Дисертації з теми "Docetaxel resistance"

Docetaxel is a chemotherapy drug used to treat breast cancer, however as with many chemotherapeutic drugs, resistance commonly occurs and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulatory mechanisms like DNA methylation, histone deacetylation and miRNA expression have been shown to play an important role in cancer drug resistance. This study investigated the role of these mechanisms in two in vitro breast cancer cell line models (MCF-7 and MDA-MB-231) of acquired docetaxel resistance. Using inhibitors to DNA methylation and histone deacetylation, response to docetaxel could be enhanced in both breast cancer cells and cDNA microarray expression analysis identified candidate genes that were re-expressed after treatment with both inhibitors, therefore being associated with docetaxel resistance. Decreased expression of one candidate gene, SERPINE1, was directly linked to docetaxel resistance whereby SERPINE1 modulation, using siRNA technology, directly altered response to docetaxel. Furthermore, miRNA expression profiling was performed in both docetaxel-sensitive and docetaxel-resistant cell lines where alterations of miRNAs were observed and associated with a docetaxel-resistant phenotype. In particular, increased expression of miR-34a was identified in docetaxel-resistant cells, which was associated with and with decreased BCL2 and cyclin D1 mRNA and protein expression in these cells. Modulation of miR-34a expression altered docetaxel response in both docetaxelsensitive and docetaxel-resistant cells, therefore identifying increased miR-34a as direct cause of docetaxel resistance in these cells. In addition, miR-34a was shown to directly target BCL2, which may present a mechanism through which miR-34a mediates docetaxel resistance. Overall, this study identified alterations in DNA methylation, histone deacetylation and miRNA expression as mechanisms through which gene expression is altered in docetaxel-resistant breast cancer cells.

A model of docetaxel resistance in breast tumours was established in two breast cancer cell lines MCF-7 and MDA-MB-231. This was the first description of docetaxel resistant breast cancer cell lines. As an initial global analysis of the genetic events involved in docetaxel resistance, comparative genomic hybridisation (CGH) was used on the DNA extracted from the resistant cell compared to the parental cells. This allowed a unique insight into the specific chromosomal regions that were modified as resistance to docetaxel developed. The resistant cells exhibited a variety of chromosomal modifications from their parental cells. Most notable was the common region of gain of chromosome 7q and loss of chromosome 10q between the two resistant cell lines. Once modified chromosomal regions were identified, the subsequent aim of the study was to further characterise the regions and to identify candidate genes contained with in them. It was demonstrated that P-glycoprotein encoded on chromosome 7q21.1 was over expressed in the resistant cells and that this over expression contributed to the resistance. However it was discovered for the first time that, in addition to P-glycoprotein, the expression of additional genes (Hsp-27 and 14.3-3&'947;) contained on chromosome 7q, was altered. In addition, decreased expression of Bcl-2 was shown to be associated with docetaxel resistance. The identification of alteration of genomic regions and the modification of gene expression identified in this study make a significant original contribution to the understanding of the molecular events involved in docetaxel resistance.

Multidrug resistance phenotype of tumor cells describes resistance to wide range of structurally unrelated anticancer agents and is a serious limitation to effective chemotherapy. It is a multifactor yet not fully elucidated phenomenon by the involvement of diverse cellular pathways. Aim of this study was to investigate the resistance mechanisms developed against docetaxel and doxorubicin that are widely used in the treatment of breast cancer in model cell line MCF-7. Resistant sublines were developed by application of drugs in dose increments and effect of docetaxel and doxorubicin on drug applied cells were investigated by cell viability assays. Expression analysis of P-gp, MRP1, BCRP, Bcl-2, Bax and &
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-tubulin isotypes were performed by RT-PCR, qPCR, Western blot and immunocytochemistry. Genome-wide expression analysis was also performed by cDNA microarray. According to cell viability assays, drug applied cells developed varying degree of resistance to docetaxel and doxorubicin. Gene expression analysis demonstrated that de novo expression of P-gp contributed significantly to drug resistance. Expression levels of class II, III and V &
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-tubulin isotypes increased in docetaxel resistant sublines. According to microarray analysis, a variety of genes showed significantly altered expression levels particularly drug metabolizing and detoxification enzymes (i.e. increased GPX1 and GSTP1 with decreased POR), survival proteins (e.g. decreased TRAIL together with increased decoy receptors and CD40), extracellular matrix components (e.g. increased integrin signaling), growth factors and cytokines (e.g. EGFR1, FGFR1, CTGF, IL6, IL8 and IL18 overexpression), epithelial-mesenchymal transition proteins (i.e. increased vimentin and N-cadherin with decreased E-cadherin and occludin) and microtubule dynamics related proteins (e.g. increased MAP1B and decreased MAP7). Development of cross-resistance and combined drug effects on resistant sublines were also studied. Results demonstrated that docetaxel and doxorubicin resistant cells developed cross-resistance to paclitaxel, vincristine, ATRA, tamoxifen and irradiation. Finally, modulatory effects of verapamil and promethazine in combined drug applications were investigated and verapamil and promethazine were shown to decrease MDR1 expression level thus reverse the MDR. They also showed synergic and additive effects in combined docetaxel and doxorubicin applications. Identification of resistance mechanisms may personalize chemotherapy potentially increasing efficacy of chemotherapy and life quality of patients.

