Cytotoxic Effects Of Docetaxel Research Articles

Increasing the radiation-induced cytotoxicity by silver nanoparticles and docetaxel in prostate cancer cells.

Radiation therapy is utilized for treatment of localized prostate cancer. Nevertheless, cancerous cells frequently develop radiation resistance. While higher radiation doses have not always been effective, radiosensitizers have been extensively studied for their ability to enhance the cytotoxic effects of radiation.So, this study aims to evaluate the possible radiosensitization effects of docetaxel (DTX) and silver nanoparticles (SNP) in LNCaP cells. The cytotoxic effects of DTX, SNP and2Gy of X-Ray radiation treatments were assessed in human LNCaP cell line using the MTT test after 24 h. Moreover, the effects of DTX, SNP and radiation on Epidermal growth factor (EGF), Caspase 3, inducible nitric oxide synthase and E-cadherin gene expression were analyzed using the Real-time PCR method. The level of Hydrogen peroxide (H2O2), an oxidative stress marker, was also detected 24h after various single and combined treatments. The combinations of SNP (in low toxic concentration) and/or DTX (0.25× IC50 and 0.5 × IC50 concentrations for triple and double combinations respectively) with radiation induced significant cytotoxicity in LNCaP cells in comparison to monotherapies. These cytotoxic effects were associated with the downregulation of EGF mRNA. Additionally, H2O2 levels increased after Radiation + SNP + DTX triple combination and double combinations including Radiation + SNP and Radiation + DTX versus single treatments. The triple combination treatment also increased Caspase 3 and and E-cadherin mRNA levels in compared to single treatments in LNCaP cells. Our results indicate that the combination of SNP and DTX with radiation induces significant anti-cancer effects. Upregulation of Caspase 3 and E-cadherin gene expression, and decreased mRNA expression level of EGF may be exerted specifically by use of this combination versus single treatments.

Treating MCF7 breast cancer cell with proteasome inhibitor Bortezomib restores apoptotic factors and sensitizes cell to Docetaxel.

Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects in this signaling pathway lead to chemoresistance. Apoptotic protease activating factor 1 regulates cellular stress evoked by chemotherapeutic agents through facilitating apoptosome assembling but can be degraded by proteasome. This study examined the role of proteasome inhibitor Bortezomib in the cytotoxic effects of Docetaxel on MCF7 cells response and its correlation with Apaf-1 expression level. MTT assay, caspase 3/7 activity assay, propidium iodide staining, adenosine triphosphate and reactive oxygen species amount measurements were utilized to demonstrate the role of Bortezomib in Docetaxel efficacy with and without Apaf-1 overexpressing. Meanwhile, two-dimensional cell migration assay was performed by scratch wound assay. The combination of Docetaxel with Bortezomib was significantly more cytotoxic compared single drug, more effectively delayed cell growth, reduced ATP level and increased ROS production. In Apaf-1 overexpressing, Docetaxel was more efficient in preventing cell migration, however, Docetaxel plus Bortezomib were not significantly effective; and fluorescence images supported the interpretation. Our findings demonstrated MCF7 resistance to Docetaxel is due in part to low Apaf-1 level and Apaf-1 overexpression resulted in the increase of cell susceptibility to Docetaxel stimulus. We assume that proteasome inhibitor may restore apoptotic proteins like Apaf-1 and prevent the degradation of cytosolic cytochrome c released by Docetaxel, consequently triggering intrinsic apoptosis and promoting cancer cell death. Collectively, treating MCF7 breast cells with proteasome inhibitor sensitizes cells to Docetaxel-induced apoptosis and possibly overcomes chemoresistance.

