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Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease where standard-of-care chemotherapeutic drugs have limited efficacy due to the development of drug resistance and poor drug delivery caused by a highly desmoplastic tumor microenvironment. Combining multiple drugs in a tumor-targeting carrier would be a favorable approach to overcome these limitations. Hence, a tumor-targeted peptide (TTP) conjugated amphiphilic tri-block copolymer was developed to make targeted polymer nanoparticles (TTP-PNPs) serving as a vehicle for carrying gemcitabine (Gem), paclitaxel (PTX), and their combination (Gem + PTX). The TTP-PNPs in the form of empty polymer (P), single drug-loaded [P(Gem) and P(PTX)], and dual drug-loaded [P(Gem + PTX)] polymer nanoformulations exhibited stable and homogenous spherical shapes with 110-160 nm size. These nanoformulations demonstrated excellent stability under in vitro physiological conditions and led to an efficient release of the drugs in the presence of reduced glutathione (GSH). The efficacy of these nanoparticles was thoroughly evaluated in vitro and in vivo, demonstrating a notable capacity to selectively target and restrict PDAC cells (PANC-1 and KPC) growth. The cellular uptake and biodistribution study showed a significantly higher tumor-targeting ability of TTP-PNPs than PNPs without TTP. Notably, P(Gem + PTX) exhibited the lowest IC50 compared to all other controls and showed heightened synergistic effects in both cell lines. Furthermore, P(Gem + PTX) showed a significantly better tumor reduction and median overall survival in mouse models than single drug-loaded TTP-PNPs or a combination of free drugs (Gem + PTX). In summary, our TTP-PNP system shows great promise as a novel platform for delivering Gem + PTX specifically to pancreatic cancer (PC), maximizing the therapeutic benefits with lower concentrations of the drugs and potentially reducing toxic side effects.
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BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.
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Carcinoma de Células Renales , Reparación del ADN , Neoplasias Renales , Survivin , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de Xenoinjerto , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/radioterapia , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Animales , Survivin/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Neoplasias Renales/patología , Neoplasias Renales/radioterapia , Neoplasias Renales/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Imidazoles/farmacología , Daño del ADN , Everolimus/farmacología , Naftoquinonas/farmacología , Naftoquinonas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Liposomas/farmacología , Inhibidores mTOR/farmacología , Inhibidores mTOR/uso terapéuticoRESUMEN
Background: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. Experimental Design: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. Results: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.
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Objective: The objective of this study is to evaluate the expression of different nicotinic acetylcholine receptors (nAChRs), programmed death ligand-1 (PD-L1), and dopamine receptor D2 (DRD2) as prognostic factors in lung cancer and any correlation among them. Since all of the above genes are typically upregulated in response to smoking, we hypothesized that a correlation might exist between DRD2, PD-L1, and nAChR expression in NSCLC patients with a smoking history and a prediction model may be developed to assess the clinical outcome. Methods: We retrospectively analyzed samples from 46 patients with primary lung adenocarcinoma who underwent surgical resection at Mayo Clinic Rochester from June 2000 to October 2008. The expression of PD-L1, DRD2, CHRNA5, CHRNA7, and CHRNA9 were analyzed by quantitative PCR and correlated amongst themselves and with age, stage and grade, smoking status, overall survival (OS), and relapse-free survival (RFS). Results: Only PD-L1 showed a statistically significant increase in expression in patients older than 65. All the above genes showed higher expression in stage IIIB than IIIA, but none reached statistical significance. Interestingly, we did not observe significant differences among never, former, and current smokers, but patients with pack years greater than 30 showed significantly higher expression of CHRNA9. We observed a strong positive correlation between PD-L1/DRD2, PD-L1/CHRNA5, and CHRNA5/CHRNA7 and a weak positive correlation between DRD2/CHRNA5 and DRD2/CHRNA7. Older age was independently associated with poor OS, whereas lower CHRNA7 expression was independently associated with better OS. Conclusions: We observed strong positive correlations among PD-L1, DRD2, and some of the nAChRs. We investigated their prognostic significance in lung cancer patients and found CHRNA7 to be an independent prognostic factor. Overall, the results obtained from this preliminary study warrant a large cohort-based analysis that may ultimately lead to potential patient-specific stratification biomarkers predicting cancer-treatment outcomes.
