Peter G. Sacks

Assembly and Initial Characterization of a Panel of 85 Genomically Validated Cell Lines from Diverse Head and Neck Tumor Sites

Purpose: Human cell lines are useful for studying cancer biology and preclinically modeling cancer therapy, but can be misidentified and cross-contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods: A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma, thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium was assembled from the collections of several individuals and institutions. Authenticity was verified by carrying out short tandem repeat analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results: Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination shows a wide range of in vitro phenotypes. Conclusions: This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies. Clin Cancer Res; 17(23); 7248–64. ©2011 AACR.

Motility-related proteins as markers for head and neck squamous cell cancer

Hypothesis Increased cell motility is a hallmark of cancer cells. Proteins involved in cell motility may be used as molecular markers to characterize the malignant potential of tumors.

Concentration dependent effects of tobacco particulates from different types of cigarettes on expression of drug metabolizing proteins, and benzo(a)pyrene metabolism in primary normal human oral epithelial cells

The ability of tobacco smoke (TS) to modulate phase I and II enzymes and affect metabolism of tobacco carcinogens is likely an important factor in its carcinogenicity. For the first time several types of TS particulates (TSP) were compared in different primary cultured human oral epithelial cells (NOE) for their abilities to affect metabolism of the tobacco carcinogen, (BaP) to genotoxic products, and expression of drug metabolizing enzymes. TSP from, reference filtered (2RF4), mentholated (MS), reference unfiltered, (IR3), ultra low tar (UL), and cigarettes that primarily heat tobacco (ECL) were tested. Cells pretreated with TSP concentrations of 0.2-10 μg/ml generally showed increased rates of BaP metabolism; those treated with TSP concentrations above 10 μg/ml showed decreased rates. Effects of TSPs were similar when expressed on a weight basis. Weights of TSP/cigarette varied in the order: MS≈IR3>2RF4>ECL>UL. All TSPs induced the phase I proteins, cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), phase II proteins, NAD(P)H dehydrogenase quinone 1 (NQO1), and microsomal glutathione S-transferase 1 (MGST1), and additionally, hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2), as assessed by qRT-PCR. The pattern of gene induction at probable physiological levels favored activation over detoxification.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

Induction of apoptosis and up-regulation of cellular proliferation in oral leukoplakia cell lines inside electric field.

OBJECTIVE In dentistry, metallic alloys are used for dentures, restorative materials, and orthodontic devices. Electric voltages up to 950 mV may occur between different dental alloys in the oral cavity. This study aimed to investigate physiologic reactions of oral leukoplakia cells in vitro to electric fields. STUDY DESIGN A human leukoplakia cell line (MSK-LEUK1), cultivated in keratinocyte growth medium (KGM-2) supplemented with growth factors in 5% CO(2) humidified air at 37°C, was exposed to electric field strength of 1-20 V/m for 24 hours in a custom-made pulse chamber. The cells were then analyzed for proliferation with the use of BrdU assay and for apoptosis with the use of TUNEL assay. Findings were assessed with the use of fluorescent microscopy. Ultrastructural changes were studied by transmission electron microscopy. RESULTS Electric field strength of 1-10 V/m led to up-regulation of cell proliferation rate from 10.64% to 44.06% (P = .0001). The apoptotic index increased significantly (P = .0001) from 20.03% at 1 V/m to 46.56% at 10 V/m. Individual cell keratinization was seen in leukoplakia cells treated with 16 V/m. CONCLUSIONS Oral galvanism induces subcellular changes in oral precancer cells in vitro that closely simulate some of the morphologic features of oral squamous cell carcinoma cells in vivo.

Physiologic Behavioural Changes of Precancerous Leukoplakia Cell lines Exerted to Extremely Low Frequency Electric Field

