Rozprawy doktorskie na temat „ALS inhibitor resistance”

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17830号
農博第2015号
新制||農||1016(附属図書館)
学位論文||H25||N4787(農学部図書室)
30645
京都大学大学院農学研究科農学専攻
(主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕
学位規則第4条第1項該当

The objective of this Ph.D. research was to identify new and novel mechanisms of wild radish (Raphanus raphanistrum L.) resistance to photosystem II (PSII) inhibitors, auxinics, and acetohydroxyacid synthase (AHAS) inhibitors. PSIIinhibitor resistance was demonstrated to be target-site based, and conferred by a Ser264 to Gly substitution of the D1 protein. Auxinic resistance was associated with reduced herbicide translocation to the meristematic regions of resistant wild radish plants. Two new resistance mutations of wild radish AHAS were discovered, including one encoding the globally rare Asp376 to Glu substitution, and another encoding an Ala122 to Tyr substitution, which has never been identified or assessed for resistance in plants previously. Characterization of the frequency and distribution of AHAS resistance mutations in wild radish from the WA wheatbelt revealed that Glu376 was widespread, and that some mutations of AHAS are more common than others. Computer simulation was used to examine the molecular basis of resistance-endowing AHAS target-site mutations. Furthermore, through the computer-aided analysis, residues were identified with the potential to confer resistance upon substitution, but which have not previously been assessed for this possibility. Results from this Ph.D. research demonstrate that diverse, unrelated mechanisms of resistance to PSII inhibitors, auxinics, and AHAS inhibitors have evolved in wild radish of the WA wheatbelt, and that these mechanisms have accumulated in some populations.

Doctor of Philosophy
Department of Agronomy
Tesfaye T. Tesso
The lack of effective post-emergence weed control options is often highlighted as one of the major factors behind dwindling acreage under sorghum (Sorghum bicolor (L.) Moench) in the United States. The discovery of herbicide resistance sources in wild sorghum population and subsequent efforts to incorporate them into cultivated sorghum was received with much optimism to change weed management practices in sorghum. As the development of the technology advances, especially of the Acetolactate synthase (ALS) resistance, concerns over the temporary interveinal chlorosis and reduced seedling vigor in some of the resistant families became heightened. This thesis research is designed to shed light on the genetic basis of seedling chlorosis and assess its impacts on yield potential. The study has three parts; the first part is focused on identifying the genetic causes and plant mechanisms associated with the chlorotic phenotype. ALS herbicide resistant sister-lines expressing normal and chlorotic phenotypes were analyzed via RNA sequencing at four time points during seedling growth. The study identified several variants of genes coding chloroplast precursors and those that cause epigenetic modifications. Once confirmed, genetic markers can be developed to track these gene variants in the breeding population and eliminate segregates genetically prone to chlorosis/yellowing. The second part of the study focuses on assessing the effect of ALS resistance associated chlorosis on agronomic and nutritional parameters of sorghum inbred lines. A set of ALS resistant lines expressing different levels of the chlorotic phenotype were evaluated in replicated field trials and laboratory methods. Results showed that interveinal chlorosis delays flowering but does not have negative effect on yield and nutritional parameters with and without herbicide treatment. The last part addresses whether there is any yield drag that may be associated with herbicide resistance traits and foliar interveinal chlorosis. For this, we synthesized a large set (182) of hybrids from ALS resistant, ACCase resistant and regular (susceptible) seed and pollinator parents. The hybrids were then evaluated in three sets at multiple locations during the 2014 and 2015 crop seasons along with commercial checks. The results revealed that resistance to both herbicides do not cause any drag to grain yield. The traits also do not have any negative impact on grain and nutritional quality of resistant hybrids.

