Resistant Human Breast Cancer Cells Research Articles

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) IandII, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Abstract 774: Role of t-Darpp in a shift to EGFR signaling in trastuzumab-resistant cells

Abstract Elucidating the molecular mechanism of both primary and acquired resistance to trastuzumab is vital to gaining insight into ways to improve therapeutic outcomes in breast cancer patients. We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant BT474 cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer resistance. We have further demonstrated that trastuzumab-resistant BT474 cells are more sensitive to the EGFR tyrosine kinase inhibitor (TKI), AG1478, in the presence of trastuzumab than in its absence (1), suggesting that these cells gain a dependence on EGFR when HER2 signaling is shut down. We have now examined the mechanism by which tDp confers resistance in HER2+ breast cancer cells. We used SK-Br-3 cells stably expressing an empty vector, tDp or flag-tDp, as well as two trastuzumab-resistant BT474 clones, BT1.0C and BT1.0E, that endogenously over-express moderate and very high levels of tDp, respectively. Cells with elevated tDp levels had a higher basal level of p-EGFR (Y1068) and we observed an increase in p-EGFR upon trastuzumab treatment in all of the HER2+ cells. To further investigate if EGFR signaling plays a role in tDp's resistance mechanism, we performed SRB assays to test the sensitivity of tDp over-expressing cells to EGFR TKIs, AG1478 and Gefitinib. We found that cells expressing high tDp are 2.5- to 7.5-fold more sensitive to EGFR inhibition compared to controls (p<0.01). Furthermore, the combination treatment of AG1478 and trastuzumab was synergistic in inhibiting the trastuzumab-resistant BT1.0E cell line that highly over-expresses tDp at an ED50, ED75 and ED90 CI of 0.61, 0.46, and 0.35, respectively. The parental BT474 cells and BT1.0C clone, which expresses lower levels of tDp, showed antagonism at the ED50 but synergism at the ED75 and ED90. Using siRNA knock-down of tDp, we will show whether tDp is required in causing an increased dependence on EGFR signaling. We will also report on similar combination experiments using tDp-transfected SK-Br-3 cells, to determine if tDp expression is sufficient for conferring synergism, and with a HER2 TKI in combination with EGFR TKI. Taken together, our results suggest that tDp is a novel molecular target that can mediate resistance to trastuzumab by inducing a compensatory shift to EGFR signaling when HER2 is inhibited. 1. Chan CT, Metz MZ, & Kane SE (2005) Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors. Breast Cancer Res Treat 91(2):187-201. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 774. doi:1538-7445.AM2012-774

Dietary canola oil and growth of implanted drug‐resistant MCF‐7 human breast cancer in Balb/c nude mice

The objective of the project was to determine the extent to which dietary canola oil (CAN) increases susceptibility of drug resistant human breast cancer cells to treatment. Treatment of doxorubicin (DOX)‐resistant MCF‐7 breast cancer cells in vitro with DOX plus CAN significantly suppressed cell proliferation as compared to the control, DOX, or CAN‐treated cells. To determine the effect of CAN on tumor growth and sensitivity to treatment in vivo, nude mice were fed either a soybean oil‐containing diet (SOY) or CAN diet and then implanted with DOX‐resistant human breast cancer cells to grow cancer. The CAN diet reduced tumor growth and also improved the tumor growth inhibitory effect of DOX on drug‐resistant cancer cells. Using qPCR, we observed that mRNA expression of both the P‐glycoprotein‐encoding multidrug resistant 1 gene and the mouse double minute 2 gene in tumor tissues were reduced by the CAN diet. Our findings suggest that a CAN diet inhibits tumor growth and sensitizes drug‐resistant breast cancer cells to treatment, due in part to alteration of gene expression. Overall, these novel findings advance the understanding of the role of dietary fat in breast cancer treatment, and future studies aimed at elucidating the detailed mechanism through which CAN affects drug resistant cancer cells are warranted. Research was funded by United States Department of Agriculture‐ National Institute of Food and Agriculture.Grant Funding Source : USDA‐NIFA

Forced expression of Hsp27 Reverses P-Glycoprotein (ABCB1) Mediated Drug Efflux and MDR1 Gene Expression in Adriamycin Resistant Human Breast Cancer Cells