Tumor cells exhibit metabolic reprogramming according to microenvironmental scenarios (i.e. stroma composition and/or anticancer drugs burden) to meet their demands for energy, rapid proliferation, metastasis and progression. In our experimental model, a vicious metabolic synergy between CAFs and prostate cancer (PCa) cells has been described as a pivotal engine allowing cancer cells to achieve aggressive features and evolve their malignancy. Such metabolic crosstalk is mainly based on the OXPHOS rewiring of PCa cells induced by highly glycolytic CAFs through the establishment of tumor:stroma lactate shuttle. In the first part of this study, we highlighted a peculiar CAFs conditioning of PCa cells in terms of OXPHOS upgrading and enhancement. Indeed, we observed that CAFs induce a SIRT1/PGC-1α axis activation in PCa cells, leading to the accumulation of mitochondrial ROS and TCA cycle oncometabolites (succinate/fumarate) that are both closely related to EMT engagement and PCa invasiveness. Indeed, we found that CAFs-exacerbated mitochondrial ROS are crucial for the oxidation of critical targets (Src, PKM2) needed for the metabolic reprogramming toward OXPHOS established in CAFs-exposed PCa cells. On the other hand, succinate is able to maintain CAFs-induced HIF-1α activation and the HIF-1- dependent malignant phenotype of PCa cells. Furthermore, we intriguingly observed a mechanism of mitochondrial transfer elicited by CAFs in order to further boost OXPHOS exploitation, mitochondrial ROS generation and invasiveness of PCa cells. Among microenvironmental cues, chemotherapy resistance has been increasingly and finely associated to the metabolic reprogramming of resistant cancer cells. In the second part of this study, we metabolically characterized docetaxel-resistant PCa cells and we clearly outlined a metabolic adaptation of resistant cancer cells compared to the sensitive counterpart. Docetaxel-resistant PCa cells undergo a Warburg escape towards OXPHOS addiction in order to ensure metabolic advantages during acquisition of resistant phenotype. Together with lactate and glucose, we also found an higher glutamine mitochondrial exploitation by docetaxel-resistant cells. Furthermore, we appreciated a role of CAFs in modulating the response to drug exposure by protecting sensitive and resistant PCa cells. We found that such metabolic/resistant phenotype can be counteracted either by metformin (or other mitocans) treatment or by overexpressing miR-205, a downregulated miRNA orchestrating prostate tumor:stroma crosstalk. Taken together, all the data obtained in our study highlight the role of OXPHOS as an important shared metabolic state between chemotherapy resistance and symbiosis with microenvironment PCa cells.

La mise au point de modèles de laboratoire est d'une importance cruciale pour comprendre la biologie du cancer de la prostate, ainsi que pour évaluer les nouveaux traitements. Le développement de tels modèles est particulièrement difficile et reste à ce jour insuffisant car la majorité de ces modèles est d'origine métastatique ou obtenu in vitro d'une façon artificielle. C'est pourquoi, nous avons entrepris au laboratoire, l'établissement de nouveaux modèles à partir d'un cancer primaire de prostate tumorale et obtenu la lignée IGR-CaP1. La lignée IGR-CaP1 constitue un modèle adapté pour étudier les étapes précoces de la cancérogenèse prostatique. De plus, sa tumoroginicité et sa capacité à induire des métastases osseuses de nature mixtes ostéoblastiques et ostéolytiques font de ce modèles un outil potentiellement intéressant pour étudier les mécanismes métastatiques et rechercher de nouvelles cibles thérapeutiques. Depuis 2004, le traitement de référence des cancers de la prostate métastatiques hormono-résistants est une chimiothérapie par le Docetaxel. Cependant, malgré le bénéfice de survie obtenu, presque la moitié des patients traités par le Docetaxel développent une résistance à la chimiothérapie. Il est donc urgent d'identifier un biomarqueur prédictif pour sélectionner les patients qui vont bénéficier de cette chimiothérapie afin de contourner cette résistance. Dans le but d'étudier les mécanismes de résistance au Docetaxel dans le cancer de la prostate, nous avons établi plusieurs clones résistants au Docetaxel à partir de la lignée IGR-CaP1. Ces clones résistants nous ont permis de réaliser une analyse génomique à haut-débit par microarray comparant l'expression génique entre la lignée sensible et les clones résistants et d'identifier une signature de gènes potentiellement impliqués dans la résistance au Docetaxel. Parmi les gènes identifiés, nous nous sommes focalisés sur le gène LZTS1 sous-exprimé dans tous les clones résistants. LZTS1 est un suppresseur de tumeur qui contrôle le cycle cellulaire en interagissant avec la cycline Cdc25C. Nos résultats suggèrent que la déplétion de LZTS1 est potentiellement impliquée dans le mécanisme de résistance au Docetaxel. La finalité de notre projet est de valider nos résultats par immunohistochimie à partir des prélèvements tumoraux obtenus dans l'essai de phase III GETUG12. Nous espérons que notre étude permettra aux cliniciens de sélectionner les sous-groupes de patients susceptibles de profiter d'un traitement par Docetaxel.

