Docetaxel is a chemotherapy drug to treat breast cancer, however as with many chemotherapeutic drugs resistance to docetaxel occurs in 50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through microRNAs (miRNA) has been shown to play an important role in cancer drug resistance. By directly targeting mRNA, miRNAs are able to inhibit genes that are necessary for signalling pathways or drug induced apoptosis rendering cells drug resistant. This study investigated the role of differential miRNA expression in two in vitro breast cancer cell line models (MCF-7, MDA-MB-231) of acquired docetaxel resistance. MiRNA microarray analysis identified 299 and 226 miRNAs altered in MCF-7 and MDA-MB-231 docetaxel-resistant cells, respectively. Docetaxel resistance was associated with increased expression of miR-34a and miR-141 and decreased expression of miR-7, miR-16, miR-30a, miR-125a-5p, miR-126. Computational target prediction revealed eight candidate genes targeted by these miRNAs. Quantitative PCR and western analysis confirmed decreased expression of two genes, BCL-2 and CCND1, in docetaxel-resistant cells, which are both targeted by miR-34a. Modulation of miR-34a expression was correlated with BCL-2 and cyclin D1 protein expression changes and a direct interaction of miR-34a with BCL-2 was shown by luciferase assay. Inhibition of miR-34a enhanced response to docetaxel in MCF-7 docetaxel-resistant cells, whereas overexpression of miR-34a conferred resistance in MCF-7 docetaxel-sensitive cells. This study is the first to show differences in miRNA expression, in particular, increased expression of miR-34a in an acquired model of docetaxel resistance in breast cancer. This serves as a mechanism of acquired docetaxel resistance in these cells, possibly through direct interactions with BCL-2 and CCND1, therefore presenting a potential therapeutic target for the treatment of docetaxel-resistant breast cancer.

Docetaxel is an effective chemotherapy drug to treat breast cancer but the underlying molecular mechanisms of drug resistance are not fully understood. DNA methylation is an epigenetic event, involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance. To investigate the role of DNA methylation in docetaxel resistance in breast cancer we used two human breast cancer cell lines (MCF-7 and MDA-MB-231) that were made resistant to docetaxel. Docetaxel-resistant sub-lines were treated with different concentrations of decitabine. Global methylation and DNA methyltransferase (DNMT) activity was measured using an ELISA-based assay. Quantitative real-time PCR was used to study DNMT gene expression. Cell viability was studied by MTT assay. Global methylation was increased in MCF-7 but not significantly changed in MDA-MB-231 docetaxel-resistant cells. Decreased DNMT activity and decreased DNMT1 and DNMT3b mRNA expression was associated with docetaxel resistance in both cell lines. To investigate how the components of the DNA methylation machinery may contribute towards docetaxel resistance, decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methylation, was used. Decitabine treatment decreased global methylation, DNMT activity and DNMT1, DNMT3a and DNMT3b mRNA expression in MDA-MB-231 docetaxel-resistant cells. In contrast, decitabine-treated MCF-7 docetaxel-resistant cells showed increased DNMT1, DNMT3a and DNMT3b mRNA expression indicating a cell line specific effect of decitabine. Decitabine treatment increased resistance in MCF-7 docetaxel-resistant cells and in the parental MCF-7 and MDA-MB231 docetaxel-sensitive cell lines, however, it did not alter response to docetaxel in MDA-MB-231 docetaxel-resistant cells. This study demonstrates that changes in the DNA methylation machinery are associated with resistance to docetaxel in breast cancer cells. The use of epigenetic therapies, as a strategy to overcome drug resistance, needs to be investigated more fully to determine their effectiveness in different cancers and for different chemotherapy drugs.

To quantify gene expression levels, appropriate controls have to be used to adjust for experimental variation. Endogenous control genes are widely used as they are stably expressed independent of cell cycle and experimental conditions, however, they can be altered upon drug treatment. DNA methylation is widely studied in chemotherapy drug resistance and the DNA methylation inhibitor decitabine showed promising results reversing drug resistance in cancer. We aimed to investigate the effect of different decitabine concentrations on the expression of selected endogenous control genes (GAPDH, 18S rRNA, PPIA, RPL13A, OAZ1) in two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) compared to untreated cells. In MCF-7 cells, 18S rRNA remained stable, however, GAPDH, PPIA and OAZ1 gene expression was increased after treatment. RPL13A was stably expressed at 8 muM decitabine but was increased at lower drug concentrations. In MDA-MB-231 cells, GAPDH levels remained relatively stable following decitabine treatment and so was PPIA expression at low decitabine concentrations. Decitabine increased 18S rRNA, RPL13A and OAZ1 gene expression. In this study, we observed cell line specific effects of decitabine and suggest that 18S rRNA is most suitable to use in MCF-7 cells, while GAPDH is recommended to use in MDA-MB-231 cells during decitabine treatment.

BACKGROUND & AIMS: Immunonutrition, containing arginine as a key component, has been shown to enhance the immune system and significantly reduce infectious complications in patients undergoing upper gastrointestinal surgery. Arginine, however, may also influence tumour cell behaviour. The aim of this study was to investigate the effects of arginine on tumour cell growth, invasion and modulation of expression of genes involved in these aspects of cell behaviour. METHODS: A human gastric cancer cell line (AGS) was grown in vitro and supplemented with arginine (2, 4, 8, 16 and 32mM) for 24, 48 and 72h. The effect of arginine on cell growth (MTT assay), apoptosis (DAPI staining), invasion (Matrigel assay), gene expression (cDNA microarray analysis and RT-PCR) and protein expression (western analysis) was determined. RESULTS: These studies demonstrated that arginine caused a decrease in AGS cell growth via induction of apoptosis. Whilst arginine decreased cell growth, no significant effect on the invasive potential of AGS cells was noted. Subsequent gene expression analysis demonstrated that arginine increased the expression of caspase 8, which was validated at the protein level. CONCLUSIONS: These results suggest that that inhibition of AGS cell growth by arginine is mediated through caspase 8 activation of apoptosis.

BACKGROUND: Evidence is growing for beneficial interactions between omega-3 fatty acids from fish and chemotherapy agents in certain human cancers. Evidence for similar effects in prostate cancer is lacking. We investigated the effects of docosahexaenoic acid (DHA-22:6n-3), a component of fish oil, on the cytotoxicity of docetaxel in prostate cancer cells. METHODS: Cell viability was studied using the MTT assay and apoptosis was evaluated by flow cytometry using PI, annexin V, and JC-1 staining. Cellular signaling mechanisms that might explain the observed pro-apoptotic effects were investigated using NF-kappaB pathway specific cDNA microarrays and RT-PCR validation. RESULTS: DHA enhanced the pro-apoptotic efficacy of docetaxel, synergistically, in hormone receptor positive and negative LNCaP, DU145 and PC3 cells, respectively. Cell cycle analysis showed an increase in G2M arrest and JC-1 staining showed a significant (P < 0.018) increase in apoptotic cells with combination treatments in LNCaP cells. Microarray and RTPCR showed decreased expression of FADD, AKT1, MAX, TRAF3, MAP2k4, TNFRSF11A, and RIPK1 in LNCaP cells. Similar results were obtained with DU145 cells; combinations were more effective than single treatments. Combination treatments suppressed NF-kappaB signaling that was induced by docetaxel alone; this is considered an anti-apoptotic response. CONCLUSION: DHA synergistically enhanced the cytotoxic effect of docetaxel in prostate cancer cells through increased apoptosis by suppression of genes involved in the NF-kappaB pathway. This highlights the possibility of developing such combination modalities for treatment of prostate cancer. Prostate (c) 2008 Wiley-Liss, Inc.

Response rates of tumours to docetaxel (DOCT) are 45-60% in advanced breast cancer but problems associated with side effects, drug resistance and high costs occur. Conjugated linoleic acids (CLAs) also have anti-tumorigenic activity that elicits similar changes in oncogene expression to DOCT and could augment DOCT efficacy. CLA isomers appear to differ in cytotoxicity toward cancer cells. Effects of two CLA isomers on cytotoxicity of DOCT in breast cancer cells (MCF-7; MDA-MB-231) in vitro were assessed. Cells were incubated up to 72h with 40muM each of LA or CLA isomers (cis-9, trans-10 CLA, or trans-10, cis-12 CLA) or a 50:50 isomer mix, alone or with DOCT (0-64muM); a pilot study determined IC(50) and IC(70) concentrations. Treatments were concurrent (CLA and DOCT together) or sequential (CLA then DOCT). MTT assay determined cell viability. Trans-10, cis-12 CLA was the most effective fatty acid (P<0.001) and increased with treatment time. IC(50) and IC(70) concentrations of DOCT were determined, concurrently or sequentially, with and without fatty acids, in the two cell types. Concurrent treatment with trans-10, cis-12 CLA and DOCT augmented inhibition of cell growth in one or both cell lines (decreased IC(50) and IC(70) in MCF-7; P<0.05 but only IC(50) in MDA-MB-231; P<0.05). CLA mix reduced IC(50) and IC(70) in MDA-MB-231 (P<0.001) but not in MCF-7. Cis-9, trans-11 CLA and LA had no effect. Sequential treatment with CLAs then DOCT reduced IC(50) and IC(70) in MCF-7 but not in MDA-MB-231. The latter had increased IC(50) and IC(70) with LA treatment (P<0.05) and increased IC(70) with cis-9, trans-11 CLA (P<0.05) with sequential but not concurrent treatment. Longer pre-incubation times with trans-10, cis-12 CLA (24-72h) elicited greater reductions in IC(50) and IC(70) in MCF-7 cells. Results show that CLA isomers augment anti-tumour effects of docetaxel in breast cancer cells and suggest possible dual treatment regimens.

This study aimed to evaluate patterns of local and distant disease recurrence in patients having primary chemotherapy and compared patterns of relapse in patients with a complete pathological response with those who had residual breast disease. This is an observational study using a sequential series of patients treated with primary chemotherapy. They were followed up for a minimum of 5 years. All data was collected prospectively. Three hundred forty-one consecutive patients with breast cancer were treated with up to eight cycles of doxorubicin-based chemotherapy. Clinical and pathological response rates were evaluated and patients were followed up for disease recurrence (local and distant) and overall survival. Fifty-two patients (16.5%) had a complete pathological response to chemotherapy. Distant disease recurrence occurred in nine patients (17.3%) but no local recurrence was observed. In patients not having a complete pathological response, 86 patients (32.6%) subsequently developed metastases. Local recurrence of disease occurred in 12 (4.5%). There was a statistically significant difference in overall survival between patients whose tumours had a complete pathological response compared with patients who had residual disease in the breast following chemotherapy (88% versus 70% at 5 years, p = 0.036). Following primary chemotherapy, about 84% of patients had residual disease in the breast. Surgery is necessary to ensure complete removal of residual tumour and excellent rates of local control are achievable. A complete pathological response is associated with fewer local and distant recurrences as well as improved survival although there are no differences in time to development of metastatic relapse.

The understanding of the role of nutrition in the surgical patient has lead to major developments in the nutritional support of patients undergoing surgery. Reductions in morbidity by ensuring that patients receive optimal nutritional support can be achieved. Furthermore, the use of nutrients to modify immune, inflammatory and metabolic processes also offers new possibilities for reducing morbidity following major surgery. However, we are only at an embryonic stage in our understanding of how nutrients and nutrition affect the genome and this knowledge offers exciting possibilities in the future for modulating many key intracellular processes, particularly in the patient with cancer.

Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the beta-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in beta-tubulin isotypes in vitro and has sequenced the beta-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231,(oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of beta-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the beta-tubulin gene. At the mRNA level, class I, II, III, and IVa beta-tubulin mRNA isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI beta-tubulin mRNA isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa beta-tubulin mRNA. However, there were increased expressions in class II, IVb, and VI beta-tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in beta-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III beta-tubulin. This study demonstrates that altered expression of mRNA beta-tubulin isotypes and modulation of beta-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. In this study high-level, docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) were created, and comparative genomic hybridisation was used to identify genomic regions associated with resistance to docetaxel. MCF-7 resistant cells showed an amplification of chromosomes 7q21.11-q22.1, 17q23-q24.3, 18, and deletion of chromosomes 6p, 10q11.2-qter and 12p. MDA-MB-231 resistant cells showed a gain of chromosomes 5p, 7q11.1-q35, 9, and loss of chromosomes 4, 8q24.1-qter, 10, 11q23.1-qter, 12q15-q24.31, 14q and 18. Whole chromosome paints confirmed these findings. Amplification of 7q21 and loss of 10q may represent a common mechanism of acquired docetaxel resistance in breast cancer cells. This study is the first description of a genomic approach specifically to identify genomic regions involved in resistance to docetaxel.