RESUMO
Lung cancer patients develop acquired resistance to tyrosine kinase inhibitors including erlotinib (ERL) after few months of primary treatment. Evidently, new chemotherapy strategies to delay or overcome the resistance are urgently needed to improve the clinical outcome in non-small cell lung cancer (NSCLC) patients. In this paper, we have investigated the cytotoxic interaction of ERL and valproic acid (VA) in ERL-resistant NSCLC cells and developed a liquisolid formulation of ERL-VA for improving oral bioavailability of ERL. ERL is weakly basic, biopharmaceutical classification system (BCS) class II drug with extremely poor aqueous solubility while VA is a branched chain fatty acid. Ionic interaction between ERL and VA (1:2 M ratio) resulted in significant enhancement in saturation solubility of ERL at different pH range. Liquisolid formulation of ERL-VA (EVLF) developed using PEG 400 and mesoporous calcium silicate was characterized for solid state and in vitro dissolution in biorelevant dissolution medium (FaSSIF and FeSSIF). Cytotoxicity of ERL was enhanced by 2-5 folds on co-incubation with VA in HCC827/ERL cell line. Flow cytometry analysis using AnnexinV-FITC assay demonstrated that VA and ERL alone have poor apoptotic effect on HCC827/ERL cells while combination showed around 69% apoptotic cells. Western blot analysis confirmed the role of survivin in overcoming resistance. In vivo pharmacokinetic studies of EVLF in rats demonstrated a 199% relative bioavailability compared to ERL suspension. Thus, EVLF could be a promising alternative to current ERL formulations in the treatment of NSCLC.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/efeitos dos fármacos , Cloridrato de Erlotinib/química , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/patologia , Ácido Valproico/química , Ácido Valproico/farmacologia , Administração Oral , Animais , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Compostos de Cálcio/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Cloridrato de Erlotinib/farmacocinética , Humanos , Neoplasias Pulmonares/metabolismo , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , Silicatos/química , Solubilidade , Ácido Valproico/farmacocinéticaRESUMO
Triple-negative breast cancer (TNBC) is the leading cancer in women. Chemotherapeutic agents used for TNBC are mainly associated with dose-dependent toxicities and development of resistance. Hence, novel strategies to overcome resistance and to offer dose reduction are warranted. In this study, we designed a novel dual-functioning agent, conjugate of cholecalciferol with PEG2000 (PEGCCF) which can self-assemble into micelles to encapsulate doxorubicin (DOX) and act as a chemosensitizer to improve the therapeutic potential of DOX. DOX-loaded PEGCCF (PEGCCF-DOX) micelles have particle size, polydispersity index (PDI), and zeta potential of 40 ± 8.7 nm, 0.180 ± 0.051, and 2.39 ± 0.157 mV, respectively. Cellular accumulation studies confirmed that PEGCCF was able to concentration-dependently enhance the cellular accumulation of DOX and rhodamine 123 in MDA-MB-231 cells through its P-glycoprotein (P-gp) inhibition activity. PEGCCF-DOX exhibited 1.8-, 1.5-, and 2.9-fold enhancement in cytotoxicity of DOX in MDA-MB-231, MDA-MB-468, and MDA-MB-231DR (DOX-resistant) cell lines, respectively. Western blot analyses showed that PEGCCF-DOX caused significant reduction in tumor markers including mTOR, c-Myc, and antiapoptotic marker Bcl-xl along with upregulation of preapoptotic marker Bax. Further, reduction in mTOR activity by PEGCCF-DOX indicates reduced P-gp activity due to P-gp downregulation as well and, hence, PEGCCF causes enhanced chemosensitization and induces apoptosis. Substantially enhanced apoptotic activity of DOX (10-fold) in MDA-MB-231(DR) cells confirmed apoptotic potential of PEGCCF. Conclusively, PEGCCF nanomicelles are promising delivery systems for improving anticancer activity of DOX in TNBC, thereby reducing its side effects and may act as a potential carrier for other chemotherapeutic agents.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Colecalciferol/química , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Polietilenoglicóis/química , Neoplasias de Mama Triplo Negativas/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Liberação Controlada de Fármacos , Feminino , Humanos , Micelas , Nanoestruturas , Tamanho da Partícula , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
