Erlotinib-Valproic Acid Liquisolid Formulation: Evaluating Oral Bioavailability and Cytotoxicity in Erlotinib-Resistant Non-small Cell Lung Cancer Cells.

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.

Therapeutic targeting of oncogenic transcription factors by natural products in eye cancer.

Carcinogenesis has a multifactorial etiology, and the underlying molecular pathogenesis is still not entirely understood, especially for eye cancers. Primary malignant intraocular neoplasms are relatively rare, but delayed detection and inappropriate management contribute to poor outcomes. Conventional treatment, such as orbital exenteration, chemotherapy, or radiotherapy, alone results in high mortality for many of these malignancies. Recent sequential multimodal therapy with a combination of high-dose chemotherapy, followed by appropriate surgery, radiotherapy, and additional adjuvant chemotherapy has helped dramatically improve management. Transcription factors are proteins that regulate gene expression by modulating the synthesis of mRNA. Since transcription is a dominant control point in the production of many proteins, transcription factors represent key regulators for numerous cellular functions, including proliferation, differentiation, and apoptosis, making them compelling targets for drug development. Natural compounds have been studied for their potential to be potent yet safe chemotherapeutic drugs. Since the ancient times, plant-derived bioactive molecules have been used to treat dreadful diseases like cancer, and several refined pharmaceutics have been developed from these compounds. Understanding targeting mechanisms of oncogenic transcription factors by natural products can add to our oncologic management toolbox. This review summarizes the current findings of natural products in targeting specific oncogenic transcription factors in various types of eye cancer.

Cholecalciferol-PEG Conjugate Based Nanomicelles of Doxorubicin for Treatment of Triple-Negative Breast Cancer.

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.

Liposomes co-Loaded with 6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3 (PFKFB3) shRNA Plasmid and Docetaxel for the Treatment of non-small Cell Lung Cancer.

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.

Noscapine chemosensitization enhances docetaxel anticancer activity and nanocarrier uptake in triple negative breast cancer.

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.

Combination Approach of YSA Peptide Anchored Docetaxel Stealth Liposomes with Oral Antifibrotic Agent for the Treatment of Lung Cancer.

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.

Novel Gefitinib Formulation with Improved Oral Bioavailability in Treatment of A431 Skin Carcinoma.

PURPOSE: Oral administration of anticancer agents presents a series of advantages for patients. However, most of the anticancer drugs have poor water solubility leading to low bioavailability. METHODS: Controlled released spray dried matrix system of Gefitinib with hydroxypropyl ß-cyclodextrin, chitosan, hydroxy propyl methyl cellulose, vitamin E TPGS, succinic acid were used for the design of formulations to improve the oral absorption of Gefitinib. Spray drying with a customized spray gun which allows simultaneous/pulsatile flow of two different liquid systems through single nozzle was used to prepare Gefitinib spray dried formulations (Gef-SD). Formulation was characterized by in vitro drug release and Caco-2 permeability studies. Pharmacokinetic studies were performed in Sprague Dawley rats. Efficacy of Gef-SD was carried out in A431 xenografts models in nude mice. RESULTS: In Gef-SD group 9.14-fold increase in the AUC was observed compared to free Gef. Improved pharmacokinetic profile of Gef-SD translated into increase (1.75 fold compared to Gef free drug) in anticancer effects. Animal survival was significantly increased in Gef formulation treated groups, with superior reduction in the tumor size (1.48-fold) and volumes (1.75-fold) and also increase in the anticancer effects (TUNEL positive apoptotic cells) was observed in Gef-SD treated groups. Further, western blot, immunohistochemical and proteomics analysis demonstrated the increased pharmacodynamic effects of Gef-SD formulations in A431 xenograft tumor models. CONCLUSION: Our studies suggested that Gefitinib can be successfully incorporated into control release microparticles based oral formulation with enhanced pharmacokinetic and pharmacodynamic activity. This study demonstrates the novel application of Gef in A431 tumor models.