Forced expression of Hsp27 Reverses P-Glycoprotein (ABCB1) Mediated Drug Efflux and MDR1 Gene Expression in Adriamycin Resistant Human Breast Cancer Cells

A Peptide of SPARC Interferes with the Interaction between Caspase8 and Bcl2 to Resensitize Chemoresistant Tumors and Enhance Their Regression In Vivo

SPARC, a matricellular protein with tumor suppressor properties in certain human cancers, was initially identified in a genome-wide analysis of differentially expressed genes in chemotherapy resistance. Its exciting new role as a potential chemosensitizer arises from its ability to augment the apoptotic cascade, although the exact mechanisms are unclear. This study further examines the mechanism by which SPARC may be promoting apoptosis and identifies a smaller peptide analogue with greater chemosensitizing and tumor-regressing properties than the native protein. We examined the possibility that the apoptosis-enhancing activity of SPARC could reside within one of its three biological domains (N-terminus (NT), the follistatin-like (FS), or extracellular (EC) domains), and identified the N-terminus as the region with its chemosensitizing properties. These results were not only confirmed by studies utilizing stable cell lines overexpressing the different domains of SPARC, but as well, with a synthetic 51-aa peptide spanning the NT-domain. It revealed that the NT-domain induced a significantly greater reduction in cell viability than SPARC, and that it enhanced the apoptotic cascade via its activation of caspase 8. Moreover, in chemotherapy resistant human colon, breast and pancreatic cancer cells, its chemosensitizing properties also depended on its ability to dissociate Bcl2 from caspase 8. These observations translated to clinically significant findings in that, in-vivo, mouse tumor xenografts overexpressing the NT-domain of SPARC had significantly greater sensitivity to chemotherapy and tumor regression, even when compared to the highly-sensitive SPARC-overexpressing tumors. Our results identified an interplay between the NT-domain, Bcl2 and caspase 8 that helps augment apoptosis and as a consequence, a tumor's response to therapy. This NT-domain of SPARC and its 51-aa peptide are highly efficacious in modulating and enhancing apoptosis, thereby conferring greater chemosensitivity to resistant tumors. Our findings provide additional insight into mechanisms involved in chemotherapy resistance and a potential novel therapeutic that specifically targets this devastating phenomenon.

Differential expression of microRNA expression in tamoxifen-sensitive MCF-7 versus tamoxifen-resistant LY2 human breast cancer cells

Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine-sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs-10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance.

Abstract 865: Fat-1 gene suppression of breast cancer growth in mice

Abstract The high ratio of n-6/n-3 polyunsaturated fatty acids (PUFAs) content in Western diets (approximately 10 to 30) may be related to the high breast cancer rate in the United States compared to Japan (approximately 4). Numerous studies in literature report about the tumor suppression effect of n-3 PUFAs, a main component of fish or fish oil. A recent study on B16 melanoma on C57BL/6 mice carrying the fat-1 gene, suppressed the melanoma by changing the n-6/n-3 ratio in tumors from 27:1 in wild type mice to 4.6:1 in fat-1 mice. In this project, a fat-1 gene, which encodes a fatty acyl desaturase that acts on substrates of 16-20 carbons and converts n-6 to n-3 fatty acids, was transfected into the human breast cancer cells MDA-MB-231, parental MCF-7 and tamoxifen-resistant MCF-7/TAM-R cells, Sk-BR3 and murine EMT6 mammary tumor cells. Both fat-1 transgenic mice and fat-1 gene treated breast cancer cells were employed to test our hypothesis that fat-1 suppresses the growth of both estrogen receptor (ER) positive and ER negative breast cancer cells that respond poorly to endocrine therapies. We have also treated the breast cancer cell lines with docosahexaenoic acid (DHA, n-3 fatty acid), arachidonic acid (AA, n-6 fatty acid) with a vehicle control. We found that for the ER negative human breast cancer cells, MDA- MB-231 and SK-BR-3 cells, the cell proliferation rate of DHA treated cells decreased 50%, compared to the corresponding control cells treated with AA and vehicle control. The fatty acid analysis of the fat-1 gene expressing breast cancer cells MDA-MB-231 and MCF-7 showed 30% and 46% reduction of n-6/n-3 ratio, respectively, and ∼30% reduction of cell proliferation rate. The fat-1 transfected MDA-MB-231 cells have ∼4-fold increase of cell apoptosis by Tunel assay. Fat-1 gene expression also suppresses the growth of tamoxifen resistant human breast cancer cells MCF-7F/Tam-R. These results indicate that fat-1 or n-3 fatty acid suppresses breast cancer cells in an ER independent way. Normal human mammary epithelial cells treated with DHA, AA and BSA showed no significant difference of growth rates, suggesting that the n-3 suppression effect is breast cancer selective. In a preliminary study, the growth rate of the EMT6 mammary tumors were significantly reduced when the cell line was treated with fat-1 gene before inoculation into the FVB/NJ mice. The transgenic mice expressing fat-1 gene are also used to investigate the breast cancer suppression effect of fat-1 gene in vivo. The underlying mechanism of the breast cancer suppression effect by fat-1 gene is currently under investigation. This study may provide a potential adjuvant therapeutic method for hormonal resistant human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2011-865