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The negative health consequences of acute ultraviolet (UV) exposure are evident, with reports of 30,000 emergency room visits annually to treat the effects of sunburn in the United States alone. The acute effects of sunburn include erythema, edema, severe pain, and chronic overexposure to UV radiation, leading to skin cancer. Whereas the pain associated with the acute effects of sunburn may be relieved by current interventions, existing post-sunburn treatments are not capable of reversing the cumulative and long-term pathological effects of UV exposure, an unmet clinical need. Here we show that activation of the vascular endothelial growth factor (VEGF) pathway is a direct and immediate consequence of acute UV exposure, and activation of VEGF signaling is necessary for initiating the acute pathological effects of sunburn. In UV-exposed human subjects, VEGF signaling is activated within hours. Topical delivery of VEGF pathway inhibitors, targeted against the ligand VEGF-A (gold nanoparticles conjugated with anti-VEGF antibodies) and small-molecule antagonists of VEGF receptor signaling, prevent the development of erythema and edema in UV-exposed mice. These findings collectively suggest targeting VEGF signaling may reduce the subsequent inflammation and pathology associated with UV-induced skin damage, revealing a new postexposure therapeutic window to potentially inhibit the known detrimental effects of UV on human skin. It is essential to emphasize that these preclinical studies must not be construed as suggesting in any way the use of VEGF inhibitors as a sunburn treatment in humans because warranted future clinical studies and appropriate agency approval are essential in that regard.
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Piel/lesiones , Rayos Ultravioleta/efectos adversos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Pelados , Piel/patología , Quemadura SolarRESUMEN
Despite recent advancements, effective treatment for pancreatic ductal adenocarcinoma (PDAC) has remained elusive. The overall survival rate in PDAC patients has been dismally low due to resistance to standard therapies. In fact, the failure of monotherapies to provide long-term survival benefits in patients led to ascension of several combination therapies for PDAC treatment. However, these combination therapies provided modest survival improvements while increasing treatment-related adverse side effects. Hence, recent developments in drug delivery methods hold the potential for enhancing therapeutic benefits by offering cocktail drug loading and minimizing chemotherapy-associated side effects. Nanoformulations-aided deliveries of anticancer agents have been a success in recent years. Yet, improving the tumor-targeted delivery of drugs to PDAC remains a major hurdle. In the present paper, we developed several new tumor-targeted dual intervention-oriented drug-encapsulated (DIODE) liposomes. We successfully formulated liposomes loaded with gemcitabine (G), paclitaxel (P), erlotinib (E), XL-184 (c-Met inhibitor, X), and their combinations (GP, GE, and GX) and evaluated their in vitro and in vivo efficacies. Our novel DIODE liposomal formulations improved median survival in comparison with gemcitabine-loaded liposomes or vehicle. Our findings are suggestive of the importance of the targeted delivery for combination therapies in improving pancreatic cancer treatment.
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Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Renales/tratamiento farmacológico , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/uso terapéutico , Animales , Femenino , Humanos , Ratones , Ratones SCIDRESUMEN
Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of mTOR and the disruption of VEGF/NRP1 axis, respectively. We also exploited a liposomal formulation decorated with a proprietary tumor-targeting-peptide (TTP) to simultaneously deliver these two agents in a tumor-targeted manner. The TTP-liposomes encapsulating both Everolimus and EG00229 (EG-L) demonstrated higher in vitro and in vivo growth retardation than the single drug-loaded liposomes (E-L and G-L) in two different ccRCC models and led to a noticeable reduction in lung metastasis in vivo. In addition, EG-L displayed remarkable inhibition of tumor growth in a highly aggressive syngeneic immune-competent mouse model of ccRCC developed in Balb/c mice. Taken together, this study demonstrates an effective approach to achieve improved therapeutic outcome in ccRCC.