Overexpression of microRNA-122 enhances in vitro hepatic differentiation of fetal liver-derived stem/progenitor cells.

MicroRNAs (miRNAs) are a versatile class of tiny non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. However, the role of miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocytes remains unclear. In this study, dual positive CD34+/CD117+ expressing human fetal liver stem/progenitor cells was enriched by magnetic cell sorting and cultured in vitro. The level of miR-122 was found to be increased at specific time intervals. Interestingly, during the differentiation process of hepatocyte-like cells, the increase in expression of miR-122 was positively correlated with expression of hepatocyte-specific genes. The status of differentiation process was improved by transfection of miR-122 into enriched stem/progenitor cells. The expression level of hepatic-specific genes as well as liver-enriched transcription factors (LETFs) was significantly increased by overexpression of miR-122 in fetal liver stem/progenitor cells. Thus, the study delineated the role of hepato-specific miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocyte-like cells which could be used as a therapeutic target molecule to generate abundant hepatocytes.

Metabolic Reprogramming by Ribitol Expands the Therapeutic Window of BETi JQ1 against Breast Cancer.

Many cancer patients still lack effective treatments, and pre-existing or acquired resistance limits the clinical benefit of even the most advanced medicines. Recently, much attention has been given to the role of metabolism in cancer, expanding from the Warburg effect to highlight unique patterns that, in turn, may improve diagnostic and therapeutic approaches. Our recent metabolomics study revealed that ribitol can alter glycolysis in breast cancer cells. In the current study, we investigate the combinatorial effects of ribitol with several other anticancer drugs (chrysin, lonidamine, GSK2837808A, CB-839, JQ1, and shikonin) in various breast cancer cells (MDA-MB-231, MCF-7, and T-47D). The combination of ribitol with JQ1 synergistically inhibited the proliferation and migration of breast cancer cells cell-type dependently, only observed in the triple-negative MDA-MB-231 breast cancer cells. This synergy is associated with the differential effects of the 2 compounds on expression of the genes involved in cell survival and death, specifically downregulation in c-Myc and other anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1), but upregulation in p53 and cytochrome C levels. Glycolysis is differentially altered, with significant downregulation of glucose-6-phosphate and lactate by ribitol and JQ1, respectively. The overall effect of the combined treatment on metabolism and apoptosis-related genes results in significant synergy in the inhibition of cell growth and induction of apoptosis. Given the fact that ribitol is a metabolite with limited side effects, a combined therapy is highly desirable with relative ease to apply in the clinic for treating an appropriate cancer population. Our results also emphasize that, similar to traditional drug development, the therapeutic potential of targeting metabolism for cancer treatment may only be achieved in combination with other drugs and requires the identification of a specific cancer population. The desire to apply metabolomic intervention to a large scope of cancer types may be one of the reasons identification of this class of drugs in a clinical trial setting has been delayed.

Mass spectrometry database of lacrimal gland adenoid cystic carcinoma and normal lacrimal gland tissue identifies extracellular matrix remodeling in these tumors.

Adenoid cystic carcinoma of the lacrimal gland (LGACC) is a slow-growing but aggressive orbital malignancy. Due to the rarity of LGACC, it is poorly understood, which makes diagnosing, treating, and monitoring disease progression difficult. The aim is to understand the molecular drivers of LGACC further to identify potential targets for treating this cancer. Mass spectrometry was performed on LGACC and normal lacrimal gland samples to examine the differentially expressed proteins to understand this cancer's proteomic characteristics. Downstream gene ontology and pathway analysis revealed the extracellular matrix is the most upregulated process in LGACC. This data serves as a resource for further understanding LGACC and identifying potential treatment targets. This dataset is publicly available.

Ribitol alters multiple metabolic pathways of central carbon metabolism with enhanced glycolysis: A metabolomics and transcriptomics profiling of breast cancer.