Antiproliferative effects of flavonoid fractions from Calendula officinalis flowers in parent and tamoxifen resistant T47D human breast cancer cells

The risk of human breast cancer is concerned to cumulative exposure of the breast cells to endogenous estrogens. Strategies aiming at reducing the production of estrogens may be useful for the prevention of estrogens-related breast cancer. Several natural products with plant origin have the potential value as chemo-preventive or therapeutic agents in cancer. Flavonoids, the natural polyphenol compounds, have shown antiviral, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. Calendula officinalis, a member of compositae family, is well known for its pharmacological effects such as anti-inflammatory, anti-viral, anti-HIV, anti-tumor, anti-mutagenic and cytotoxic properties. Mechanism of the possible chemo-preventing action of flavonoids has not been yet completely understood. They may exert their chemo-preventive and anti-carcinogenic effects in estrogen-dependent cells by inhibiting aromatase, 17?-hydroxysteroid oxidoreductase and other enzymes involved or by possessing estrogenic and anti-estrogenic activities. In this study, three major flavonoid fractions were separated from a methanol extract of Calendula officinalis flowers by preparative TLC. These fractions were evaluated for inhibition of parent and tamoxifen resistant T47D human breast cancer. The fractions did not have inhibitory effect. We also examined the effect of quercetin and isorhamnetin on the growth of parent and resistant T47D cells in the presence or absence of tamoxifen. It was found that quercetin increased cell proliferation of the resistant T47D cells at the presence of tamoxifen but no effect was detected by using quercitin itself. In consequence, isorhamnetin had not any proliferative or anti-proliferative activity on the both cell lines. Many mechanisms can contribute to the affects obtained on the cell growth.

Abstract LB-340: Inhibition of autophagy enhances the anticancer activity of the retinoic acid derivative VN/12-1 in endocrine resistant human breast cancer cell lines

Abstract Breast cancer is the most common cancer and the second leading cause of cancer related deaths among women in the United States. Although endocrine therapies such as anti-estrogens and aromatase inhibitors have improved the overall survival, resistance to this therapy remains a major concern. Autophagy is a unique, regulated mechanism of cell survival by catabolism of proteins under nutrient deprivation or chemotherapeutic stress. It is an important cause of resistance to chemotherapeutic agents. VN/12-1, a novel 4-imidazolyl methyl ester of retinoic acid synthesized in our laboratory, showed excellent anti-proliferative activity (as shown by MTT assay) in endocrine resistant human breast cancer cell lines SK-BR-3 and MDA-MB-231. Its IC50 in SK-BR-3 cells was 5.2 μM compared to 15.2 μM (all-trans retinoic acid-ATRA) and 45.6 μM (letrozole). For MDA-MB-231 cells, IC50 of VN/12-1 was 7.3 μM compared to 53.7 μM (ATRA) and 74.3 μM (letrozole). At low micromolar concentrations of VN/12-1 treatment on these cells, western blot analysis and imaging studies confirmed the presence of autophagosomes. This was associated with up-regulation of proteins involved in endoplasmic reticulum (ER) stress, markers for DNA damage and downregulation of cyclin D1 and phosphorylated Akt. Co-treatment with chloroquine (an autophagy inhibitor) resulted in increased sensitivity of cells to VN/12-1 as indicated by decrease in cell viability from 71.22% for VN/12-1 (3 μM) alone, 90.18% chloroquine (3 μM) alone to 34.31% for the combination of VN/12-1 (3 μM) and chloroquine (3 μM) in SK-BR-3 cells. It also resulted in induction of apoptosis which was caspase mediated. In summary, ER stress and DNA damage induced by VN/12-1 resulted in cell cycle arrest and induction of autophagy. Inhibition of autophagy by chloroquine overwhelmed this cellular stress and resulted in apoptotic cell death in endocrine resistant breast cancer cell lines. Significance: VN/12-1 and its combination with autophagy inhibitor chloroquine is a novel therapeutic alternative to treat endocrine resistant breast cancer and hence warrants further pre-clinical and clinical development. This is the first in vitro study which showed an enhancement of anti-cancer activity of a retinoic acid derivative by an autophagy inhibitor in endocrine resistant human breast cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-340.