Solid tumors are complex biological structures which are composed of cellular and matrix components, everything being perfused by blood vessels. During tumor development, modifications of both biochemical and mechanical parameters are observed and can feedback on one another. Cancer cells constantly interact with their mechanical environment and the whole tissue is mostly confined by its surrounding. Compressive mechanical stress develops in part from cell proliferation and could eventually result in the clamping of blood vessels leading to increased interstitial fluid pressure (hydrostatic pressure). The consequent hypoperfusion poses important obstacles to drug delivery and nanomedicines. In fact, the tortuous tumor microvasculature has blood velocities up to one order of magnitude lower compared to healthy capillary networks. Moreover, the fast angiogenesis during tumor progression leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs (Enhanced Permeation Retention – EPR – effect). These effects have served as a basis for the development of drug delivery systems which are aimed at enhancing tumor tissue targeting and drug therapeutic effectiveness. Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet, drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation of nanoparticle-loaded agents - nanomedicines - are sub-optimal. Starting from these considerations, during my PhD, I studied two complementary approaches: in the first two years my work was focused on implementing the characteristics of Discoidal Polymeric Nanoconstructs designed with controlled geometries and mechanical properties. In the last year, I investigated the role of mechanical stress on chemotherapeutic efficacy. More precisely, this work first reviews the use of deformable discoidal nanoconstructs (DPNs) as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. In this work, the synthesis, drug loading and release, and physico-chemical characterization of DPNs were enhanced with particular emphasis on the ability to independently control size, shape, surface properties, and mechanical stiffness. Two different loading strategies were tested, namely the straightforward “direct loading” and the “absorbance loading”. In the former case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the latter case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of three different molecules and their corresponding prodrugs were systematically assessed (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine lipid chains or 1 kDa PEG chains were directly conjugated with Cy5.5 or methotrexate and Doxorubicin). Moderately hydrophobic compounds with low molecular weight showed encapsulation efficiencies of 80%, with absorption loading (direct loading has efficiencies around 1%). The DOX-DPN showed on triple negative breast cancer cells a toxicity comparable to free DOX. Preliminary in vivo preliminary studies conducted with directly loaded Cy5-DPN demonstrated a fairly solid integration of the imaging compound with the polymer matrix of the particles. The second part of the work dissect what happens to free drugs or to drugs carried by nanovectors once they reach the tumor site. As we mention above, the elevated mechanical stress derived from tumor progression could result in blood vessels clamping with consequent reduction of drug efficacy. It is quite obvious to imagine that if the drug fails to reach the tumor it cannot act on it. Indeed, mechanical stress within the tumor site is present from the early stages of the disease. Our goal was to understand what happens when mechanical stress is not yet so large enough to fully collapse the blood vessels. Are there mechanical alterations that can affect the efficacy of a chemotherapeutic? We studied how mechanical perturbations of the tumor microenvironment could contribute to the mechanical-form of Gemcitabine drug resistance. Specifically, we developed a new in vitro strategy to mimic the mechanical compression stress induced by the stroma during tumor progression. We embedded pancreatic tumor spheroids into agarose polymeric matrix in order to demonstrate the effect of mechanical compressive stress on tumor proliferation. Then, we validated our results with other types of mechanical stresses. Finally, we investigated the therapeutic efficacy of a proliferation-based chemotherapy: Gemcitabine. Collectively, having the physical cues of cancer in mind, it can be important to cross-fertilize the fields of physical oncology and nanomedicine.