Objectives: Oral galvanism arising from the presence of two different metallic fillings in the mouth that may reach up to 16V/m, induces several changes in oral environment such as gingival swelling and erythema, mucosal pain, lichenoid reactions and leukoplakia. Study design: To investigate in vitro the physiologic reactions of oral leukoplakia caused by electrostatic corrosion potentials, human leukoplakia cell lines (MSK-LEUK1), cultivated in KGM-2 supplemented with bullet kit in 5% CO2 humidified air at 37°C, were exposed to electric field strength of 1-20 V/m for 24 hours in a pseudo realistic apparatus called “Pulse chamber”. Following this, the cells were analysed for proliferation using BrdU assay, and for apoptosis using TUNEL assay. Findings were assessed utilizing fluorescent microscopy. Ultra structural changes were studied by TEM. Data was evaluated statistically using non parametric Chi-square test. Results: Electric field strength of 1-10V/m led to upregulation of cell proliferation rate between 10.64% and 44.06% (p=0.0001). The apoptotic index increased significantly (p=0.0001) from 20.03% at 1V/m to 46.56% at 10V/m. Leukoplakia cells treated with 16V/m show individual cell keratinization. Conclusion: Oral galvanism induces subcellular changes in oral precancerous leukoplakia cells in vitro that resemble some of the histopathologic features of oral squamous cell carcinoma cells in vivo.

Abstract 1582: Comparative transforming effects of ultra low tar (ULT) and full flavor low tar (FFLT) cigarette smoke particulate extracts on human oral epithelial cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA ULT cigarettes produce much lower levels of tars and particulate matter than FFLT cigarettes, but are generally not safer alternatives with respect to carcinogenesis, possibly because of compensatory smoking behavior. In a previous study (P.G. Sacks, et al., Food Chem Toxicol 49 (2011) 2348-2355) we reported that tobacco smoke particulate (TSP) extracts from FF, FFLT and ULT cigarettes exerted similar effects on the induction of phase I and II proteins and the activation of the tobacco carcinogen, benzo(a)pyrene (BaP) to genotoxic metabolites in primary oral epithelial cells when compared on a concentration basis, although ULT cigarettes were less effective on a per cigarette basis. Here we report that both a FFLT and an ULT TSP extract transform a human oral epithelial cell line to growth independence in soft agar, and enhance the transforming effects of BaP. At equal concentrations the ULT extract was more effective than the FFLT extract. A human oral leukoplakia cell line, MSKleuk1, was employed in these studies. Cells were treated twice over a two week period or 3 times over a 3 week period with: a) one uM BaP, b) 4ug/ml TSP extract, c) TSP extract + BaP, or d) vehicle control. One week after the last treatment cells were seeded on soft agar and were assayed either by counting colonies 3 weeks after seeding, or 8 days after seeding using an anchorage-independent growth assay (CytoSelect 96-Well Cell Transformation Assay Kit, Cell Biolabs). Results in initial experiments for the two week treatment were assayed by counting colonies, and the fraction of growth-independent cells for groups a-d resp. were: a, 5.5 +/- 0.2; b, 6.0 +/- 0.6; c, 6.6 +/- 0.3 and d, 0.78 +/- 0.3 (all in units of 10E-4). For the three week treatment the results were: a, 9.1 +/- 0.5; b, 9.2 +/- 1.2; c, 11.9, +/- 2.5; d, 1.4 +/- 0.7. The TSP extract was from a reference filtered cigarette, 2RF4. A further soft agar assay was carried out using the cell transformation assay; and TSP extracts from a commercial ULT and 2RF4 cigarettes were compared, in the presence and absence of BaP. The groups were a) 1 uM BaP, b) 4ug/ml 2RF4 extract, c) 4ug/ml 2RF4 extract + 1 uM BaP, d) 4ug/ml ULT extract, e) 4ug/ml ULT extract + 1uM BaP and f) vehicle control. All groups were treated 2x as described above. Results are expressed relative to the vehicle control which is set as 1. a, 1.9 +/- 0.4; b, 1.4 +/- 0.3; c, 2.2 +/- 0.4; d, 2.1 +/ 0.3; e, 2.6 +/- 0.6.The results of these show: 1) human oral epithelial cells can be transformed by BaP, and TSP extracts from FFLT and ULT cigarettes, 2) the effects of BaP and TSP extracts are approximately additive, 3) when compared on a concentration of particulate basis, ULT TSP extracts were more effective than FFLT TSP extracts. Unlike previous studies relating primarily to carcinogenesis initiation, ULT TSP extracts appear to more effective at transformation than FFLT TSP extracts. Citation Format: Tianzhen Han, Peter Sacks, Joseph B. Guttenplan. Comparative transforming effects of ultra low tar (ULT) and full flavor low tar (FFLT) cigarette smoke particulate extracts on human oral epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2014-1582