Combination Regimen of Amino-Noscapine and Docetaxel for Evaluation of Anticancer Activity

Docetaxel (DOX) is one of the clinically used chemotherapeutic agents for the treatment of breast cancer, instead of its severe side effects. Noscapine, an antitussive opium alkaloid, on the other hand, has shown antitumor activity against a number of cancers without any significant side effects. Since both the compounds targeted tubulin, it is interesting to see the combined effect of both the compounds for the management of breast cancer. The binding energy of -3.49 and -4.18 kcal/mol respectively was noted by the molecular docking of amino-noscapine and DOX on the microtubule. In contrast, the binding energy was improved significantly (-6.27 kcal/mol) when the DOX was docked to the co-complex of amino-noscapine and tubulin, indicating the combined effect of both the ligands. The cell killing potential represented in terms of IC50 value was 38.07 μM and 28.4 μM for a treatment duration of 48 h and 72 h for amino-noscapine. Parenthetically the IC50 value was 0.61 μM and 0.08 μM for DOX respectively for the treatment duration of 48 h and 72 h. The cytotoxic effect of DOX was reduced significantly (to 0.05 μM) in combined treatment with amino-noscapine (20 μM). Apropos to the cytotoxic effect, both the compounds induced apoptosis to cancer cell by interfering with the cell cycle progression. Again, the induction of apoptosis was more effective in the combined treatment of both the compounds. This investigation offers a promising concept for the combination of DOX and aminonoscapine treatment to improve effectiveness in the treatment of breast cancer.

Altered Expression of CD44, SIRT1, CXCR4, miR-21, miR-34a, and miR-451 Genes in MKN-45 Cell Line After Docetaxel Treatment.

Today it is known that the gene expression profile of cancer stem cells differs from other cancer cells, which may lead to the resistance to routine treatments. The aim of this study was to investigate the effect of docetaxel (DOC) treatment on CD44+ cell frequency in human gastric cancer (GC) MKN-45 cell line and its effect on expression levels of SIRT1, CXCR4, microRNA (miR)-21, miR-451, and miR-34a that are closely correlated with the chemoresistance or self-renewal of cancer stem cells (CSCs). The cytotoxic effect of DOC on MKN-45 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay. The frequency of CD44+ cells was measured by flow cytometry in the treated and control groups. The expression level of SIRT1, CXCR4, miR-21, miR-451, and miR-34a was assessed in DOC-treated and non-treated cells using quantitative real-time PCR. Data were analyzed using Statistical Package for the Social Sciences (SPSS) software. The half-maximal inhibitory concentration (IC50) of DOC was 10μg/ml after 48h. Flow cytometry showed a significant increase in CD44+ cells after treatment with DOC (94.3%) when compared with non-treated cells (84.6%) (P < 0.01). The expression of SIRT1, CXCR4, and miR-21 was up-regulated (1.4-fold, 6.7-fold, and 1.22-fold, respectively, P < 0.05) in DOC-treated cells relative to non-treated cells, while miR-451 and miR-34a were down-regulated (0.14-fold and 0.36-fold, respectively, P < 0.05). DOC treatment affected CD44+ cell frequency in MKN-45 cell line and induced significant changes in the expression of SIRT1, CXCR4, miR-21, miR-451, and miR-34a that are implicated in stemness and chemo-radioresistance, which might offer new insights for future GC therapies.

Abstract 4803: CXCR6-directed therapeutic approach potentiates efficacy of docetaxel in prostate cancer

