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In the original publication [...].
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An active fluidic microenvironment governs peritoneal metastasis in epithelial ovarian cancer (EOC), but its critical functional/molecular cues are not fully understood. Utilizing co-culture models of NIH3T3 cells (differentially overexpressing Jagged1) and SKOV3 cells expressing a Notch3 luciferase reporter-sensor (SNFT), we showed that incremental expression of Jagged1 led to proportional Notch3 activation in SNFT. With no basal luciferase activity, this system efficiently recorded dose-dependent Notch3 activation by rh-Jag1 peptide and the non-appearance of such induction in co-culture with NIH3T3Δjag1 cells indicates its sensitivity and specificity. Similar Notch3 modulation was shown for the first time in co-cultures with HGSOC patients' ascites-derived cancer-associated fibroblasts and Jagged1-expressing EOC cell lines. NIH3T3J1-A and OVCAR3 co-cultured SNFT cells showed maximum proliferation, invasion, and cisplatin resistance among all the heterotypic/homotypic cellular partners. VEGFA and CDKN1A are the two most upregulated genes identified across co-cultures by the gene profiler array. Co-culture induced VEGFA secretion from SNFT cells which also reduced cancer stem cell differentiation in platinum-resistant A2780 cells. rh-Jag1-peptide promoted enhanced nuclear-cytoplasmic p21 expression. Additionally, metastatic HGSOC tumors had higher VEGFA than corresponding primary tumors. This study thus demonstrates the tumoral and non-tumoral cell-mediated differential Notch3 activation imparting its tumorigenic effects through two critical molecular regulators, VEGFA and p21, during EOC progression.
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Chemoresistance, the ability of cancer cells to overcome therapeutic interventions, is an area of active research. Studies on intrinsic and acquired chemoresistance have partly succeeded in elucidating some of the molecular mechanisms in this elusive phenomenon. Hence, drug-resistant cellular models are routinely developed and used to mimic the clinical scenario in-vitro. In an attempt to identify the underlying molecular mechanisms that allow ovarian cancer cells to gradually acquire chemoresistance, we have developed isogenic cellular models of cisplatin and paclitaxel resistance (singularly and in combination) over six months, using a clinically relevant modified pulse method. These models serve as important tools to investigate the underlying molecular players, modulation in genetics, epigenetics, and relevant signaling pathways, as well as to understand the role of drug detoxification and drug influx-efflux pathways in development of resistance. These models can also be used as screening tools for new therapeutic molecules. Additionally, repurposing therapeutic agents approved for diseases other than cancer have gained significant attention in improving cancer therapy. To investigate the effect of metformin on acquirement of chemoresistance, we have also developed a combinatorial model of metformin and platinum-taxol, using two different strategies. All these models were subsequently used to study modulation in receptor tyrosine kinase pathways, cancer stem cell functionalities, autophagy, metastasis, metabolic signatures, and various biological processes during development of chemoresistance. Herein, we outline the protocols used for developing these intricate resistant cellular models.
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Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin-paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome-lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum-taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum-taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Animales , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular Tumoral , Activación Enzimática , Femenino , Humanos , Cinética , Ratones Desnudos , Proteína Quinasa 3 Activada por Mitógenos/genética , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fosforilación , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Development of resistance poses a significant challenge to effective first-line platinum based therapy for epithelial ovarian cancer patients. Cancer Stem Cells are envisaged as a critical underlying factor for therapy resistance. Thus, there is a critical need for developing approaches to diminish the enrichment of cancer stem cells and acquirement of resistance. Administration of metformin, a commonly prescribed drug against Type II diabetes exhibited promising effect in the management of ovarian cancer. However, the effect of long term administration of low dose of metformin as an adjuvant to cisplatin and paclitaxel during acquirement of chemoresistant phenotype has not been investigated so far. Using two isogenic cellular chemoresistant models (A2780 and OAW42) developed in the presence or absence of metformin, we demonstrated the ability of metformin to impede the development of resistance through increased drug sensitivity, increased proliferation, and reduced migratory abilities of the resistant cells. Metformin introduction also decreased the cancer stem cell population, expression of specific biomarkers and pluripotent genes. Further metabolic profiling of these cells using 1H-Nuclear Magnetic Resonance spectroscopy revealed significant modulation in taurine and histidine levels in resistant cells developed in the presence of metformin. Intriguingly, taurine treatment considerably reduced the cancer stem cell population and chemoresistance in resistant cells, indicating a novel role of taurine in differentiation of ovarian cancer stem cells. Altogether this is the first report on the potential role of metformin for targeting the cancer stem cell population via up regulation of taurine, leading to impediment in the acquirement of chemoresistance.
