DG-MNPs in all cell types, whereas dramatic cell death was observed at higher concentrations. In addition, the gene expression levels of four drug-metabolizing enzymes, two Phase I (CYP1A1, CYP1B1) and two Phase II (GSTM3, GSTZ1) were also increased with the high concentrations of 2DG-MNPs.

Furanodiene enhances the anti-cancer effects of doxorubicin on ERα-negative breast cancer cells in vitro

Furanodiene is a natural product isolated from Rhizoma curcumae, and exhibits broad-spectrum anti-cancer activities in vitro and in vivo. Our previous study proved that furanodiene could increase growth inhibition of steroidal agent in ERα-positive breast cancer cells, but whether furanodiene can influence ER status is not clear. In this study, we confirmed that furanodiene down-regulated the ERα protein expression level and inhibited E2-induced estrogen response element (ERE)-driven reporter plasmid activity in ERα-positive MCF-7 cells. Actually, ERα-knockdown cells were more sensitive than ERα positive cells to furanodiene on the cytotoxicity effect. So the anti-cancer effects of furanodiene and non-steroidal agent in breast cancer cells still requires further investigation. Our results showed that furanodiene exposure could enhance growth inhibitory effects of doxorubicin in ERα-negative MDA-MB-231 cells and ERα-low expression 4T1 cells. However, furanodiene did not increase the cytotoxicity of doxorubicin in ERα-positive breast cancer cells, non-tumorigenic breast epithelial cells, macrophage cells, hepatocytes cells, pheochromocytoma cells and cardiac myoblasts cells. Furanodiene enhances the anti-cancer effects of doxorubicin in ERα-negative breast cancer cells through suppressing cell viability via inducing apoptosis in mitochondria-caspases-dependent and reactive oxygen species-independent manners. These results indicate that furanodiene may be a promising and safety natural agent for cancer adjuvant therapy in the future.

Involvement of p53 in insulin-like growth factor binding protein-3 regulation in the breast cancer cell response to DNA damage.

Chemotherapy drugs that induce apoptosis by causing DNA double-strand breaks, upregulate the tumor suppressor p53. This study investigated the regulation of the growth-regulatory protein insulin-like growth factor binding protein-3 (IGFBP-3), a p53 target, by DNA-damaging agents in breast cancer cells. IGFBP-3 was upregulated 1.4- to 13-fold in response to doxorubicin and etoposide in MCF-10A, Hs578T, MCF-7 and T47D cells, which express low to moderate basal levels of IGFBP-3. In contrast, IGFBP-3 was strongly downregulated by these agents in cells with high basal levels of IGFBP-3 (MDA-MB-231, MDA-MB-436 and MDA-MB-468). In MDA-MB-468 cells containing the R273H p53 mutation, reported to display gain-of-function properties, chemotherapy-induced suppression of IGFBP-3 was not reversed by the p53 reactivating drug, PRIMA-1, or by p53 silencing, suggesting that the decrease in IGFBP-3 following DNA damage is not a mutant p53 gain-of-function response. SiRNA-mediated downregulation of endogenous IGFBP-3 modestly attenuated doxorubicin-induced apoptosis in MDA-MB-468 and Hs578T cells. IGFBP-3 downregulation in some breast cancer cell lines in response to DNA-damaging chemotherapy may have clinical implications because suppression of IGFBP-3 may modulate the apoptotic response. These observations provide further evidence that endogenous IGFBP-3 plays a role in breast cancer cell responsiveness to DNA damaging therapy.

The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acid-treated MCF-7 breast cancer cell line.