Abstract Development of chemo-resistance negatively impacts therapeutic outcome of advanced prostate cancer (PCa). Molecular mechanism used by cancer cells to overcome therapeutic effect is still undefined. Here, we demonstrate the influence of CXCR6/CXCL16 mediated molecular signals on the efficacy of docetaxel (DTX). Out data showed poor DTX efficacy on PCa cells (LNCaP, PC3, and DU145) after stimulating CXCR6 with soluble CXCL16, while significant improvement in cytotoxic effect of DTX was observed after CXCR6 blockade using anti-CXCR6 monoclonal antibody. Interestingly, CXCR6, CXCL16, and ADAM-10, a metalloproteinase involved in CXCL16 cleavage from PCa cell membranes, were significantly elevated in response to DTX treatment. This was further confirmed on xenograft tumor by immunohistochemistry. Further, phospho-proteomic profiling and western blot analysis showed activation of pro-survival molecules: GSK-3β, NFκB, ERK/1/2, and survivin following soluble CXCL16 treatment. Activation of CXCR6-CXCL16 axis also inhibited expression of the epithelial marker (E-cadherin) while increased expression of mesenchymal markers (β-catenin, α-SMA, vimentin, snail), which highlights the role of CXCR6 in promoting mesenchymal phenotype that is known to have a significant impact on cancer progression and therapeutic outcome. Thus, our study shows that CXCR6-CXCL16 signaling instigates chemotherapeutic resistance by increasing pro-survival signaling and promoting mesenchymal phenotype. Hence, combining CXCR6 blockade with DTX would be a better treatment option for advanced, drug-resistant PCa. Citation Format: Neeraj Kapur, Hina Mir, Guru Sonpavde, James W. Lillard, Shailesh Singh. CXCR6-directed therapeutic approach potentiates efficacy of docetaxel in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4803.

The Combination of Omega-3 Stearidonic Acid and Docetaxel Enhances Cell Death over Docetaxel Alone in Human Prostate Cancer Cells.

Background: Docetaxel (DOC), or Taxotere, is an anthracycline antibiotic used to treat multiple types of cancer. It is a first-line chemotherapy treatment for patients with metastasized, hormone-resistant prostate cancer (PCa) or for patients with high-risk, localized PCa that could benefit from early chemotherapy treatment. Previously, we showed that stearidonic acid (SDA), an omega-3 fatty acid, enhances the cytotoxicity of doxorubicin (DOX) in human PCa cells. This observation suggests that PCa therapies using SDA and chemotherapeutic drugs in combination offer attractive possibilities for developing treatments that ameliorate toxic side effects of some commonly used chemotherapy drugs.Objectives: We used androgen-resistant PC3 and DU 145 cells derived from human prostate cancer to quantify the effects of combined SDA and DOC on proliferation/viability and on the production of pro-apoptotic caspases 9 and 3. We also compared the effects of SDA with those of BAY, a pharmacological inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), in androgen-sensitive LNCaP cells. Finally, we qualitatively and quantitatively assessed the drug combination on androgen receptor (AR) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in LNCaP and PC3 cells, respectively.Methods: The half maximal inhibitory concentration (IC50) and combination indices of SDA and DOC in PC3 and DU 145 cells were determined using the MTT cell viability assay. To quantify the effects of SDA and BAY on NF-ĸB activity, we used luciferase reporter assays in LNCaP cells that were stably transduced with lentiviral vectors carrying NF-ĸB response element sequence upstream of the luciferase gene sequence. AR and PPARγ expression were assessed by western blotting and immunocytochemistry. We considered caspase 9 and 3 cleavage to be apoptosis markers and determined the drug combination effect on the extent of that cleavage by western blot analysis.Results: The cytotoxic effects of DOC were synergistically enhanced by SDA when the two were added to DU145 and PC3 cell cultures. Combination index (CI) analyses based on the Chou-Talalay method and mass action law showed synergistic interaction with CI <1. SDA suppressed TNFα-induced NF-κB activity similarly to BAY. The SDA/DOC combination down regulated testosterone (T)-induced AR and troglitazone-induced PPARγ protein expression when compared to using the drugs singly. Similarly, the SDA/DOC combination induced caspase 9 and 3 production and cleavage suggesting apoptosis induction. Like our DOX studies, this work provides proof-of-concept for using SDA and DOC in combination to reduce the dose, and therefore the toxicity, of DOC and possibly increasing the survival benefit in DOC clinical translation studies.

Cytotoxic effects of docetaxel as a candidate drug of drug-eluting stent on human umbilical vein endothelial cells and the signaling pathway of cell migration inhibition, adhesion delay and shape change.