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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Nanomedicine, however, offers new opportunities to facilitate drug delivery in PDAC. Our previous work has shown that poly(ethylene glycol)-functionalized nanodiamond (ND) mediated drug delivery offered a considerable improvement over free drug in PDAC. Inspired by this result and guided by molecular simulations, we opted for simultaneous loading of irinotecan and curcumin in ultra-small PEGylated NDs (ND-IRT + CUR). We observed that ND-IRT + CUR was more efficacious in killing AsPC-1 and PANC-1 cells than NDs with single drugs. Using NDs functionalized with a near-infrared (NIR) dye, we demonstrated the preferential localization of the NDs in tumors and metastatic lesions. We further demonstrate that ND-IRT + CUR is capable of producing pronounced anti-tumor effects in two different clinically relevant, immune-competent genetic models of PDAC. Cytokine profiling indicated that NDs with or without drugs downregulated the expression of IL-10, a key modulator of the tumor microenvironment. Thus, using a combination of in silico, in vitro, and in vivo approaches, we show for the first time the remarkable anti-tumor efficacy of PEGylated NDs carrying a dual payload of irinotecan plus curcumin. These results highlight the potential use of such nano-carriers in the treatment of patients with pancreatic cancer.
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Curcumina , Portadores de Fármacos , Nanodiamantes , Neoplasias Pancreáticas , Animales , Línea Celular Tumoral , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Humanos , Ratones , Ratones Mutantes , Nanodiamantes/química , Nanodiamantes/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates among cancers. Chemotherapy is the standard first-line treatment, but only modest survival benefits are observed. With the advent of targeted therapies, epidermal growth factor receptor (EGFR) has been acknowledged as a prospective target in PDAC since it is overexpressed in up to 60% of cases. Similarly, the tyrosine-protein kinase Met (cMET) is also overexpressed in PDAC (27-60%) and is a prognostic marker for poor survival. Interestingly, EGFR and cMET share some common signaling pathways including PI3K/Akt and MAPK pathways. Small molecule inhibitors or bispecific antibodies that can target both EGFR and cMET are therefore emerging as novel options for cancer therapy. We previously developed a dual EGFR and cMET inhibitor (N19) that was able to inhibit tumor growth in nonsmall cell lung cancer models resistant to EGFR tyrosine kinase inhibitors (TKI). Here, we report the development of a novel liposomal formulation of N19 (LN19) and showed significant growth inhibition and increased sensitivity toward gemcitabine in the pancreatic adenocarcinoma orthotopic xenograft model. Taken together, our results suggest that LN19 can be valued as an effective combination therapy with conventional chemotherapy such as gemcitabine for PDAC patients.
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Adenocarcinoma/patología , Diseño de Fármacos , Liposomas/química , Neoplasias Pancreáticas/patología , Polietilenglicoles/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacología , Composición de Medicamentos , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , Proteolisis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina , Neoplasias PancreáticasRESUMEN
BACKGROUND: Renal cell carcinoma (RCC) is notorious for its resistance towards chemotherapy and radiation therapy in general. Combination therapy is often helpful in alleviating the resistance mechanisms by targeting multiple signaling pathways but is usually more toxic than monotherapy. Co-encapsulation of multiple therapeutic agents in a tumor-targeted drug delivery platform is a promising strategy to mitigate these limitations. METHODS: A tumor-targeted liposomal formulation was prepared using phospholipids, cholesterol, DSPE-(PEG)2000-OMe and a proprietary tumor-targeting-peptide (TTP)-conjugated lipopeptide. An efficient method was optimized to encapsulate everolimus and vinorelbine in this liposomal formulation. Single drug-loaded liposomes were also prepared for comparison. Finally, the drug-loaded liposomes were tested in vitro and in vivo in two different RCC cell lines. RESULTS: The tumor-targeted liposomal formulation demonstrated excellent tumor-specific uptake. The dual drug-loaded liposomes exhibited significantly higher growth inhibition in vitro compared to the single drug-loaded liposomes in two different RCC cell lines. Similarly, the dual drug-loaded liposomes demonstrated significantly higher suppression of tumor growth compared to the single drug-loaded liposomes in two different subcutaneous RCC xenografts. In addition, the dual drug-loaded liposomes instigated significant reduction in lung metastasis in those experiments. CONCLUSION: Taken together, this study demonstrates that co-delivery of everolimus and vinorelbine with a tumor-targeted liposomal formulation is an effective approach to achieve improved therapeutic outcome in RCC.
