Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: a favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is worldwide health problem associated with high morbidity and mortality. From both the patient and socioeconomic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full-thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation, i.e. nuclear import via binding to cellular RA binding protein-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation [Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting] upregulation of a transglutaminase enzyme essential for cornified envelope formation (transglutaminase 3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

Mucopenetrating Janus Nanoparticles For Field-Coverage Oral Cancer Chemoprevention.

INTRODUCTION: Oral squamous cell carcinoma (OSCC), is associated with high morbidity and mortality. Preemptive interventions have been postulated to provide superior therapeutic options, but their implementation has been restricted by the availability of broadly applicable local delivery systems. METHODS: We address this challenge by engineering a delivery vehicle, Janus nanoparticles (JNP), that combine the dual mucoadhesive properties of a first cationic chitosan compartment with a second hydrophobic poly(lactide-co-glycolide) release compartment. JNP are designed to avoid rapid mucus clearance while ensuring stable loading and controlled release of the IL-6 receptor antagonist, tocilizumab (TCZ). RESULTS: The JNP featured defined and monodispersed sizes with an average diameter of 327 nm and a PDI of 0.245, high circularities above 0.90 and supported controlled release of TCZ and effective internalization by oral keratinocytes. TCZ released from JNP retained its biological activity and effectively reduced both, soluble and membrane-bound IL-6Rα (71% and 50%). In full-thickness oral mucosal explants, 76% of the JNP breached the stratum corneum and in 41% were observed in the basal cell layer indicating excellent mucopenetrating properties. When tested in an aggressive OSCC xenograft model, TCZ-loaded JNP showed high levels of xenograft inhibition and outperformed all control groups with respect to inhibition of tumor cell proliferation, reduction in tumor size and reduced expression of the proto-oncogene ERG. CONCLUSION: By combining critically required, yet orthogonal properties within the same nanoparticle design, the JNP in this study, demonstrate promise as precision delivery platforms for intraoral field-coverage chemoprevention, a vastly under-researched area of high clinical importance.

Detection of DNA adducts derived from the tobacco carcinogens, benzo[a]pyrene and dibenzo[def,p]chrysene in human oral buccal cells.

Polycyclic aromatic hydrocarbons (PAHs) are recognized as potential etiological agents in the development of oral cancer in smokers. In particular, benzo[a]pyrene (B[a]P) and dibenzo[def,p]chrysene (DB[a,l]P) are detected in cigarette smoke and the environment and can induce DNA damage, mutagenesis and carcinogenesis in the oral cavity of rodents. Consequently, DNA adducts are regarded as the most direct markers of genotoxicity and can be used as biomarkers of cancer risk. Thus, this study used LC-MS/MS analysis with isotope labeled internal standard to detect and quantify DNA adducts derived from B[a]P and DB[a,l]P in buccal cells of cigarette smokers and non-smokers. Participants in this study include 21 smokers and 16 non-smokers. Our data are the first to report that levels (mean ± SD) of BPDE-N2-dG were significantly (P < 0.001) higher in smokers (20.18 ± 8.40 adducts/108 dG) than in non-smokers (0.84 ± 1.02 adducts/108 dG). Likewise, levels of DBPDE-N6-dA in smokers (5.49 ± 3.41 adducts/108 dA) were significantly higher (P = 0.019) than non-smokers (2.76 ± 2.29 adducts/108 dA). Collectively, the results of this clinical study support that PAHs in tobacco smoke can contribute to the development of oral cancer in humans.

Fenretinide combines perturbation of signaling kinases, cell-extracellular matrix interactions and matrix metalloproteinase activation to inhibit invasion in oral squamous cell carcinoma cells.

