Koenimbin, a natural dietary compound of Murraya koenigii (L) Spreng: inhibition of MCF7 breast cancer cells and targeting of derived MCF7 breast cancer stem cells (CD44+/CD24-/low): an in vitro study

BACKGROUND: Inhibition of breast cancer stem cells has been shown to be an effective therapeutic strategy for cancer prevention. The aims of this work were to evaluate the efficacy of koenimbin, isolated from Murraya koenigii (L) Spreng, in the inhibition of MCF7 breast cancer cells and to target MCF7 breast cancer stem cells through apoptosis in vitro. METHODS: Koenimbin-induced cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release were observed using high-content screening. Cell cycle arrest was examined using flow cytometry, while human apoptosis proteome profiler assays were used to investigate the mechanism of apoptosis. Protein expression levels of Bax, Bcl2, and heat shock protein 70 were confirmed using Western blotting. Caspase-7, caspase-8, and caspase-9 levels were measured, and nuclear factor kappa B (NF-κB) activity was assessed using a high-content screening assay. Aldefluor™ and mammosphere formation assays were used to evaluate the effect of koenimbin on MCF7 breast cancer stem cells in vitro. The Wnt/β-catenin signaling pathway was investigated using Western blotting. RESULTS: Koenimbin-induced apoptosis in MCF7 cells was mediated by cell death-transducing signals regulating the mitochondrial membrane potential by downregulating Bcl2 and upregulating Bax, due to cytochrome c release from the mitochondria to the cytosol. Koenimbin induced significant (P<0.05) sub-G0 phase arrest in breast cancer cells. Cytochrome c release triggered caspase-9 activation, which then activated caspase-7, leading to apoptotic changes. This form of apoptosis is closely associated with the intrinsic pathway and inhibition of NF-κB translocation from the cytoplasm to the nucleus. Koenimbin significantly (P<0.05) decreased the aldehyde dehydrogenase-positive cell population in MCF7 cancer stem cells and significantly (P<0.01) decreased the size and number of MCF7 cancer stem cells in primary, secondary, and tertiary mammospheres in vitro. Koenimbin also significantly (P<0.05) downregulated the Wnt/β-catenin self-renewal pathway. CONCLUSION: Koenimbin has potential for future chemoprevention studies, and may lead to the discovery of further cancer management strategies by reducing cancer resistance and recurrence and improving patient survival

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.

peer reviewedEukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions

Kinesin-13, tubulins and their new roles in DNA damage repair

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules.Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers

Isolation and differentiation of vibrio species from seafood and molecular characterisation of vibrio parahaemolyticus / Mohammadjavad Paydar