Abstract 5419: Effects of selective aryl hydrocarbon receptor modulators (SAhRMs) in enhancing activity of tamoxifen against doxorubicin resistant MCF-7 Cells: Role of BRCA1

Abstract Recent reports indicate that SAhRMs such as diindolylmethane (DIM) inhibit α-estrogen receptor (ER) expression and estrogen signaling in human breast cancer cells. There is also increasing evidence that SAhRMs demonstrate anti estrogenic and antitumorigenic activity in ER positive human breast adenocarcinoma (MCF-7) cells via cross talk between aromatic hydrocarbon receptor (AhR) and ER pathways. Additional reports suggest that BRCA1 transcriptional activity is enhanced by interactions with AhR and the levels of BRCA1 expression play a role in tamoxifen sensitivity/resistance in breast cancer cells by altering estrogen receptor-coregulator interactions. However, the effects of AhR and its agonists and antagonists, and their modulatory effect on BRCA1 in resistant human breast cancer cells have not been investigated. We have tested the hypothesis that DIM, an AhR agonist may inhibit cell growth in a concentration and time dependant manner against doxorubicin resistant MCF-7 (MCF-7/dox) cells and may enhance the activity of 4-OH tamoxifen (TAM) against both MCF-7 and MCF-7/dox cells by enhancing the expression of BRCA1. Initially we determined the cytotoxic effect of DIM against the two cell lines by utilizing the MTT assay. Both DIM and TAM were equally effective against MCF-7 and MCF-7/dox cells with the IC50 values of approximately 90 μM and 20 μM, respectively at 72 hr. However, a combination of DIM and TAM resulted in an increase in cytotoxcity and statistically significant lowering of the IC50 concentration to 50 μM and 10 μM for each drug, respectively. We also investigated the effects of AhR antagonist, α-naphthoflavone (NF) which had a similar IC 50 value of approximately 112 μM against both these cell lines, whereas a combination of NF and TAM required only half the concentrations of 50 μM and 10 μM to inhibit 50 % of the cell growth of each cell line. Since BRCA1, a tumor suppressor gene, has been implicated in the mechanism of tamoxifen induced growth inhibition, we determined the expression of this protein upon exposure of these cells to DIM. Results from western blot analysis show that there is a significant increase in BRCA1 and AhR protein levels in these cells upon treatment with DIM when compared to protein levels from untreated MCF-7 and MCF-7 /dox cells. Thus activation of BRCA1 by SAhRMs appears to be one of the mechanisms responsible for enhanced growth inhibitory effect of tamoxifen against these cells. These data suggest that either an AhR agonist or an antagonist can be conveniently employed in combination with TAM to increase the activity of TAM against both MCF-7 cells and doxorubicin resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5419.

Effect of methyl nutrients on the growth of multidrug resistant MCF-7 human breast cancer cells

Effect of methyl nutrients on the growth of multidrug resistant MCF-7 human breast cancer cells

Characterization of a novel trastuzumab resistant human breast cancer cell line

HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.

