Introduction

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Tobacco derived carcinogens such as PAHs are reported sources of anthraquinones (anthraquinonoids) and other ROS, which enhance oxidative metabolism by cytochrome P450. This activity in keratinocytes increases risk for oral squamous cell carcinoma (OSCC).1,2 However, OSCC does not have a high risk for HPV 16 infection.3 In contrast, epidemiology for a specific type of carcinoma which is HPV 16 related oropharynx carcinoma (OPC) is not often associated with tobacco product exposure.3,4 Moreover, presence of HPV 16 in a carcinoma suggests a clinical benefit in terms of growth behavior, treatment response and survival.57 These clinical relationships between HPV 16 entry, and exposure environment that includes exposure to tobacco product chemistry and a possible physiology change in oral keratinocytes require more mechanistic study to enhance understanding of clinical significance.

We test in this study some of the molecular basis for us to suggest presence of HPV 16 and mucotropism at an oropharynx site in contrast to an oral cavity site may reflect interaction with commensal microbes derived from the Actinomycete order that are identified at carcinoma sites.1316

Actinomycete phylum, and class are gram positive rods that branch and resemble fungal hyphae (255 genera; 1906 species). In addition, many species from the class or order of Actinomycetales produce antibiotics such as streptomycin, from Streptomyces spp.. We hypothesize that this antibiotic capacity modifies the biofilm in which they participate and alters proliferation of other species of microbes to result in changes in oral hygiene and risk for HNSCC through an induction of a ROS.8

The genus, Actinomyces is found in the aquatic environments in the world marine locations and in the oral cavity. A variety of Actinomycete species are identified in human oral mucosa or jaw disease sites such as osteonecrosis (ONR); root canals; intra-oral cysts; abscesses of tongue, and neck.911 These sites are areas of high levels of ROS induction because of inflammatory activities. The subgroup 3 contains oral Actinomyetes found among oral isolates and forms a complex to include: A. israelli; A.vicosus; gerencseriae; naeslundii; odontolyticus and 17 other oral microbes with genetic similarities. Other actinomyces species identified in dental periodontal and mucosa are Aggregatibacter actinomycetemcomitans that predominant in the periodontal sulcus. Moreover, genetically related Actinomyces viscosus and Rhodococcus spp, exists in both marine and terrestrial environments.12 In addition, Actinomycetes genera; Mycobacteria and Rhodococcus are found in sites which generate ROS; for example, OSCC,1315 and in tobacco leaf used for combustible or smokeless tobacco.1619 Microbe species from this genus also metabolize PAH such as phenanthracene and degrade PAH to produce anthraquinones.1419

Another strain of marine derived Actinomycete, Salinispora arenicola does not have genetic machinery to produce specific types of anthraquinones but there is a high degree of variation among Actinomycetes.20 It is also recognized that these microbes show a high degree of plasticity in their secondary metabolome that maximize metabolite diversity (e.g., 15,400 secondary metabolites) within a limited number of pathways.21 However, many terrestrial and marine Actinomycetes species degrade PAHs to quinones though oxidation which is identified by a variety of syntheses of anthraquinones.1,22 Species such as, marine Salinispora pacifica and Streptomyces spp. also show a high degree of diversity in quinone metabolism.22 In addition, in the oral cavity Actinomycetes work in concert with Veillonella and Rothia genera and these microbes convert nitrate to nitrite and nitrous oxide to release ROS which can change vascular hemodynamics.2326 The combined release of ROS from several sources; such as, anthraquinones, nitrosation/ ammonification and urea metabolism produces a change in epithelial cell redox state as hydroxyl; superoxide anion, and other oxidative radicals are released to cause a change in enzymatic reduction typified by catalase (e.g.,Haber-Weiss reaction), superoxide dismutase, glutathione reductases, glucuronosyltransferases and Cu(I) cytochrome P450 reductase activities.26 Furthermore, Actinomycetes participate in infectious and repair disorders that accelerate release of ROS. An example is chronic granulomatous disease; a disorder of defective NADPH oxidase and inappropriate regulation of ROS.27 Release of ROS, in the form of anthraquinones change epithelial physiology and phosphorylation of connexin-43, an epithelial bridge binding protein to stimulate epithelial turnover.32,34 In addition, glutathione reduction (e.g. glutathione reductases) quenches ROS, and increases tyrosine phosphatase activity to balance tyrosine kinases activity. These examples of ROS regulation of phosphorylation also involve EGFR and FAS Ligand binding to FAS receptor to mediate apoptosis.33 Moreover, specific chemical action at cysteine rich sites of EGFR is expected to affect sites of homology in insulin growth and tumor necrosis factor receptors, laminin factor families and furin convertase (FC) which will influence HPV 16 entry into epithelial cells.28 Anthraquinone derivatives are also potent inhibitors of c-Met kinase which control a wider range of extracellular signaling pathways37 and are presently classified as indeterminate carcinogens for induction of lung carcinoma among dye workers.38 In this context we anticipate a clinical significance derived from a positive correlation between presence of Actinomycete; induction of Anthraquinone, ROS and HPV 16 entry.3,4,6,7