Basement membrane invasion defines malignant transformation of surface premalignancy. Treatment of oral squamous cell carcinoma (OSCC) cells with the synthetic vitamin A derivative, fenretinide (4HPR), induces numerous cancer-preventive effects including suppression of basement membrane invasion, elimination of anchorage-independent growth, disruption of actin cytoskeletal components and inhibition of the invasion-enabling focal adhesive kinase. The purpose of this study was to elucidate 4HPR's effects on additional invasion-relevant mechanisms including matrix metalloproteinase (MMP) activation and function, cell-extracellular matrix (ECM) attachments and interaction with a kinase that is essential for the epithelial-myoepithelial transformation i.e. c-Jun NH2-terminal kinase (JNK). Our data revealed that 4HPR binds with high affinity to the ATP-binding site of all three JNK isoforms with concurrent suppression of kinase function. Additional studies showed 4HPR treatment inhibited both OSCC cell-ECM adhesion and MMP activation and function. JNK downregulation and induced expression studies confirmed that the JNK3 isoform conveyed that largest impact on OSCC migration and invasion. Biodegradable polymeric implants formulated to preserve 4HPR's function and bioavailability were employed to assess 4HPR's chemopreventive impact on an OSCC tumor induction model. These studies revealed 4HPR local delivery significantly inhibited OSCC tumor size, mitotic indices and expression of the endothelial marker, erythroblast transformation-specific-related gene with concurrent increases in tumor apoptosis (cleaved caspase-3). Collectively, these data show that 4HPR suppresses invasion at multiple sites including 'outside-in' signaling, cell-ECM interactions and suppression of MMPs. These functions are also essential for physiologic function. Regulation is therefore essential and reinforces the pharmacologic advantage of local delivery chemopreventive formulations. .

Inherent phenotypic plasticity facilitates progression of head and neck cancer: endotheliod characteristics enable angiogenesis and invasion.

The presence of the EMT (epithelial-mesenchymal transition), EndMT (endothelial-mesenchymal transition) and VM (vasculogenic mimicry) demonstrates the multidirectional extent of phenotypic plasticity in cancers. Previous findings demonstrating the crosstalk between head and neck squamous cell carcinoma (HNSCC) and vascular endothelial growth factor (VEGF) imply that HNSCC cells share some functional commonalities with endothelial cells. Our current results reveal that cultured HNSCC cells not only possess endothelial-specific markers, but also display endotheliod functional features including low density lipoprotein uptake, formation of tube-like structures on Matrigel and growth state responsiveness to VEGF and endostatin. HNSCC cell subpopulations are also highly responsive to transforming growth factor-ß1 and express its auxiliary receptor, endoglin. Furthermore, the endotheliod characteristics observed in vitro recapitulate phenotypic features observed in human HNSCC tumors. Conversely, cultured normal human oral keratinocytes and intact or ulcerated human oral epithelia do not express comparable endotheliod characteristics, which imply that assumption of endotheliod features is restricted to transformed keratinocytes. In addition, this phenotypic state reciprocity facilitates HNSCC progression by increasing production of factors that are concurrently pro-proliferative and pro-angiogenic, conserving cell energy stores by LDL internalization and enhancing cell mobility. Finally, recognition of this endotheliod phenotypic transition provides a solid rationale to evaluate the antitumorigenic potential of therapeutic agents formerly regarded as exclusively angiostatic in scope.

Salivary gland carcinosarcoma ex pleomorphic adenoma: a case report with molecular characterization.

True malignant mixed tumors, also known as salivary gland carcinosarcoma (SCS), are uncommon yet highly aggressive lesions associated with a poor prognosis. These tumors exhibit a distinctive biphasic structure characterized by both epithelial and mesenchymal components. Recent research has shown that the majority of SCS cases stem from pre-existing pleomorphic adenomas (PAs), suggesting a stepwise developmental pattern. In this report, we present a case of a 73-year-old female with SCS and describe the clinical, radiographic, and pathologic observations. Notably, the SCS was associated with a residual PA. The SCS displayed a CTNNB1::PLAG1 gene rearrangement, providing a molecular basis for its origin from the PA. Further DNA genomic analysis exposed mutations in BAP1, PER1, and LRPB1. Our findings provide support to the theory that SCS emerges from a pre-existing PA while highlighting the multiple genetic changes that could contribute to malignant transformation.

The Effects of Black Raspberry as a Whole Food-Based Approach on Biomarkers of Oxidative Stress in Buccal Cells and Urine of Smokers.