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Diferenciación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Metformina/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/patología , Taurina/biosíntesis , Aminoácidos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Femenino , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Paclitaxel/farmacología , Factores de TiempoRESUMEN
Deregulated IGF-1R-AKT signaling influences multiple nodes of cancer cell physiology and assists in migration, metastasis and acquirement of radio/chemoresistance. Enrichment of cancer stem cells (CSC) positively correlates with radio/chemoresistance development in various malignancies. It is unclear though, how IGF-1R-AKT signalling shapes CSC functionality especially in ovarian cancer. Previously we showed that upregulated IGF-1R expression is essential to initiate platinum-taxol resistance at early stage which declines with elevated levels of activated AKT at late resistant stage in ovarian cancer cells. Here, we investigated the effect of this oscillatory IGF-1R-AKT signalling upon CSC functionality during generation of chemoresistance. While gradual increase in CSC properties from early (ER) to late (LR) resistant stages was observed in three different (cisplatin/paclitaxel/cisplatin-paclitaxel) cellular models created in two ovarian cancer cell lines, the stemness gene expressions (oct4/sox2/nanog) reached a plateau at early resistant stages. Inhibition of IGF-1R only at ER and AKT inhibition only at LR stages significantly abrogated the CSC phenotype. Interestingly, real time bioluminescence imaging showed CSCs of ER stages possessed faster tumorigenic potential than CSCs belonging to LR stages. Together, our data suggest that IGF-1R-AKT signalling imparts functional heterogeneity in CSCs during acquirement of chemoresistance in ovarian carcinoma.
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Resistencia a Antineoplásicos , Células Madre Neoplásicas/patología , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Femenino , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Transducción de SeñalRESUMEN
Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cisplatino/farmacología , Factor 88 de Diferenciación Mieloide/deficiencia , FN-kappa B/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Paclitaxel/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma Epitelial de Ovario , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Cisplatino/administración & dosificación , Femenino , Humanos , Ratones , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Transducción de Señal , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Both chronic and acute inflammatory circuits are known to be associated with malignancy and drug resistance indicating that many antiinflammatory agents can potentially act as chemotherapeutic drugs. A series of new class of propanediones with good antiinflammatory activity were shown to possess moderate cytotoxic activities. AIM: The aim of the study was to evaluate this new series of 1-(2',4'-difluorophenyl)-3-(substituted phenyl)-1,3 propanediones (PR 1-7) for their caspase dependent apoptotic activity by using a reporter gene mediated caspase-3 sensor in chemo sensitive and paclitaxel resistant ovarian cancer cells. MATERIALS AND METHODS: A cellular model of paclitaxel resistance was developed in OAW42 cells stably expressing the caspase 3 sensor. The activity of caspase 3 after single and combinatorial drug treatments was determined using western blot and luciferase activity. Cell viability and cell cycle analysis were determined by MTT 3 (4,5- dimethyl thiazol-2 yl-2,5- diphenyl tetrazolium bromide (MTT) and Flow cytometric analysis (FACS) analysis. High Performance Liquid Chromatography (HPLC) analysis was performed to assess cellular uptake of the propanediones. RESULTS: Both nitro/methoxy (Group I) and halogen substituted propandiones at ortho, meta and para positions (Group II) showed a moderate increase in caspase-3 activity by 1.5- to 3.3-fold as compared with controls. However, no noticeable change in apoptotic cells percentage was observed. Increased intracellular uptake of Paclitaxel was observed during combinatorial treatment with one of the propanediones (PR2). Intriguingly, PR2 alone or in combination with Paclitaxel could induce a 2.5- to 2.9-fold increase in caspase-3 activity in Paclitaxel resistant cells. CONCLUSION: Our study reports a new class of propanediones that can augment the cytotoxic effect of Paclitaxel, and potentially can be used for treating Paclitaxel-resistant cancers.