Breast cancer is heterogenous in development and cell population with prognoses being highly dependent on numerous factors from driving mutations, biomarker expression and variation in extracellular environment, all affecting response to therapies. Recently, much attention has been given to the role of metabolic alteration in cancers, expanding from the Warburg effect to highlight unique patterns in different cancer cell populations for improving diagnostic and therapeutic approaches. We recently reported on modulation of mannosylation of α-dystroglycan with the metabolite ribitol in breast cancer lines. Here we investigate the effects of pentose sugars ribitol, ribose, and xylitol media supplementation in breast cancer cells by metabolomics and differential gene expression profiling. This combined approach revealed distinctive patterns of alterations in metabolic pathways by ribitol, contrasted with the closely related pentose ribose and pentitol xylitol. Significantly, ribitol supplementation enhances utilization of glucose by glycolysis, whereas ribose improves oxidative phosphorylation and fatty acid synthesis. Ribitol supplementation also increased levels of reduced glutathione (associated with a decrease in oxidative phosphorylation, gluconeogenesis), where ribose supplementation elevated levels of oxidized glutathione (GSSG) indicating an increase in oxidative stress. Treatment with ribitol also enhanced nucleotide biosynthesis. The apparent TCA cycle dysregulation, with distinctive pattern in response to the individual pentitol and pentose, such as ribitol increasing succinate and fumarate while decreasing citrate, demonstrate the adaptive capability of cancer cells to nutritional environment. This metabolic reprogramming presents new avenues for developing targeted therapies to cancers with metabolites, especially in combination with other drug treatments.

Establishment and Characterization of a Novel Human Ocular Adnexal Sebaceous Carcinoma Cell Line.

Purpose: Sebaceous carcinoma (SC) is a malignant eyelid tumor of the ocular adnexa that is primarily treated via surgical excision. Few therapies exist in advanced cases, and medical therapy is limited because of our incomplete understanding of SC biology. Herein, we describe a technique to culture human ocular adnexal SC for use as an in vitro model. Methods: Human ocular adnexal SC tumor cells were isolated from a patient undergoing orbital exenteration surgery and named Bascom Palmer 50 (BP50). They were cultured in Dulbecco's modified Eagle medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum and antibiotics and were maintained at 37°C in humidified 5% CO2. The cells were characterized by immunohistochemistry, exome sequencing, and short tandem repeats analysis. In vitro drug screening against mitomycin-C (MMC) was performed using a cell viability assay. Results: BP50 grew past 40 passages with a doubling time of 52.3 hours. Immunocytochemical staining revealed expression of SC-associated markers adipophilin, epithelial membrane antigen, p53, and androgen receptor. Whole exome sequencing showed a significant carryover in somatic mutations between the tumor tissue and corresponding cell line, revealing genetic markers consistent with SC. MMC affected cell viability in a dose-dependent manner. Conclusions: BP50 displays characteristics of ocular adnexal SC and therefore may facilitate improved understanding of SC biology and the high throughput assessment of novel therapeutic compounds and new drug combinatorial approaches targeted for this disease. Translational Relevance: Drug screening with MMC against these cells shows in vitro evidence to support its continued clinical use in SC.

Fibroblast growth factor receptor 1 (FGFR1) as a therapeutic target in adenoid cystic carcinoma of the lacrimal gland.