Cigarette smoke is a rich source of free radicals that can promote oxidative stress and carcinogenesis, including head and neck squamous cell carcinoma (HNSCC) development; importantly, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-iso-prostaglandin F2α (8-isoprostane) are biomarkers of oxidative stress. Several mechanisms, including the antioxidant properties of black raspberry (BRB), account for their chemopreventive effects. In the present clinical trial, we tested the hypothesis that BRB administration reduces biomarkers levels of oxidative stress in buccal cells and urine of smokers. One week after enrolling 21 smokers, baseline buccal cells and urine samples were collected before the administration of BRB lozenges for 8 weeks (5/day, 1 gm BRB/lozenge). Buccal cells and urine samples were collected at the middle and the end of BRB administration. The last samples were collected after the BRB cessation (washout period). We analyzed levels of 8-oxodG and 8-isoprostane (LC/MS-MS), urinary cotinine (ELISA), and creatinine (spectrophotometry). BRB significantly reduced the levels of 8-oxodG by 17.08% (P = 0.00079) in buccal cells and 12.44% (P = 0.034) in urine at the middle of BRB administration as compared with baseline; the corresponding values at the end of BRB administration were 16.46% (P = 0.026) in buccal cells and 25.72% (P = 0.202) in urine. BRB had no significant effect on the levels of urinary 8-isoprostane. BRB's capacity to inhibit 8-oxodG formation of smokers' buccal cells and urine is clearly evident and the reduction in 8-oxodG suggests that antioxidant abilities are central to BRB's HNSCC chemopreventive properties. PREVENTION RELEVANCE: Cigarette smoke contains highly active components namely free radicals that can promote oxidative stress and oral cancer. We found that black raspberry (BRB) inhibited the formation of oxidative stress markers in the oral cavity and urine of smokers suggesting the antioxidant abilities of BRB in preventing oral cancer.

Clinical teaching in dentistry: Evaluating a clinical oral pathology rotation while looking to the future of dental education.

PURPOSE/OBJECTIVES: Clinical teaching in dentistry has cycled through specialty-based "departmental" teaching to group practice scenarios. The aims of this study were to ascertain third-year dental students' perceptions of a specialty-based rotation supplemented by online educational tools and to compare their Objective Structured Clinical Exam (OSCE) scores to those of the students in the previous year. METHODS: This retrospective research design included the analysis of OSCE scores, and student responses to survey questions regarding their perceptions of the clinical oral pathology (COP) rotation. This study was completed in 2022. It included data from 2020 to 2021 and 2021 to 2022 corresponding to input from the Classes of 2022 and 2023, respectively. The response rate was 100%. RESULTS: The students evaluated the focused COP rotation and the online teaching modules as a positive experience. The results of the OSCE were similar to those of the previous class and showed a high average score. CONCLUSIONS: This study shows that students had a positive perception to specialty-based learning with online educational tools and that it enhanced the education they received in the comprehensive care clinic. The OSCE scores were similar to those of the previous class. These findings suggest a method of maintaining high-quality dental education through challenges as it continues evolving.

Evaluation of a mucoadhesive fenretinide patch for local intraoral delivery: a strategy to reintroduce fenretinide for oral cancer chemoprevention.

Systemic delivery of fenretinide in oral cancer chemoprevention trials has been largely unsuccessful due to dose-limiting toxicities and subtherapeutic intraoral drug levels. Local drug delivery, however, provides site-specific therapeutically relevant levels while minimizing systemic exposure. These studies evaluated the pharmacokinetic and growth-modulatory parameters of fenretinide mucoadhesive patch application on rabbit buccal mucosa. Fenretinide and blank-control patches were placed on right/left buccal mucosa, respectively, in eight rabbits (30 min, q.d., 10 days). No clinical or histological deleterious effects occurred. LC-MS/MS analyses of post-treatment samples revealed a delivery gradient with highest fenretinide levels achieved at the patch-mucosal interface (no metabolites), pharmacologically active levels in fenretinide-treated oral mucosa (mean: 5.65 µM; trace amounts of 4-oxo-4-HPR) and undetectable sera levels. Epithelial markers for cell proliferation (Ki-67), terminal differentiation (transglutaminase 1-TGase1) and glucuronidation (UDP-glucuronosyltransferase1A1-UGT1A1) exhibited fenretinide concentration-specific relationships (elevated TGase1 and UGT1A1 levels <5 µM, reduced Ki-67 indices >5 µM) relative to blank-treated epithelium. All fenretinide-treated tissues showed significantly increased intraepithelial apoptosis (TUNEL) positivity, implying activation of intersecting apoptotic and differentiation pathways. Human oral mucosal correlative studies showed substantial interdonor variations in levels of the enzyme (cytochrome P450 3A4-CYP3A4) responsible for conversion of fenretinide to its highly active metabolite, 4-oxo-4-HPR. Complementary in vitro assays in human oral keratinocytes revealed fenretinide and 4-oxo-4-HPR's preferential suppression of DNA synthesis in dysplastic as opposed to normal oral keratinocytes. Collectively, these data showed that mucoadhesive patch-mediated fenretinide delivery is a viable strategy to reintroduce a compound known to induce keratinocyte differentiation to human oral cancer chemoprevention trials.