Development of radioresistance in drug resistant human MCF-7 breast cancer cells

AbstractBackground and purpose: Radiotherapy is used for the treatment of malignant tumours, and may be used as the primary therapy. It is also common to combine radiotherapy with surgery, chemotherapy, hormone therapy or some combination of them. Even if the tumour is treated intensively, women diagnosed with breast cancer may develop a recurrence. Most recurrences may be in the form of distant metastases, development of multi-drug resistance phenotype or both together. This study demonstrated that some of the multi-drug resistant cancer cells may also become radioresistant.Materials and Methods: Chemoresistance in paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), vincristine (MCF-7/Vinc), doxorubicin (MCF-7/Dox) and zoledronic acid (MCF-7/Zol) resistant MCF-7 cells were demonstrated by XTT assay. MDR1 gene expression was detected by real-time PCR in human MCF-7 breast cancer cells. Drug resistant and sensitive cells were exposed to γ-radiation and development of radioresistance was investigated.Results: Results have indicated that paclitaxel, docetaxel, vincristine, doxorubicin and zoledronic acid–selected cells gained varying degrees of resistance to their selective drugs when compared with original MCF-7/S. MCF-7/Pac, MCF-7/Doc, MCF-7/Vinc and MCF-7/Dox cells have all acquired MDR1 expression. Among the resistant sub-lines, MCF-7/Pac and MCF-7/Doc cells were significantly cross-resistant to irradiation compared to the sensitive cells.Conclusion: MCF-7/Pac and MCF-7/Doc cell lines were found radioresistant to γ-radiation. On the contrary, doxorubicin, vincristine and zoledronic acid resistant cancer cells were still sensitive to radiation.

Deterministic Effects Propagation Networks for reconstructing protein signaling networks from multiple interventions

BackgroundModern gene perturbation techniques, like RNA interference (RNAi), enable us to study effects of targeted interventions in cells efficiently. In combination with mRNA or protein expression data this allows to gain insights into the behavior of complex biological systems.ResultsIn this paper, we propose Deterministic Effects Propagation Networks (DEPNs) as a special Bayesian Network approach to reverse engineer signaling networks from a combination of protein expression and perturbation data. DEPNs allow to reconstruct protein networks based on combinatorial intervention effects, which are monitored via changes of the protein expression or activation over one or a few time points. Our implementation of DEPNs allows for latent network nodes (i.e. proteins without measurements) and has a built in mechanism to impute missing data. The robustness of our approach was tested on simulated data. We applied DEPNs to reconstruct the ERBB signaling network in de novo trastuzumab resistant human breast cancer cells, where protein expression was monitored on Reverse Phase Protein Arrays (RPPAs) after knockdown of network proteins using RNAi.ConclusionDEPNs offer a robust, efficient and simple approach to infer protein signaling networks from multiple interventions. The method as well as the data have been made part of the latest version of the R package "nem" available as a supplement to this paper and via the Bioconductor repository.

The effect of poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity

The aim of this work was to investigate the effect of triblock copolymer poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity. Docetaxel-loaded nanoparticles were prepared by oil-in-water emulsion/solvent evaporation technique using biodegradable poly(lactic- co-glycolic acid) (PLGA) with or without addition of poloxamer 188, respectively. The resulting nanoparticles were found to be spherical with a rough and porous surface. The nanoparticles had an average size of around 200 nm with a narrow size distribution. The in vitro drug-release profile of both nanoparticle formulations showed a biphasic release pattern. An increased level of uptake of PLGA/poloxamer 188 nanoparticles in the docetaxel-resistant MCF-7 TAX30 human breast cancer cell line could be found in comparison with that of PLGA nanoparticles. In addition, the docetaxel-loaded PLGA/poloxamer 188 nanoparticles achieved a significantly higher level of cytotoxicity than that of docetaxel-loaded PLGA nanoparticles and Taxotere ( P < .05). In conclusion, the results showed advantages of docetaxel-loaded PLGA nanoparticles incorporated with poloxamer 188 compared with the nanoparticles without incorporation of poloxamer 188 in terms of sustainable release and efficacy in breast cancer chemotherapy. From the Clinical Editor The effects of poloxamer 188, a triblock copolymer were studied on nanoparticle morphology, size, cancer cell uptake and cytotoxicity. An increased level of uptake of PLGA/poloxamer 188 nanoparticles in resistant human breast cancer cell line was demonstrated, resulting in a significantly higher level of cytotoxicity.