Previously we showed oral commensal bacteria, Streptococcus spp. participate in HPV 16 entry into epithelial cells.28,29 Reported are also clinical associations between Firmicutes phylum members such as, Streptococcus spp. and Actinobacteria phylum members, for example, Actinomycetes at sites of oral disease to produce ROS.1316 Taken together, a laboratory testing approach is presented between Actinomycetes, anthraquinone and ROS generators such as PAH, TSNA, and nicotine mediated by FC and EGFR to reflect regulation of HPV 16 entry. Understanding and detecting this relationship is anticipated to enhance novel approaches for patient prevention, and treatment of HNSCC related to HPV 16 infection.

Materials and Methods

Cells: 293 cells are human renal epithelial cells30 and human telomerase (hTERT) induced immortalized human oral keratinocyte; HPV 16 negative.31

Chemicals: Chloromethyl ketone (CMK) (1M) (Bachem, ENZO, Life Sciences. Farmingdale, NY; stock: 100microMolar, working concentration 1.0 microMolar) is an irreversible cell permeable competitive inhibitor of proprotein convertases: furin /SpC1; SPC2/PC2; SPC4/PACE4; SPC6/PC5/PC6 and SPC7/LPC/PC7/PCB. Dibenzo [a,l]pyrene dissolved in methylene chloride (Sigma Chemical, Supelco, BellefontePA ) a working stock solution of 222 microgram/ml (734nM). Benzo{a]pyrene dissolved in dimethylsulfoxide (DMSO) (168 micro- Molar, working stock solution; Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (579.3 nM) (NNK) dissolved in methanol (320 microMolar; 37.3 micrograms/ml.); 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (6.9 micromolar; 144 micrograms/mL)Sigma Chemical Co. MO) (NNAL); anthraquinone dissolve in DMSO (98nM; 20.4 micrograms/ml.; Supelco; Sigma Chemical Co, St. Louis); Collagen (human); 2.5-3.5 mg/mL protein (Sigma Aldrich Chemical Co. St. Louis MO.); Epidermal growth factor (EGF), Sigma Chemical, St. Louis MO, was used at a working concentration of 4 microgram/ml.

Antibodies: anti-phosphotyrosine epidermal growth factor antibody (rabbit polyclonal, Sigma Chemical, St Louis, MO (1:400 (0.4 microgram/ml.) and Anti-furin antibody (AbCam rabbit polyclonal 28547 reacts with amino-terminal end) used at 1:200 for cell immunohistochemistry and western immunoblotting.

Dilutions used for immunodetection were between 1:50 to 1:400 and detection used chemiluminescence (ECL, Amersham, Piscataway, NJ).

Extracts: A broth from the marine Actinomycete Salinispora belongs to the family Micromonosporacae and contains three species Salinispora arenicola, tropica and pacifica.

In prior studies, fermentation extracts from the marine Actinomycete spp. were produced.13 Other publications indicated the presence of a diverse collection of 60 marine-sediment-derived Actinobacteria representing 52 operational taxonomic units. We initially examined 100 extracts from a variety of marine Actinomycetes sources that contain diverse bioactive products such as, anthraquinones-γ-pyrones; diazepinomicin; thiscoraline; abissomicia C; enterscin; lynamicin B; mansouramycin C; proximicin C; ML-449, and salinoporamide A.

Capability for metabolite synthesis of marine Actinomycete spp. has been published using 75 strains of marine Actinomycete genus Salinispora for pathways linkage to polyketide and non-ribosomal peptide biosynthesis.21 (Figure. 0I)

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Fig. 1: 01 Scheme for Actinomycete Extraction

Dilutions used for each extract were either: 1:50; 1:100 or 1;200 for each well that was assayed as described below with an original concentration of 10mg/ml (initial dilutions were 1:40 then further diluted). Some of the bioactive substances in this extract are stated above and additional novel metabolites are provided by Dr. Lam (2006) in his review of novel metabolites from marine actinomycetes. For Salinispora spp, the candidate for active cellular activity on human cells is anthraqunione-γ-pyrones, and salinosporamide A (β-lactone-γ-lactam; (1R,4R,5S)-4-(2-Chloroethyl)-1-[(S)-(1S)-2-cyclohexen-1-yl(hydroxy)methyl]-5-methyl-6-oxa-2-azabicyclo[ 3.2.0]heptane-3,7-dione); irreversible proteasome inhibitor (NPI-0052 (Marizomib/Carfilzomib) (Nereus Pharmaceuticals Inc., San Diego, CA ) and sporolides ( A and B) synthesized from two different polyketides.