Mucoadhesive fenretinide patches for site-specific chemoprevention of oral cancer: enhancement of oral mucosal permeation of fenretinide by coincorporation of propylene glycol and menthol.

The objective of this study was to enhance oral mucosal permeation of fenretinide by coincorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Coincorporation of 5-10 wt % PG (mean J(s) = 16-23 µg cm⁻² h⁻¹; 158-171 µg of fenretinide/g of tissue) or 1-10 wt % PG + 5 wt % menthol (mean J(s) = 18-40 µg cm⁻² h⁻¹; 172-241 µg of fenretinide/g of tissue) in fenretinide/Eudragit RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt % in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (∼43 µg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt % PG + 5 wt % menthol) relative to permeation obtained from enhancer-free patch (∼17 µg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by coincorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch coincorporated with 2.5 wt % PG + 5 wt % menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide.

Optimizing therapeutic efficacy of chemopreventive agents: A critical review of delivery strategies in oral cancer chemoprevention clinical trials.

Due to its characterized progression from recognized premalignant oral epithelial changes (i.e., oral epithelial dysplasia) to invasive cancer, oral squamous cell carcinoma represents an optimal disease for chemopreventive intervention prior to malignant transformation. The primary goal of oral cancer chemoprevention is to reverse, suppress, or inhibit the progression of premalignant lesions to cancer. Over the last several decades, numerous oral cancer chemoprevention clinical trials have assessed the therapeutic efficacy of diverse chemopreventive agents. The standard of care for more advanced oral dysplastic lesions entails surgical excision and close clinical follow-up due to the potential (~33%) for local recurrence at a similar or more advanced histological stage. The purpose of this review was to identify prominent oral cancer chemoprevention clinical trials, assess their overall therapeutic efficacy, and delineate effects of local versus systemic drug administration. In addition, these compiled clinical trial data present concepts for consideration in the design and conduction of future clinical trials.

Development and in vitro-in vivo evaluation of fenretinide-loaded oral mucoadhesive patches for site-specific chemoprevention of oral cancer.

PURPOSE: To develop fenretinide oral mucoadhesive patch formulations and evaluate their in vitro and in vivo release performance for future site-specific chemoprevention of oral cancer. METHODS: Solubilization of fenretinide in simulated saliva (SS) was studied by incorporating nonionic surfactants (Tween® 20 and 80, and Brij® 35 and 98), bile salts (sodium salt of cholic, taurocholic, glycocholic, and deoxycholic acids), phospholipid (lecithin), and novel polymeric solubilizer (Souplus®). Adhesive (polycarbophil: hydroxypropyl methylcellulose 4KM) and drug release (Fenretinide/Eudragit® RL PO with or without solubilizers) layers were prepared by solvent casting. Oral mucoadhesive patches were formed by attaching drug and adhesive layers onto backing layer (Tegaderm™ film). Physical state of drug in Eudragit® films was examined by X-ray diffraction (XRD). Evaluation of in vitro and in vivo fenretinide release from the patch was conducted in SS containing 5%w/v sodium deoxycholate and rabbits, respectively. Fenretinide was quantified by HPLC. RESULTS: Tween® 20 and 80, Brij® 98, and sodium deoxycholate exhibited the highest fenretinide solubilization potential among the solubilizers. Drug loading efficiency in Eudragit® films was 90%-97%. XRD suggested fenretinide was amorphous in solubilizer-free and solubilizer-loaded films. Solubilizer-free patch exhibited poor in vitro and in vivo controlled drug release behavior. Increases in drug loading (5-10 wt%) or changes in polymeric matrix permeability did not provide continuous drug release. Co-incorporation of either single or mixed solubilizers in fenretinide/Eudragit® patches, (20 wt% Tween® 20, Tween® 80 and sodium deoxycholate or 20 wt% Tween® 80 + 40 wt% sodium deoxycholate solubilizers) led to significantly improved continuous in vitro/in vivo fenretinide release. CONCLUSION: Fenretinide/Eudragit® RL PO patches with 20 wt% Tween® 80 + 40 wt% sodium deoxycholate solubilizers exhibit excellent release behavior for further preclinical and/or clinical evaluation in oral cancer chemoprevention.