The development of high-throughput sequencing technologies has prompted the evaluation of large cohorts of different tumor types. The results from these comprehensive genomic studies have revealed the most commonly mutated genes across human cancers, providing further understanding of the pathogenesis, molecular classification, and therapeutic strategies for this disease. PIK3CA, the gene encoding the PI3Kα isoform, is among the most frequently mutated genes in breast, head and neck, colorectal, and lung cancer, among others. Activating mutations in PIK3CA promote hyperactivation of the PI3K/AKT pathway, leading to increased proliferation, cell growth, survival, and metabolism. Current efforts are aimed to develop PI3K inhibitors as an effective therapy for PIK3CA mutated cancers and, despite promising clinical responses, the emergence of drug resistance is a clear limitation. In this doctoral thesis, we have explored these mechanisms of resistance in order to provide a better understanding of tumor evolution upon therapy, define subpopulations of patients that are likely to respond to PI3K inhibitors, and provide novel pharmacological combinations to overcome therapy refractoriness. The loss of the tumor suppressor PTEN was found to play an important role in the resistance of PI3Kα inhibitors in preclinical models and patients, mainly by reactivating the PI3K/AKT pathway as a result of an increased dependency on the PI3Kβ isoform. Our work also demonstrated the notion of tumor evolution and phenotypic convergent evolution in response to therapeutic pressure. Moreover, we have established that intrinsic resistance to PI3Kα inhibitors occurs as a result of incomplete inhibition of the mammalian target of rapamycin complex 1 (mTORC1), a downstream effector of the PI3K/AKT pathway. PI3Kα inhibitor-resistant cells could be re-sensitized through the blockade of phosphoinositide-dependent kinase (PDK1), a constitutively active kinase, using genetic or pharmacologic inhibition. Further experiments showed that the downstream effector of PDK1 is the serum and glucocorticoid-induced kinase 1 (SGK1), which promotes cell survival through the phosphorylation of key proteins such as FOXO3 and TSC2. Accordingly, the resistant phenotype could be also reverted by inhibiting SGK1, a novel pharmacological approach that has revealed interesting roles of this kinase in tumor biology. Genetically engineered mouse models represent reliable tools for investigating the etiology, biology, and progression of human diseases, as well as for exploring novel therapeutic approaches. By serendipity, we discovered the role of PIK3CA mutations in the genesis of venous malformations, an aberration of normal venous development that currently lacks effective treatments. Our mouse models recapitulated the histopathologic features of the disease and provided an experimental platform to test novel pharmacological approaches. PI3Kα inhibitors were effective at reducing the morbidity and mortality of mice carrying venous malformations. The results from this thesis highlight the importance of defining the molecular determinants of sensitivity and resistance to PI3K inhibitors, a therapy that will most likely benefit PIK3CA mutant patients.
El desenvolupament de noves tècniques de seqüenciació massiva ha fomentat l’estudi d’un gran nombre de mostres de diversos tipus tumorals. Els resultats d’aquests estudis genòmics exhaustius ha revelat els gens que es troben mutats en major prevalença, contribuint a una millor comprensió dels processos de patogènesis, classificació molecular i estratègies terapèutiques per a aquesta malaltia. PIK3CA, el gen que codifica per a la isoforma PI3Kα, es troba entre els gens mes freqüentment mutats en el carcinoma de mama, cap i coll, colorectal, pulmó, entre d’altres. Les mutacions activadores a PIK3CA promouen la hiperactivació de la via de senyalització de PI3K/AKT, donant lloc a un increment en la proliferació, la supervivència, i el metabolisme de les cèl·lules tumorals. Els esforços actuals es centren en el desenvolupament d’inhibidors de l’enzim PI3K com a una possible teràpia efectiva en tumors que presenten mutacions a PIK3CA. Tot i que els assajos clínics inicials son prometedors, l’emergència de resistència a aquestes teràpies és una clara limitació. En aquesta tesis doctoral s’han explorat els possibles mecanismes de resistència per intentar entendre com els tumors evolucionen enfront d’aquest fàrmacs, poder definir les subpoblacions de pacients que respondran als inhibidors de PI3K i proporcionar noves combinacions farmacològiques per combatre el fenomen de la resistència. Hem demostrat que la pèrdua del supressor tumoral PTEN juga un paper important en la resistència als inhibidors de PI3Kα, tant en models preclínics com en pacients, mitjançant la reactivació de la via de PI3K/AKT que és resultat d’un increment en la dependència de la isoforma PI3Kβ. El nostre treball també ha evidenciat la noció d’evolució tumoral i ha demostrat el concepte d’evolució convergent fenotípica en resposta a la pressió terapèutica. També s’ha demostrat que la resistència intrínseca als inhibidors de PI3Kα es pot donar com a resultat d’una inhibició incompleta del complex 1 de mTOR (mTORC1), un efector clau de la via de PI3K/AKT. Cèl·lules resistents a inhibidors de PI3Kα es van poder sensibilitzar amb el bloqueig genètic o farmacològic de PDK1, una quinasa constitutivament activa. Experiments addicionals van poder demostrar que l’efector molecular de PDK1 era la quinasa SGK1, la qual promou la supervivència cel·lular a través de la fosforilació de proteïnes clau com FOXO3 i TSC2. El fenotip resistent es va poder revertir mitjançant la inhibició farmacològica d’aquesta proteïna, una aproximació terapèutica que ha revelat un rol interessant en la biologia tumoral. Els models murins modificats genèticament representen una eina segura per a l’estudi de la etiologia, biologia i progressió de malalties humanes, així com per explorar noves aproximacions terapèutiques. Com a resultat d’un descobriment imprevist, també hem pogut revelar el rol de les mutacions de PIK3CA en la formació de malformacions venoses, una aberració del desenvolupament normal de les venes que actualment no tenen un tractament específic. El nostre model animal de malformació venosa recapitula les característiques histopatològigues de la malaltia i proporciona una plataforma experimental única per a l’estudi de noves teràpies. En aquests models animals, els inhibidors de PI3Kα han demostrat ser efectius en la reducció de la morbiditat de les malformacions venoses.