Valproic acid (VPA), a drug used in the treatment of neurological disorders, has been shown to have cytotoxic effects on cancer cells through different mechanisms. Telomerase, a ribonucleoprotein reverse transcriptase, is responsible for elongation of the telomere and is activated in cancers. A relation between telomerase activity and resistance to apoptosis has been established. This study focused on probable effects of VPA on MCF-7 cancer cells. In particular, we investigated VPA effects on viability, apoptosis and telomerase activity. Cytotoxicity effects of VPA on MCF-7 cells were determined by neutral red uptake assay. Cells were treated with different concentrations of VPA (0-32 mM) and telomerase activity and Bax and Bcl-2 protein levels were determined using TRAP assay (PCR-ELISA) method and ELISA method, respectively. The cytotoxic effects of different concentration of VPA on MCF-7 cells were observed as a reduction in cell viability and telomerase activity and altered expression of Bcl-2 family protein levels. The results also showed that there is a significant correlation between reduction of telomerase activity and increase in Bax/Bcl-2 ratio (P=0.001). Our study demonstrated that cell viability of MCF-7 cells was decreased after treatment with VPA, probably through a reduction of telomerase activity and an increase in Bax/bcl-2 ratio. Therefore, it could be concluded that VPA is a potent anti-cancer agent for breast cancer cells through inhibition of telomerase activity and induction of apoptosis.

Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells

Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.

Radiation promotes malignant phenotypes through SRC in breast cancer cells.

Despite the fact that ionizing radiation (IR) is widely used as a standard treatment for breast cancer, much evidence suggests that IR paradoxically promotes cancer malignancy. However, the molecular mechanisms underlying radiation-induced cancer progression remain obscure. Here, we report that irradiation activates SRC signaling among SRC family kinase proteins, thereby promoting malignant phenotypes such as invasiveness, expansion of the cancer stem-like cell population, and resistance to anticancer agents in breast cancer cells. Importantly, radiation-activated SRC induced SLUG expression and caused epithelial–mesenchymal cell transition through phosphatidylinositol 3-kinase/protein kinase B and p38 MAPK signaling. In agreement, either inhibition of SRC or downstream signaling of p38 MAPK or protein kinase B effectively attenuated radiation-induced epithelial–mesenchymal cell transition along with an increase in the cancer stem-like cell population. In addition, downregulation of SRC also abolished radiation-acquired resistance of breast cancer cells to anticancer agents such as cisplatin, etoposide, paclitaxel, and IR. Taken together, our findings suggest that combining radiotherapy with targeting of SRC might attenuate the harmful effects of radiation and enhance the efficacy of breast cancer treatment.

Abstract 2731: PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance

Abstract Estrogen Receptor positive breast cancer is commonly treated with hormonal therapy; however, many patients acquire therapy resistance, a major clinical problem. Recent evidence suggests that the accumulation of chromosomal abnormalities via increased error-prone Alternative-Non-Homologous End Joining (A-NHEJ) DNA repair pathway could contribute to breast cancer therapy resistance. Proline, Glutamic acid, Leucine rich Protein-1 (PELP1), an oncogenic co-regulator for transcription factors, is commonly overexpressed in breast cancer, and its deregulation has been implicated in therapy resistance. Our recent studies identified PELP1 as a novel substrate of the DNA damage response (DDR) kinases. In this study, we discovered that PELP1 regulates the A-NHEJ pathway, and we developed a novel peptide drug that targets PELP1-A-NHEJ axis for the treatment of therapy resistance. Our results suggest that PELP1 is phosphorylated at Ser1033 by DDR kinases and phosphorylated PELP1 co-localizes with the gamma-H2AX foci. Using Homologous Recombination (HR) and NHEJ pathway specific reporter assays, we found that PELP1 knockdown decreases NHEJ, with no apparent effect on the HR pathway. Further, using A-NHEJ specific reporter cell line, we found that PELP1 knockdown decreased the frequency of A-NHEJ, while PELP1 overexpression increased the repair frequency. PELP1's ability to regulate A-NHEJ pathway was further confirmed by sequencing repaired plasmids and by quantitating the degree of end resection. More importantly, metaphase chromosome spreads revealed gross chromosomal abnormalities in PELP1 deregulated breast cancer cells. Mechanistic studies revealed that PELP1 interacts with Mre11, and modulates the degree of end resection at the DNA double strand breaks. Mapping studies identified the C-terminus of PELP1 as the binding site for Mre11. Using yeast based genetic screen composed of 10 million peptides, we identified a unique peptide, PIP3 that binds the C-terminus of PELP1. We developed a stapled peptide of PIP3 (sPIP3), which functioned as a potent cytotoxic agent for breast cancer cells with little activity on normal cells. Further, sPIP3 efficiently inhibited the A-NHEJ pathway and significantly, reduced survival and promoted apoptosis of therapy resistant model cells. Collectively, these findings suggest that PELP1 plays a critical role in the A-NHEJ pathway and sPIP3 represents a novel drug for the treatment of therapy resistant breast cancer. Citation Format: Samaya R. Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2014-2731