Docetaxel (DTX), a paclitaxel analogue, can efficiently inhibit proliferation of vascular smooth muscle cells and has broadly been used as an antiangiogenesis drug. However, as a candidate drug of drug-eluting stent, the effects of DTX on human umbilical vein endothelial cells (HUVECs) are still not well understood. Herein, we investigated the effects of DTX on proliferation, apoptosis, adhesion, migration and morphology of HUVECs in vitro. We found that DTX had the cytostatic and cytotoxic effects at low and high concentrations, respectively. DTX could inhibit the proliferation and migration of HUVECs, induce HUVECs apoptosis, delay HUVECs adhesion and decrease spreading area and aspect ratio of individual cells. The signaling pathway that DTX led to the migration inhibition, adhesion delay and shape change of HUVECs is the VE-cadherin mediated integrin β1/FAK/ROCK signaling pathway. The study will provide a theoretical basis for the clinical application of DTX.

Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells.

As Notch receptors have been shown to induce chemoresistance, we hypothesized that delta-like ligand-4 (DLL4), a central Notch signalling ligand, might also participate in chemoresistance in breast cancer. To investigate this issue, overexpression of DLL4 was induced by transfection with expression vectors for DLL4 in the human breast cancer cell line Michigan cancer foundation-7 (MCF-7). It was found that DLL4 could be adaptively upregulated by docetaxel (DOC) treatment in a dose-dependent manner, but Notch1 was unaffected. Overexpression of DLL4 could significantly attenuate the cytotoxic effects of DOC by increasing Bcl-2 expression, while decreasing Bax expression, apoptosis rate and DNA damage. The protective effects of DLL4 made cells acquire chemoresistance against DOC and resulted in cancer cell survival. DLL4 is normally regarded as a regulator of vascular development. Our results expanded the understanding of DLL4. Since DLL4 may play an important role in the process of acquiring chemoresistance, it may be a promising target in overcoming chemoresistance in breast cancer.

MP84-20 BIOINFORMATIC ANALYSIS UTILIZING THE ANDROGENIC SUPPRESSIVE EFFECT IDENTIFIED PROMISING CANDIDATE DRUGS FOR REPROGRAMMING DOCETAXEL-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-20 BIOINFORMATIC ANALYSIS UTILIZING THE ANDROGENIC SUPPRESSIVE EFFECT IDENTIFIED PROMISING CANDIDATE DRUGS FOR REPROGRAMMING DOCETAXEL-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER Hiroshi Hongo, Takeo Kosaka, Yota Yasumizu, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Hiroshi HongoHiroshi Hongo More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yota YasumizuYota Yasumizu More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2244AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent evidence suggests CRPC cells may have an unknown regulation system to protect themselves from the androgenic suppressive effect. In this study, we investigated gene expression profiles of androgen-treated cells using a whole human genome microarray, and identified candidate drugs for reprogramming of docetaxel-resistant castration-resistant prostate cancer. METHODS Two cell lines were used: LNCaP and C4-2AT6, a castration-resistant prostate cancer cell line. Gene expression profiles of androgen-treated cells were analyzed and compared using CMAP to identify candidate drugs with the potential to revert an inverse gene signature pattern. RESULTS We investigated and compared the viability of prostate cancer cells treated with DHT at various concentrations. When treated with 0.1 nM DHT, LNCaP cells demonstrated significantly increased cell viability. On the other hand, C4-2AT6 cells showed significantly decreased cell viability when treated with 0.1 nM DHT. We next investigated and compared the gene signatures between LNCaP and C4-2AT6 cells treated with 0.1 nM DHT. CMAP analysis identified candidate drugs for reprogramming the gene expression changes. Based on the CMAP analysis data, candidate drugs were screened and tested for anti-tumor activity. The cytotoxic effect of docetaxel was first investigated by a cell viability assay in LNCaP and C4-2AT6 cells; relative cell viability when treated with 1nM docetaxel was 48.8 ± 0.1 % and 87.5 ± 1.9 %, respectively, suggesting increased docetaxel resistance in C4-2AT6 cells. Among the listed drugs, KOSH035 was identified as a candidate for reprogramming the castration resistance of docetaxel-resistant C4-2AT6 cells. We investigated the anti-tumor activity of KOSH035 on LNCaP and C4-2AT6 cells, and observed that relative cell viability was 72.2 ± 1.2 % and 72.8 ± 4.4 %, respectively. We further investigated the synergic effect of docetaxel and KOSH035. Relative cell viabilities of LNCaP and C4-2AT6 cells after treatment with both 1nM docetaxel and 10uM KOSH035 were 51.4 ± 0.5 % and 68.1 ± 2.0 %, respectively. The combination of docetaxel and KOSH035 showed a synergic effect on CRPC cells. CONCLUSIONS KOSH035 may be promising drug for reprogramming docetaxel-resistant PC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1097 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hiroshi Hongo More articles by this author Takeo Kosaka More articles by this author Yota Yasumizu More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modelling of tumour growth and cytotoxic effect of docetaxel in xenografts