Abstract 4834: Microbiome in Oral Epithelial Dysplasia and Squamous Cell Carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oral squamous cell carcinoma (OSCC) is a complex malignancy representing 90% of all oral cancers having its origin from mucosal epithelium. The progression of cancer is associated with multiple factors such as social habits, environmental, genetic alterations, poor oral health, viral or chronic bacterial infections. It involves the step-wise transition from normal epithelia to pre-malignant lesions to metastatic tumor. The mucosal-associated bacterial infections and chronic inflammation are implicated in OSCC pathogenesis. Alterations to the integrity of epithelial barrier initiate polymicrobial infections by shifting the indigenous commensals to opportunistic pathogens. We hypothesize that specific bacteria colonize epithelial dysplasia and OSCC site. We investigated the dysbiosis in complex oral microbial communities present at dysplasia and tumor sites as compared to non-diseased sites using high throughput pyrosequencing. Bacterial genomic DNA was purified from the brush biopsy samples of 11 patients with epithelial dysplasia and from the tissue samples of 18 patients with OSCC along with its corresponding non-dysplasia and non-tumor controls collected ∼5cm distant to lesion or contralaterally. PCR amplicons targeting V3-V5 region of 16S rRNA gene were sequenced by 454 pyrosequencing. The bacterial community structures were evaluated using QIIME analysis pipeline. Phylum Firmicutes and genus Streptococcus was found to be predominant in all non-diseased and diseased oral sites. Bacteroidetes and Fusobacteria were elevated at tumor sites as compared to non-tumor controls, dysplasia and non-dysplasia controls. Genera, Actinomyces, unclassified Methylobacteriaceae, unclassified Comamonadaceae and Acenitobacter were prevalent in dysplasia site (p<0.1) as compared to non-dysplastic site. Rothia and Parascardovia were higher in dysplasia samples whereas Prevotella, Capnocytophaga, unclassified Clostridiaceae, Fusobacteria and Wolbachia showed higher frequency in tumor samples. Thirty eight named species-level OTUs were common to all four sites suggesting the co-existence of this microbiota with their specific environment. The species richness was higher in tumor samples which reflect more diverse bacterial communities in OSCC lesions. s-diversity showed that most of the bacterial communities were similar within the site with some overlap between the sites signifying altered microbiome. The dysbiosis in the bacterial community structure from non-diseased sites to dysplasia to tumor sites may indicate site specificity of certain bacteria to colonize OSCC and dysplasia lesions. These bacteria can prove to be the potential targets for further studies. This work is supported by grants DE019178 and DE020891. Citation Format: Deepak Saxena, Smruti Pushalkar, Arun Devotta, Yihong Li, Bhuvanesh Singh, Zoya Kurago Kurago, Alexander Kerr, Wenbo Yan, Peter Sacks, Xin Li. Microbiome in Oral Epithelial Dysplasia and Squamous Cell Carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4834. doi:10.1158/1538-7445.AM2014-4834