Vibrio species are significant causes of gastroenteritis in humans associated with consumption of raw or under-cooked seafood. Pathogenic vibrios are common causes of seafood-borne illness in Southeast Asian countries including Malaysia, where the temperature is optimal for their growth. The objectives of this study were to investigate the prevalence of Vibrio spp. in raw seafood using conventional and molecular methods; to compare these methods based on their sensitivity and discriminatory power; and to analyse pathogenicity and genetic variability of V. parahaemolyticus isolates. One-hundred and fifty seafood samples, including fish, shrimps, prawns, cockles, oysters, clams and squids, were collected from retail stores and hypermarkets in Kuala Lumpur, Petaling Jaya and Seri Kembangan. Colony appearance on CHROMagarTM Vibrio and thiosulfate-citrate-bile salts-sucrose (TCBS), followed by conventional biochemical tests including oxidase, Triple Sugar Iron (TSI), Sulfur reduction – Indole – Motility (SIM), Methyl Red (MR) and Voges-Proskauer (VP), and salt tolerance tests were used for preliminary identification of Vibrio species.API 20E test strip, was applied for biochemical confirmation of vibrios. A multiplex polymerase chain reaction (PCR) targeting gyrB gene for detection of Vibrio spp., and pntA genes for V. cholerae, V. parahaemolyticus and V. vulnificus was further evaluated on spiked fish, shrimp and oyster, respectively. Sensitivity of the multiplex PCR on spiked seafood was 2.0x103 CFU ml-1 for V. cholerae and V. parahaemolyticus, and 9.0x103 CFU mL-1 for V. vulnificus. The multiplex PCR indicated 100% accuracy and 100% specificity, hence it was used for confirmation of the isolates. Based on the multiplex PCR results, 63% (93/150) of the seafood samples, purchased from the retail stores, harboured Vibrio spp., where the prevalence of V. cholerae, V. parahaemolyticus and V. vulnificus was 5.3% (8/150), 29% (43/150) and 8% (12/150), respectively. PCR determination of the iv virulence genes in V. parahaemolyticus isolates showed that all of the isolates tested (n =50) contained toxR gene, while 4% (2/5) and 12% (6/50) contained thermostable direct hemolysin (tdh) gene and thermostable direct hemolysin-related (trh) gene, respectively. Repetitive Extragenic Palindromic PCR (REP-PCR) was performed to genetically characterize the V. parahaemolyticus isolates. Forty-one REP profiles were observed and V. parahaemolyticus isolates were categorized into 10 distinct clusters at 80% similarity. In summary, detection of Vibrio spp. was done more efficiently by CHROMagarTM Vibrio rather than TCBS. Comparison of the results of biochemical tests with multiplex PCR indicated that API 20E had higher discriminatory power (83%) to differentiate V. cholerae, V. parahaemolyticus and V. vulnificus, compared to the conventional biochemical tests (56%). The gyrB/pntA genes-based multiplex PCR was confirmed as an accurate and efficient screening tool for detection of Vibrio spp. Virulent V. parahaemolyticus isolates were isolated from seafood and it implies a potential risk to consumers if such seafoods were consumed uncooked. High genetic diversity of the V. parahaemolyticus isolates was shown by REP-PCR and it was able to distinguish the isolates with different virulotypes

Evidence for conformational change-induced hydrolysis of β-tubulin-GTP

ABSTRACTMicrotubules, protein polymers of α/β-tubulin dimers, form the structural framework for many essential cellular processes including cell shape formation, intracellular transport, and segregation of chromosomes during cell division. It is known that tubulin-GTP hydrolysis is closely associated with microtubule polymerization dynamics. However, the precise roles of GTP hydrolysis in tubulin polymerization and microtubule depolymerization, and how it is initiated are still not clearly defined. We report here that tubulin-GTP hydrolysis can be triggered by conformational change induced by the depolymerizing kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We provide biochemical evidence that conformational change precedes tubulin-GTP hydrolysis, confirming this process is mechanically driven and structurally directional. Furthermore, we quantitatively measure the average size of the presumptive stabilizing “GTP cap” at growing microtubule ends. Together, our findings provide the molecular basis for tubulin-GTP hydrolysis and its role in microtubule polymerization and depolymerization.</jats:p

Prevalence and characterisation of potentially virulent Vibrio parahaemolyticus in seafood in Malaysia using conventional methods, PCR and REP-PCR

A previously developed multiplex PCR targeting gyrB of Vibrios at genus level and pntA genes for specific detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus was evaluated. The sensitivity of the multiplex PCR on spiked seafood was 1.5 × 103 CFU g−1. One hundred and fifty seafood samples were collected from retail stores and hypermarkets in different locations in Kuala Lumpur, Petaling Jaya and Seri Kembangan. The prevalence of V. parahaemolyticus was 29% (43/150). The pntA primers for V. parahaemolyticus detection were 100% specific and comparable to the toxR gene-based PCR. Six (12%) and 2 (4%) isolates contained trh and tdh genes, respectively. Repetitive Extragenic Palindromic PCR (REP-PCR) was used to genetically characterize the V. parahaemolyticus isolates in which 41 REP profiles were observed and all the isolates were categorized into 11 distinct clusters at the similarity of 80%. tdh-positive isolates shared a low level of similarity with trh-positive isolates. The prevalence of V. parahaemolyticus and particularly the presence of virulent gene such as trh and tdh among the isolates reiterate a high risk of contamination for seafood consumers in Malaysia. DNA fingerprinting of V. parahaemolyticus in this study indicates a high genetic diversity among the isolates and REP-PCR was able to distinguish the isolates with different virulotypes