A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer

Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration‐resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen‐insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel‐resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel‐resistant (DU145‐TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145‐TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial‐to‐mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans‐differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients. J. Cell. Biochem. 114: 1286–1293, 2013. © 2012 Wiley Periodicals, Inc.     

Lin28 mediates paclitaxel resistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.     

BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability

The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.     

Differential Response to α-Oxoaldehydes in Tamoxifen Resistant MCF-7 Breast Cancer Cells

Tamoxifen is the standard adjuvant endocrine therapy for estrogen-receptor positive premenopausal breast cancer patients. However, tamoxifen resistance is frequently observed under therapy. A tamoxifen resistant cell line has been generated from the estrogen receptor positive mamma carcinoma cell line MCF-7 and was analyzed for putative differences in the aldehyde defence system and accumulation of advanced glycation end products (AGE). In comparison to wt MCF-7 cells, these tamoxifen resistant cells were more sensitive to the dicarbonyl compounds glyoxal and methylglyoxal and displayed increased caspase activity, p38-MAPK- and IκBα-phosphorylation. However, mRNA accumulation of the aldehyde- and AGE-defence enzymes glyoxalase-1 and -2 (GLO1, GLO2) as well as fructosamine-3-kinase (FN3K) was not significantly altered. Tamoxifen resistant cells contained less free sulfhydryl-groups (glutathione) suggesting that the increased sensitivity towards the dicarbonyls was due to a higher sensitivity towards reactive oxygen species which are associated with dicarbonyl stress. To further analyse, if these data are of more general importance, key experiments were replicated with tamoxifen resistant MCF-7 cell lines from two independent sources. These cell lines were also more sensitive to aldehydes, especially glyoxal, but were different in their cellular signalling responses to the aldehydes. In conclusion, glyoxalases and other aldehyde defence enzymes might represent a promising target for the therapy of tamoxifen resistant breast cancers.     

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR‐145‐5p‐mediated regulation of AKAP12

Our present work was aimed to study on the regulatory role of MALAT1/miR‐145‐5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX‐resistant PCa cell lines (DU‐145‐DTX and PC‐3‐DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT‐PCR analysis was performed to measure MALAT1 expression in DTX‐sensitive and DTX‐resistant tissues/cells. The human DTX‐resistant cell lines DU145‐PTX and PC3‐DTX were established as in vitro cell models, and the expression of MALAT1, miR‐145‐5p and AKAP12 was manipulated in DTX‐sensitive and DTX‐resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual‐luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR‐145‐5p, as well as between miR‐145‐5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR‐145‐5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up‐regulated in clinical DTX‐resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR‐145‐5p as a target of MALAT1. MiR‐145‐5p overexpression in PC3‐DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR‐145‐5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX‐chemoresistance in vivo. There was an lncRNA MALAT1/miR‐145‐5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy.     

Mathematical deconvolution uncovers the genetic regulatory signal of cancer cellular heterogeneity on resistance to paclitaxel

Drug resistance remains a major problem in combating malignancies, resulting critical the resistance to paclitaxel used in the treatment of many different cancers. Elucidating the cellular heterogeneity composition of tumours may be relevant to designing more effective treatment strategies on drug resistance. In particular, such heterogeneity correlates with the measurement of gene expression below the population level. However, experimental assays capturing differential response are limited and cannot discern the variation in gene expression specific to different cellular types in tumour populations. These limitations led us to consider a mathematical modelling approach, in which the gene expression of cellular subpopulations is recovered by deconvolution. Mathematically, the deconvolution is a multi-linear regression-based problem. We combined herein data on cellular subpopulation frequency composition with gene expression values from 16 tumour lines (8 resistant and 8 sensitive to paclitaxel treatment) to find genes that are differentially expressed between paclitaxel resistant and paclitaxel sensitive tumour lines in different cellular subpopulations. The results indicate that many genes differentially expressed between paclitaxel resistant and sensitive cancer lines are only detected when considering their heterogeneous cellular composition. Overall, our methodology is thought to keep in mind phenotypic heterogeneity improving our resolution in the identification of biomarkers on resistance to chemo-therapeutic agents.     

miR-17-5p Downregulation Contributes to Paclitaxel Resistance of Lung Cancer Cells through Altering Beclin1 Expression