Le cancer de la prostate (CaP) est l’un des cancers le plus mortel chez les hommes. Le CaP avancé est traité avec les inhibiteurs du récepteur des androgènes ou la chimiothérapie (chimiothérapie), mais la plupart des patients développent des résistances. Ainsi, des nouvelles stratégies thérapeutiques sont nécessaires pour améliorer la prise en charge du CaP. Les faibles niveaux circulants de la vitamine D ou de son récepteur VDR dans les cellules épithéliales prostatiques (PECs) sont corrélés avec la gravité du CaP, mais le mécanisme sous-jacent n'est pas décrit. Cette étude montre que VDR réduit la prolifération des PECs dans un modèle murin de CaP, les souris Pten(i)pe-/-, via modération du stress oxydant. De plus, le VDR dans les PECs réduit le recrutement des neutrophiles, qui sont une cible thérapeutique pour la dissémination du CaP. Par ailleurs, la combinaison d'un agoniste de VDR avec le docétaxel réduit efficacement les volumes tumoraux du CaP chimiorésistant. Pour conclure, ce travail met en évidence comment la vitamine D ralentit la progression du CaP et suggère de nouvelles stratégies thérapeutiques pour cette maladie
Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in men. Androgen receptor signaling inhibitors are the gold standard treatment for advanced PCa, but most patients develop castration-resistant prostate cancer (CRPC). The treatment of choice for CRPC is the chemotherapy (docetaxel), but the overall survival is only about one year. Thus, novel therapeutic strategies are required to improve PCa care. Low circulating vitamin D levels and reduced expression of its receptor VDR in prostatic epithelial cells (PECs) correlate with PCa severity, but the underlying mechanism is unclear. This study shows that VDR in PECs of Pten(i)pe-/- mice, a model of PCa, reduces cell proliferation via oxidative stress attenuation. Furthermore, VDR in PECs limits the recruitment of neutrophils, that are shown to be therapeutic target for PCa dissemination. Additionally, combining a VDR agonist with docetaxel effectively reduces tumor volumes in chemoresistant CRPC xenografts. Overall, this work highlights how vitamin D signaling slows PCa progression and suggests new therapeutic strategies for advanced PCa

碩士
高雄醫學大學
醫學研究所碩士班
105
Docetaxel is the first-line chemotherapeutic agent for patients with castration resistant prostate cancer (CRPC). Unfortunately, clinical treatment with docetaxel often encounters a number of undesirable side effects, including drug resistance. AMP-activated protein kinase (AMPK) is the cellular energy sensor, which can regulate metabolism and maintain energy homeostasis involving glycolysis. Recently, we found AMPK was associated with the development of docetaxel resistance in PC. However, the mechanisms of AMPK-mediated docetaxel-resistance in PC were remained unclear. Our results showed that the level of phospho-AMPK (S487) was significantly higher expression in PC/DX25 cells (a docetaxel resistance PC cell line) than in parental PC3 cells by Western blotting analysis. The expression of phospho-AMPK (S487) was gradually increased by docetaxel treatment in a dose-dependent manner in PC3 cells. Knockdown of AMPK expression reversed docetaxel sensitivity in PC/DX25 cells by MTT assay. However, using the AMPK agonist 2-Deoxy-D-glucose (2DG) and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) enhanced the docetaxel resistance in PC/DX25 cells. We also found the expression of HIF-1α and PFKFB4 were reduced via AMPK in PC/DX25 cells. Downregulation of HIF-1α and PFKFB4 were associated with PC/DX25 cell proliferation. The phospho-AMPK (S487) was overexpressed in clinical cancer samples of castration-resistant prostate cancer (CRPC). According to the above results, AMPK may play an important role in regulating chemoresistane in docetaxel-resistant prostate cancer.