Meeting Report: Translational Advances in Cancer Prevention Agent Development Meeting.

The Division of Cancer Prevention of the National Cancer Institute (NCI) and the Office of Disease Prevention of the National Institutes of Health co-sponsored the Translational Advances in Cancer Prevention Agent Development Meeting on August 27 to 28, 2020. The goals of this meeting were to foster the exchange of ideas and stimulate new collaborative interactions among leading cancer prevention researchers from basic and clinical research; highlight new and emerging trends in immunoprevention and chemoprevention as well as new information from clinical trials; and provide information to the extramural research community on the significant resources available from the NCI to promote prevention agent development and rapid translation to clinical trials. The meeting included two plenary talks and five sessions covering the range from pre-clinical studies with chemo/immunopreventive agents to ongoing cancer prevention clinical trials. In addition, two NCI informational sessions describing contract resources for the preclinical agent development and cooperative grants for the Cancer Prevention Clinical Trials Network were also presented.

Nanoparticles for local drug delivery to the oral mucosa: proof of principle studies.

PURPOSE: To determine if solid lipid nanoparticles represent a viable strategy for local delivery of poorly water soluble and unstable chemopreventive compounds to human oral tissues. METHODS: Nanoparticle uptake and compound retention evaluations employed monolayer-cultured human oral squamous cell carcinoma (OSCC) cell lines and normal human oral mucosal explants. Feasibility of nanoparticle delivery was also evaluated with respect to the presence of phase-III efflux transporters in normal oral mucosal tissue and OSCC tissues. RESULTS: Functional uptake assays confirmed significantly greater internalization of nanoparticle-delivered fluorescent probe relative to free-fluorescent probe delivery, while concurrently demonstrating nanoparticle uptake rate differences among the OSCC cell lines and the phagocytic control human monocyte cell line. Mucosal explants exhibited nanoparticle penetration and internalization in the spinous and basal epithelial layers (7/10 specimens), and also exhibited the presence of the phase-III efflux transporters multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). CONCLUSIONS: These data confirm nanoparticle internalization by OSCC cells and support the premise that nanoparticle-based delivery provides higher final intracellular levels relative to bolus administration. Furthermore, the penetration and subsequent internalization of nanoparticles within the proliferating basal layer cells demonstrates the feasibility of nanoparticle formulations for local delivery and stabilization of oral chemopreventive compounds.

Formulation and in vitro-in vivo evaluation of black raspberry extract-loaded PLGA/PLA injectable millicylindrical implants for sustained delivery of chemopreventive anthocyanins.

PURPOSE: The objective of this study was to formulate and evaluate freeze-dried black raspberry (FBR) ethanol extract (RE) loaded poly(DL-lactic-co-glycolic acid) (PLGA) and poly(DL-lactic acid) (PLA) injectable millicylindrical implants for sustained delivery of chemopreventive FBR anthocyanins (cyanidin-3-sambubioside (CS), cyanidin-3-glucoside (CG) and cyanidin-3-rutinoside (CR)). METHODS: Identification and quantitation of CS, CG, and CR in RE was performed by mass spectroscopy and HPLC. RE:triacetyl-beta-cyclodextrin (TA-beta-CD) inclusion complex (IC) was prepared by a kneading method and characterized by X-ray diffraction (XRD), nuclear magnetic resonance spectroscopy (NMR) and UV-visible spectroscopy. RE or RE:TA-beta-CD IC-loaded PLGA or PLA implants were prepared by a solvent extrusion method. In vitro and in vivo controlled release studies were conducted in phosphate-buffered saline Tween-80 (pH 7.4, 37 degrees C) and after subcutaneous administration in male Sprague-Dawley rats, respectively. Anthocyanins were quantified by HPLC at 520 nm. RESULTS: The content of CS, CG, and CR in RE was 0.2, 1.5, and 3.5 wt%, respectively. The chemical stability of anthocyanins in solution was determined to be pH-dependent, and their degradation rate increased with an increase in pH from 2.4 to 7.4. PLGA/PLA millicylindrical implants loaded with 5 or 10 wt% RE exhibited a high initial burst and short release duration of anthocyanins (35-52 and 80-100% CG + CR release after 1 and 14 days, respectively). The cause for rapid anthocyanins release was linked to higher polymer water uptake and porosity associated with the high osmolytic components of large non-anthocyanin fraction of RE. XRD, (1)H NMR and UV-visible spectroscopy indicated that the non-anthocyanin fraction molecules of RE formed an IC with TA-beta-CD, decreasing the hydrophilicity of RE. Formation of an IC with hydrophobic carrier, TA-beta-CD, provided better in vitro/in vivo sustained release of FBR anthocyanins (16-24 and 97-99% CG + CR release, respectively, after 1 and 28 days from 20 wt% RE:TA-beta-CD IC/PLA implants) over 1 month, owing to reduced polymer water uptake and porosity. CONCLUSION: PLA injectable millicylindrical implants loaded with RE:TA-beta-CD IC are optimal dosage forms for 1-month slow and continuous delivery of chemopreventive FBR anthocyanins.