Les analyses moléculaires et la classification des adénocarcinomes bronchiques ont conduit au développement de thérapies ciblées sélectives visant à améliorer le contrôle de la maladie et la survie des patients. ALK (anaplastic lymphoma kinase) est un récepteur tyrosine kinase de la famille des récepteurs de l'insuline. Des réarrangements chromosomiques impliquant le domaine kinase d’ALK sont présents dans environ 3 à 6% des patients atteints d'un adénocarcinome bronchique. La protéine de fusion provoque une activation du domaine kinase de manière constitutive et indépendante du ligand. Lorlatinib est un inhibiteur d’ALK de troisième génération avec une efficacité et une sélectivité optimale, ainsi qu’une pénétration élevée vers le système nerveux central. Lorlatinib peut vaincre la résistance induite par plus de 16 mutations secondaires dans le domaine kinase d’ALK acquises lors de la progression aux ALK TKI de première et deuxième générations. Le traitement par lorlatinib est donc efficace chez les patients préalablement traités par un ALK TKI de première ou deuxième génération, et est actuellement approuvé pour cette indication. Le spectre complet de mécanismes de résistance au lorlatinib chez les patients reste à élucider. Il a récemment été rapporté que l'acquisition séquentielle de deux mutations ou plus dans le domaine kinase, également appelées mutations composées, est responsable de la progression de la maladie chez environ 35% des patients traités par le lorlatinib, principalement en altérant sa liaison au domaine kinase d’ALK. Cependant, l’effet de ces mutations sur la sensibilité aux différents inhibiteurs d’ALK peut varier, et les autres mécanismes de résistance survenant chez la plupart des patients restent inconnus. Mon travail de thèse avait pour but d’explorer la résistance au lorlatinib chez des patients atteints d'un cancer du poumon ALK réarrangé par la mise en œuvre de biopsies spatiales et temporelles et le développement de modèles dérivés de patients. Dans le cadre de l’étude institutionnelle MATCH-R (NCT02517892), nous avons effectué un séquençage à haut débit de l’exome, de l’ARN et ciblé, ainsi qu’un séquençage des ctDNA afin d’identifier les mécanismes de résistance. Nous avons établi des lignées cellulaires dérivées de patients et caractérisé de nouveaux mécanismes de résistance et identifiés de nouvelles stratégies thérapeutiques in vitro et in vivo. Nous avons identifié trois mécanismes de résistance chez quatre patients avec des biopsies appariées. Nous avons étudié l'induction de la transition épithélio-mésenchymateuse (EMT) par l'activation de SRC dans une lignée cellulaire, dérivée d’un patient, exposée au lorlatinib. Les cellules mésenchymateuses étaient sensibles à l’inhibition combinée de SRC et d'ALK, montrant que même en présence d'un phénotype agressif, des stratégies de combinaison peuvent surmonter la résistance aux ALK TKI. Nous avons identifié deux nouvelles mutations composées du domaine kinase d’ALK, F1174L / G1202R, C1156Y / G1269A survenues chez deux patients traités par le lorlatinib. Nous avons développé des modèles de cellules Ba / F3 exprimant les mutations simples et composées pour étudier leur effet sur la résistance au lorlatinib. Enfin, nous avons caractérisé un nouveau mécanisme de résistance provoqué par la perte de fonction de NF2 au moment de la progression du lorlatinib par l’utilisation de PDX et de lignées cellulaires dérivées de patients, et par CRISPR / CAS9 knock-out de NF2. Nous avons constaté que l'activation de mTOR par la perte de fonction de NF2 provoquait la résistance au lorlatinib et qu'elle pouvait être surmontée par le traitement avec des inhibiteurs de mTOR.Cette étude montre que les mécanismes de résistance au lorlatinib sont plus divers et complexes que prévu. Nos résultats démontrent également comment les études longitudinales de la dynamique tumorale permettent de déchiffrer la résistance aux TKI et d'identifier des stratégies thérapeutiques
The molecular study and classification of lung adenocarcinomas has led to the development of selective targeted therapies aiming to improve disease control and survival in patients. The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor from the insulin tyrosine kinase receptor family, with a physiologic role in neural development. Gene rearrangements involving the ALK kinase domain occur in ~3-6% of patients with lung adenocarcinoma. The fusion protein dimerizes leading to transactivation of the ALK kinase domain in a ligand-independent and constitutive manner. Lorlatinib is a third generation ALK inhibitor with high potency and selectivity for this kinase in vitro and in vivo, and elevated penetrance in the central nervous system. Lorlatinib can overcome resistance mediated by over 16 secondary kinase domain mutations occurring in 13 residues upon progression to first - and second - generation ALK TKI. In addition, treatment with lorlatinib is effective for patients who have been previously treated with a first and a second generation or a second generation ALK TKI upfront and is currently approved for this indication. The full spectrum of biological mechanisms driving lorlatinib resistance in patients remains to be elucidated. It has been recently reported that the sequential acquisition of two or more mutations in the kinase domain, also referred as compound mutations, is responsible for disease progression in about 35% of patients treated with lorlatinib, mainly by impairing its binding to the ALK kinase domain. However, the effect of these compound mutations on the sensitivity to the repertoire of ALK inhibitors can vary, and other resistance mechanisms occurring in most patients are unknown. My PhD thesis aimed at exploring resistance to lorlatinib in patients with ALK-rearranged lung cancer through spatial and temporal tumor biopsies and development of patient-derived models. Within the institutional MATCH-R study (NCT02517892), we performed high-throughput whole exome, RNA and targeted next-generation sequencing, together with plasma sequencing to identify putative genomic and bypass mechanisms of resistance. We developed patient-derived cell lines and characterized novel mechanisms of resistance and personalized treatment strategies in vitro and in vivo. We characterized three mechanisms of resistance in four patients with paired biopsies. We studied the induction of epithelial-mesenchymal transition (EMT) by SRC activation in a patient-derived cell line exposed to lorlatinib. Mesenchymal cells were sensitive to combined SRC and ALK co-inhibition, showing that even in the presence of an aggressive and challenging phenotype, combination strategies can overcome ALK resistance. We identified two novel ALK kinase domain compound mutations, F1174L/G1202R, C1156Y/G1269A, occurring in two patients treated with lorlatinib. We developed Ba/F3 cell models harboring single and compound mutations to study the differential effect of these mutations on lorlatinib resistance. Finally, we characterized a novel mechanism of resistance caused by NF2 loss of function at the time of lorlatinib progression through the development of patients derived PDX and cell lines, and in vitro validation of NF2 knock-out with CRISPR/CAS9 gene editing. Downstream activation of mTOR was found to drive lorlatinib resistance by NF2 loss of function and was overcome by providing treatment with mTOR inhibitors.This study shows that mechanisms of resistance to lorlatinib are more diverse and complex than anticipated. Our findings also emphasize how longitudinal studies of tumor dynamics allow deciphering TKI resistance and identifying reversing strategies

Recent whole-exome sequencing studies of hundreds of high-risk neuroblastoma (hNB) patients have identified Anaplastic Lymphoma Kinase (ALK) as the only directly ‘druggable’ target with a significant mutation rate (9%). ALK is a receptor tyrosine kinase whose dysregulation has been implicated as the driver lesion in a variety of cancer types, including Non-Small Cell Lung Cancer (NSCLC) and various paediatric malignancies. As a kinase normally only expressed during early development in the foetal brain, ALK is an ideal therapeutic target and it has proven relatively simple to target therapeutically. However, resistance to ALK-targeted therapy, particularly in ALK+ NSCLC patients has frequently been observed. The majority of the acquired resistance mechanisms noted in NSCLC patients rely on bypass signalling pathways, which are tissue-context dependent. To proactively identify and develop strategies to counter these varied yet expected resistance mechanisms in other ALK-driven tumours, we must gain a better insight of the bypass-track mechanism(s) in a tumour-specific manner. The present study aimed to functionally identify putative resistance mechanisms against ALK inhibitors via extensive CRISPR/Cas9-based genome-wide knockout (GeCKO) or overexpression screens (SAM) in the human neuroblastoma cell line, SHSY-5Y, to develop novel therapeutic strategies for ALK mutant NBs. The GeCKO screen identified a total of 39 genes and miRNAs, and the SAM overexpression screen identified 25 genes that induce resistance to ALK inhibitors. These putative resistance-inducing candidates were then aligned with a publicly available expression dataset of hNB patients (n = 476) to identify those with prognostic significance (Kaplan-Meier event-free survival analysis), specifically those that are indicative of relapse risk. Furthermore, all candidates identified from the screen were individually validated in vitro. Two of the candidates, one from each of the knockout and overexpression screens, were further investigated. Inhibition of hsa-miR-1304-5p, identified from GeCKO screen, induced resistance to ALK inhibitors. Interestingly, interference of has-miR-1304-5p, in the absence of ALK inhibitors, also enabled enhanced cell viability whilst the transfection of its mimic led to a significant reduction of viability across 17 distinct NB cell lines. Through genome-wide cDNA microarrays, in silico predictions, and UTR-luciferase assays, this study identified hsa-miR-1304-5p to be a major regulator of the Ras/MAP Kinase pathway. Overexpression of PIM1, identified from the SAM screen, in NB cell lines induced resistance to ALK inhibitors and this phenotype could be reversed on transducing cells with RNAi against PIM1. Interestingly, inhibition of PIM1 in wild-type cell lines via RNAi or pharmacological compounds led to substantially enhanced potency of ALK inhibitors suggesting PIM1 inhibitors as combinatorial agents with ALK inhibitors for the therapy of treatment-naive hNB. Through protein analysis of all identified downstream targets of PIM1, this study revealed NB-specific actions of the PIM1 oncoprotein that include the inactivation of the pro-apoptotic protein BAD. In summary, this study has identified mechanisms of resistance to ALK inhibitors as well as novel front-line therapeutic strategies for hNB patients that should be implemented clinically.

