Ping Pei

Topical Application of a Bioadhesive Black Raspberry Gel Modulates Gene Expression and Reduces Cyclooxygenase 2 Protein in Human Premalignant Oral Lesions

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation.

Nanoparticles for Local Drug Delivery to the Oral Mucosa: Proof of Principle Studies

ABSTRACTPurposeTo determine if solid lipid nanoparticles represent a viable strategy for local delivery of poorly water soluble and unstable chemopreventive compounds to human oral tissues.MethodsNanoparticle 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.ResultsFunctional 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).ConclusionsThese 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.

Effects of human oral mucosal tissue, saliva and oral microflora on intraoral metabolism and bioactivation of black raspberry anthocyanins

Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried components of black raspberries (BRB) affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRBs chemopreventive effects. Consequently, functional assays, immunoblotting, and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were conducted. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles showed the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated, whereas immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. β-Glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC/MS-MS analyses showed retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, postrinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data show that comparable to the small intestine, the requisite hydrolytic, phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, interpatient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness. Cancer Prev Res; 4(8); 1209–21. ©2011 AACR.

Topical Application of a Mucoadhesive Freeze-Dried Black Raspberry Gel Induces Clinical and Histologic Regression and Reduces Loss of Heterozygosity Events in Premalignant Oral Intraepithelial Lesions: Results from a Multicentered, Placebo-Controlled Clinical Trial

Purpose: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions. Experimental Design: This placebo-controlled multicenter study investigated the effects of topical application of bioadhesive gels that contained either 10% w/w freeze-dried black raspberries (BRB) or an identical formulation devoid of BRB placebo to biopsy-confirmed OIN lesions (0.5 g × q.i.d., 12 weeks). Baseline evaluative parameters (size, histologic grade, LOH events) were comparable in the randomly assigned BRB (n = 22) and placebo (n = 18) gel cohorts. Evaluative parameters were: histologic grade, clinical size, and LOH. Results: Topical application of the BRB gel to OIN lesions resulted in statistically significant reductions in lesional sizes, histologic grades, and LOH events. In contrast, placebo gel lesions demonstrated a significant increase in lesional size and no significant effects on histologic grade or LOH events. Collectively, these data strongly support BRBs chemopreventive impact. A cohort of very BRB-responsive patients, as demonstrated by high therapeutic efficacy, was identified. Corresponding protein profiling studies, which demonstrated higher pretreatment levels of BRB metabolic and keratinocyte differentiation enzymes in BRB-responsive lesions, reinforce the importance of local metabolism and differentiation competency. Conclusions: Results from this trial substantiate the LOH reductions identified in the pilot BRB gel study and extend therapeutic effects to significant improvements in histologic grade and lesional size. Clin Cancer Res; 20(7); 1910–24. ©2014 AACR.

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 VEGFs 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 VEGFs primary mitogenic receptors, VEGFR‐2/KDR and VEGFR‐1/Flt‐1 in cultured HNSCC cells. Subsequent studies evaluated VEGFs effects on HNSCC intracellular signaling, mitogenesis, invasive capacities, and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF165 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 VEGFs 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 hrVEGF165 (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. J. Cell. Biochem. 105: 1202–1210, 2008.

A rapid and sensitive LC-MS/MS method for quantification of four anthocyanins and its application in a clinical pharmacology study of a bioadhesive black raspberry gel.