OBJECTIVE: To determine the effect of clinically used zoledronate (ZOL) and docetaxel on breast cancer cells and bone biology under both bone remodeling stages and the rate of tumor dissemination and state of dormancy. METHODS: The effect of clinically used zoledronate (ZOL) was examined on MDA-MB-231 and MDA-BO cells in a roller tube system under bone resorption and formation conditions. Three groups; calvaria alone, calvarial co-cultured with tumor cells, and calvaria with tumor cells treated with four repeat doses of 2 µM of ZOL were cultured for 8, 14 and 20 days. The formation groups were supplemented with 150 µg/ml ascorbic acid. Cell counts were performed on trypsinized calvaria harvested at 2, 8 and 14 days. Media was changed every 2 days and the changed media was re-seeded in a 24-well for 20 days. To test the impact of chemotherapy agents on cancer-bone metastasis the effect of 10 µM of docetaxel was tested on breast cancer cells under formation and resorption conditions using the above design. Tumor burden was assessed at 8 days. RESULTS: Tumor burden: no statistically significant difference between ZOL treated and untreated groups under resorption and formation conditions in both cell lines. Exposure to docetaxel revealed that ~30% of the cells were affected by chemotherapy in formation model, while ~70% was affected in resorption model in both cell types. Dissemination model: the dissemination rate for MDA and BO cells under formation condition is significantly less than for resorption conditions. Fluorescent microscopy: MDA and BO tumor-calvaria were treated with Ki 67 antibody showed that under bone resorption conditions the cancer-bone cells colony were predominantly in proliferation stage while under formation conditions cancer cells were in dormancy. Confocal microscopy: observation confirmed the relation of the mode of cancer cell attachment to bone endosteal cell layer with the dormancy and cell proliferation states. CONCLUSIONS: Both cancer cell lines showed resistance to ZOL under formation and resorption conditions. Drug resistance to docetaxel was more evident under formation condition, where cells are dormant and not proliferating. The dissemination rate is significantly higher in resorption condition, suggesting that cells in formation are dormant with lower dissemination rate.
2019-07-23T00:00:00Z

Prostate cancer is the second most common cancer in men and acording to the Globocan report from 2012, there will be over 1.2 million new cases and over 300 000 deaths, worldwide. Organ-defined prostate cancer is a curable disease, but then it develops into metastatic castration resistant prostate cancer, and it is usually a matter of time until the patient develops resistance to chemotherapy and becomes an incurable disease. This evokes the need for biomarkers that can predict disease progression and response to therapy. This would allow for a better stratification of the patients to receive the most efficacious, therapeutic treatment. Cancer cell derived exosomes, with their molecular cargo that represents the tumor cells, have been demonstrated to be a good source of such prognostic and predictive biomarkers. In this study we isolated and characterized exosomes derived from prostate cancer cells that are resistant to docetaxel and abiraterone acetate. One interesting finding is that the cells that are resistant to these two drugs secrete more exosomes than their sensitive counterparts. We compared the proteomic profile of these exosomes with exosomes from the parental, drug-sensitive cell lines. We have identified a number of putative markers that may constitute a predictive signature and are currently under validation. From our pilot studies we have identified that p-gp positive exosomes may function as predictive biomarkers for response to docetaxel, and that exosomes containing the splice variant 7 of the androgen receptor may have the potential to predict response to abiraterone acetate and enzalutamide. However, both of these biomarkers have to be validated in larger patient cohorts along with the newly identified proteins. In addition to proteomics, the exosomes are currently being analysed by transcriptomics, mRNA and miRNA arrays. These combined molecular data will provide valuable information in finding predictive signatures for response to therapy, in order to provide the patients with a more personalized medical care and stratified course of treatment, leading to improved overall survival.

碩士
高雄醫學大學
生物化學研究所
97
Clinically, for metastatic prostate cancer who received hormone therapy can improve symptoms, but most patients die in a few years after the recurrence of hormone-resistant prostate cancer, recent studies found that docetaxel-based therapy can significantly extend the survival time of patients, but the use of docetaxel treatment after a period of time often caused by drug-resistant.This study hopes to explore through the docetaxel-resistant prostate cancer have a mechanism with a view to identify the future policy of the treatment. First, we establishment of a Docetaxel-resistant cell lines, through the microscopic observation drug-resistant strain was found to change the cell type, the use of colony analysis experiment to prove their faster cell growth, and the use of the Western blot method to prove cell skeleton and the morphology-related protein: α, γ-tublin and lamin A / C performance increase, E-cadherin expression decreased, and its downstream molecules α and β-catenin activation.Use of cell migration analysis found that drug-resistant strain has become easier to transfer, and cell migration of related protein p-Src, FAK, HO-1, Caveolin 1 and VEGF protein expression increased, and its downstream Roh A, paxillin protein expression decreased, resulting in resistance change the overall structure of strains have become more vicious, more malignant.in addition, resistant strains can be found in the epidermal growth factor receptor (EGFR) and chemokine IL-8 expression increased, and further analysis of EGFR-related downstream signaling molecules can be found STAT3, AKT pathway, ERK pathway activation, and to promote apoptosis of P-P38, caspase 3 activity decreased, resulting in docetaxel-resistant cell growth more vigorous and more easily killed.in order to more clearly identify the mechanism of drug resistance caused by docetaxel, we used EGFR siRNA molecules inhibit EGFR signaling, are known to inhibit EGFR signaling molecule when will respond to drug-resistant cells of the docetaxel sensitivity, and FAK, Caveolin 1, α , β-catenin, p-cRaf, pERK and IL-8 mRNA expression was inhibited, and p-PDK1, p-AKT and STAT3 no significant difference in the future will be to further explore the molecular signals after EGFR inhibition of the docetaxel resistance affected cells. In addition we also proceed from the other signal molecules, such as the inhibition of STAT3 to explore the docetaxel effect of drug-resistant cells, with a view to have a resistance to the treatment of patients to find a better approach.