Viability Assay (Trypan Blye Dye Exclusion): Trypan blue dye exclusion is a standard assay to test viability. Extracts at dilutions of <1:100, for example 1:50 using Trypan blue (0.25%) were assessed for viability using a calibrated micrometer (Fisher Scientific. USA.Pittsburgh, PA) using 293TT and hTERT HOK targets.

Production of Human Papilloma Virus Pseudovirus (Psv): HPV 16 entry has been optimized in 293TT cells that are an adenovirus transformed human embryonic kidney cell line with a stably integrated SV40 genome with high levels of large T antigen grown in DMEM+ Enhanced GLU (GIBCO, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis MO.) SV40 and large T antigen is used in this cell line to maintain biologic integrity. For example, a comparison of the amino acids encoded by adenovirus Ela; HPV16 E7 and sv40LT, a third viral oncogene whose transforming protein binds Rb to reveal a region of homology that contains a stretch of 17 residues within this region essential for the biological activity of all three proteins.

PsV production and an Optiprep purification method or a maturation method using overnight incubation of crude cell lysate at 370C is reported (Buck CB Curr Protocol. 2007) and is available at http://ccr.cancer.gov/ staff/staff.asp?profileid=5637. http://home.ccr.cancer.gov/lco/pseudovirusproduction.htm). A map of HPV 16 PsV packaging plasmid (p16L1-GFP and/or pfwB) and expression vectors for luciferase (pCLucf) driven by the cytomegalovirus (CMV) promoter is also found at this website. Our system relies upon co-propagation of L1/L2 expression plasmid together with a reporter plasmid (green fluorescent protein (GFP)) to generate high titer of mature PsV stocks for visualization.

Detection of Fluorescence and: Pseudovirus (PsV) Infection: The initial assays performed involved incubating epithelial-engineered target cells (293TT) on a 96-well plate for at least a 24 hour period, and then exposing these cells to each of the bacterial extracts along with a sample of HPV pseudoviral (PsV) particles that lacked oncogene activity. Specifically, these assays involved a 1:100 dilution of 100 extracts of Actinomycete spp. -derived extract and at least triplicate wells were involved for each of the extract samples. Following a 48 hour period of incubation with both the bacterial extract and the HPV PsV, we recorded fluorescent units compared to triplicate control wells using a fluorometer (550-60 nm emission). Specifically, we determined the percent difference of fluorescence between the 293TT cells treated with the extract and HPV PsV and the cells that were only treated with HPV PsV. We also assessed percentage of HPV 16 Psv entry into 293TT cells, by visual counting of total fluorescent target epithelial cells in a 6 well plate.

In vitro Condition Effects on HPV 16 Entry: Numbers of 293TT cells and titer of HPV 16 PsV were examined along with presence of coated, collagen wells and time of incubations of 293TT cells with HPV 16 PsV evaluated for randomly selected extracts of Actinomycetes spp. origin.

Re-examination of Selected Extracts: After these initial screenings were completed, we selected the 12 most effective extracts that exhibited the most significant changes in terms of increasing or decreasing viral entry into the epithelial target cells. Similar assays described above were then performed to confirm the activity of these extracts. Variations of the initial assays were also performed that aimed to examine changes in PsV uptake by epithelial cells based on experimental variables. Incubating the 293TT cells on a 6-well plate rather than a 96- well plate allowed for an assessment of the effects of differences in cell density. Additionally, similar experiments were performed in which the 293TT cells were allowed to grow on collagen-coated 6-well plates prior to performing the initial assay. Finally, different experiments were performed in which the effect of extract dilution was reexamined. This was done by treating the cells 1:50 extract dilutions and 1:200 extract dilutions and comparing the level HPV PsV entry to that obtained from the standard 1:100 extract dilution assays.