Abstract 4876: Effects of a combination of resveratrol and sulforaphane on mutagenesis and DNA adduct formation in rat mammary tissue and a rat mammary fibroblast cell line.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In this study we investigated the effects of the combination sulforaphane (Sul) and resveratrol (Rev) on carcinogen metabolism to DNA adducts in rat mammary fibroblasts, and on DMBA-induced mutagenesis in mammary tissue from rats treated in vivo. The use of combinations of potential cancer chemopreventive agents has become one strategy to optimize chemoprevention. By targeting more than one pathway/process it may be possible to impact prevention by additive or synergistic effects, and utilize lower doses of agents that might have toxic effects at higher doses. We have previously tested a number of potential inhibitors of mutagenesis induced by the rat mammary carcinogen, 7,12-dimethylbenzanthrace (DMBA) in rat mammary epithelial and fibroblast cell lines (isolated by Dr. P.D. Josephy from lacI rats, Mutat Res, 497: 39-47, 2001). We had compared binary combinations of the agents, sulforaphane (Sul), resveratrol (Res), lipoic acid, epigallocatechin gallate, a vitamin C and E mix, and N-acetylcysteine. The agents alone had minor effects, but the combination of Sul and Res inhibited mutagenesis by about 40%. The current study extended this finding. In the metabolism study, HPLC of metabolites was employed; benzo(a)pyrene (BaP), rather than DMBA was used as the carcinogen, as standards of BaP metabolites, but not those of DMBA were available. BaP like DMBA is a carcinogenic polycyclic aromatic hydrocarbon. Fibroblasts were used, as these metabolized BaP much more effectively than epithelial cells. In the presence of 4uM BaP, in the absence of inhibitors, the cells generated 498 ± 83 pg/ml medium of the BaP-7,8 dihydrodiol -9,10 epoxide decomposition product, BaP 7,8,9,10 tetrol. This was reduced by 0.18uM Sul to 153 ± 48, by 4uM Res to 416 ± 87 and by a combination of the agents to 83 ± 37 pg/ml. The reduction was significant for Sul and for the combination. For the in vivo study rats were treated (6/group) by gavage with a single dose of 80 mg/kg DMBA. Two weeks before the DMBA treatment and continuing for 4 wks, rats received 50 mg/L Res, 120 mg/L Sul, and combinations of the agents at 50 and 120 mg/L or ½ the doses in drinking water. Eight wks after the DMBA treatment the rats were euthanized and mutagenesis in the cII gene was measured. The treatments and mutant fractions resp. were: DMBA alone, 9.1 ± 5.0; DMBA + Res, 8.9 ± 3.4; Sul, 8.2 ± 2.0; Res + Sul, 7.1 ± 2.8; Res + Sul, ½ dose, 7.9 ± 3.4; control, 1.3 ± 1.0. DMBA-induced mutagenesis was inhibited by both combination treatments, and Sul alone, although the inhibitions did not reach statistical significance, possibly because of large inter-animal variations. Taken together the results support the use of combinations of agents to enhance chemoprotection. Supported by the Susan Komen Foundation grant # KG080836. Citation Format: Peter G. Sacks, Wieslawa Kosinska, Mitaliben Contractor, Tian-Zhen Han, Joseph B. Guttenplan. Effects of a combination of resveratrol and sulforaphane on mutagenesis and DNA adduct formation in rat mammary tissue and a rat mammary fibroblast cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2013-4876

Abstract 3701: Dimethylbenzanthracene (DMBA) and DMBA dihydrodiol mutagenicity in rat epithelial and fibroblast cell lines, and its inhibition by combinations of nutraceuticals

7,12-Dimethylbenzanthracene (DMBA) is a potent mammary carcinogen in rats. Combinations of non-toxic nutraceutical agents, administered at or near physiological levels, were investigated for their abilities to inhibit the mutagenicity of DMBA or DMBA-dihydrodiol (DMBAD, a primary metabolite and proximate mutagen of DMBA) in vitro, in rat mammary epithelial and fibroblast cells derived from a lacI (BigBlue) Fischer rat (McDiarmid, H.M., Douglas, G.R., Coomber, B.L., and Josephy, P.D. Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay. Mutat. Res., 497: 39-47, 2001). In the epithelial cells, DMBA was not appreciably mutagenic at concentrations up to 4 µM, but DMBAD, was significantly mutagenic at ten-fold lower concentrations. These results indicate that the epithelial cells can bioactivate the intermediate, DMBAD, but cannot effect the complete biotransformation of DMBA to its ultimate mutagenic metabolite, DMBA-dihydrodiolepoxide. In the fibroblast cell line, in contrast, DMBA was mutagenic at concentrations as low as 20 nM and DMBAD was even more potent than DMBA. Several combinations of nutraceuticals (dietary components providing health benefits) were tested for their abilities to inhibit mutagenesis in these cell lines; the concentrations tested were based on reported serum concentrations and these were used to establish 1x concentrations. The agents and their 1x concentrations were: resveratrol (Res), 2.4 μM; sulforaphane (Sul), 0.06 μM; antioxidant mix (α- and γ-tocopherol, 30 μM, plus vitamin C, 68 μM – VCE); α-lipoic acid (LA), 2 μM; epigallocatechin gallate (EGCG), 0.7 μM; and N-acetylcysteine (NAC), 12 μM. None of the agents or their combinations, tested at 1 – 3 x concentrations, showed any cytotoxicity. In both epithelial and fibroblast cells, Sul and Res alone slightly inhibited mutagenesis at 2x concentrations; no other agents had observable effects. All binary combinations of Res, LA, and Sul, at 2x concentrations, inhibited mutagenesis; the Res + Sul combination was particularly effective (ca. 50% inhibition). These results suggest a role for fibroblast cells in the bioactivation of carcinogens, implicating the microenvironment, and indicate that combinations of nutraceuticals can inhibit mutagenesis by polycyclic aromatic hydrocarbons. Supported by the Susan Komen Foundation grant # KG080836 (JBG) and NSERC Canada (PDJ). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2011-3701