One of the major sources of information on physiological and pathophysiological effects in pre-clinical oncology studies is the xenografted tumour animal model. However, measurement of tumour volume over time potentially masks a range of biological changes that the xenograft is undergoing. In this paper a mechanistic model of tumour growth in xenografts is presented that can be used to investigate the mode of drug action with respect to phenotypic changes. The model encapsulates key histological biomarkers and spatial constraints. The unknown model parameters are first shown to be uniquely identifiable from the proposed experimental studies, and then estimated from the resulting data using the anti-cancer agent docetaxel.

Caffeic acid phenethyl ester synergistically enhances docetaxel and paclitaxel cytotoxicity in prostate cancer cells

Evidence is growing for the beneficial role of selective estrogen receptor modulators (SERM) in prostate diseases. Caffeic acid phenethyl ester (CAPE) is a promising component of propolis that possesses SERM activity. This study aimed at investigating the modulatory impact of CAPE on docetaxel (DOC) and paclitaxel (PTX) cytotoxicity in prostate cancer cells and exploring the possible underlying mechanisms for this chemomodulation. CAPE significantly increased DOC and PTX potency in PC-3, DU-145 and LNCaP prostate cancer cells. Combination index calculations showed synergistic interaction of CAPE/DOC and CAPE/PTX cotreatments in all the tested cell lines. Subsequent mechanistic studies in PC-3 cells indicated that cyclin D1 and c-myc were significantly reduced in the combined treatment groups with concurrent increase in p27kip. DNA-ploidy analysis indicated a significant increase in the percentage of cells in pre-G1 in CAPE/DOC and CAPE/PTX cotreatments. Decreased Bcl-2/Bax ratio together with increased caspase-3 activity and protein abundance were observed in the same groups. Estrogen receptor-β (ER-β) and its downstream tumor suppressor forkhead box O1 levels were significantly elevated in CAPE and combination groups compared to DOC or PTX-alone. ER-α and insulin-like growth factor-1 receptor protein abundance were reduced in the same groups. CAPE significantly reduced AKT, ERK and ER-α (Ser-167) phosphorylation in PC-3 cells. CAPE-induced inhibition of AKT phosphorylation was more prominent (1.7-folds higher) in cells expressing ER-α such as PC-3 compared to LNCaP. In conclusion, CAPE enhances the antiproliferative and cytotoxic effects of DOC and PTX in prostate cancer cells. This can be, at least partly, attributed to CAPE augmentation of DOC and PTX proapoptotic effects in addition to CAPE-induced alterations in ER-α and ER-β abundance.

Abstract 4895: Caveolin-1 modulates docetaxel activity by inducing p53 expression in breast cancer.