Detection of Furin Convertase Activity and HPV 16 PSV Entry: After the effects of the selected 12 Actinomycete- derived extracts were confirmed, similar experiments were again performed in which CMK, a known inhibitor of FC, was administered to the experimental wells prior to treating the 293TT cells with the extract samples and HPV PsV. These experiments were performed on 6-well plates and the CMK was allowed a 1 hour incubation time prior to treating the cells with the extract samples and HPV 16 PsV. The remainder of the experiment was identical to the initial assays involved in our study.

Western Immunoblot for Phosphorylated EGFR: A linkage between HPV epithelial entry and epidermal growth factor receptor (EGFR), previously identified as a possible regulator of FC, was also assessed through Western immunoblotting. To do this, the 4 Actinomycete-derived extract samples that exhibited the most significant effect in terms of increase of HPV PsV entry into the 293TT cells were selected. 293TT cells were grown on a 6-well plate and then starved in serum-free media for 3 hours at 37C. Four experimental wells were then exposed to these 4 extracts (at the same 1:100 dilution that had been previously used). At the same time, a control well was also prepared in which epidermal growth factor (EGF) (50 nM) was administered to the cells instead of an extract sample. A control well of untreated 293TT cells was also prepared. Following an incubation period of 10 minutes, the media was removed from both the control and experimental wells and the cells were lysed in 200 ul of RIPA buffer. The samples obtained from cell lysis were later used to perform Western immunoblotting involving an anti-EGFR antibody. Figure .01 features a flow chart that summarizes the methods that were involved in our study.

Statistical Analysis: Statistical significance was demonstrated through a Students T test at a level of confidence of p<.001 for HPV 16 entry ( 5%) compared to controls on the identical 96 well plate.

Results

Initial triplicate well plating: The initial incubation for 100 different extracts showed increases and decreases in comparison to the untreated controls for HPV 16 entry into 293 TT cells. 51% were increased while 49% showed decreases below untreated levels (no extract). The highest level was 988.58% greater than the untreated control, while -81.88% was the lowest value for any extract below untreated baseline. (Figures. 0I, 02) Viability assay demonstrated that all extracts at 1:25 and >50% at 1:50 showed 85-90% loss of viability compared to controls after 24h (p<0.0001). Furthermore, 30% of extracts exhibited loss of viability within 1h of treatment at 1:25 dilutions and 45% of extracts at 1h with 1:50 dilutions.

Dilution to reduce expected cytotoxicity: To reduce the range and possible cytotoxic damage a dilution of 1:100 of extracts was used. Recorded were increases in 56% of extracts while 44% were below untreated controls values for HPV 16 entry.

Method scheme presented to evaluate HPV 16 entry into 293TT cells.

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Fig. 2: Scheme For Evaluation of Marine Actinomycete Extracts Capacity to Alter Human Papillomavirus 16 Entry (HPV 16) into Epithelial Cells

We present a scheme to determine the treatment response from marine Actinomycetes extracts (e.g., Salinispora arenicola) in two epithelial cell populations and a capacity for HPV 16 entry.

Effect of CMK Inhibition on HPV 16: 12 extracts were chosen based on the original results of increased or decreased HPV 16 entry into 293TT cells. 12 extracts were incubated using a 1:100 of extract to well volume and results demonstrated that 12 selected extracts altered HPV 16 uptake through a range of -2.23% to +204.3% (p<.001) as we compared no CMK and no extract to extract present results, or as later described CMK treatment response to extract or no extract.

To each well CMK (1.0μM) was added before the addition of extract or with no addition of extract. In these non-collagen treated wells addition of CMK reduced HPV 16 entry 83% in comparison to CMK without extracts, while 16.6% showed an increase and 8% were unchanged from the above control.

293TT cells have a tendency to detach and to overcome this problem we increased precision counting by automated fluorometry with the addition of collagen to wells. We therefore repeated the above assays with collagen coated plates.

Effect of collagen on HPV 16: Using these identical 12 extracts we examined the effects of time (48 h) and the presence of collagen on HPV 16 entry into 293TT cells.

Plating on to culture dishes of 6 or 96 well plates is performed because of differences in cell density results in differences in HPV 16 entry.

6 well tissue culture plate: The untreated wells demonstrated a 35.2% +/-5.6%; in comparison to extracts that demonstrated a range from 6.3%+/- 5.5% (82.1% reduction) to 62.6%+/-8.1% (a 77.8% increase). An example of the 6 well plate HPV 16 PsV results are seen in Figure. 03.

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Fig. 3: Microphotographs of HPV 16 Pseudoparticles (PsV) Entry into 293TT Cells Following Exposure to marine Actinomycetes (48h).