Identification of molecular targets is the first step in developing efficacious therapeutic strategies for tumors. A tumors' biological response to perturbagens yields important information on the molecular determinants for tumor growth. The aim of this study was to characterize the response of adenoid cystic carcinoma of the lacrimal gland (LGACC) to intra-arterial cytoreductive chemotherapy (IACC) in order to identify novel targets to enhance therapy. We performed high-throughput proteomic analysis on paired samples from pre-IACC diagnostic biopsies and post-IACC excised tumor samples from 6 LGACC patients. This proteomic analysis provides a comprehensive landscape of the cellular compartments contained within the excised tumors. Interestingly, we found a strong upregulation across the fibroblast growth factor (FGF) signaling pathway, with FGF receptor 1 (FGFR1) exhibiting a consistent and significant upregulation in all post-IACC samples. We thus evaluated the therapeutic efficacy of a novel FGFR1 selective inhibitor, AZD4547, in combination with cisplatin on LGACC cells in-vitro. The combination index (CI) value (<0.895) demonstrated synergistic effect of AZD4547 and cisplatin in inhibiting cell growth and viability (p<0.02), with a differential response seen in post-IACC cultures when compared to pre-IACC cultures. The combination approach showed synergy of the drugs in the migration assay. Western blot analysis indicated a significant upregulation of cleaved caspase-3 and downregulation the expression of FGFR1 (p<0.05) with the combination treatment as compared to either agent independently. Our findings demonstrate that FGFR1 inhibition potentiates the cytoreductive effects of cisplatin and suggest a potential therapeutic benefit of using AZD4547 in the management of LGACC.

The Effect of Prostaglandin Analogue Bimatoprost on Thyroid-Associated Orbitopathy.

Purpose: We characterize the effect of bimatoprost on orbital adipose tissue in thyroid-associated orbitopathy (TAO) with clinicopathologic correlation. Methods: Orbital adipose-derived stem cells (OASCs) from types 1 and 2 TAO and control patients with and without exposure to 1 µm bimatoprost were examined via immunohistochemistry, RT-PCR, and Western blot for cell viability, migration capacity, lipid content, adipocyte morphology, mitochondrial content, and levels of adipogenic markers. A retrospective chart review was performed for clinicopathologic correlation. In mice, optical coherence tomography and pattern electroretinography were performed at baseline and at 1 month following a retrobulbar injection of bimatoprost, followed by orbital exenteration for histopathologic examination. Results: Types 1 and 2 TAO-derived cells had a significantly higher migration capacity and lipid content than those of healthy controls. With the addition of bimatoprost, types 1 and 2 TAO and control adipocytes exhibited a significant decrease in lipid content with morphologic transformation into smaller and multilocular lipid droplets, and an increase in mitochondrial load and UCP-1 expression consistent with an increase in brown adipose tissue turnover. Retrobulbar injection of bimatoprost in mice did not alter the gross morphology, retinal thickness, or ganglion cell function in vivo. Conclusions: Bimatoprost inhibits adipogenesis in OASCs and upregulates pathways involved in the browning of adipocytes. Furthermore, retrobulbar injection of bimatoprost is tolerated without immediate adverse effects in mice. Our results suggest a potential future application of prostaglandin analogues in the treatment of TAO.

Honokiol nanomicellar formulation produced increased oral bioavailability and anticancer effects in triple negative breast cancer (TNBC).

Triple negative breast cancer (TNBC), owing to its aggressive behavior and toxicity associated with available chemotherapy; currently no suitable therapy is available. Honokiol (HNK) is a promising anticancer drug but has poor bioavailability. In the current study, we evaluated the anticancer effects of an oral Honokiol nanomicellar (NM) formulation (size range of 20-40nm) in vitro against various TNBC cells lines. Cytotoxicity, clonogenic and wound healing assays demonstrated the promising anticancer effects. In vitro Caco-2 permeability studies suggested increased absorption of Honokiol. Compared to HNK-FD, nanomicellar formulations resulted in significant increase in the oral bioavailability. Cmax (4.06 and 3.60-fold) and AUC (6.26 and 5.83-fold) were significantly increased in comparison to oral 40 and 80mg/kg free drug respectively. Further, anticancer effects of these formulations were studied in BALB/c nude mice transplanted with orthotopic MDA-MB-231 cell induced xenografts. After 4 weeks of daily administration of HNK-NM formulation, significant reduction in the tumor volumes and weights compared to free drug (p<0.001) treated groups was observed. Surprisingly, in some of the animals (25%), the treatment resulted in complete eradication of tumors. Increased apoptosis and antiangiogenic effect was observed in HNK-NM groups compared to free drug and untreated control animals. This is the first report demonstrating that HNK-FD possesses anticancer effects against TNBC.