Non- small- cell lung cancer (NSCLC) is one of the most leading causes of cancer-related deaths worldwide. Paclitaxel based combination therapies have long been used as a standard treatment in aggressive NSCLCs. But paclitaxel resistance has emerged as a major clinical problem in combating non-small-cell lung cancer and autophagy is one of the important mechanisms involved in this phenomenon. In this study, we used microRNA (miRNA) arrays to screen differentially expressed miRNAs between paclitaxel sensitive lung cancer cells A549 and its paclitaxel-resistant cell variant (A549-T24). We identified miR-17-5p was one of most significantly downregulated miRNAs in paclitaxel-resistant lung cancer cells compared to paclitaxel sensitive parental cells. We found that overexpression of miR-17-5p sensitized paclitaxel resistant lung cancer cells to paclitaxel induced apoptotic cell death. Moreover, in this report we demonstrated that miR-17-5p directly binds to the 3′-UTR of beclin 1 gene, one of the most important autophagy modulator. Overexpression of miR-17-5p into paclitaxel resistant lung cancer cells reduced beclin1 expression and a concordant decease in cellular autophagy. We also observed similar results in another paclitaxel resistant lung adenosquamous carcinoma cells (H596-TxR). Our results indicated that paclitaxel resistance of lung cancer is associated with downregulation of miR-17-5p expression which might cause upregulation of BECN1 expression.     

miR-337-3p and its targets STAT3 and RAP1A modulate taxane sensitivity in non-small cell lung cancers

NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC.     

Characterization of two independent, exposure-time dependent paclitaxel-resistant human ovarian carcinoma cell lines

This experiment was conducted to address the question of whether acquired paclitaxel resistance is dependent upon whether it is given as a single brief exposure or as a long-term exposure. PX2 and PX24 were established from 2008 human ovarian cancer cells by 2-h single exposure or 24-h continuous exposure to paclitaxel. PX2 acquired paclitaxel resistance faster than PX24 by twofold. Drug resistant pattern was exposure-time dependent. In 2-h exposure, PX2 showed 53.86 ± 4.96 (mean ± standard deviation [SD]) fold paclitaxel resistance while PX24 showed 9.51 ± 1.01 fold resistance (P = 0.002). In 24-h exposure, PX2 showed 2.31 ± 0.3 fold paclitaxel resistance while PX24 showed 28.17 ± 0.98 fold resistance (P = 0.040). PX2 and PX24 acquired cross-resistance to docetaxel and SN38 and the resistance degrees were significantly higher in PX2 than PX24. They displayed approximately twofold cisplatin collateral sensitivity. PX24 also displayed sensitivity to other platinum drugs, oxaliplatin and ZD0473, whereas PX2 acquired significant resistance to both of them. Although differential tubulin-isotype expressions were noted among 2008, PX2 and PX24, they were not significant. In electron microscopy, prominent, densely stained lysosomes were observed more in the resistant cells than 2008. Two independent, exposure-time dependent paclitaxel-resistant human ovarian carcinoma cell lines were established. Understanding the characteristics of the differential resistance pattern could be clinically beneficial for the selection of second line chemotherapy for relapsed ovarian cancer.     

Rapid assessment of drug resistance of cancer cells to gefitinib and carboplatin using optical imaging

The overall goal of this study was to evaluate optical molecular imaging approaches to determine the drug response of chemotherapy and molecular targeted agents in drug sensitive and drug resistant cell lines. The optical molecular imaging approaches selected in this study were based on changes in intracellular uptake and retention of choline and glucose molecules. The breast cancer cell lines were treated with a molecular targeted anti-EGFR therapy. The bladder cancer cell lines were treated with a conventional chemotherapy approach. Sensitivity of optical molecular imaging approach was also compared with conventional cell viability and cell growth inhibition assays. Results demonstrate that optical molecular imaging of changes in intracellular uptake of metabolites was effective in detecting drug susceptibility for both molecular targeted therapy in breast cancer cells and chemotherapy in bladder cancer cells. Between the selected metabolites for optical molecular imaging, changes in glucose metabolic activity showed higher sensitivity in discrimination between the drug sensitive and drug resistant cell lines. The results demonstrated that optical molecular imaging approaches more significantly sensitive as compared to the conventional cell viability and growth assays. Overall, the results demonstrate potential of optical molecular imaging of metabolic activity to improve sensitivity of in-vitro drug response assays.     

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.     