Abstract 3358: Significant increase in survival of triple negative breast cancer animal model in response to BPM31510 alone or in combination with standard of care: BPM31510 mediated dynamic metabolic (Warburg) shift in breast cancer as potential mechanism

Abstract This study demonstrates that BPM 31510, a Coenzyme Q10 containing proprietary formulation to influences cellular metabolism that is deregulated in cancers and is now considered to be a “hallmark of cancer”. These studies demonstrate that BPM 31510 alone or in combination with standard of care significantly improved survival of triple negative breast cancer (TNBC) animal model. To further tease out beneficial effects of BPM31510, human breast cancer cells of varying receptor status (SKBR3, MDA-MB231) were subjected to either (a) pretreatment with BPM 31510 (6 h) followed co-incubation with chemotherapeutic agents for 48-72 h or (b) co-treatment with BPM 31510 and chemotherapeutic agents, and cancer cell responses were compared to non-tumorigenic mammary cells (MCF12A). Cellular bioenergetics profiling revealed that BPM 31510 shifted cellular metabolism from glycolysis to mitochondrial metabolism, and this metabolic shift was associated with significant increases in reactive oxygen species (ROS). Both BPM 31510 alone or pretreatment and cotreatment strategies with BPM 31510 plus standard of care resulted in significant decreases in viable breast cancer cells when compared to chemotherapeutic agents; however, minimal effects were observed in MCF12A cells. In contrast, BPM 31510 in combination with chemotherapeutic agents amplified caspase 3 activation and apoptotic cell death, indicating BPM 31510 enhances apoptotic signaling. Taken together, these data demonstrate that BPM 31510 is a novel agent that reengages the cellular metabolic and apoptotic machinery of cancer cells independent of genetic make-up underlying malignancy. In addition, BPM 31510 enhances the cytotoxicity of standard-of-care chemotherapeutic agents in breast cancer cells through regulation of mitochondrial metabolism and oxidative stress. These findings confirm that BPM 31510 is a novel, paradigm shifting agent with multiple utility (as a single agent or in combination) in breast cancer including TNBCs that otherwise have poor prognosis and limited therapeutic options. Citation Format: Niven R. Narain, Anne R. Diers, Tony E. Walshe, Arleide Lee, Rakib Ouro-Djobo, Vivek K. Vishnudas, Ely Benaim, Rangaprasad Sarangarajan. Significant increase in survival of triple negative breast cancer animal model in response to BPM31510 alone or in combination with standard of care: BPM31510 mediated dynamic metabolic (Warburg) shift in breast cancer as potential mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2014-3358

Evaluation of wild yam (Dioscorea villosa) root extract as a potential epigenetic agent in breast cancer cells.