The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in the model plant, mouse-ear cress, was reported in winter wheat fields in Westmoreland County, Virginia. A putative resistant (R) mouse-ear population was assessed for ALS resistance relative to a putative susceptible (S) and a susceptible lab population Columbia (C). Results indicated that the R population needed 23 to >2400 fold rate of thifensulfuron relative to S or C population, and it has evolved cross-resistance to sulfonylureas (SU), triazolopyrimidine sulfonanilides (TP), and sulfonylaminocarbonyltriazolinones (SCT). Further studies sequenced the whole genome for four field populations, representing two locations and two resistance levels (high and low) per location, to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained mutations in the ALS gene at the Pro197 site, although the Pro was substituted by Phe in one location and Thr in the other. Also, both high- and low-level resistant plants at one location had additional mutations (Trp574Leu or Asp376Glu) known to confer resistance to ALS inhibiting herbicides. Patterns of herbicide cross-resistance also varied among the populations. Additionally, research was conducted to assess preemergent (PRE) and postemergent (POST) alternative herbicide options for control of ALS resistant mouse-ear cress and its interference with winter wheat. Results indicate flumioxazin, pyroxasulfone, and metribuzin can be used for effective PRE control whereas 2,4-D, dicamba, and metribuzin can be effective post control options. No mouse-ear cress interference with winter wheat was observed at density of more than 300 plants m-2.
Master of Science in Life Sciences

Els inhibidors de la transcriptassa inversa no anàlegs de nucleòsids (ITINAN) s’utilitzen sovint en el tractament antiretroviral (TAR) per la seva eficàcia i simplicitat. La resistència està causada per mutacions específiques a posicions de resistència. L’ús clínic dels ITINAN de primera generació (nevirapina i efavirenz) s’ha vist limitat pels efectes adversos, i la baixa barrera en front a la resistència, pel que s’han desenvolupat ITINAN de segona generació (etravirina i rilpivirina), ambdós recentment aprovats. Rilpivirina mostra també una baixa barrera a la resistència. Al igual que nevirapina o efavirenz, pot perdre l’activitat amb només una o dues mutacions. Hi ha un elevat grau de resistència creuada entre tots els ITINAN, pràcticament complerta entre nevirapina i efavirenz, i més limitada entre els ITINAN de primera i segona generació. Per tant, el coneixement del impacte de les mutacions de resistència seleccionades per ITINAN de primera generació sobre els de segona generació té una importància cabdal. Els llistats de mutacions de resistència front als ITINAN estan ja ben definits a l’actualitat, permetent el seu anàlisi en mostres clíniques, oferint una oportunitat única per a la investigació del impacte de la resistència del VIH-1 en la resposta al TAR de inici, rescat o simplificació. En aquesta tesi es discuteix la rellevància de les mutacions de resistència sobre la resposta al tractament, s’identifiquen les mutacions i patrons de mutacions seleccionades en el fracàs virològic (FV) amb determinats ITINAN, i s’analitza el risc de FV posterior a tractaments basats en ITINAN. El primer capítol avalua la efectivitat de etravirina en el rescat de FV atesos a 4 hospitals de referència de Barcelona. Els tractaments van ser ben tolerats i assolir taxes de supressió virològica superiors a les observades en els seus assaigs clínics de registre, probablement degut a la inclusió de un nombre més elevat de fàrmacs actius als règims. Hem identificat l’associació de recomptes de cèl·lules CD4+ >200 cèls/mm3 i l’ús de raltegravir i darunavir a taxes menors de FV en l’anàlisi multivariant. No hem trobat relació entre interrupció o FV previs a nevirapina o efavirenz, i resposta a etravirina. El segon capítol analitza el impacte sobre rilpivirina de mutacions de resistència seleccionades en FV als altres ITINAN (nevirapina, efavirenz o etravirina) a 22 centres hospitalaris de España. Es va demostrar resistència a rilpivirina en el 20% de FV a ITINAN, més freqüent en FV a etravirina i nevirapina que a efavirenz. Les mutacions de resistència a rilpivirina més freqüents van ser Y181C, K101E/P, H221Y i E138A/G/K. E138K/M184I, la combinació mes freqüentment seleccionada amb rilpivirina en naives, va estar absent en aquesta població pretractada. L100I i V108I van ser significativament més freqüents en fracassos a efavirenz. Contràriament, Y181C/I, V106A, H221Y i F227L ho van ser amb nevirapina. Finalment el tercer capítol estima la eficàcia de un règim de simplificació de TAR amb nevirapina, tenofovir i emtricitabina/lamivudina, realitzat a la nostra Unitat a Barcelona. No es van aïllar patrons de mutacions inesperats en els FV. A les 48 setmanes el 90% de malalts tenien una càrrega viral < 50 còpies/mL, el FV va ser infreqüent, i 25 (7.4%) pacients van suspendre el tractament per toxicitat. Els factors associats amb FV a l’anàlisi multivariant van ser drogadicció intravenosa, temps amb càrrega viral indetectable abans de la simplificació, nombre de NRTIs i NNRTIs rebuts, i les interrupcions no programades de tractaments previs amb nevirapina o efavirenz. En base a aquestes dades s’hauria de desaconsellar el reinici del TAR amb nevirapina, tenofovir i emtricitabina/lamivudina en interrupcions no programades del TAR, encara que la càrrega viral sigui indetectable al moment de la suspensió. Inesperadament, hem trobat una taxa significativament més elevada de FV amb lamivudina que amb emtricitabina amb aquesta combinació, així com una major selecció de M184V. Aquests resultats suggereixen precaució en la substitució de emtricitabina per lamivudina, al menys en règims basats en nevirapina i tenofovir.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are popular components of antiretroviral therapy due to their efficacy and simplicity. Resistance is caused only by specific mutations at drug-resistance positions. Despite its proven efficacy, the clinical use of first-generation NNRTIs (nevirapine and efavirenz) has been limited by side effects and low barrier to resistance. To overcome these limitations, a second-generation of NNRTIs has been developed including etravirine and rilpivirine, both recently approved. Rilpivirine also depicts a low barrier to resistance development. Like nevirapine and efavirenz, complete drug resistance can arise with only one or two resistance-associated mutations (RAMs). In addition, there is a considerable degree of class cross-resistance among all NNRTIs, nearly complete between nevirapine and efavirenz, and more limited from first to second generation NNRTIs. Therefore, the knowledge of RAMs selected by first-generation NNRTIs that have a potential to impact both rilpivirine or etravirine in subsequent treatments is of paramount importance. Genotypic scores are now fully developed for all these drugs, therefore allowing resistance analyses in clinical samples and offering a unique opportunity to investigate the clinical impact of HIV-1 resistance on treatment response both in initial, salvage or simplification treatment. In this PhD thesis, we discuss the relevance of RAMs on treatment response; we pinpoint the patterns of RAMs selected at virologic failure (VF) with specific NNRTIs, and the consequent risk of failure to salvage or simplification with NNRTIs. The first chapter evaluates the effectiveness of etravirine in salvage regimens in VF recruited at four acute-care University hospitals in Barcelona. These regimens were generally well tolerated and achieved rates of virological suppression that exceed those observed in etravirine’s pivotal clinical trials, probably due to the inclusion of a higher number of active drugs in the regimens. We identified baseline CD4+ T cell count >200 cells/mm3 and use of raltegravir and darunavir as factors associated with lower treatment failure rates using a multivariate analysis. We found no relationship between prior interruption or VF with nevirapine or efavirenz and response to etravirine. The second chapter assesses the RAMs selected in subjects failing NNRTI-based treatments (with nevirapine, efavirenz or etravirine) at 22 clinics in Spain and the potential impact on rilpivirine’s activity. Rilpivirine resistance was recognized in 20% of these patients, more commonly following etravirine or nevirapine failures than efavirenz. The most prevalent rilpivirine RAMs in subjects failing other NNRTIs were Y181C, K101E/P, H221Y and E138A/G/K. E138K/M184I, the most frequently selected combination in initial treatment with rilpivirine, was absent in this treatment-experienced population. L100I and V108I were significantly more frequent in efavirenz failures. Conversely, Y181C/I, V106A, H221Y and F227L were more prevalent in nevirapine ones. Finally, the third chapter estimates the effectiveness of a nevirapine-based switch regimen in subjects with suppressed viremia, combined with tenofovir and emtricitabine (or lamivudine). The analysis has been done in our clinic in Barcelona. No unexpected RAMs or patterns of RAMs were selected in treatment failures to this regimen. At week 48, nearly 90% of the subjects had HIV-1 RNA <50 copies/mL, VF was uncommon, and 25 (7.4%) subjects discontinued the treatment due to toxicity. Factors independently associated with VF in multivariate analysis were intravenous drug use, time with undetectable viral load before the switch, number of prior NRTIs or NNRTIs, and previous nevirapine or efavirenz unscheduled interruptions. Reinitiation of nevirapine plus tenofovir plus emtricitabine (or lamivudine) should be discouraged in subjects experiencing unplanned treatment interruptions, even with an undetectable plasma viral load at the time of treatment withdrawal. Unexpectedly, we found a significantly higher rate of VF with lamivudine instead of emtricitabine with this regimen, with a significantly higher selection of M184V as well. Our findings suggest caution against substituting emtricitabine for lamivudine, at least in nevirapine- and tenofovir-based regimens.