碩士
中山醫學大學
醫學研究所
99
Lung cancer is the leading cause of death worldwide from all cancers, and chemotherapy is the common treatment for lung cancer patients. The development of drug resistance is the single most important cause of treatment failure in patients with chemotherapy. When patients become resistant to chemotheraputic drugs, it is commonly developed cross-resistance to other anti-cancer drugs. In previous experiments, we have used A549 cells to select docetaxel resistant cell lines and two sublines of A549/D16 and A549/D32 were established. We have found these two sublines were more sensitive to Alimta than the parental A549 cells. Therefore, we further characterize Alimta sensitivity in these two A549/D16 and A549/D32 cells. We applied MTT assay, Clonogenic assay, Flow cytometry to determine the Alimta sensitivity in A549/D16 and A549/D32 cells. The results demonstrated that A549/D16 and A549/D32 are 4-fold more sensitive to Alimta when compared with the parental cells by IC50 of MTT assay. Furthermore, When A549/D16 cells were treated with Alimta, the thymidylate synthase (TS) protein was induced on the first day then decreased the expression with time-dependence; p53 and p21 proteins were induced on the first day and maintained the protein level until the fourth day, along with the increased apoptotic sub-G1 cells. The target genes of Alimta: TS, dihydrofolate reductase (DHFR), glycinaminde ribonucleotide formyl transferase (GARFT) and drug metabolism-related genes: reduced folate carrier (RFC), gamma-glutamyl hydrolase (GGH) expression were decreased in A549/D16 and A549/D32 cells. These results suggested that sensitivity of Alimta in A549/D16 and A549/D32 cells may be closely associated with these genes. Then we cloned the open reading frame of TS and GGH to p3XFlag-CMV-10 vector, and transfection to cell to examine their expression. Overexpression of TS and GGH in A549/D16 and A549/D32 cells to reduce Alimta sensitivity are required for research in the future. Moreover, we also found that Lipocalin-2 (LCN2) expression was increased in A549/D16 and A549/D32 cells, but inhibibted LCN2 expression by shRNA has no significantly effects in the sensitivity of Alimta in A549/D16 and A549/D32 cells. Our results demonstrated that it is not cross-resistant, but more sensitive to Alimta in docetaxel resistant cell lines of A549/D16 and A549/D32.

碩士
高雄醫學大學
生物化學研究所
99
Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for prostate cancer. Unfortunately, clinical treatment with docetaxel often encounters a number of undesirable side effects, including drug resistance. Therefore, it has become essential to identify molecular events that may be associated with the development of docetaxel resistance. Tubulin isoforms have been previously examined for their resistant ability to docetaxel in many cancers, but the real mechanisms remained unclear. In this study, we evaluated the feasibility of employing docetaxel as a cytotoxic agent for PC3 cells and to examine the role of acetyl-tubulin in docetaxel-resistant prostate cancer. Human prostate cancer cell lines(PC3) and docetaxel-resistant cell subclones(PC/DX), to investigate the expression of acetyl-tubulin, alpha-tubulin, beta-tubulin, gamma-tubulin and beta III-tubulin were significantly higher expression in PC/DX cells than in parental PC3 cells and up-regulation of acetyl-tubulin, alpha-tubulin, beta-tubulin and gamma-tubulin expression with different concentrations of docetaxel PC/DX cells by western blotting analysis. The expression of acetyl-tubulin was gradually increased by docetaxel in a dose- and time-dependent manner in PC3 cells. In the docetaxel-resistant prostate cancer patient tissue samples were also up-regulation of acetyl-tubulin. Histone deacetylase 6, a deacetyl enzyme of tubulin, mRNA and protein levels were significantly decreased in PC/DX than in PC3 cells. Further, we used siRNA to inhibit HDAC6 expression. Up-regulation of acetyl-tubulin in HDAC6 knockdown PC3 cells became more resistant to docetaxel. In addition, we also found up-regulation of kinesin (KIF5B、KIF2C) and stathmin (STMN1) in the PC/DX cells that may be as a function of docetaxel-resistant. Microtubule have more acetylation and KIF2C in the PC/DX cells, and tubulin acetylation also stimulates KIF2C binding to microtubules. Moreover, up-regulation of acetyl-tubulin protein expression after recombinant epidermal growth factor treatment and reducing docetaxel cytotoxicity in PC3 cells, and inhibition of EGFR in the PC/DX cells to cause down-regulation of acetyl-tubulin. This study highlights the role of acetyl-tubulin in docetaxel-resistant prostate cancer cells and that may be regulated by EGFR signaling pathway. Moreover, KIF2C may be required for this resistant factor. The detailed mechanisms for which will be further explored.