PURPOSE: Non-small cell lung cancer is the leading cause of cancer related deaths globally. Considering the side effects and diminishing chemosensitivity to chemotherapy, novel treatment approaches are sought. Hence, we aim to develop a liposomal co-delivery system of pDNA expressing shRNA against PFKFB3 (pshPFKFB3) and docetaxel (DTX). METHODS: Cationic DTX liposomes complexed with pshPFKFB3 (PSH-DL) were developed. In vitro cell line studies were performed to evaluate transfection, PFKFB3 mRNA silencing, cytotoxicity, pGP inhibition, and protein markers expression. In vivo efficacy study was performed in A549 xenograft nude mice model. RESULTS: Cytotoxicity studies showed significantly enhanced anticancer activity of PSH-DL against individual treatment alone confirming the chemoenhancing effect of pshPFKFB3 on DTX activity. Fluorescence microscopy and RT-PCR showed effective transfection and RNAi by pshPFKFB3. pGP inhibition assay and western blotting revealed that PFKFB3 downregulation caused diminution of pGP activity leading to changes in cell cycle (Cdk2), survival (survivin), apoptosis (Bcl2 and cleaved caspase 3) and stress (p-JNK and p-p38) markers so that induces apoptosis by PSH-DL in NSCLC cells. PSH-DL also showed ~3.8-fold reduction in tumor volume in A549 xenograft model which was significantly higher than individual treatments alone. CONCLUSION: Targeting PFKFB3 through shRNA based RNAi is a promising approach for potentiating activity of DTX in NSCLC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Lipossomos/química , Neoplasias Pulmonares/tratamento farmacológico , Fosfofrutoquinase-2/genética , RNA Interferente Pequeno/genética , Taxoides/farmacologia , Animais , Antineoplásicos/química , Apoptose , Caspase 3/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Docetaxel , Combinação de Medicamentos , Inativação Gênica , Técnicas de Transferência de Genes , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Nus , Tamanho da Partícula , Fosfofrutoquinase-2/metabolismo , Plasmídeos , Complexo de Inativação Induzido por RNA/metabolismo , Propriedades de Superfície , Taxoides/química , Carga Tumoral/efeitos dos fármacosRESUMO
Chemosensitization and enhanced delivery to solid tumor are widely explored strategies to augment the anticancer efficacy of existing chemotherapeutics agents. The aim of current research was to investigate the role of low dose Noscapine (Nos) in potentiating docetaxel cytotoxicity and enhancing tumor penetration of nanocarriers. The objectives are; (1) To evaluate the chemo-sensitizing effect of Nos in combination with docetaxel (DTX), and to elucidate the possible mechanism (2) To investigate the effect of low dose Nos on tumor stroma and enhancing nanocarrier uptake in triple negative breast cancer (TNBC) bearing nude mice. Cytotoxicity and flow cytometry analysis of DTX in Nos (4µM) pre-treated MDA-MB-231 cells showed 3.0-fold increase in cell killing and 30% increase in number of late apoptotic cells, respectively. Stress transducer p38 phosphorylation was significantly upregulated with Nos exposure. DTX showed remarkable downregulation in expression of bcl-2, survivin and pAKT in Nos pre-treated MDA-MB-231 cells. Nos pre-sensitization significantly (p<0.02) enhanced the anti-migration effect of DTX. In vivo studies in orthotopic TNBC tumor bearing mice showed marked reduction in tumor collagen-I levels and significantly (p<0.03) higher intra-tumoral uptake of coumarin-6 loaded PEGylated liposomes (7-fold) in Nos treated group. Chemo-sensitization and anti-fibrotic effect of Nos could be a promising approach to increase anticancer efficacy of DTX which can be used for other nanomedicinal products.
Assuntos
Antineoplásicos/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Noscapina/farmacologia , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Migração Celular , Movimento Celular/efeitos dos fármacos , Cumarínicos/metabolismo , Docetaxel , Ativação Enzimática/efeitos dos fármacos , Imunofluorescência , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipossomos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Coloração e Rotulagem , Tiazóis/metabolismo , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and -3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (p < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (p < 0.05). Substantially lower expression (p < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer.