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Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/métodos , Everolimus/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Vinorelbina/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Antígeno Ki-67/metabolismo , Liposomas , Neoplasias Pulmonares/secundario , Ratones SCID , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Programmed death ligand 1 (PD-L1) is an immune checkpoint protein; however, emerging data suggest that tumor cell PD-L1 may regulate immune-independent and intrinsic cellular functions. We demonstrate regulation of PD-L1 by oncogenic BRAFV600E and investigated its ability to influence apoptotic susceptibility in colorectal cancer (CRC) cells. Endogenous or exogenous mutant vs. wild-type BRAF were shown to increase PD-L1 messenger RNA (mRNA) and protein expression that was attenuated by MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase) inhibition or c-JUN and YAP knockdown. Deletion of PD-L1 reduced tumor cell growth in vitro and in vivo. Loss of PD-L1 was also shown to attenuate DNA damage and apoptosis induced by diverse anti-cancer drugs that could be reversed by restoration of wild-type PD-L1, but not mutants with deletion of its extra- or intracellular domain. The effect of PD-L1 on chemosensitivity was confirmed in MC38 murine tumor xenografts generated from PD-L1-knockout vs. parental cells. Deletion of PD-L1 suppressed BH3-only BIM and BIK proteins that could be restored by re-expression of PD-L1; re-introduction of BIM enhanced apoptosis. PD-L1 expression was significantly increased in BRAFV600E human colon cancers, and patients whose tumors had high vs. low PD-L1 had significantly better survival. In summary, BRAFV600E can transcriptionally upregulate PD-L1 expression that was shown to induce BIM and BIK to enhance chemotherapy-induced apoptosis. These data indicate an intrinsic, non-immune function of PD-L1, and suggest the potential for tumor cell PD-L1 as a predictive biomarker.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Antígeno B7-H1/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , Antígeno B7-H1/genética , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.
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Nanodiamantes/química , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Liberación de Fármacos , Endocitosis/efectos de los fármacos , Humanos , Hidrodinámica , Masculino , Ratones , Nanodiamantes/ultraestructura , Neoplasias Pancreáticas/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/química , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Distribución Tisular/efectos de los fármacos , Resultado del TratamientoRESUMEN
Aggregated amyloid ß (Aß) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aß peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aß1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aß oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aß1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aß1-42 monomers, and 5 µM Aß1-42 fibrils, respectively. Brain ECs treated with Aß1-42 oligomers showed increased senescence-associated ß-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aß1-42 monomer or Aß1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aß1-42 oligomer-treated ECs, and these cells showed significantly increased VEGFR-1 expression and decreased VEGFR-2 levels. Overexpression of VEGFR-1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR-1 signaling events in this paradigm. More importantly, small interfering RNA-mediated knockdown of VEGFR-1 expression in brain ECs was able to prevent up-regulation of p21 protein expression and significantly reduced induction of senescence following Aß1-42 oligomer treatment. Our studies show that exposure to Aß1-42 oligomers may impair vascular functions by altering VEGFR-1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR-1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.-Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.