碩士
高雄醫學大學
生物化學研究所
101
Hormone therapy for hormone-sensitive prostate cancer patients slows down the growth of the cancer for few years but it will finally turn into castration-resistant prostate cancer (CRPC). The chemotherapeutic agent docetaxel has been used as a prostate chemotherapy drug for years. Significant outcome has been observed in CRPC patients, but for the most part them will ultimately get docetaxel resistant cancer. Without any widely-used or promoted clinical drug for docetaxel resistant patients, to find the mechanism behind the drug resistance has become imperative and of utmost importance. In our previous studies, we found that the docetaxel resistant cell line PC/DX expresses a higher Interleukin-8 (IL-8) mRNA level than its parental androgen-insensitive prostate cancer cell line (AIPC) PC-3. In order to further this finding, we were hoping to confirm that IL-8 plays a role in docetaxel resistance and to locate the possible mechanism behind it. At the start, we constructed another docetaxel resistant cell lines DU/DX from DU145. This higher IL-8 secretion cell line expressed a 43.3 times stronger docetaxel resistant ability than the DU145. Treating DU145 and PC-3 with docetaxel induced IL-8 cell secretion. We also showed that IL-8 plays a role in drug resistance by treating both DU145 and PC-3 with a recombinant IL-8 and knocking down an innate IL-8. Considerable IL-8 expression DU145 stable clones we constructed in this study also displayed higher docetaxel resistance ability. We also proved that NF-κB and AP-1 are transcription factors of AIPC cell lines by transfecting IL-8 promoter which contains different mutated transcription factors binding sites into AIPC cells, and detecting with Luciferase reporter assay. An over-expressed p-EGFR protein was observed in DU/DX cell lines, corresponding to former research of PC/DX done in our lab. To make sure that EGFR had some relations to IL-8, we treated DU145 and PC-3 with EGF. A more highly secreted IL-8 and stronger IL-8 promoter activity were observed while the EGF was binding to these cells. To simulate the more highly secreted IL-8 appearance in the DU/DX cell line, we treated DU145 and PC-3 with a recombinant IL-8. We found that the recombinant IL-8 stimulating cell expressed higher p-EGFR and p-AKT both in DU145 and PC-3 with a time dependence, which corresponded to circumstances in DU/DX and IL-8 stable clones. In this study, we have proved the role of IL-8 in docetaxel resistance and found the possible pathway activated by IL-8 in AIPC cell lines. These results may modestly provide a new aspect of the role of cytokines in multiple drug resistance and views to aid future research.

碩士
中山醫學大學
醫學研究所
103
Chemotherapy is the common treatment for lung cancer patients. The development of drug resistance is the most important cause of treatment failure. When cancer resistant to chemotheraputic drugs, it is commonly developed cross-resistance to other anti-cancer drugs. In previous experiments, we have used docetaxel to select A549 cells and two sublines of A549/D16 and A549/D32 have been established. We have found these two sublines were more sensitive to Alimta than the parental A549 cells. Therefore, we plan to further characterize the mechanism of Alimta sensitivity in these two A549/D16 and A549/D32 cells. We used MTT assay, Clonogenic assay, Western blot to determine the Alimta sensitivity and protein expression in these docetaxel-resistant cells. The data showed that thymidylate synthase (TS), dihydrofolate reductase (DHFR) and gamma-glutamyl hydrolase (GGH) protein expression were downregulated. We further showed that the docetaxel resistant lung cancer line with low TS determined higher Alimta sensitivity. When TS was overexpressed by transfection resulted in lower Alimta sensitivity. In addition, we also found that when A549 cells treated with docetaxel, the upregulated p53 was associated with a decline in the TS. The data indicated that p53 may downregulate TS expression. The genes of p53 and TS that mediate Alimta sensitivity in docetaxel-resistant A549 cells could be used as a biomarker for the second line chemotherapy. Our study may benefit those docetaxel-resistant lung cancer patients to have a tailed-made anti-cancer therapy.