Cyanidin 3-glucoside (C3GLU), cyanidin 3-rutinoside (C3RUT), cyanidin 3-sambubioside (C3SAM) and cyanidin 3-(2(G)-xylosyl) rutinoside (C3XRUT) are the four constituent black raspberry anthocyanins that contribute significantly to the chemopreventive effects of freeze-dried black raspberries (FBR). A highly sensitive and specific LC-MS/MS assay was developed and validated to simultaneously quantify these four FBR anthocyanins in human saliva, plasma and oral tissue homogenates. In saliva, the lower limit of quantification (LLOQ) for these anthocyanins was 1.0 ng/mL. The within-run and between-run coefficients of variations (CVs) at the quality control concentrations (1.0, 5.0, 50 and 500 ng/mL) were all <12%, except for C3SAM and C3RUT at the LLOQ, which showed a within-run CV of 18.3% and between-run CV of 16.0%, respectively. The accuracy values ranged from 87.5 to 110%. In plasma, the LLOQ for C3GLU and C3RUT was 1.0 ng/mL and for C3SAM 5.0 ng/mL. The CVs at the above concentrations were <15%, except for C3GLU at the LLOQ, which showed the between-run CV of 16.9%. The accuracy values ranged from 90.7% to 112.7% except for C3GLU at the LLOQ, which showed 119.3%. In tissue homogenates, the LLOQ for C3GLU and C3RUT was 2.0 ng/mL, and C3SAM 5.0 ng/mL. The CVs and accuracy values at concentrations (2.0, 5.0, 50 and 500 ng/mL) were similar to those in human plasma. This assay was subsequently used in a pilot pharmacology study to evaluate the effects of topical application of a 10% (w/w) FBR bioadhesive gel to selected mucosal sites in the posterior mandibular gingiva. Measurable saliva and tissue levels of the FBR anthocyanins confirmed that gel-delivered anthocyanins are readily distributed to saliva and easily penetrate human oral mucosa.

THIOL REDOX MODULATION OF DOXORUBICIN MEDIATED CYTOTOXICITY IN CULTURED AIDS-RELATED KAPOSI'S SARCOMA CELLS

The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS‐ related Kaposis sarcoma (AIDS‐KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single‐agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicins mechanism of action against AIDS‐KS is unknown, we hypothesized that doxorubicins ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic‐associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV− aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV− donor cells are significantly less susceptible to doxorubicins cytotoxic effects relative to AIDS‐KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N‐acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicins cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS‐KS cells but deleterious effects in the HIV− control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS‐KS cells, as well as an impairment of endogenous defenses in the HIV− donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS‐KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicins clinical efficacy in AIDS‐KS treatment. J. Cell. Biochem. 73:259–277, 1999.

AIDS‐related Kaposi's sarcoma cells rapidly internalize endostatin, which co‐localizes to tropomysin microfilaments and inhibits cytokine‐mediated migration and invasion

AIDS‐related Kaposis sarcoma (KS) is the most common HIV‐related malignancy. In some respects, KS is analogous to other angioproliferative diseases, in that KS lesions are highly vascularized and promoted by inflammatory cytokines. However, unlike other cancers or inflammatory mediated vascular diseases, KS is unique in that the KS lesional cells both express and respond to the complete angiogenic cytokines vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Therefore, the angiogenic phenotype, which is crucial for cancer progression, is inherent to KS tumor cells. Due to the recognized importance of angiogenesis in cancer progression, numerous angiostatic agents are being investigated as potential therapeutic agents. One such agent is endostatin, which is a 20‐kDa carboxyl‐terminal fragment of collagen XVIII that has demonstrated potent angiostatic activities at both the in vivo and in vitro levels. Since endostatin is recognized as a potent angiostatic agent, the majority of in vitro endostatin studies have evaluated its effects on endothelial cells. Although KS cells are speculated to arise from endothelial cell precursors and KS lesions are highly vascularized, no previous studies have investigated endostatin–KS cell interactions. This present study evaluated endostatins effects on KS tumor cell: (i) signal transduction (endostatin internalization and transcription factor activation), and (ii) migration and invasion (functional activity assays and tropomysin co‐localization). Our results show that KS cells rapidly internalize endostatin and that endostatin initiates activation of the transcription activating factors nuclear factor‐κB (NF‐κB) and activating protein 1 (AP‐1). Our data also show that internalized endostatin co‐localizes to tropomysin microfilaments and acts to inhibit KS cell migration and invasion in response to the clinically relevant angiogenic cytokines VEGF and bFGF. As a consequence of its combined angiostatic and antitumorigenic activities, endostatin could provide dual therapeutic benefits for patients with mucocutaneous KS. J. Cell. Biochem. 89: 133–143, 2003. Published 2003 Wiley‐Liss, Inc.

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-HPRs 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.

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-β1 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.