Abstract Caveolin-1 is reported to be down-regulated in breast cancers compared with normal mammary tissue. More recent data showed that high caveolin-1 expression is more associated with basaloid cancers than ER and/or PR or HER-2 expressing cancers. Investigatirs have reported that Caveolin-1 promoted resistance to chemotherapy-induced apoptosis in Ewing's sarcoma cells. However, the biologic relevance of caveolin-1 in breast cancer remains unclear. Thus, the aim of this study is to investigate its potential biologic relevance of Caveolin-1 to cell survival after docetaxel chemotherapy, one of the most important drug in breast cancer treatment. In this current study, we first showed that Caveolin-1 protein expression was suppressed in the ER(+) human breast cancer cell lines (MCF-7, T47D, and ZR75-1), but not in triple negative cancer cells (MDA-MB-231 and HS578T) and one of the HER-2 type cell (HCC1954) using Western blot analysis. Better cytotoxic effect of docetaxel was observed in the caveolin-1-expressing triple negative cells and HER-2 type cell than the other caveolin-1-deficient HER-2 type and luminal type of cancer cells. Next, caveolin-1 gene was reintroduced into caveolin-1 deficient luminal type breast cancer cells (MCF-7 and ZR75-1) to investigate its potential modulatory effect on docetaxel-induced cytotoxicity. The cell proliferation and MTT assay showed that the overexpressed caveolin-1 had further modulated docetaxel-induced anti-proliferative and cytotoxic effect in the luminal type cells. The anti-proliferative effect of docetaxel in the caveolin-1 gene over-expressed breast cancer cell lines was accompanied by decreasing cyclin D1 expression and inducing cyclin B1 accumulation, then resulting in cell cycle arrest at G2/M phase in the FACS analysis. In addition, re-introduction of caveolin-1 further increased docetaxel-induced cell death by synergistically inducing p53 expression and subsequent induction of p21, then finally apoptosis of breast cancer cells. These results were verified in the mouse model using SCID mice implated with either Caveolin-1-MCF-7 cells or control MCF-7 cells. When each group of tumor-bearing mice were treated by docetacel, mice with Caveolin-1-MCF-7 tumors showed a signiflcantly smaller tumors than those of control group. These results suggest that caveolin-1 may have a modulating factor for docetaxel activity by inducing p53 expression in breast cancer patients. 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 4895. doi:1538-7445.AM2012-4895

Abstract 2194: SLC5A8, a novel transporter with tumor suppressor function, mediates its effect through survivin depletion

Abstract Introduction: SLC5A8 is a Na+-coupled transporter for lactate, pyruvate, nicotinate and ketone bodies and is down-regulated in a wide variety of cancers. It is expressed in normal cells whereas tumor cells suppress its expression by methylation of CpG islands in its promoter. Studies from our laboratory have shown that (i) pyruvate (but not lactate) is a tumor suppressor through its ability to inhibit HDAC1/3, and that ectopic expression of SLC5A8 in breast cancer cells suppresses expression of the anti-apoptotic protein survivin. Apart from its role in protecting tumor cells from apoptosis, survivin also serves as an important determinant of chemoresistance. Based on these findings, we hypothesized that forced expression of SLC5A8 in breast cancer cells will deplete survivin and consequently enhance chemosensitivity. Methods: The estrogen receptor-positive human breast cancer cell line MCF7 does not express SLC5A8. These cells were engineered to express SLC5A8 by a doxycycline-inducible promoter. The expression of the transporter was monitored by Na+-coupled nicotinate uptake and by immunocytochemistry. The interaction between SLC5A8 and survivin was investigated by immunocytochemical analysis for co-localization. The chemosensitivity was studied by monitoring the cytotoxic effects of docetaxel in SLC5A8-negative control cells and SLC5A8-expressing cells. Results: MCF7 cells transfected with vector alone did not show Na+-coupled nicotinate uptake, indicating the absence of functional expression of SLC5A8. In contrast, MCF7 cells transfected with the SLC5A8 construct under a doxycycline-inducible promoter exhibited marked activity for Na+-coupled nicotinate uptake but only when exposed to doxycycline. This was complemented with immunocytochemical evidence of SLC5A8 protein expression in the plasma membrane. In SLC5A8-expressing control MCF7 cells, survivin was expressed robustly and the expression was restricted predominantly to the nucleus. In contrast, in SLC5A8-expressing MCF7 cells, the cellular levels of survivin were depleted. Furthermore, the location of survivin was shifted from the nucleus to the plasma membrane. Co-localization studies revealed merging of immuno-positive signals for SLC5A8 and survivin at the plasma membrane. Chemosensitivity studies showed that SLC5A8-expressing MCF7 cells were more susceptible to docetaxel than SLC5A8-negative control MCF7 cells. Conclusions: MCF7 cells are SLC5A8-negative, but express survivin with its localization found predominantly in the nucleus. Ectopic expression of SLC5A8 in these cells depletes survivin as well as changes the localization of survivin from the nucleus to the plasma membrane through interaction with SLC5A8. This is accompanied with a marked increase in chemosensitivity. 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 2194. doi:10.1158/1538-7445.AM2011-2194