Ce travail a eu pour but premier d’étudier le risque d’évolution de la résistance aux herbicides inhibiteurs de l’acétolactate synthase (ALS) chez l’ambroisie à feuilles d’armoise (Ambrosia artemisiifolia L.) à travers quatre points : (i) la pression de sélection (étude de l’efficacité d’une gamme d'herbicides inhibiteurs de l’ALS), (ii) la capacité de réponse adaptative de l’adventice (détermination de la variation de la sensibilité aux inhibiteurs de l’ALS entre plantes et mise en place d’un programme de sélection récurrente), (iii) une étude de terrain (recherche de résistance aux inhibiteurs de l’ALS au champ en France), (iv) l’étude des mécanismes de résistance (liée à la cible – RLC – et non liée à la cible – RNLC – par une approche de transcriptomique). Le second objectif fut d’étudier la connectivité des populations d’A. artemisiifolia dans des paysages agricoles à l’aide de marqueurs microsatellites développés lors de ce travail afin de déterminer les facteurs qui pourraient faciliter la dispersion de cette espèce et de la résistance à l’échelle du paysage agricole.En ce qui concerne la résistance aux herbicides :-La réponse de d’A. artemisiifolia aux herbicides inhibiteurs de l’ALS est très variable entre substances.-Des plantes ayant survécu à la dose maximale autorisée et à des doses supérieures de metsulfuron ont été sélectionnées pour débuter un programme de sélection récurrente. Après deux cycles de sélection, on observe une intensification de la résistance au metsulfuron et une émergence de la résistance à l’imazamox et au tribénuron.-Trois cas de résistance à l’imazamox ont été identifiés au champ dont deux cas de pure RNLC et un cas de coexistence RLC – RNLC.-Un transcriptome d’A. artemisiifolia a été généré grâce à la technique de séquençage PacBio pour rechercher des gènes impliqués dans les mécanismes de RNLC (approche RNAseq). 62 gènes candidats ont été identifiés dont des transporteurs ABC, des cytochromes P450 ainsi que des glutathione-S-transférases connus pour être impliqués dans la dégradation des herbicides.Pour l’étude de la connectivité des populations agricoles :-26 marqueurs microsatellites ont été développés et ont révélé une forte variabilité génétique. La structuration génétique a été étudiée à grande échelle pour des populations d’A. artemisiifolia d’Europe (aire d’invasion) et d’Amérique du Nord (aire d’origine).-À une échelle plus fine (paysage agricole), la structure génétique des populations reste influencée par les événements de colonisation. Les événements de migration qui ont été identifiés entre zones de présence de l’ambroisie suggèrent des flux de gènes (pollen/semences) et une connectivité modérés à l’échelle d’un territoire agricole. Dans les environnements agricoles, la dispersion des allèles de résistance aux herbicides pourrait se faire facilement de proche en proche via les flux de pollen, et également à plus longue distance via des dispersions de graines. Les activités anthropiques jouent un rôle majeur dans la dispersion des semences (machineries agricoles, lots de semences contaminés…).-L’analyse du système de reproduction a confirmé que cette espèce est allogame ce qui entraîne des flux de gènes intra- et inter-populations importants.Les connaissances acquises au cours de ce travail pourront aider à développer des stratégies de contrôle mieux adaptées, pour lutter efficacement contre A. artemisiifolia afin de limiter son expansion, telles que :-Des stratégies de désherbage diversifiées : combinaison de lutte mécanique (dont faux semis) et chimique (diversification des modes d’action herbicides).-Un allongement et une diversification des rotations de cultures en favorisant des cultures d’hiver et/ou des cultures couvrantes et compétitrices.Ces connaissances pourront aussi être utilisées dans la lutte contre une autre espèce adventice du genre Ambrosia, Ambrosia trifida.Mots-clés (6) : Ambrosia artemisiifolia L., ambroisies, résistance aux herbicides
The first aim of this work was to investigate the risk for the evolution of resistance to acetolactate synthase inhibitor (ALS) herbicides in the common ragweed (Ambrosia artemisiifolia L.) through four points: (i) the selection pressure (effectiveness of a range of ALS inhibitor herbicides), (ii) the adaptive response of Ambrosia artemisiifolia (recurrent selection experiment), (iii) a resistance monitoring in fields in France, and (iv) the investigation of the mechanisms underlying herbicide resistance (target-site (TSR) and non-target-site resistance (NTSR) using transcriptomic analyses). The second aim was to study the connectivity of A. artemisiifolia populations in agricultural landscapes using microsatellite markers developed during this work, to determine factors that could facilitate the spread of this invasive weed species and the spread of herbicide resistance.In regards to herbicide resistance:-The sensitivity of A. artemisiifolia to ALS-inhibiting herbicides is variable between active ingredients.-Plants that survived the French maximum authorized field rate and higher rates of metsulfuron were selected to implement a recurrent breeding program. After two selection cycles, the resistance level to metsulfuron increased and resistance to imazamox and tribenuron emerged.-Three cases of imazamox resistance were identified in the field, including two cases of pure NTSR and one case of TSR - NTSR coexistence.-A transcriptome for A. artemisiifolia, AMBELbase, was generated using the PacBio sequencing technology to search for genes involved in NTSR mechanisms (RNAseq approach). 62 candidate contigs were identified including ABC transporters, cytochromes P450 and glutathione S-transferases known to be involved in the degradation of herbicides.In regards to population connectivity:-26 microsatellite markers were developed and revealed high genetic variability. Genetic structuring has been studied on a large scale for populations of A. artemisiifolia from Europe (invasion range) and North America (native range).-On a finer scale (agricultural landscape), the genetic structure of populations was influenced by colonization events. Migration events detected among the areas colonized by A. artemisiifolia suggested moderate pollen/seed flows and connectivity at the farmland scale. In agricultural environments, herbicide resistant alleles could be easily spread among neighbouring populations via pollen flow, and also at longer distances via seed dispersal. Human-related activities play a major role in the dispersal of seeds (agricultural machinery, contaminated seed lots, etc.).-The mating system analysis confirmed that A. artemisiifolia is an obligate outcrossing species which leads to important intra- and inter-population gene flow.The knowledge acquired during this work may help to foster the development of better management strategies to effectively control A. artemisiifolia to limit its spread, such as:-Diversified weed control strategies: combination of mechanical (including false-seed) and chemical weeding (diversification of herbicide modes of action).-Longer diversified crop rotations including more winter crops and/or cover and competitive crops to break the life cycle of A. artemisiifolia.These knowledge may also be used to better control of another weed species of the genus Ambrosia, Ambrosia trifida L