Codominance of cisplatin resistance in somatic cell hybrids

Intrinsic or acquired resistance to cisplatin in cancer cells remains a major obstacle to successful chemotherapy. The clinically relevant genetic and molecular mechanisms of resistance have not yet been identified. Cisplatin-resistant (CP-r) human KB epidermoid carcinoma cell lines (HeLa) resistant to varying levels of cisplatin after single and multiple selection steps are cross-resistant to other platinum compounds and to methotrexate. Intraspecies hybrids of the sensitive and KB CP-r cells were fused with HeLa D98(OR) CP-s, hypoxanthine-aminopterin-thymidine (HAT) sensitive, ouabain resistant, to determine whether cisplatin resistance is dominant or recessive. Cell-cell hybridization between the sensitive cells and single-step or two-step KB CP-r cells both indicated codominance of cisplatin resistance compared to hybrids between sensitive cell lines (D98(OR)xKB). The hybrids between sensitive cell lines (D98xKB) and a single-step CP-r KB cell line (D98xKB-CP.5) also were cross-resistant to carboplatin and methotrexate. In addition, the relatively slower growth rate of CP-r cells appears to be dominant. In the two-step CP-r KB cell line, KB-CP1, resistance is no more dominant than in the single-step CP-r KB cell line, KB-CP.5, suggesting that one of the two steps of resistance in KB-CP1 may not be dominant. These dominance data suggest that it might be possible to identify one or more genes responsible for cisplatin resistance by gene transfer from a resistant cell line to a sensitive cell line.     

Resistance to paclitaxel in hepatoma cells is related to static JNK activation and prohibition into entry of mitosis

Hepatocellular carcinoma (HCC) generally shows chemoresistant features to anticancer agents. Paclitaxel has been clinically used in the treatment of various cancers. However, effect of paclitaxel on HCC has not been adequately addressed. Here, we found two categories of hepatoma cells in response to paclitaxel. Paclitaxel effectively decreased the cell viability of SNU475, Hep3B, and SNU387 HCC cells and Chang liver cells (death prone). In contrast, the other five hepatoma cell lines (SNU449, SNU398, SUN368, SNU354, and HepG2 cells) were resistant to paclitaxel (death reluctant). In response to paclitaxel, Bcl-2 was highly phosphorylated in death-prone cells, whereas much less Bcl-2 was phosphorylated in death-reluctant cells. Cotreatment with SP600125, an inhibitor JNK, significantly reduced the phosphorylated Bcl-2 in death-prone cells and caused a significant reduction in cell death. The reduced cell death was due to prohibition into mitotic entry as evidenced by low cyclin B(1)/Cdk1 kinase activity. In death-reluctant cells, inbuild-phospho-JNK levels were high but no longer activated in response to paclitaxel. We found that paclitaxel combined with caffeine or UCN-01, inhibitors of G(2) DNA damage checkpoint, was able to partially overcome resistance to paclitaxel in these cells. Thus our data provide the molecular basis of paclitaxel resistance in hepatoma cells, and appropriate combination therapy may increase treatment efficacy.     

Glycoproteomics of paclitaxel resistance in human epithelial ovarian cancer cell lines: Towards the identification of putative biomarkers

Glycosylation, one of the most common post translational modifications (PTMs) of proteins, is often associated with carcinogenesis and tumor malignancy. Ovarian cancer is the sixth cause of cancer-related death in Western countries. Currently, it is treated by debulking surgery followed by chemotherapy based on paclitaxel, alone or in combination with other drugs. However, chemoresistance represents a major obstacle to positive clinical outcome. We used two approaches, Multiplexed Proteomics (MP) technology and Multilectin Affinity Chromatography (MAC) to characterize the glycoproteome of the human ovarian cancer cell line A2780 and its paclitaxel resistant counterpart A2780TC1. Furthermore proteins were separated by traditional 2DE or DIGE and identified by MS (MALDI TOF or LC MS/MS). Seventy glycoproteins were successfully identified in ovarian cancer cells and 10 were found to be differentially expressed between sensitive and resistant cell lines. We focused on four glycoproteins (tumor rejection antigen (gp96) 1, triose phosphate isomerase, palmitoyl-protein thioesterase 1 precursor and ER-associated DNAJ) which were remarkably upregulated in A2780TC1 compared to A2780 cell line and which may represent biomarkers for paclitaxel resistance in ovarian cancer.     