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Péptidos beta-Amiloides/farmacología , Senescencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Encéfalo/irrigación sanguínea , Capilares/citología , Supervivencia Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
NAD salvage is one of the pathways used to generate NAD in mammals. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in this pathway, uses nicotinamide (NAM) to generate nicotinamide mononucleotide (NMN). NMN is one of the main precursors of NAD synthesis in cells. Our previous study showed the importance of NAMPT in maintaining NAD levels in pancreatic ductal adenocarcinoma cells (PDAC), and that the NAMPT inhibitor FK866 decreased pancreatic cancer growth. We now tested the effect of STF-118804, a new highly specific NAMPT inhibitor, in models of pancreatic ductal adenocarcinoma. STF-118804 reduced viability and growth of different PDAC lines, as well as the formation of colonies in soft agar. In addition, STF-118804 decreased glucose uptake, lactate excretion, and ATP levels, resulting in metabolic collapse. STF-118804 treatment activated AMPK and inhibited of mTOR pathways in these cells. This effect was significantly potentiated by pharmacological AMPK activation and mTOR inhibition. Exogenous NMN blocked both the activation of the AMPK pathway and the decrease in cell viability. Panc-1 cells expressing GFP-luciferase were orthotopically implanted on mice pancreas to test the in vivo effectiveness of STF-118804. Both STF-118804 and FK866 reduced tumor size after 21 days of treatment. Combinations of STF-118804 with chemotherapeutic agents such as paclitaxel, gemcitabine, and etoposide showed an additive effect in decreasing cell viability and growth. In conclusion, our preclinical study shows that the NAMPT inhibitor STF-118804 reduced the growth of PDAC in vitro and in vivo and had an additive effect in combination with main current chemotherapeutic drugs.
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An astute modification of the plectin-1-targeting peptide KTLLPTP by introducing a C-terminal cysteine preceded by a tyrosine residue imparted a reducing property to the peptide. This novel property is then exploited to fabricate gold nanoparticles (GNP) via an in situ reduction of gold(iii) chloride in a one-pot, green synthesis. The modified peptide KTLLPTPYC also acts as a template to generate highly monodispersed, spherical GNPs with a narrow size distribution and improved stability. Plectin-1 is known to be aberrantly expressed in the surface of pancreatic ductal adenocarcinoma (PDAC) cells while showing cytoplasmic expression in normal cells. The synthesized GNPs are thus in situ surface modified with the peptides via the cysteine residue leaving the N-terminal KTLLPTP sequence free for targeting plectin-1. The visual molecular dynamics based simulations support the experimental observations like particle size, gemcitabine conjugation and architecture of the peptide-grafted nanoassembly. Additionally, GNPs conjugated to gemcitabine demonstrate significantly higher cytotoxicity in vitro in two established PDAC cell lines (AsPC-1 and PANC-1) and an admirable in vivo antitumor efficacy in a PANC-1 orthotopic xenograft model through selective uptake in PDAC tumor tissues. Altogether, this strategy represents a unique method for the fabrication of a GNP based targeted drug delivery platform using a multifaceted peptide that acts as reducing agent, template for GNP synthesis and targeting agent to display remarkable selectivity towards PDAC.
