Inhibition of Pro-inflammatory and Anti-apoptotic Biomarkers during Experimental Oral Cancer Chemoprevention by Dietary Black Raspberries.

Oral cancer continues to be a significant public health problem worldwide. Recently conducted clinical trials demonstrate the ability of black raspberries (BRBs) to modulate biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. However, it is essential that a preclinical animal model of black raspberry (BRB) chemoprevention which recapitulates human oral carcinogenesis be developed, so that we can validate biomarkers and evaluate potential mechanisms of action. We therefore established the ability of BRBs to inhibit oral lesion formation in a carcinogen-induced rat oral cancer model and examined potential mechanisms. F344 rats were administered 4-nitroquinoline 1-oxide (4NQO) (20 µg/ml) in drinking water for 14 weeks followed by regular drinking water for 6 weeks. At week 14, rats were fed a diet containing either 5 or 10% BRB, or 0.4% ellagic acid (EA), a BRB phytochemical. Dietary administration of 5 and 10% BRB reduced oral lesion incidence and multiplicity by 39.3 and 28.6%, respectively. Histopathological analyses demonstrate the ability of BRBs and, to a lesser extent EA, to inhibit the progression of oral cancer. Oral lesion inhibition by BRBs was associated with a reduction in the mRNA expression of pro-inflammatory biomarkers Cxcl1, Mif, and Nfe2l2 as well as the anti-apoptotic and cell cycle associated markers Birc5, Aurka, Ccna1, and Ccna2. Cellular proliferation (Ki-67 staining) in tongue lesions was inhibited by BRBs and EA. Our study demonstrates that, in the rat 4NQO oral cancer model, dietary administration of BRBs inhibits oral carcinogenesis via inhibition of pro-inflammatory and anti-apoptotic pathways.

Suppression of Proinflammatory and Prosurvival Biomarkers in Oral Cancer Patients Consuming a Black Raspberry Phytochemical-Rich Troche.

Black raspberries (BRB) demonstrate potent inhibition of aerodigestive tract carcinogenesis in animal models. However, translational clinical trials evaluating the ability of BRB phytochemicals to impact molecular biomarkers in the oral mucosa remain limited. The present phase 0 study addresses a fundamental question for oral cancer food-based prevention: Do BRB phytochemicals successfully reach the targeted oral tissues and reduce proinflammatory and antiapoptotic gene expression profiles? Patients with biopsy-confirmed oral squamous cell carcinomas (OSCC) administered oral troches containing freeze-dried BRB powder from the time of enrollment to the date of curative intent surgery (13.9 ± 1.27 days). Transcriptional biomarkers were evaluated in patient-matched OSCCs and noninvolved high at-risk mucosa (HARM) for BRB-associated changes. Significant expression differences between baseline OSCC and HARM tissues were confirmed using a panel of genes commonly deregulated during oral carcinogenesis. Following BRB troche administration, the expression of prosurvival genes (AURKA, BIRC5, EGFR) and proinflammatory genes (NFKB1, PTGS2) were significantly reduced. There were no BRB-associated grade 3-4 toxicities or adverse events, and 79.2% (N = 30) of patients successfully completed the study with high levels of compliance (97.2%). The BRB phytochemicals cyanidin-3-rutinoside and cyanidin-3-xylosylrutinoside were detected in all OSCC tissues analyzed, demonstrating that bioactive components were successfully reaching targeted OSCC tissues. We confirmed that hallmark antiapoptotic and proinflammatory molecular biomarkers were overexpressed in OSCCs and that their gene expression was significantly reduced following BRB troche administration. As these molecular biomarkers are fundamental to oral carcinogenesis and are modifiable, they may represent emerging biomarkers of molecular efficacy for BRB-mediated oral cancer chemoprevention.

Chemoprevention of oral cancer by topical application of black raspberries on high at-risk mucosa.

OBJECTIVE: To evaluate the preclinical efficacy of topical administration of freeze-dried black raspberries (BRBs) to inhibit the progression of premalignant oral lesions and modulate biomarkers of cancer development in high at-risk mucosa (HARM). STUDY DESIGN: Hamster cheek pouches (HCPs) were treated with carcinogen for 6 weeks to initiate a HARM microenvironment. Subsequently, right HCPs were topically administered a BRB suspension in short-term or long-term studies. After 12 weeks, squamous cell carcinoma (SCC) multiplicity, SCC incidence, and cell proliferation rates were evaluated. mRNA expression was measured in short-term treated pouches for selected oral cancer biomarkers. RESULTS: SCC multiplicity (-41.3%), tumor incidence (-37.1%), and proliferation rate (-6.9%) were reduced in HCPs receiving BRBs. Topical BRBs correlated with an increase in RB1 expression in developing oral lesions. CONCLUSIONS: Topical BRBs inhibit SCC development when targeted to HARM tissues. These results support the translational role of BRBs to prevent oral cancer development in humans.

Cyclooxygenase-2 and the inflammogenesis of breast cancer.

Cohesive scientific evidence from molecular, animal, and human investigations supports the hypothesis that constitutive overexpression of cyclooxygenase-2 (COX-2) is a ubiquitous driver of mammary carcinogenesis, and reciprocally, that COX-2 blockade has strong potential for breast cancer prevention and therapy. Key findings include the following: (1) COX-2 is constitutively expressed throughout breast cancer development and expression intensifies with stage at detection, cancer progression and metastasis; (2) essential features of mammary carcinogenesis (mutagenesis, mitogenesis, angiogenesis, reduced apoptosis, metastasis and immunosuppression) are linked to COX-2-driven prostaglandin E2 (PGE-2) biosynthesis; (3) upregulation of COX-2 and PGE-2 expression induces transcription of CYP-19 and aromatase-catalyzed estrogen biosynthesis which stimulates unbridled mitogenesis; (4) extrahepatic CYP-1B1 in mammary adipose tissue converts paracrine estrogen to carcinogenic quinones with mutagenic impact; and (5) agents that inhibit COX-2 reduce the risk of breast cancer in women without disease and reduce recurrence risk and mortality in women with breast cancer. Recent sharp increases in global breast cancer incidence and mortality are likely driven by chronic inflammation of mammary adipose and upregulation of COX-2 associated with the obesity pandemic. The totality of evidence clearly supports the supposition that mammary carcinogenesis often evolves as a progressive series of highly specific cellular and molecular changes in response to induction of constitutive over-expression of COX-2 and the prostaglandin cascade in the "inflammogenesis of breast cancer".

Chemoprevention of oral cancer by lyophilized strawberries.

BACKGROUND/AIM: Oral cancer represents approximately 2.5% of all cancers in the United States, with five- and 10-year survival rates of 62% and 51%. In the present study, lyophilized strawberries (LS) were evaluated for their potential to inhibit tumorigenesis in the hamster cheek pouch (HCP) model of oral cancer and for their ability to modify expression of several genes relevant to oral cancer development. MATERIALS AND METHODS: HCPs were painted three times a week for six weeks with 0.2% 7,12-dimethylbenz(a)anthracene (DMBA). Hamsters were given 5% or 10% LS in their diet prior to, during, and after, or only after carcinogen treatment. Animals were sacrificed 12 weeks from the beginning of DMBA treatment and the number of total lesions and tumors was determined. RESULTS: A significant difference (p<0.01-0.04) in the number of tumors was found between the LS-treated groups and the carcinogen controls. Histological examination of HCPs revealed a significant reduction in mild and severe dysplasia following 12 weeks of treatment with LS. Molecular analysis revealed that genes related to tumor development were modulated by LS. CONCLUSION: These experiments support previous studies in HCP that demonstrated a chemopreventive activity by black raspberries and show, to our knowledge for the first time, that strawberries can inhibit tumor formation in an animal model of oral cancer.

Chemoprevention of mouse lung and colon tumors by suberoylanilide hydroxamic acid and atorvastatin.

Atorvastatin and suberoylanilide hydroxamic acid (SAHA) were evaluated for chemoprevention of mouse lung tumors. In Experiment 1, lung tumors were induced by vinyl carbamate in strain A/J mice followed by 500 mg/kg SAHA, 60 or 180 mg/kg atorvastatin, and combinations containing SAHA and atorvastatin administered in their diet. SAHA and both combinations, but not atorvastatin, decreased the multiplicity of lung tumors, including large adenomas and adenocarcinomas with the combinations demonstrating the greatest efficacy. In Experiment 2, lung tumors were induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol in strain A/J mice followed by 180 mg/kg atorvastatin, 500 mg/kg SAHA, or both drugs administered in the diet. SAHA and the combination of both drugs, but not atorvastatin alone, decreased the multiplicity of lung tumors and large tumors, with the combination demonstrating greater efficacy. In Experiment 3, lung tumors were induced by 1,2-dimethylhydrazine in Swiss-Webster mice followed by 160 mg/kg atorvastatin, 400 mg/kg SAHA, or a combination of both drugs administered in the diet. SAHA and the combination, but not atorvastatin, decreased the multiplicity of lung tumors with the combination demonstrating greater efficacy. The multiplicity of colon tumors was decreased by SAHA, atorvastatin, and the combination, without any significant difference in their efficacy. mRNA expression analysis of lung tumor bearing mice suggested that the enhanced chemopreventive activity of the combination is related to atorvastatin modulation of DNA repair, SAHA modulation of angiogenesis, and both drugs modulating invasion and metastasis pathways. Atorvastatin demonstrated chemoprevention activity as indicated by the enhancement of the efficacy of SAHA to prevent mouse lung tumors.

Prevention of mouse lung tumors by combinations of chemopreventive agents using concurrent and sequential administration.

BACKGROUND: Concurrent and sequential administration of combinations of budesonide, bexarotene, suberoylanilide hydroxamic acid (SAHA) and atorvastatin were evaluated in A/J mice for prevention of lung tumors initiated by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol, NNK). MATERIALS AND METHODS: Individual drugs and their combinations were administered for 26 weeks after NNK initiation. For sequential administration, budesonide was given for 21 weeks followed by a second drug. RESULTS: Alone, budesonide, bexarotene, and SAHA caused a significant decrease in total and large tumors at 21 and 26 weeks. Concurrent treatment with budesonide and bexarotene or SAHA caused a significantly greater decrease in total tumors and large tumors than either drug administered alone. Sequential administration of all combinations (except budesonide/atorvastatin) gave a significant reduction in total and large tumors. Budesonide followed by SAHA and SAHA with atorvastatin yielded a greater reduction in large tumors. CONCLUSION: Combinations of drugs demonstrated a greater efficacy in preventing mouse lung tumors than did the individual agents.

Oral cancer in Appalachia.

Oral cancer accounts for 2.3% of malignancies in the U.S. and has one of the lowest five-year survival rates. An examination of oral cancer in Appalachia was motivated by the high incidence of lung and bronchial cancers in Appalachian states, the risk factors for which overlap with those for oral cancer. The incidence and mortality rates for oral cancer in 13 Appalachian states and the relative frequency of presumptive risk factors were examined and compared with national rates, using data from the National Program of Cancer Registries, Surveillance Epidemiology and End Results, Behavioral Risk Factor Surveillance System, the Appalachian Regional Commission, and the National Health Interview Survey. Combined incidence rates for oral cancer were higher in six of 12 Appalachian states, and mortality rates higher in 10 of 13, compared with the national average. Smoking was more prevalent than the national average in nine of 13 states, whereas alcohol consumption was the same or less in 11 Appalachian states. Only five of 13 states averaged fewer than the recommended five or more servings per day of fruits and vegetables.

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.

Tumor suppressor p16(INK4A)/Cdkn2a alterations in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced hamster cheek pouch tumors.

The prevalence of p16(INK4A)/Cdkn2a genetic alterations in human oral cancers indicates that the p16 gene could be a potent and appropriate target for novel intervention. While chemically induced hamster cheek pouch (HCP) tumors are regarded as an appropriate surrogate model for human oral cancers because of their similarities to human oral cancers in both histology and genetics, little is known about the genetic events in the p16 gene in the HCP tumor model. The purpose of this study was to evaluate chemically induced HCP tumor specimens for potential inactivating p16 alterations. HCP tumors were induced with 7, 12-dimethylbenz(a)anthracene (DMBA), and DNA extracted from 34 such specimens were analyzed for homozygous/hemizygous deletions, aberrant methylation of 5' CpG islands, and point mutations using real-time multiplex PCR, methylation-specific PCR, and direct sequencing/cold single strand conformation polymorphism (SSCP), respectively. Homozygous deletions, hemizygous deletions, aberrant methylation of 5'-CpG islands, and point mutation were identified in 11, 4, 9, and 1 of 34 specimens, respectively. While the overall incidence of p16 alterations was 70.6% (24 of 34 specimens), the majority of inactivating events (67.6%) stemmed from deletion or methylation, which is consistent with the observations found in human oral SCCs. Our results show the resemblance between chemically induced HCP tumors and their human counterparts in p16 genetic alterations, and strongly support the use of DMBA-induced HCP tumor model in evaluating novel p16-targeted therapy and prevention of human oral SCCs.

Frequent p16INK4A/CDKN2A alterations in chemically induced Syrian golden hamster pancreatic tumors.

The p16INK4A/CDKN2A (p16) tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphological and biological similarities with human pancreatic tumors and represent a potentially suitable model for the evaluation of therapies targeting p16. The purpose of this study was to evaluate primary hamster pancreatic tumor specimens for potentially inactivating p16 alterations. Tumors were induced with N-nitroso-bis-(2-oxopropyl) amine, followed by two cycles of augmentation pressure, and were harvested on day 100. Foci of tumor cells were identified by light microscopy after staining with hematoxylin and eosin, and corresponding tumor tissues were excised for DNA extraction. The techniques of multiplex real-time PCR, direct sequencing and methylation-specific PCR were used to evaluate 30 tumor specimens for homozygous deletions, mutations and aberrant methylation of 5' CpG islands, respectively. Homozygous deletions were identified in 11 of 30 (36.7%) specimens, mutations were identified in four of 30 (13.3%) specimens, and aberrant methylation of 5' CpG islands was found in 14 of 30 (46.7%) specimens. The overall frequency of p16 alterations was 93.3% (28 of 30 specimens) and the majority of changes (83.3%) were noted to be secondary to methylation or homozygous deletion. The four mutations significantly impaired cyclin-dependent kinase 4 inhibitory activity, and two resulted in perturbation of the global structure of P16 protein. These findings indicate that p16 inactivation is a common event in chemically induced hamster tumors, and that this animal model is appropriate for comparative studies evaluating pancreatic cancer therapeutic strategies targeting p16.

Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia.

A number of genetic aberrations have been reported in end-stage squamous cell carcinoma of the head and neck, including p16(INK4a) and p14(ARF) (INK4a/ARF) inactivation rates of 70-85%. Still, the cell cycle-regulatory genes p16(INK4a) and p14(ARF) remain poorly understood in oral cavity premalignant lesions. This study evaluated INK4a/ARF locus alterations in 26 patients (28 samples) deemed to be at increased risk for malignant transformation to squamous cell carcinoma due to the diagnosis of severe oral epithelial dysplasia. Microscopically confirmed dysplastic oral epithelium and matching normal tissue were laser capture-microdissected from paraffin sections, DNA was isolated, and molecular techniques were used to evaluate p16(INK4a) and p14(ARF) gene deletion, mutation, loss of heterozygosity (LOH), and hypermethylation events. Deletion of exon 1beta, 1alpha, or 2 was detected in 3.8%, 11.5%, and 7.7% of patients, respectively. INK4a and ARF mutations were detected in 15.4% and 11.5% of patients with severe dysplasia of the oral epithelium. All identified mutations occurred in the INK4a/ARF conserved exon 2. Allelic imbalance was assessed using three markers previously reported to show high LOH rates in head and neck tumors. LOH was found in 42.1%, 35.0%, and 82.4% of patients for the markers IFNalpha, D9S1748, and D9S171, respectively. Hypermethylation of p16(INK4a) and p14(ARF) was detected in 57.7% and 3.8% of patients, respectively, using nested, two-stage methylation-specific PCR. The highest rates of p16(INK4a) hypermethylation occurred in lesions of the tongue and floor of the mouth. In addition, p16(INK4a) hypermethylation was significantly linked to LOH in two or more markers. These data support that INK4a/ARF locus alterations are frequent events preceding the development of oral cancer and that p16(INK4a) inactivation occurs to a greater extent in oral dysplasia than does p14(ARF) inactivation.

Chemoprevention of oral cancer by black raspberries.

Oral cavity cancers represent 2.5% of the cancers that occur in the United States and are ranked sixth worldwide. Since current therapeutic protocols are relatively ineffective, alternative strategies for prevention need to be developed and tested in appropriate animal models. In the study reported herein, the hamster cheek pouch (HCP) was used to evaluate the ability of black raspberries to inhibit oral cavity tumors. Male Syrian Golden hamsters, 3-4 weeks of age, were fed 5% and 10% lyophilized black raspberries (LBR) in the diet for two weeks prior to treatment with 0.2% 7,12-dimethylbenz(a) anthracene in dimethylsulfoxide and for 10 weeks thereafter. HCPs were painted 3X/week for eight weeks. The animals were sacrificed 12-13 weeks from the beginning of DMBA treatment and the number and volume of tumors (mm3) determined. There was a significant difference (p = 0.02) in the number of tumors between the 5% LBR and control groups (27 tumors/14 animals and 48 tumors/15 animals, respectively) and an intermediate number of tumors in the 10% berry-treated animals (39 tumors/15 animals). These experiments support previous studies from our laboratories showing the chemopreventive activity of black raspberries and show, for the first time, that dietary black raspberries will inhibit tumor formation in the oral cavity.