碩士
高雄醫學大學
醫學研究所碩士班
105
In clinical, docetaxel is used in the first-line treatment for patients with castration‑resistant prostate cancer （CRPC）, significantly extended overall survival of patients. However, the treatment is usually limited when gradual development of docetaxel-resistance. It has become imperative and important to find the mechanism of the docetaxel-resistance in prostate cancer. In previous studies have suggested that epidermal growth factor receptor （EGFR） and Interleukin-8 （IL-8） are required for prostate cancer progression and drug resistance. The molecular mechanism of how EGFR and IL-8 mediated migration in prostate cancer cells is still elusive. Thus, this study was aimed to investigate the molecular mechanisms of EGFR and IL-8 regulated migration in the docetaxel-resistant prostate cancer cells and to search the possible therapeutic strategies for docetaxel-resistant metastatic CRPC in future. We have established PC3 docetaxel-resistant cell line (PC/DX), PC/DX25 maintain in 25 nM docetaxel. We found that PC/DX25 metastatic developed faster than PC3 cellls, and expressed the higher protein levels of EGFR, IL-8 and CXCR1 than PC3 and Immortalized prostate cells. In this study, the cell migration, protein levels of EGFR, p-EGFR(Y1068), IL-8, NF-κB p65 and Epithelial-Mesenchymal Transition (EMT) were induced by the treatment with EGF and IL-8 recombinant proteins. In contrast, the cell migration, protein levels of EGFR, IL-8, NF-κB p65 and EMT were inhibited by the treatment with EGFR and IL-8 siRNA. And we also found that nuclear p-p38 was inhibited by the treatment with EGFR siRNA. Interestingly, the PC3 cells were induced EGFR and IL-8 protein levels by the treatment with docetaxel. We also found that treated the PC3 cells with docetaxel induced IL-8 promoter activative and IL-8 cytokine secretion. These results suggest that EGFR and IL-8 have key roles in regulation of the migration of docetaxel-resistant prostate cancer cells, and molecular mechanism of cell migration EGFR/p-EGFR（Y1068）/NF-κB p65/IL-8 signaling pathway. Our study revealed EGFR and IL-8 signaling pathway regulated cell migration in docetaxel-resistant prostate cancer cells, and suggested that combined EGFR and IL-8 inhibitor with docetaxel may used to treatment for prevention of potential drug-resistance and metastasis.

碩士
高雄醫學大學
香粧品學系碩士班
105
In this study, BV001 is a new synthetic quinoline derivative with the cytotoxic effects in prostate cancer(PCa) cells. BV001 has been found to have the anti-proliferation in PCa cell lines(PC3 and PC/DX25) by arresting cell cycle in intra-S phase. Nowadays, the docetaxel-base therapies are used as a first-line chemotherapy for castration-resistant prostate cancer(CRPC) patients. However, almost all patients died from docetaxel-resistant CRPC during three to five months. Hence, the development of effective chemotherapeutic agents for CRPC patients has become imperative and important. Cytotoxicity of BV001 was examined in PC3 and PC/DX25(a docetaxel-resistant subline) by MTT assay. IC50 of BV001 in PC3 and PC/DX25 were 1.75 ± 0.32 and 1.6 ± 0.28 μM, respectively, indicating that BV001 was with the cytotoxic effects in both cell lines. BV001 increased the production of ROS in PC3 and PC/DX25 cells, and the ROS generation was 1.5 times than control group. ROS generation was one of the factors that caused DNA damage. Cell cycle analysis showed that BV001-treated PC3 and PC/DX25 leaded to DNA damage inducing intra-S phase arrest and increasing the deaths of apoptotic cell in time increasing. The results showed that the expression of p-H2AX confirmed DNA damage, and the modulation of intra-S and G2/M cell cycle regulators, such as cyclin A, CDK2, cdc25c, cyclin B1 and CDK1, confirmed intra-S phase arrest. The results of annexin V-FITC/PI double staining assay also confirmed BV001-induced apoptotic prostate cancer. Simultaneously, the results showed BV001 medicated apoptosis of PCa cells through the down-regulation of Bcl-2, the up-regulation of Bax and Bad, the releasing of cytosol cyto c from mitochondria, the degradation of PARP and the activation of caspase 3 by Western blot. In conclusion, BV001 induces apoptosis of prostate cancer cells through cell cycle intra-S arrest because intracellular ROS production resulted in DNA damage which regulated cell cycle intra-S-related protein and mitochondrial-dependent signaling pathway.