Microencapsulation of amorphous solid dispersions of fenretinide enhances drug solubility and release from PLGA in vitro and in vivo.

The purpose of this study was to develop solid dispersions of fenretinide(4HPR), incorporate them into poly(lactic-co-glycolic)(PLGA) millicylindrical implants, and evaluate the resulting implants in vitro and in vivo for future applications in oral cancer chemoprevention. Due to the extreme hydrophobicity of 4HPR, 4HPR-polyvinylpyrrolidone (PVP) amorphous solid dispersions(ASDs) were prepared for solubility enhancement. The optimal PVP-4HPR ratio of 9/1(w/w) provided a 50-fold solubility enhancement in aqueous media, which was sustained over 1 week. PVP-4HPR ASD particles were loaded into PLGA millicylinders and drug release was evaluated in vitro in PBST and in vivo by recovery from subcutaneous injection in rats. While initial formulations of PLGA PVP-4HPR millicylinders only released 10% 4HPR in vitro after 28 days, addition of the plasticizer triethyl-o-acetyl-citrate(TEAC) into PVP-4HPR ASDs resulted in a 5.6-fold total increase in drug release. Remarkably, the TEAC-PVP-4HPR PLGA implants demonstrated slow, continuous, and nearly complete release over 1 month in vivo compared to a 25% release for our previously reported formulation incorporating solubilizers and pore-forming agents. Hence, a combination of PLGA plasticizer and ASD formation provides an avenue for long-term controlled release in vivo for the exceptionally difficult drug to formulate, 4HPR, and a suitable formulation for future evaluation in rodent models of oral cancer.

Effects of a topically applied bioadhesive berry gel on loss of heterozygosity indices in premalignant oral lesions.

PURPOSE: The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene-associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties. EXPERIMENTAL DESIGN: Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes. RESULTS: Confirming earlier phase I data, none of the 27 participants developed FBR gel-associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene-associated loci. CONCLUSIONS: These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.

Fenretinide, Tocilizumab, and Reparixin Provide Multifaceted Disruption of Oral Squamous Cell Carcinoma Stem Cell Properties: Implications for Tertiary Chemoprevention.

Locoregional recurrence of oral squamous cell carcinoma (OSCC) dramatically reduces patient survival. Further, as many OSCC recurrences are inoperable, radiotherapy and chemotherapy with or without biological adjuncts are the remaining treatment options. Although the tumors may initially respond, radiotherapy- and chemotherapy-resistant cancer stem cells (CSC) can readily repopulate OSCC tumors. Currently, following the initial OSCC treatment, patients are closely monitored until a recurrence or a second primary is detected. Identification of agents with complementary mechanisms to suppress CSC tumorigenic functions could change this passive approach. The goals of this study were twofold: (1) develop and validate CSC-enriched (CSCE) OSCC cell lines and (2) identify chemopreventive agents that obstruct multiple CSCE protumorigenic pathways. CSCE cultures, which were created by paclitaxel treatment followed by three tumorsphere passes, demonstrated CSC characteristics, including increased expression of stem cell and inflammatory genes, increased aldehyde dehydrogenase (ALDH) activity, and enhanced in vitro/in vivo proliferation and invasion. Three chemopreventives, fenretinide, tocilizumab, and reparixin, were selected due to their distinct and complementary CSC-disruptive mechanisms. The CSCE selection process modulated the cells' intermediate filaments resulting in an epithelial-predominant (enhanced cytokeratin, proliferation, IL6 release) line and a mesenchymal-predominant (upregulated vimentin, invasive, IL8 release) line. Our results confirm that 4HPR binds with appreciably higher affinity than Wnt at the Frizzled binding site and significantly inhibits CSC-enabling Wnt-ß-catenin downstream signaling. Notably, combination fenretinide-tocilizumab-reparixin treatment significantly suppressed IL6 and IL8 release, stem cell gene expression, and invasion in these diverse CSCE populations. These promising multiagent in vitro data provide the basis for our upcoming in vivo CSCE tertiary chemoprevention studies.

Human head and neck squamous cell carcinoma cells are both targets and effectors for the angiogenic cytokine, VEGF.

Former vascular endothelial growth factor (VEGF)-head and neck squamous cell carcinoma (HNSCC) studies have focused on VEGF's contributions toward tumor-associated angiogenesis. Previously, we have shown that HNSCC cells produce high levels of VEGF. We therefore hypothesized that VEGF serves a biphasic role, that is, pro-angiogenic and pro-tumorigenic in HNSCC pathogenesis. Western blots confirmed the presence of VEGF's primary mitogenic receptors, VEGFR-2/KDR and VEGFR-1/Flt-1 in cultured HNSCC cells. Subsequent studies evaluated VEGF's effects on HNSCC intracellular signaling, mitogenesis, invasive capacities, and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF(165) initiated ROS-mediated intracellular signaling, resulting in kinase activation and phosphorylation of KDR and Erk1/2. As high endogenous VEGF production rendered HNSCC cells refractory to exogenous VEGF's mitogenic effects, siRNA was employed, inhibiting endogenous VEGF production for up to 96 h. Relative to transfection vector matched controls, siRNA treated HNSCC cells showed a significant decrease in proliferation at both 30 and 50 nM siRNA doses. Addition of exogenous hrVEGF(165) (30 and 50 ng/ml) to siRNA-silenced HNSCC cells resulted in dose-dependent increases in cell proliferation. Cell invasion assays showed VEGF is a potent HNSCC chemoattractant and demonstrated that VEGF pre-treatment enhanced invasiveness of HNSCC cells. Conditioned media from VEGF challenged HNSCC cells showed a moderate increase in gelatinase activity. Our results demonstrate, for the first time, that HNSCC cells are both targets and effectors for VEGF. These data introduce the prospect that VEGF targeted therapy has the potential to fulfill both anti-angiogenic and anti-tumorigenic functions.

Frequent alterations of p16INK4a and p14ARF in oral proliferative verrucous leukoplakia.

Proliferative verrucous leukoplakia (PVL) represents a rare but highly aggressive form of oral leukoplakia with > 70% progressing to malignancy. Yet, PVL remains biologically and genetically poorly understood. This study evaluated the cell cycle regulatory genes, p16INK4a and p14ARF, for homozygous deletion, loss of heterozygosity, and mutation events in 20 PVL cases. Deletion of exon 1beta, 1alpha, or 2 was detected in 40%, 35%, and 0% of patients, respectively. Deletions of exons 1alpha and 1beta markedly exceed levels reported in non-PVL dysplasias and approximate or exceed levels reported in oral squamous cell carcinomas. Allelic imbalance was assessed for markers reported to be highly polymorphic in squamous cell carcinomas and in oral dysplasias. Loss of heterozygosity was detected in 35.3%, 26.3%, and 45.5% of PVLs for the markers IFNalpha, D9S1748, and D9S171, respectively. INK4a and ARF sequence alterations were detected in 20% and 10% of PVL lesions, accordingly. These data show, for the first time, that both p16INK4a and p14ARF aberrations are common in oral verrucous leukoplakia; however, the mode and incidence of inactivation events differ considerably from those reported in non-PVL oral premalignancy. Specifically, concomitant loss of p16INK4a and p14ARF occurred in 45% of PVL patients greatly exceeding loss reported in non-PVL dysplastic oral epithelium (15%). In addition, p14ARF exon 1beta deletions were highly elevated in PVLs compared with non-PVL dysplasias. These data illustrate that molecular alterations, even within a specific genetic region, are associated with distinct histologic types of oral premalignancy, which may affect disease progression, treatment strategies, and ultimately patient prognosis.