Assuntos
Antineoplásicos/farmacologia , Lipossomos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Peptídeos/farmacologia , Taxoides/farmacologia , Células A549 , Animais , Antineoplásicos/química , Caderinas/metabolismo , Linhagem Celular Tumoral , Colágeno/metabolismo , Docetaxel , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Peptídeos/química , Polietilenoglicóis/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptor EphA2/metabolismo , Taxoides/química , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Multidrug resistance (MDR) is a major impediment to cancer treatment. Here, for the first time, we investigated the chemo-sensitizing effect of Noscapine (Nos) at low concentrations in conjunction with docetaxel (DTX) to overcome drug resistance of triple negative breast cancer (TNBC). In vitro experiments showed that Nos significantly inhibited proliferation of TNBC wild type (p < 0.01) and drug resistant (p < 0.05) TNBC cells. Nos followed by DTX treatment notably increased the cell viability (~1.3 fold) markedly (p < 0.05) in 3D models compared to conventional 2D systems. In vivo oral administration of Nos (100 mg/kg) followed by intravenous DTX (5 mg/kg) liposome treatment revealed regression of xenograft tumors in both wild type (p < 0.001) and drug-resistant (p < 0.05) xenografts. In wild type xenografts, combination of Nos plus DTX group showed 5.49 and 3.25 fold reduction in tumor volume compared to Nos and DTX alone groups, respectively. In drug-resistant xenografts, tumor volume was decreased 2.33 and 1.41 fold in xenografts treated with Nos plus DTX significantly (p < 0.05) compared to Nos and DTX alone respectively and downregulated the expression of anti-apoptotic factors and multidrug resistance proteins. Collectively, chemo-sensitizing effect of Nos followed by DTX regime provide a promising chemotherapeutic strategy and its significant role for the treatment of drug-resistant TNBC.
Assuntos
Docetaxel/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Noscapina/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/efeitos adversos , Feminino , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Camundongos , Taxoides/administração & dosagem , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gambogic acid (GA) is a naturally derived potent anticancer agent with extremely poor aqueous solubility. In the present study, positively charged PEGylated liposomal formulation of GA (GAL) was developed for parenteral delivery for the treatment of triple-negative breast cancer (TNBC). The GAL was formulated with a particle size of 107.3 ± 10.6 nm with +32 mV zeta potential. GAL showed very minimal release of GA over 24 h period confirming the non-leakiness and stability of liposomes. In vitro cytotoxicity assays showed similar cell killing with GA and GAL against MDA-MB-231 cells but significantly higher inhibition of HUVEC growth was observed with GAL. Furthermore, GAL significantly (p < 0.05) inhibited the MDA-MB-231 orthotopic xenograft tumor growth with >50% reduction of tumor volume and reduction in tumor weight by 1.7-fold and 2.2-fold when compared to GA and controls, respectively. Results of western blot analysis indicated that GAL significantly suppressed the expression of apoptotic markers, bcl2, cyclinD1, survivin and microvessel density marker-CD31 and increased the expression of p53 and Bax compared to GA and control. Collectively, these data provide further support for the potential applications of cationic GAL in its intravenous delivery and its significant role in inhibiting angiogenesis against TNBC.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Polietilenoglicóis/química , Xantonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Cátions , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Lipossomos , Tamanho da Partícula , Neoplasias de Mama Triplo Negativas , Xantonas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: Therapeutic efficacy of anticancer nanomedicine is compromised by tumor stromal barriers. The present study deals with the development of docetaxel loaded PEGylated liposomes (DTXPL) and to investigate the effect of tumor stroma disrupting agent, telmisartan, on anticancer efficacy of DTXPL. METHODS: DTXPL was prepared using proprietary modified hydration method. Effect of oral telmisartan treatment on tumor uptake of coumarin-6 liposomes and anticancer efficacy of DTXPL was evaluated in orthotopic xenograft lung tumor bearing mice. RESULTS: DTXPL (105.7 ± 3.8 nm) showed very high physical stability, negligible hemolysis, 428% enhancement in bioavailability with significantly higher intratumoral uptake. Marked reduction in collagen-I, MMP2/9 and lung tumor weight were observed in DTXPL+telmisartan group. CONCLUSION: Combination of DTXPL with telmisartan could significantly enhance clinical outcome in lung cancer.