La sintasa d’àcids grassos (FASN), enzim que realitza la síntesi de novo dels àcids grassos, es sobreexpressa en alguns càncers, sobretot en càncer de mama, i s’associa a un mal pronòstic. La seva regulació està relacionada amb la via de senyalització d’HER2, receptor cel.lular de membrana que pertany a la família dels receptors de creixement epidèrmic (HER), tot i que encara es desconeixen els mecanismes moleculars d’aquesta relació. El coneixement de la implicació del paper del metabolisme dels àcids grassos en la iniciació i la progressió del càncer de mama i el desenvolupament d’inhibidors farmacològics de l’activitat de FASN té un gran interès científico-mèdic. L’ús clínic d’inhibidors de FASN, com la cerulenina o el C75, presenten una baixa estabilitat i efectes col.laterals com l’anorèxia i la pèrdua de pes, ja que activen la Carnitina Palmitoil Transferasa 1 (CPT1), enzim regulador de la β-oxidació dels àcids grassos. Anteriorment el nostre grup va demostrar que el (-)-3-galat d’epigalocatequina (EGCG), un polifenol present en el té verd, inhibeix FASN, té efectes antitumorals i és més selectiu que la cerulenina i el C75 perquè no afecta la CPT1, i en conseqüència no produeix pèrdua de pes in vivo. L’ús terapèutic de l’EGCG com agent antitumoral es veu limitat per l’elevat valor de la IC50 (~ 150 >M) i també per la inestabilitat en condicions fisiològiques. En base a aquests resultats, en aquest treball es presenta el disseny d’una família de compostos polifenòlics relacionats estructuralment amb l’EGCG per millorar-ne la seva activitat antitumoral, la biodistribució i l’estabilitat. In vitro s’ha realitzat l’avaluació de l’activitat biològica i molecular d’aquests compostos per tal de valorar-ne l’especificitat, la capacitat inhibidora específica de FASN i l’activitat citotòxica en diferents models de càncer de mama. S’ha determinat que el compost G28UCM presenta una inhibició de l’activitat de FASN d’un 90% i una elevada citotoxicitat en cèl.lules de càncer de mama (IC50= 30 >M). Per altra banda, s’han analitzat les interaccions cel.lulars i moleculars del composta G28UCM en combinació amb les teràpies anti-HER existents (trastuzumab, lapatinib, erlotinib i cetuximab) en un model cel.lular de càncer de mama FASN/HER2 positiu. El G28UCM mostra un clar sinergisme farmacològic amb els fàrmacs anti-HER, trastuzumab, lapatinib i erlotinib, a diferència de l’EGCG que tant sols mostra additivisme amb el trastuzumab. A més al laboratori s’han desenvolupat models cel.lulars de càncer de mama resistents als fàrmacs anti-HER2, trastuzumab i lapatinib, i s’ha avaluat si el G28UCM té activitat antitumoral en aquests models cel.lulars. Els resultats obtinguts ens mostren que el G28UCM segueix mostrant activitat citotòxica amb les cèl.lules resistents als fàrmacs anti-HER2. In vivo, primer es va desenvolupar el millor model murí de carcinoma de mama FASN i HER2+ utilitzant els compostos inhibidors de FASN de referència EGCG i C75. Un cop establert el model animal es va caracteritzar l’activitat antitumoral i el perfil de toxicitat del compost seleccionat G28UCM. Es va obtenir una inhibició del creixement tumoral en un terç dels ratolins tractats amb el G28UCM, els quals no varen mostrar cap tipus de toxicitat (renal, hepàtica, cardíaca) ni pèrdua de pes. Aquests resultats proporcionen el fonament pel desenvolupament preclínic del G28UCM, ja sigui sol o en combinació amb les teràpies anti-HER existents en els tumors de mama que sobreexpressen HER2 i els que han progressat al tractament.
Fatty Acid Synthase (FASN), an enzyme responsible for the de novo synthesis of fatty acids is overly manifested in certain types of cancer, especially in breast cancer, and it is associated with a poor diagnosis. Its regulation is associated with the signalling of HER2, a cellular receptor belonging to the human epidermal growth receptors family. However, the molecular mechanisms associated with this relationship are not well understood. FASN inhibitors, such as cerulenin or C75, offers low levels of stability and present side effects, like anorexia and weight loss, since they activate Carnitina Palmitoil Transferasa 1 (CPT1), a regulator enzyme of fatty acids’ β-oxidation. Previously our research group has proven that epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, inhibits FASN, has anti-tumoral effects and since it does not affect CPT1, is more selective than both cerulenin and C75. The therapeutic use of EGCG as an anti-tumoral agent is limited by its high IC50 value, as well as by its instability in physiological conditions. Following these results, a family of polyphenol compounds structurally related to EGCG has been designed with the goal of enhancing its anti-tumoral activity, its biodistribution and its stability. In vitro, we evaluated the biological and molecular activity of these compounds to assessing their specificity, their FASN-specific inhibiting capacity and their cytotoxic activity in different models of breast cancer. G28UCM shows a 90% inhibition of FASN activity and a high cytotoxicity levels in breast cancer cells (IC50= 30 9M). Also, G28UCM were analyzed in combination with already existing anti-HER therapies (trastuzumab, lapatinib, erlotinib, and cetuximab) in a cellular model of FASN/HER2- positive breast cancer. G28UCM shows synergysm with trastuzumab, lapatinib, and erlotinib, as opposed to EGCG, which only shows additivism with trastuzumab. Further, we also developed cellular models of breast cancer, which were resistant to trastuzumab, and lapatinib, and assessed whether G28UCM presents anti-tumoral activity in said cellular models. G28UCM presents cytotoxic activity with anti-HER2 drug resistant cells. In vivo, the tumoral activity and the toxicity profile of G28UCM were characterized in the murine model of FASN and HER2+ breast carcinoma. The results showed an inhibition of tumor growth in mice treated with a third G28UCM and any toxicity or weight loss. In summary, our findings provide the basis for the pre-clinical development of G28UCM.