The present study was designed to evaluate the efficacy of wild yam root extract (WYRE) as a potential demethylating agent using two breast cancer cell lines, MCF-7 (estrogen receptor positive; ER(+)) and MDA-MB-231 (Estrogen receptor negative; ER(-)), and a methylated gene, GATA3, as a potential marker of breast cancer development. The cells were treated with WYRE (0-50 μg/mL) for 72 h and used for viability, mRNA, and methylation analyses. WYRE significantly reduced viability of both cell lines and enhanced mRNA content of GATA3 in a concentration-dependent manner; however, DNMT mRNAs (DNMT1, 3A, 3B) were found to increase significantly only in MDA-MB-231 cells. Global DNA methylation, analyzed as 5'-methyl-2'-deoxycytidine (5-mC) and 5-hydroxymethylcytosine (5-hmC), showed a concentration-dependent enhancement of 5-mC with no alteration in 5-hmC level in MCF-7 cells; however, in MDA-MB-231 cells, in contrast to MCF-7 cells, 5-mC remained unaltered but 5-hmC reduced significantly in all WYRE concentrations (10-50 μg/mL) used in this study. Since 5-hmC is generated from 5-mC by ten-eleven-translocation (TET) enzymes, analysis of TET mRNAs (TET1, TET2, and TET3) in MDA-MB-231 cells indicated a concentration-dependent reduction in TET1 and induction of TET3; however, TET2 remained unaltered. No alterations in any of the TET mRNAs were found in MCF-7 cells. Methylation analysis of GATA3 promoter at specific locus indicates probable demethylating activity of WYRE in MDA-MB-231 cells. We conclude that activation of GATA3 gene in ER(-) MDA-MB-231 cells may occur by altering DNA methylation pattern on the promoter region which may be different from the mechanisms operated in ER(+) MCF-7 cells.

Regulation of apoptosis by long non-coding RNA GAS5 in breast cancer cells: implications for chemotherapy

The putative tumour suppressor and apoptosis-promoting gene, growth arrest-specific 5 (GAS5), encodes long ncRNA (lncRNA) and snoRNAs. Its expression is down-regulated in breast cancer, which adversely impacts patient prognosis. In this preclinical study, the consequences of decreased GAS5 expression for breast cancer cell survival following treatment with chemotherapeutic agents are addressed. In addition, functional responses of triple-negative breast cancer cells to GAS5 lncRNA are examined, and mTOR inhibition as a strategy to enhance cellular GAS5 levels is investigated. Breast cancer cell lines were transfected with either siRNA to GAS5 or with a plasmid encoding GAS5 lncRNA and the effects on breast cancer cell survival were determined. Cellular responses to mTOR inhibitors were evaluated by assaying culture growth and GAS5 transcript levels. GAS5 silencing attenuated cell responses to apoptotic stimuli, including classical chemotherapeutic agents; the extent of cell death was directly proportional to cellular GAS5 levels. Imatinib action in contrast, was independent of GAS5. GAS5 lncRNA promoted the apoptosis of triple-negative and oestrogen receptor-positive cells but only dual PI3K/mTOR inhibition was able to enhance GAS5 levels in all cell types. Reduced GAS5 expression attenuates apoptosis induction by classical chemotherapeutic agents in breast cancer cells, providing an explanation for the relationship between GAS5 expression and breast cancer patient prognosis. Clinically, this relationship may be circumvented by the use of GAS5-independent drugs such as imatinib, or by restoration of GAS5 expression. The latter may be achieved by the use of a dual PI3K/mTOR inhibitor, to improve apoptotic responses to conventional chemotherapies.

Anti-angiogenic effects of trabectedin (Yondelis; ET-743) on human breast cancer cells.

Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata, has been shown to have antitumor effects. In this study, we assessed the possible anti-angiogenic effects of trabectedin on human umbilical vein endothelial cells (HUVECs) and breast cancer cell lines. An XTT cell viability assay was used to determine cytotoxicity. A scratch assay was used to detect the migration of cells after trabectedin treatment. Angiogenic cytokine profiles of breast cancer cell lines, before and after treatment with trabectedin, were investigated using an angiogenesis antibody array. Changes in mRNA expression levels of VEGF were evaluated using qRT-PCR. Trabectedin inhibited the viability of HUVECs and breast cancer cells in a concentration- and time-dependent manner. The migration of both HUVECs and breast cancer cells was suppressed by trabectedin treatment. Angiogenic cytokines which are known to regulate tumorigenicity and angiogenesis, such as GM-CSF, IGFBP-2, VEGF, and uPA, were inhibited, while several anti-angiogenic cytokines such as TIMP-1 and Serpin E1were induced in breast cancer cells. Furthermore, expression levels of VEGF mRNA were inhibited in all breast cancer cells tested. Although additional studies are needed to elucidate the molecular mechanisms underlying the anti-angiogenic activity of trabectedin, our results suggest that trabectedin may act as a potential anti-angiogenic agent in breast cancer cells.