Reversal of drug-resistance by noscapine chemo-sensitization in docetaxel resistant triple negative breast cancer.

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.

Novel amphiphilic lipid augments the co-delivery of erlotinib and IL36 siRNA into the skin for psoriasis treatment.

In this study, we demonstrate for the first time the concurrent transdermal delivery of erlotinib and IL36α siRNA as a potential dual therapy for psoriasis. The objectives were to develop and evaluate lipid nanocarriers (CYnLIP) using a novel pyrrolidinium lipid to disrupt the skin barrier for enhanced transdermal delivery. CYnLIP (132.00±6.23nm) had encapsulation efficiency of 49.04±2.54% for erlotinib. DSC confirmed encapsulation of erlotinib within CYnLIP. Atomic Force Microscopy demonstrated notable topographical changes in the stratum corneum of skin permeated with CYnLIP that were absent in skin hydrated with water. Peak force distance curves also exhibited a more permeable membrane for CYnLIP-incubated skin than hydrated skin. Permeation studies showed enhanced (p<0.01) skin retention of erlotinib by CYnLIP (40.76-fold) than solution and more pronounced fluorescence at deeper layers of the skin for fluorescein-labeled siRNA-CYnLIP than solution. The enhanced co-transdermal delivery of erlotinib and IL36α siRNA by CYnLIP efficaciously treated psoriatic-like plaques in C57BL/6 mice (PASI score of 1) compared to imiquimod-only treatment (PASI score of 4). IHC and western blotting revealed reduction in epidermal hyperplasia (Ki67) and in the dermal infiltration of inflammatory cytokines (IL36α, pSTAT3, TNFα, NFκB, IL23 and IL17) for erlotinib/IL36α siRNA-CYnLIP (p<0.05) comparable to Tacrolimus but markedly less than imiquimod-only treatment.

Tumor neovasculature-targeted cationic PEGylated liposomes of gambogic acid for the treatment of triple-negative breast cancer.

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.

Novel diindolylmethane derivatives based NLC formulations to improve the oral bioavailability and anticancer effects in triple negative breast cancer.

The present study demonstrates the promising anticancer effects of novel C-substituted diindolylmethane (DIM) derivatives DIM-10 and DIM-14 in aggressive TNBC models. In vitro studies demonstrated that these compounds possess strong anticancer effects. Caco-2 permeability studies resulted in poor permeability and poor oral bioavailability was demonstrated by pharmacokinetic studies. Nano structured lipid carrier (NLC) formulations were prepared to increase the clinical acceptance of these compounds. Significant increase in oral bioavailability was observed with NLC formulations. Compared to DIM-10, DIM-10 NLC formulation showed increase in Cmax and AUC values by 4.73 and 11.19-folds, respectively. Similar pattern of increase was observed with DIM-14 NLC formulations. In dogs DIM-10 NLC formulations showed an increase of 2.65 and 2.94-fold in Cmax and AUC, respectively. The anticancer studies in MDA-MB-231 orthotopic TNBC models demonstrated significant reduction in tumor volumes in DIM-10 and DIM-14 NLC treated animals. Our studies suggest that NLC formulation of both DIM-10 and 14 is effective in TNBC models.

NR4A1 Antagonists Inhibit ß1-Integrin-Dependent Breast Cancer Cell Migration.

Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor ß (TGF-ß) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-ß independent and dependent on regulation of ß1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-ß-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-ß-induced cell migration. We also observed that NR4A1 regulates expression of both ß1- and ß3-integrins, and unlike other ß1-integrin inhibitors which induce prometastatic ß3-integrin, NR4A1 antagonists inhibit expression of both ß1- and ß3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis.