Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance

Identification and characterization of the ecdysteroid UDPglucosyltransferase gene of the \u3ci\u3eLymantria dispar\u3c/i\u3e multinucleocapsid nuclear polyhedrosis virus

We have located, cloned, sequenced and characterized the ecdysteroid UDP-glucosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus,(LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) disrupts the hormonal balance of the host larva by galactosylating ecdysone, which prevents moulting. The location of the LdMNPV egt gene, determined by hybridization analysis using a cloned coding segment of the AcMNPV egt gene, was mapped to between 79.1 and 80.2 map units on the viral genome. This region contains an open reading frame of 1464 nucleotides capable of encoding a 55K polypeptide. This predicted protein exhibits a 42% amino acid identity with the AcMNPV egt polypeptide. Transcripts of the egt gene were analysed by Northern blot and primer extension. The egt gene is transcribed from approximately 12 to 48 h, and maximally at about 16h post-infection. Transcription occurred in the presence of aphidicolin, a viral DNA synthesis inhibitor, but not in the presence of cycloheximide, a protein synthesis inhibitor. Therefore the LdMNPV egt gene is classified as a delayed early gene. The egt gene is transcribed in a clockwise direction with respect to the circular map, and transcription initiates at a single site. Comparisons between the two baculoviral egt proteins and mammalian UDP-glucuronosyltransferases reveal areas which are conserved between the mammalian and baculoviral genes, as well as areas that are only conserved in the viral egt proteins. The LdMNPV protein sequence appears to include a signal peptide, which would allow the protein to be secreted into the haemolymph

Error-promoting DNA synthesis in ovarian cancer cells

AbstractObjectiveThe objective of this study is to determine whether an altered DNA replication process is responsible for some of genetic damage observed in ovarian cancer.MethodsThe replication fidelity of the DNA synthetic process was evaluated in both malignant and non-malignant human ovarian cells. The types of replication errors produced were identified. In addition, kinetic analyses of the efficiency of ovarian cancer DNA polymerases for misincorporating nucleotides were performed.ResultsWe report for the first time that ovarian cancer cells harbor an error promoting DNA replication apparatus which contributes to the decrease in DNA synthetic fidelity exhibited by these cells. Our study also shows that the decrease in DNA replication fidelity was not a result of an increased DNA replication activity. In addition, it was observed that the higher rate of DNA replication errors does not result in significant differences in the type of DNA replication-errors made during the DNA replication process; just the relative abundance. A detailed kinetic analysis of the efficiency of misincorporating nucleotides demonstrated that the DNA polymerases within the ovarian cancer cells exhibited a significant propensity for creating purine–pyrimidine nucleotide mismatches relative to non-malignant ovarian cells, while being only slightly more efficient at incorrectly pairing a purine nucleotide with a purine nucleotide.ConclusionsAll together, these data suggest that the systematic analysis of the DNA replication process in ovarian cancer could uncover information on some of the molecular mechanisms that drive the accumulation of genetic damage, and probably contribute to the pathogenesis of the disease

Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance.

Tumor Necrosis Factor alpha (TNF-α) has been shown to be released by tumor cells in response to docetaxel, and lipopolysaccharides (LPS), the latter through activation of toll-like receptor 4 (TLR4). However, it is unclear whether the former involves TLR4 receptor activation through direct binding of the drug to TLR4 at the cell surface. The current study was intended to better understand drug-induced TNF-α production in tumor cells, whether from short-term drug exposure or in cells selected for drug resistance. ELISAs were employed to measure cytokine release from breast and ovarian tumor cells in response to several structurally distinct chemotherapy agents and/or TLR4 agonists or antagonists. Drug uptake and drug sensitivity studies were also performed. We observed that several drugs induced TNF-αrelease from multiple tumor cell lines. Docetaxel-induced cytokine production was distinct from that of LPS in both MyD88-positive (MCF-7) and MyD88-deficient (A2780) cells. The acquisition of docetaxel resistance was accompanied by increased constitutive production of TNF-αand CXCL1, which waned at higher levels of resistance. In docetaxel-resistant MCF-7 and A2780 cell lines, the production of TNF-α could not be significantly augmented by docetaxel without the inhibition of P-gp, a transporter protein that promotes drug efflux from tumor cells. Pretreatment of tumor cells with LPS sensitized MyD88-positive cells (but not MyD88-deficient) to docetaxel cytotoxicity in both drug-naive and drug-resistant cells. Our findings suggest that taxane-induced inflammatory cytokine production from tumor cells depends on the duration of exposure, requires cellular drug-accumulation, and is distinct from the LPS response seen in breast tumor cells. Also, stimulation of the LPS-induced pathway may be an attractive target for treatment of drug-resistant disease

Basal cytokine production changes during selection for resistance to docetaxel.

<p>Levels of TNF-α (A and B), CXCL8 (C and D), and CXCL1 (E and F) in media as measured by ELISA, from MCF-7 (A, C, and E) and A2780 cells (B, D, and F) after 72 hours of cell culture. Cell lines to the right of the vertical broken grey line exhibit acquired resistance to docetaxel, as confirmed in clonogenic assays. Each value represents the mean of three replicates (+/-SEM). Two-tailed T-tests were used to assess the significance of differences in cytokine levels between docetaxel-selected cell lines and their respective co-cultured control cell lines (MCF-7_CC10 or A2780_CC12); ***p<0.0001, **p<0.001, *p<0.01.</p

The effect of TLR4 inhibition on secreted TNF-α levels induced by docetaxel or LPS.

<p>MDA-MB-231 cells were treated for 72 hours with either 2.5 nM docetaxel (TXT) or 0.1 μg/ml LPS, after pretreatment with either 100 μg/ml LPS-RS (A) or 0.1 μg/ml TAK-242 (B). The data represent the mean of three replicates (+/-SEM). The significance of differences between treated and untreated cells were assessed using a two-tailed T-test; ***p<0.001, **p<0.01, *p<0.02.</p

Media cytokine profiles for tumor cells after LPS or docetaxel exposure.

<p>The levels of TNF-α (A and B), CXCL8 (C and D), and CXCL1 (E and F) in cell media of either MCF-7 (A, C and E) or A2780 (B, D and F) cells were measured by ELISA after 72 hours of LPS or docetaxel treatment. The data represents the mean of three replicates (+/-SEM). All treatments were 10 μg/ml and 2.5 nM for LPS and docetaxel, respectively; The significance of differences in TNF-α levels between treated and untreated cells were determined using a two-tailed T-test; *** for p<0.0001, ** for p<0.01, and * for p<0.01.</p

Levels of TLR4 and adaptor proteins during acquisition of resistance to docetaxel.

<p>Immunoblots were performed from extracts of MCF-7 and A2780 drug-naive and drug-resistant cell lines (A and B) in order to confirm the presence or absence of TLR4 and adaptor proteins MyD88 and TRIF. Changes in protein levels of drug-naive and drug-resistant cell lines were assessed by densitometry (C, D, E, and F). Statistical analysis consisted of two-tailed T-tests; *p<0.05.</p

Effect of Marimastat on TNF-α levels in the medium of MCF-7_TXT10 cells.

<p>Cells were treated for 72 hours with media only (NT), 200 nM Marimastat, 2.5 nM docetaxel (TXT), 10 μg/ml LPS, or a combination thereof. The data represents the mean of three replicates (+/-SEM) and the significance of differences in TNF-α levels between treatments was determined using a T-test; *p<0.01, **p<0.001, ***p<0.0001.</p