Effects of psoralens as anti-tumoral agents in breast cancer cells.

This review examines the biological properties of coumarins, widely distributed at the highest levels in the fruit, followed by the roots, stems and leaves, by considering their beneficial effects in the prevention of some diseases and as anti-cancer agents. These compounds are well known photosensitizing drugs which have been used as pharmaceuticals for a broad number of therapeutic applications requiring cell division inhibitors. Despite this, even in the absence of ultraviolet rays they are active. The current paper mainly focuses on the effects of psoralens on human breast cancer as they are able to influence many aspects of cell behavior, such as cell growth, survival and apoptosis. In addition, analytical and pharmacological data have demonstrated that psoralens antagonize some metabolizing enzymes, affect estrogen receptor stability and counteract cell invasiveness as well as cancer drug resistance. The scientific findings summarized highlight the pleiotropic functions of phytochemical drugs, given that recently their target signals and how these are modified in the cells have been identified. The encouraging results in this field suggest that multiple modulating strategies based on coumarin drugs in combination with canonical chemotherapeutic agents or radiotherapy could be a useful approach to address the treatment of many types of cancer.

Abstract P2-09-07: Inhibition of aurora-A by MLN8237 decreases SMAD5 expression and increases effectiveness of chemotherapeutic agents in breast cancer cells

Abstract BACKGROUND: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis in patients with breast cancer. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A in breast cancer cells will lead to increased sensitivity to chemotherapy. RESULTS: Increased Aurora-A expression, as assessed by IHC staining, in patients with locally advanced disease was associated with worse prognosis. We show that in ER+ cell line, MCF7, activating MAPK pathway through overexpression of Raf1 leads to increase in Aurora-A activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to untransfected cells in MTT assays. This effect is even more exacerbated with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by specific inhibitor, Alisertib. This was further explored in triple negative (MDA-MB-231) or Her-2/neu expressing (SUM149) cell lines. These cells express higher levels of Aurora-A. Inhibition of Aurora-A activity in combination with paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are looking at a role of Aurora-A in activating SMAD5 and its role in resistance to chemotherapy. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. CONCLUSIONS: Inhibition of Aurora-A by a specific inhibitor, MLN8237, resulted in decreased SMAD5 expression and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be very useful in developing more effective treatments for breast cancer patients in neo-adjuvant, adjuvant and metastatic settings. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-07.

Fucoidan extract enhances the anti-cancer activity of chemotherapeutic agents in breast cancer cells

Background Fucoidan, a fucose-rich polysaccharide isolated from brown alga, is currently under investigation as a new anticancer compound [1-4]. In the present study, fucoidan extract (FE) from Cladosiphon navae-caledoniae Kylin was prepared by enzymatic digestion. We investigated whether a combination of FE with chemotherapeutic agents had the potential to improve the therapeutic efficacy of cancer treatment.

Repositioning of zoledronic acid for breast cancer using transferrin-conjugated liposome