A novel doxorubicin-mitomycin C co-encapsulated nanoparticle formulation exhibits anti-cancer synergy in multidrug resistant human breast cancer cells

Anthracycline-containing treatment regimens are currently the most widely employed regimens for the management of breast cancer. These drug combinations are often designed based on non-cross resistance and minimal overlapping toxicity rather than drug synergism. Moreover, aggressive doses are normally used in chemotherapy to achieve a greater therapeutic benefit at the cost of more acute and long-term toxic effects. To increase chemotherapeutic efficacy while decreasing toxic effects, rational design of drug synergy-based regimens is needed. Our previous work showed a synergistic effect of doxorubicin (DOX) and mitomycin C (MMC) on murine breast cancer cells in vitro and improved efficacy and reduced systemic toxicity of DOX-loaded solid polymer-lipid hybrid nanoparticles (PLN) in animal models of breast cancer. Herein we have demonstrated true anticancer synergy of concurrently applied DOX and MMC, and have rationally designed PLN to effectively deliver this combination to multidrug resistant (MDR) MDA435/LCC6 human breast cancer cells. DOX-MMC co-loaded PLN were effective in killing MDR cells at 20-30-fold lower doses than the free drugs. This synergistic cell killing was correlated with enhanced induction of DNA double strand breaks that preceded apoptosis. Importantly, co-encapsulation of dual agents into a nanoparticle formulation was much more effective than concurrent application of single agent-containing PLN, demonstrating the requirement of simultaneous uptake of both drugs by the same cells to enhance the drug synergy. The rationally designed combination chemotherapeutic PLN can overcome multidrug resistance at a significantly lower dose than free drugs, exhibiting the potential to enhance chemotherapy and reduce the therapeutic limitations imposed by systemic toxicity.

Potent modulation of P‐glycoprotein activity by naturally occurring phenylbutenoids from Zingiber cassumunar

Five phenylbutenoid derivatives from the rhizomes of Zingiber cassumunar Roxb. (Zingiberaceae) were evaluated for their P-glycoprotein (P-gp) inhibitory effects in a P-gp over-expressing multidrug resistant (MDR) human breast cancer cell line, MCF-7/ADR. As a result, a phenylbutenoid dimer, (+/-)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (1), exhibited highly potent P-gp inhibitory activity, decreasing the IC(50) value of daunomycin (DNM) to 4.31 +/- 0.40 microm in the cells (DNM IC(50) = 37.1 +/- 0.59 microm). The positive control, verapamil decreased the IC(50) value of DNM to 6.94 +/- 0.40 microm. Three phenylbutenoid monomers, 2-4 from this plant, also resulted in a significant decrease in the IC(50) values of DNM compared with the control. In particular, compound 1 markedly enhanced [(3)H]-DNM accumulation and significantly reduced [(3)H]-DNM efflux compared with the control, and this effect was more potent than that of verapamil, a well-known P-gp inhibitor. These results suggest that compound 1 of Z. cassumunar can be developed as a potent chemo-sensitizing agent that reverses P-gp-mediated MDR in human cancer chemotherapy.

Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

IntroductionDespite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.MethodsTo compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.ResultsProteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.ConclusionsThis study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Experimental study of adenovirus-mediated p53 gene on the reversal of multidrug resistance in breast cancer

To explore the effects of adenovirus-mediated wild-type p53 gene (Ad-p53) on the reversal of multidrug resistance (MDR) and multidrug resistance gene-1 (MDR1) in adriamycin (ADM) resistant human breast cancer cell. Ad-p53 was transfected into the human breast cancer cells of the line MCF-7/ADM. p53 protein expression was detected by flow cytometry (FCM). The cell proliferation was assessed by trypan blue dye exclusion. The effects of Ad-p53 on ADM mediated drug resistance were observed by MTT assay. The expression levels of MDR1 mRNA were detected with real-time RT-PCR. The expression rate of p53 protein of the MCF-7/ADM cell was increased from 10.24% to 36.20% after transfected by MOI(50) Ad-p53 for 48 hours. The growth inhibition rate was 7.4 % (P = 0.003). The chemosensitivity of MCF-7/ADM to ADM increased by 11.6 times (P = 0.001). The result of real-time RT-PCR reveales that MDR1 mRNA was decreased from 1.25 +/- 0.01 to 0.91 +/- 0.01 (P = 0.011) after the MCF-7/ADM cells were transfected by Ad-p53. Ad-p53 can inhibit the expression of MDR1 gene and partially reverse the MDR of MCF-7/ADM cells, thus enhancing the chemosensitivity of human breast cancer cells to ADM.