Les leucémies aiguës (LA) sont un groupe hétérogène d'hémopathies malignes dues à latransformation oncogénique clonale des cellules souches hématopoïétiques (CSH). On distingue les LA myéloblastiques etlymphoblastiques (LAL). Les LAL sont classées selon le type de précurseur lymphoïde atteint, leur degré de maturité et leurs anomalies cytogénétiques.Le traitement permettant d'obtenir 80 à 90 % de rémission complète (RC) comporte une chimiothérapie d'induction, une consolidation et une intensification(intensification retardée ou allogreffe de CSH selon la situation pronostique). Néanmoins la survie globale à long terme n'est que de 40 à 50 %, du fait de la survenue de rechutes et de la toxicité des traitements. Différents groupes pronostiques basés sur la cytogénétique et la biologie moléculaire se dégagent,pouvant bénéficier de thérapeutiques adaptées. Dans les LAL à chromosome philadelphie (LAL à Ph),antérieurement de mauvais pronostic, les inhibiteurs de tyrosine kinase (ITK) permettent d'obtenir 80% de RCavec cependant un taux de rechute non négligeable. Nous avons démontré qu'une intensification thérapeutique par autogreffe chez des patients avec une maladie résiduelle indétectable permettait une survie à long terme prolongée avec une toxicité moindre que celle de l'allogreffe. En montrant l'implication de l'autotaxine dans les mécanismes de résistance aux ITK dans les LAL à Ph, nous ouvrons la voie à l'utilisation potentielle de nouvelles thérapeutiques. Dans les LAL T, groupe considéré de bon pronostic, un tiers des patients rechute. Nous avons démontré que l'absence de mutation de Notch et/ou FBXW7 ou la présence de mutations de RAS ou PTEN était de mauvais pronostic identifiant un sous-groupe de LAL T dont le traitement devait être renforcé. Nos travaux ont ainsi contribué à l'identification des groupes pronostiques dans les LAL et à l'adaptation des traitements afin d'améliorer les chances de survie
Acute leukemias are a heterogeneous groups of malignant hematological diseases due to the clonaloncogenic transformation of hematopoietic stem cells(HSTs). We distinguish acute myeloblastic leukaemiafrom acute lymphoblastic leukemia (ALL). ALLs are classified according to the type of lymphoid precursoraffected, its degree of maturity, and with associated cytogenetic abnormalities.Treatment incorporating induction therapy,consolidation, and intensification – delayedintensification or allogeneic stem cell transplantation(SCT) according to prognostic factors – enable 80 to 90% of complete remission (CR). Nevertheless, long-termoverall survival is only 40 to 50% because of relapseand treatment-related toxicity. Different prognosticgroups based on cytogenetic abnormalities andmolecular biology are emerging and patients from eachprognostic group can benefit from adapted therapies.In chromosome Philadelphia-positive ALL (Ph+ ALL) which used to be of particular bad prognosis, tyrosinekinase inhibitors (TKIs) enables 80% of CR but with ahigh-relapse risk. We demonstrated that high-dosetherapy followed by autologous SCT enables prolongedlong-term survival with less drug-related toxicity ascompared to allogeneic SCT in patients with undetectable minimal residual disease. By showing the implication of autotaxine in the resistance to TKIs inPh+ LAL, we enable the use of novel therapeutics inclinical practice.T-cell ALL is considered of poor prognosis as one thirdof patients relapse. In this group of patients we showedthat the absence of a Notch and/or a FBXW7 mutation or the presence of mutations in RAS or PTEN identified a subgroup of patients in whom the treatmentmust be intensified. Our research has contributed to the identification of prognostic groups in ALL and to theadjustment of treatment according to potential survival

Acetolactate synthase (ALS) inhibitors are a widely used class of selective herbicides used to control grass and broadleaf weeds. The repeated use of ALS-inhibiting herbicides has selected for biotypes of weeds resistant to ALS inhibitors, especially in the weeds most problematic to growers in the Midwest. While ALS inhibitor use seems futile, new mechanisms of herbicide action are not predicted to be commercialized in the near future to solve this problem. This leads to the main objective of this research, determining what value ALS inhibitors provide in controlling populations of weeds with resistance to ALS inhibitors.