Zoledronic acid (ZOL) is one of the most potent nitrogen containing bisphosphonates (N-BP) which are used for cancer-induced skeletal disease by inhibiting osteoclast-mediated bone resorption. It acts by inhibiting the farnesyldiphosphate synthase which is one of several key enzymes in the mevalonate pathway consequently leading to osteoclast apoptosis. From recent studies, direct anti-cancer effects of bisphosphonates, particularly ZOL, was demonstrated in breast cancers. The synergic anti-cancer effects of ZOL treated with other anti-cancer agents in breast cancer cells were also reported. However, pharmacokinetic properties of ZOL have limited its cytotoxic activities because almost 60 % of injected ZOL are disposed into bone and 40 % of the dose cleared rapidly from blood, so that not enough dose of ZOL delivered to the cancer cells. For this reason, we introduced transferrin-conjugated long-circulating liposomes as targeted delivery system for enhancing the anti-cancer effect of ZOL. Transferrin receptors have been explored as a target to deliver therapeutics into cancer cells due to their increased expression on malignant cells. Liposomes were prepared by a reverse-phase evaporation vesicle method with aqueous ZOL solution. Transferrins were attached to the neutral liposomes via free thiols (–SH) to the PDP-end of the pyridylthiopropionoylamino-PEG-distearoyl-phosphatidyethanolamine (PDP-PEG-DSPE). Liposomes were characterized by measuring size distribution and zeta potential. The mean diameter of liposomes was 190 nm, and the encapsulation efficiency of ZOL was about 10 %, which was determined by HPLC analysis. Based on the 50 % inhibitory concentration (IC50) of ZOL against breast cancer cell MDA-MB-231, increased anti-cancer effect of transferrin-conjugated liposomal ZOL was evaluated compared to free ZOL and non-conjugated liposomal ZOL. In conclusion, transferrin-conjugated liposome can enhance the anti-cancer effect of ZOL and it is suggested that ZOL can be used as an anti-cancer agent with this formulation in breast cancer patients.

Abstract A55: Histone deacetylase inhibitors as differentiation agents in breast cancer cells

Abstract FOXA1 and GATA3 are two luminal lineage transcription factors that form a cross-regulatory transcriptional network that controls the morphogenesis of the mammary gland and regulates estrogen receptor (ER) signaling. Approximately two thirds of breast tumors overexpress ER at the time of diagnosis. Antiestrogen therapy has been effective in blocking the proliferative effects of ER, but unfortunately a significant proportion of patients will relapse due to resistance. Histone deacetylase inhibitors (HDACis) have been shown to suppress ER expression in ER+ breast cancer cells and are currently being tested in clinical trials in combination with antiestrogens for treatment of ER+ breast tumors. Here, we report that the HDACi Trichostatin A (TSA) abrogates the expression of FOXA1 and GATA3, as well as that of ER in MCF-7 breast cancer cells. Using gene expression microarrays, we observed that several markers of lactogenic differentiation including the cholesterol biosynthesis pathway were induced by TSA treatment. Moreover, expression of ER, FOXA1 and GATA3 is reduced during lactation in the mouse mammary gland. Finally, overexpression of GATA3 in MCF-7 cells reduced the TSA-mediated induction of lactogenic markers, suggesting that GATA3 may act as a repressor of lactogenic differentiation. Altogether, our studies suggest that HDACi treatment partially mimics differentiation events taking place in ER+ cells during lactogenesis. Future experiments will examine the mechanisms and clinical relevance of the differentiation properties of HDACis. Citation Format: Houssam Ismail, Martine Bail, David Laperrière, Khalid Hilmi, Sylvie Mader. Histone deacetylase inhibitors as differentiation agents in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A55.

SMAD5 expression and inhibition of the mitotic kinase aurora-A on sensitivity of breast cancer cells to chemotherapy.

e13516 Background: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A will lead to increased sensitivity to chemotherapy. Methods: The hypothesis was tested in vitro. Cellular proliferation was tested using MTT assay and protein levels were determined on Western blots. Results: In ER+ cell line, MCF7, constitutive activation of Raf-1/MAPK signaling pathway leads to aberrant activation of Aurora-A kinase activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to parental MCF7 cells. This effect is even more pronounced with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by novel inhibitor, Alisertib. This was further explored in triple negative cells (MDA-MB-231) and cells expressing Her-2/neu (BT474) which express higher levels of Aurora-A. Combination of Alisertib and paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are investigating a role of Aurora-A in developing chemoresistance through activating SMAD5. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. Conclusions: Inhibition of Aurora-A by Alisertib resulted in decreased SMAD5 nuclear phosphorylation and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be extremely useful in developing more effective treatments for breast cancer patients both in neo-adjuvant, adjuvant and metastatic settings.