Abstract PD02-09: Effects of an mTOR Inhibitor RAD001 on Human Breast Cancer Stem Cells In Vitro

Abstract Background: Recent evidences have suggested that there are a rare population of cells called cancer stem cells (CSCs) in breast cancer, which have the ability of extensive self-renewal and contribute to metastasis and treatment resistance. This study evaluated the effects of an mTOR inhibitor, RAD001 (Everolimus) on breast CSCs of primary breast cancer cells and 2 breast cancer cell lines (MCF-7, MDA-MB-231) in vitro. Methods: Primarily, we isolated primary breast cancer cells from the 1cm3 axenic tissue from breast cancer patients, which was been digested in collagenase typelV in 1-3 hours. After filtration, the cells from the organoid were plated in DMEM/F12(1:1) containing 10% FBS ,100U/ml penicillin,100µg/ml streptomycin and some growth factors overnight. The next day, we sorted CD44 +, CD24-/low, ESA + cells as breast CSCs by flow cytometry from primary breast cancer cells, breast cancer cell lines MCF-7 and MDA-MB-231. And then the three CSCs were treated with different concentrations of docetaxel alone(0,20µM,40µM,80µM, 160µM), mTOR inhibitor RAD001 alone (0,10nM,100nM,1µM,10µM), or in combination with docetaxel(20µM).the Inhibition of the drugs on different Breast CSCs was quantified by methyl thiazolyl tetrazolium (MTT) assay. All employed CSCs were divided into four groups, given respectively 24h treatment of RAD001(100nM), docetaxel(20µM), combination or contrals. Apoptosis and distribution of cell cycles were examined with flow cytometry. Experiments were performed in triplicate. Result: All three kinds of breast CSCs were resistant to the standard treatment doses of docetaxel in vitro(IC50=80 µM±5µM),compared with this drug inhibited normal breast cancer cell lines(IC50=16µM±2µM). Treatment with RAD001 resulted in growth inhibition of all employed CSCs in a dose-dependent manner, which is more effective than the treatment with docetaxel alone(P&amp;lt;0.001), and CSCs of MDA-MB-231 proved to be most sensitive to this drug(IC20=8nM).Combination studies showed that there was an additive growth inhibitory effect of a combination treatment on three CSCs in vitro compared with treatment with RAD001 alone (P&amp;lt;0.001)or docetaxel alone(P&amp;lt;0.001). In addition, an increase in G2-M cell cycle arrest were seen in the combination treatment group when compared with controls(P&amp;lt;0.05), suggesting that cell cycle arrest may contribute to the increased growth inhibitory effects of combination treatment seen in this study. In primary CSCs, combined treatment induced a mount-up population of early apoptosis, the phenomenon was not taken place in the treatment of RAD001 alone, however, it's no statistical significance(P&amp;gt;0.05). Discussion: We conclude that RAD001 has more effective inhibitory effect than docetaxel, and enhances the cytotoxic effects of docetaxel in models of breast CSCs in vitro by inducing cell cycle arrest, indicating that combination treatment with RAD001 and docetaxel may be an efficient therapy for refractory and metastatic breast cancer. But the drug interaction of RAD001 and docetaxel in the clinical situation has to be evaluated in further studies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-09.