Field experiments with soil-applied (PRE) applications of ALS inhibitors on horseweed (Erigeron canadensis) and tall waterhemp (Amaranthus tuberculatus var. rudis) exhibited higher efficacy than would be expected given the frequency of the ALS resistance trait in the population. Whereas control of these species with POST-applied applications was similar or less than the proportion of the population characterized as susceptible using molecular techniques. Soil-applied applications, therefore, resulted in relatively greater control than POST applications in populations with known ALS-inhibitor-resistance mechanisms.

Greenhouse experiments showed that overall resistance ratios were higher for PRE applications of ALS inhibitors in horseweed, tall waterhemp, and Palmer amaranth (Amaranthus palmeri). However, GR₅₀ values decreased for both susceptible and resistant biotypes for the PRE applications compared to POST, suggesting the biologically effective dose of these herbicides is lower in soil residual applications. This research found that PRE applications of ALS inhibitors resulted in some level of control on horseweed and tall waterhemp classified as resistant to ALS inhibitors due to the higher efficacy of PRE herbicide applications.

Genetic analysis assessing the amino acid substitutions that confer resistance to ALS inhibitors in tall waterhemp confirmed a difference in selection pressure between PRE and POST applications and between ALS active ingredients in tall waterhemp. Applications of chlorimuron PRE at 11 g ai ha^-1 selected for 35% homozygous W574L genotypes and at 44 g ha^-1 selected for 70% homozygous W574L genotypes. An increase of homozygous W574L individuals along with a decrease in heterozygous individuals from 65 (11 g ha^-1) to 29% (44 g ha^-1) suggests that W574L is semi-dominant in tall waterhemp and that high labeled rates of chlorimuron applied PRE can partially overcome the heterozygous W574L-resistance mechanism. In horseweed, no difference in selection pressure was observed between application timing or between chlorimuron or cloransulam. A new mutation conferring ALS-inhibitor resistance in horseweed was discovered, a Pro197Leu amino acid substitution, with resistance ratios of 21X to chlorimuron and 8.6X to cloransulam. These resistance ratios are slightly less than those reported for the Pro197Ala and Pro197Ser amino acid substitutions in conferring ALS-inhibitor resistance in horseweed.

Finally, a survey of 42 populations of tall waterhemp in Indiana counties with confirmed ALS-inhibitor resistant populations of tall waterhemp found that all populations contained at least 16% individuals with the W574L amino acid substitution, 35 populations contained at least 1% individuals with the S653N substitution, and 9 populations contained at least 1% individuals with the S653T substitution. Taking into consideration the three mutations tested, 8 of the 42 populations tested contained <50% ALS-inhibitor resistant individuals within the population. Using the same tall waterhemp populations as collected in the survey, Next-Generation Sequencing was used to determine if other amino acid substitutions conferring resistance to ALS inhibitors existed. Results from WideSeq revealed that 10 other amino acid substitutions in the ALS protein may be conferring resistance in tall waterhemp in Indiana: A122T, A122N, A122S, P197T, P197L, P197H, D376E, and G654F. Further research from this survey also suggests that metabolic resistance to ALS inhibitors is likely a contributor to resistance in tall waterhemp in Indiana.

This research suggests that ALS-inhibiting herbicides, more specifically chlorimuron, would provide the greatest contribution to management of tall waterhemp. Chlorimuron would perform best when used in soil residual applications and in populations of tall waterhemp containing either individuals susceptible to chlorimuron or individuals heterozygous for ALS inhibitor resistance conferred by the W574L mutation. This research also demonstrates the specificity of the amino acid substitutions in the ALS protein and by weed species to realize the benefit of these herbicides for management of weeds resistant to ALS inhibitors. Molecular characterization of target site resistance to ALS inhibitors has traditionally been considered relatively simple. However, we found 11 new amino acid substitutions that confer resistance to ALS inhibitors in horseweed and tall waterhemp. The complexity of ALS inhibitor resistance calls for the use of methods such as NGS to detect all potential resistance mutations in a timely manner and for the use of tests detecting metabolic resistance. Overall, this research demonstrates that ALS inhibitors still provide some utility for management of weed populations classified as resistant to ALS inhibitors and that the resistance mechanisms in horseweed and tall waterhemp are more numerous than previously reported.

碩士
大仁科技大學
藥學系碩士班
107
Pterostilbene is known to possess antioxidant activity and induces cell death in various types of cancer cells. Here, the effects of pterostilbene on cell viability in cisplatin-resistant AGS gastric cancer cells were investigated. This study demonstrated that pterostilbene was able to inhibit cell proliferation and induce cell death in concentration- and time-dependent manners. Pterostilbene-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation by TUNEL staining. The molecular mechanism of pterostilbene induced apoptosis was also investigated. The results show the caspase-9 and caspase-3 are activated, and using the pan-caspase inhibitor carbobenzoxyvalyl-alanyl-aspartyl fluoromethyl ketone by pterostilbene in cisplatin-resistant AGS gastric cancer cells. In summary, pterostilbene induced apoptosis in cisplatin-resistant AGS gastric cancer cells through activating the caspase cascade. The induction of apoptosis by pterostilbene may provide a pivotal mechanism of the anti-cancer effects and for treatment of human gastric cancer.

碩士
大仁科技大學
藥學系碩士班
107
Cisplatin is the leading therapeutic agent for gastric cancer therapy. In our study, Kaempferol suppressed cell viability in cisplatin-resistant AGS gastric cancer cells through inhibiting cell proliferation and causing cell death. The results demonstrated that cisplatin-resistant AGS gastric cancer cells exhibited marked cell shrinkage, cell membrane breakage and apoptotic bodies following treatment with kaempferol. Kaempferol also effectively suppressed cell confluence in a time- and concentration-dependent manner. The DAPI/TUNEL double staining determined that DNA condensation, a characteristic of apoptosis, was enhanced following treatment with kaempferol, which confirmed by the pan-caspase inhibitor. In addition, kaempferol increased caspase-3 and caspase-9 activities in cisplatin-resistant AGS gastric cancer cells. Overview, Kaempferol may elicit an anti-cancer response in cisplatin-resistant AGS gastric cancer cells and may have a good chemotherapeutic adjuvant to treat gastric cancer.