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Desoxicitidina/análogos & derivados , Portadores de Fármacos/síntesis química , Oro , Nanopartículas del Metal , Neoplasias Pancreáticas/tratamiento farmacológico , Plectina/metabolismo , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Humanos , Péptidos , GemcitabinaRESUMEN
Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015-27. ©2016 AACR.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Azetidinas/farmacología , Neoplasias Colorrectales/genética , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Alelos , Animales , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many cancers including the late stage ones become drug-resistant and undergo epithelial-to-mesenchymal transition (EMT). These lead to enhanced invasion, migration, and metastasis toward manifesting its aggressiveness and malignancy. One of the key hallmarks of cancer is its overdependence on glycolysis as its preferred energy metabolism pathway. The strict avoidance of alternate energy pathway gluconeogenesis by cancer cells points to a yet-to-be hoisted role of glucocorticoid receptor (GR) especially in tumor microenvironment, where cells are known to become drug-sensitive through induction of gluconeogenesis. However, since GR is involved in metabolism, anti-inflammatory reactions, immunity besides inducing gluconeogenesis, a greater role of GR in tumor microenvironment is envisaged. We have shown previously that GR, although ubiquitously expressed in all cells; afford to be an effective cytoplasmic target for killing cancer cells selectively. Herein, we report the therapeutic use of a newly developed GR-targeted liposomal concoction (DXE) coformulating a lipophilic drug (ESC8) and an anti-Hsp90 anticancer gene against aggressive tumor models. This induced drug-sensitivity and apoptosis while reversing EMT in tumor cells toward effective retardation of aggressive growth in pancreas and skin tumor models. Additionally, the ESC8-free lipid formulation upon cotreatment with hydrophilic drugs, gemcitabine and doxorubicin, could effectively sensitize and kill pancreatic cancer and melanoma cells, respectively. The formulation-triggered EMT-reversal was GR-dependent. Overall, we found a new strategy for drug sensitization that led to the advent of new GR-targeted anticancer therapeutics.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Liposomas/química , Receptores de Glucocorticoides/metabolismo , Animales , Células COS , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/química , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Transición Epitelial-Mesenquimal/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Liposomas/administración & dosificación , Ratones , Reacción en Cadena de la Polimerasa , Receptores de Glucocorticoides/genética , GemcitabinaRESUMEN
BACKGROUND: Studies over the past decade and half have identified cancer stem cells (CSCs) to be responsible for tumorigenesis, invasion, sustenance of metastatic disease, radio- and chemo-resistance and tumor relapse. Recent reports have described the plasticity of breast CSCs (BCSCs) to shift between the epithelial and mesenchymal phenotypes via Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) states as the reason for their invasive capabilities. Additionally, BRCA1 has been found to be a mammary stem cell fate determinant. However, it is not clear what would be the best marker that can be used for identifying CSCs in BRCA1 mutated cancers. Also, anticancer agents that can reduce CSC population in a BRCA1 defective condition have not been addressed so far. METHODS: Putative BCSCs were identified based on Hoechst exclusion, CD44(+)/24(-/low) expression and Aldehyde Dehydrogenase 1 (ALDH1) positivity using flow cytometry. The 'stemness' of the isolated ALDH1+ cells were analysed by immunofluorescence, western blotting for stem cell and EMT markers as well as in vitro mammosphere assays. Induction of Reactive Oxygen Species (ROS) by Plumbagin (PB) in BCSCs was assayed by Dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. Ovarian cancer xenografts treated with PB were subjected to immunohistochemical analysis to study the ability of PB to target CSCs. RESULTS: We have confirmed that ALDH1 positivity is the best marker for the identification of BCSCs in BRCA1-defective breast cancer cell lines when compared to the CD marker profile and Side Population (SP) analysis. BRCA1 status was observed to be a determinant of the abundance of epithelial-like (ALDH1+) or mesenchymal-like (CD44(+)/24(-/low)) BCSCs, and the reconstitution of a full length, wild type BRCA1 in HCC1937 breast cancer cells possessing a mutated BRCA1, transforms them from 'stem-like' to more 'mesenchymal'. For the first time we have shown that Plumbagin (PB), a naturally occurring naphthoquinone which is predominantly a ROS inducer, could reduce BCSCs specifically in BRCA1-defective, basal-like cancer cells. CONCLUSIONS: The best marker for identifying BCSCs in BRCA1 defective condition could be ALDH1 and that BRCA1 mutated BCSCs would be mostly 'stem like' than 'mesenchymal'. Also ROS inducers like PB could reduce BCSCs in BRCA1 defective cancers.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteína BRCA1/genética , Neoplasias de la Mama/patología , Naftoquinonas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Proteína BRCA1/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Carboplatino/farmacología , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Concentración 50 Inhibidora , Ratones SCID , Naftoquinonas/uso terapéutico , Células Madre Neoplásicas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.