Collateral Chemoresistance to Anti-Microtubule Agents in a Lung Cancer Cell Line with Acquired Resistance to Erlotinib

Various alterations underlying acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been described. Although treatment strategies specific for these mechanisms are under development, cytotoxic agents are currently employed to treat many patients following failure of EGFR-TKIs. However, the effect of TKI resistance on sensitivity to these cytotoxic agents is mostly unclear. This study investigated the sensitivity of erlotinib-resistant tumor cells to five cytotoxic agents using an in vitro EGFR-TKI-resistant model. Four erlotinib-sensitive lung adenocarcinoma cell lines and their resistant derivatives were tested. Of the resistant cell lines, all but one showed a similar sensitivity to the tested drugs as their parental cells. HCC4006ER cells with epithelial mesenchymal transition features acquired resistance to the three microtubule-targeting agents, docetaxel, paclitaxel and vinorelbine, but not to cisplatin and gemcitabine. Gene expression array and immunoblotting demonstrated that ATP-binding cassette subfamily B, member 1 (ABCB1) was up-regulated in HCC4006ER cells. ABCB1 knockdown by siRNA partially restored sensitivity to the anti-microtubule agents but not to erlotinib. Moreover, the histone deacetylase inhibitor entinostat sensitized HCC4006ER cells to anti-microtubule agents through ABCB1 suppression. Our study indicates that sensitivity of tumor cells to cytotoxic agents in general does not change before and after failure of EGFR-TKIs. However, we describe that two different molecular alterations confer acquired resistance to EGFR-TKIs and cytotoxic agents, respectively. This phenomenon should be kept in mind in selection of subsequent therapy after failure of EGFR-TKIs.     

Orphan nuclear receptor RORγ confers doxorubicin resistance in prostate cancer

Prostate cancer (PCa) is a malignant tumor with an extremely high prevalence. Doxorubicin is the first‐line clinical treatment for castration‐resistant PCa. Clinically, relapse is almost inevitable due to the cancer cells' increasing resistance to doxorubicin. Our previous studies have revealed that retinoic acid‐related orphan nuclear receptor γ (RORγ) is a key protein for cancer progression and a promising target for PCa therapy. Though, RORγ's role and mechanism in doxorubicin‐resistant PCa remain unclear. To study the mechanism of doxorubicin resistance, we generated a doxorubicin‐resistant PCa cell line C4‐2B (C4‐2B DoxR) in this study, by culturing cells in an increasing doxorubicin concentration. Here, we show that RORγ expression was upregulated in C4‐2B DoxR cells compared with that in normal C4‐2B cells. The RORγ‐stably‐overexpressing PCa cell line constructed by lentiviral transfection showed an obvious improvement in doxorubicin resistance and a trend toward castration resistance. Furthermore, RORγ‐specific small molecule inhibitors XY018, GSK805, and SR2211 can significantly inhibit the proliferation of C4‐2B DoxR cells and promote their apoptosis. Collectively, these results have demonstrated the correlation between the upregulation of RORγ and the development of PCa's doxorubicin resistance, thus providing new ideas for solving the problem of chemotherapy drug resistance in PCa.     

Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.     

mTOR-independent 4E-BP1 phosphorylation is associated with cancer resistance to mTOR kinase inhibitors

ATP -competitive mTO R kinase inhibitors (mTorKIs) are a new generation of mTO R-targeted agents with more potent anticancer activity than rapamycin in several tumor models. However, the sensitivity and resistance of cancer cells to mTorKIs remain poorly understood. In this study, we tested mTorKIs against a large panel of colorectal cancer (CRC) cell lines, and found that mTorKIs displayed broader anti-CRC activity than rapamycin, including CRC cells with K-Ras or B-Raf mutations, suggesting that these mTorKIs are particularly useful for CRCs resistant to EGFR inhibitors. Unexpectedly, we found that 40% CRC cell lines were intrinsically drug resistant. Moreover, we discovered an mTO R-independent 4E? BP1 phosphorylation that was correlated with mTorKI resistance. Altogether, our findings provide compelling preclinical support for testing mTorKIs in human CRC clinical trials. They further reveal the existence of significant intrinsic mTorKI drug resistance in cancer cells and suggest that 4E-BP1 phosphorylation is a predictive biomarker for mTorKI sensitivity and resistance.     

Extreme sensitivity to Yondelis (Trabectedin, ET-743) in low passaged sarcoma cell lines correlates with mutated p53

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.