6642 Gap junctional intercellular communication influences the cytotoxic effect of docetaxel in esophageal cancer cells

6642 Gap junctional intercellular communication influences the cytotoxic effect of docetaxel in esophageal cancer cells

Molecular mechanisms of underlying synergistic effect of combinations of histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) with docetaxel and trastuzumab against human breast cancer cells

560 Background: Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor (HDI), which inhibits class I and II HDACs, induces hyperacetylation of the core nucleosomal histones, increases p21WAF1, and induces growth arrest and apoptosis of human breast cancer cells. In the present studies, we determined the effects of SAHA on Her-2, AKT and c-Raf (all hsp90 client proteins) levels, as well as on the cytotoxic effects of docetaxel and trastuzumab on human breast cancer BT-474 and SKBR-3 cells containing Her-2 amplification. Methods: The cells were treated with SAHA (1.0 to 5.0 μM) and/or trastuzumab (10 to 20 μg/ml) or docetaxel (5 to 20 nM) for 24 to 48 hours. Following this, % of apoptotic cells was determined or Western blot analyses of p21, p27, acetylated histones H3 and H4, AKT, c-Raf, and Her-2, as well as of Bcl-2, Bcl-xL, Bak, Bax, Bim, XIAP and survivin were performed. Attenuation of HDAC6 levels was achieved by siRNA to HDAC6 and estimated by immunoblot analysis. Synergistic interaction between drugs was evaluated by median dose-effect and isobologram analysis. Results: Treatment with SAHA upregulated p21 and p27 levels, increased the % of cells in G2M phase of the cell cycle, as well as induced apoptosis of the cells. This was associated with upregulation of pro-death Bak and Bim, as well as attenuation of the levels of pro-survival XIAP, survivin, Bcl-2, Bcl-xL and Her-2 proteins. SAHA treatment induced acetylation of hsp90. Attenuation of HDAC6 levels by siRNA to HDAC6 also caused acetylation of hsp90, indicating the mechanistic role of HDAC6 in SAHA-mediated acetylation of hsp90. This reduced the chaperone association of Her-2 with hsp90, which promoted the degradation of Her-2 by the proteasome. Co-treatment with SAHA and docetaxel or trastuzumab exerted synergistic apoptotic effects. Co-treatment with SAHA and trastuzumab caused greater attenuation of Her-2, p-AKT, as well as greater induction of p27 and Bim. Conclusions: These pre-clinical findings support the development of SAHA in combination with docetaxel and/or trastuzumab against Her-2 amplified breast cancer. No significant financial relationships to disclose.

Decreased response to paclitaxel versus docetaxel in HER-2/neu transfected human breast cancer cells.

Taxanes are effective in the treatment of metastatic breast cancer. Docetaxel has been shown to be more potent than paclitaxel in inducing bcl-2 phosphorylation and apoptosis and is clinically active in some paclitaxel-resistant breast tumors. HER-2/neu overexpression has been shown to correlate with resistance to hormonal therapy as well as chemotherapy. Using a HER-2/neu transfected MCF-7 human breast cancer cell line, we investigated the role of HER-2/neu overexpression on resistance to paclitaxel and docetaxel treatment. A control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were treated with various concentrations of docetaxel or paclitaxel. Cell number was assessed using the MTT tetrazolium dye assay. In the control vector transfected MCF/neo cell line, paclitaxel and docetaxel gave similar dose-dependent growth inhibition ( p = 0.175). In HER-2/neu transfected MCF/18 cells, docetaxel treatment resulted in a dose-dependent inhibition similar to that seen in MCF/neo cells. Paclitaxel, however, gave significantly less growth inhibition than docetaxel in the HER-2/neu overexpressing MCF/18 cells (p = 0.0003). These data suggest that HER-2/neu overexpression may contribute to paclitaxel resistance. In contrast, the cytotoxic effects of docetaxel in these breast carcinoma cells are not affected by HER-2/neu expression. Therefore, docetaxel may be the preferred taxane therapy in HER-2/neu overexpressing breast tumors.

Cytochrome P450 CYP1B1 protein expression:: A novel mechanism of anticancer drug resistance

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line ( P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.