A. Untreated 293TT cells: Bright field (grey-scale) and comparative dark-field (fluorescence). Cells containing replication of plasmid contained in HPV 16 PsV (GFP).A count (% of fluorescent cells/total number of cells present in at least three fields (10X) is obtained from fluorimeter and visual counting.

B. Extract B. Counted as stated above. Slight increase compared to control (A.) (15.63%)

C. Extract E. Counted as stated above. Slight increase compared to control (A.) (1.04%)

92 Well Microtiter plate: After 48 hour incubation on a collagen coated microtiter plate and fluorimeter (550- 60 nm emission) readings were obtained as shown in Figure. 04 A.; for 12 extracts. The % changes from untreated baseline ranged from -2.46% to an increase of 145.61%.

We repeated these assays with incubation for 72 h and found a similar pattern of enhanced and reduced HPV 16 entry for each extract.

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Fig. 4: Values and calculation of marine Actinomycetes for HPV 16 Entry into 293TT cells with or without additional treatment with CMK

12 extracts of marine Actinomycete origin are examined using fluorimeter for change in HPV 16 (145% to -2.26%). Addition of CMK, inhibitor for FC expression generally reduces HPV 16 (3.79% to -4.76%)

CMK Effect on 12 Extracts: To establish the relationship between these HPV 16 entry responses and furin convertase (FC), we used the inhibitor CMK in some wells with extracts while in others CMK was applied without extracts as stated above. (Figure. 03.B.)

Only 3 extracts showed an increase in HPV 16 entry after CMK treatment, but about a 10-15% reduction in HPV 16 entry was still recorded in comparison to CMK treatment with no extract application. (Figure. 03.B.)

Immunocytochemistry for FC expression: (Figure. 05) 293TT cells were used to compare to untreated cells to 293TT cells after exposure to an extract that showed a prior capacity to increase HPV 16 entry. A non-extract exposed 293TT cell population (17.5%+/-5.8%) in comparison to two extract treated 293TT groups is described below. Using identical extracts shown in Figure .03. The % of FC in expressed cells (shown as red fluorescence): Extract B. 43.1%+/-7.3% (p<0.0001) and Extract E(figure C): 67.9%+/-4.4% (p<0.0001) compared to control. In addition we note CMK results for Extract B: 3.00% and for Extract C. 3.79%; following treatment of CMK results in depression of FC function.

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Fig. 5: Immunocytohemistry for FC Expression in 293TT Cells Exposed to Marine Actinomycetes (48h)

A. Untreated cells: Bright field (grey-scale; estimate of cell size with bar scale 5.6 microns) and comparative dark-field (red fluores cence). Cells not exposed to extracts. (% of cells fluorescent/total number of cells present in at least 3 fields) (17.5%+/-5.8%) B. Extract

B. Examples of FC expression following exposure to marine Actinomycete. (63.1%+/-7.3%). This increase in expression may not be a product of enhanced transcription of protein but also a change in cell cycling for an accumulation of FC on the membrane.

C. Extract E. A similar effect for FC expression following exposure to marine Actinomycete. (37.9%+/-4.4%). In general a trend was noted with FC levels corresponded to HPV 16 entry.

These assays led us to conclude that FC is an important regulatory factor for HPV 16 entry following Actinomycete extract exposure.

Role for EGFR: Previous studies have identified a chemical; cysteine rich region homology between FC and EGFR.35

A Western immunoblotting was performed with 4 extracts (Figure. 06). EGFR expressions were: Extract 1(B) (142.2% increase), 3 (C) (50.4% increase), and 4(D) (139.7% increase) levels of phosphorylated EGFR expressions while Extract 2 (A) (8.4% increase), showed a level approximately equal to untreated control.

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Fig. 6: Western Immunoblot For Expression of Phosphorylated EGFR

Lanes: I. 293TT (110 kD) band for EGFR expression is identified (arrow) (4.50 densitometry units)

II. 293TT + EGF stimulation of 293TT cells. (increase X/control: 57.3% increase)

III. Extract (B): (142.2% increase)

IV. Extract (E): (8.4% increase)

V. Extract (C): (50.4% increase)

VI. Extract (D): (139.7% increase)

We concluded assays demonstrate a high degree of variability for Actinomycete extract effects upon FC, EGFR and HPV 16 entry into epithelial cells. However, FC is an important regulator of this process. Using another cell line analogous to HOK cells we examined whether extract treatment would produce a similar response for HPV 16 entry.

hTERT HOK Assay: (Figure. 07) The control no extract level was as stated above, 8.5%+/- 5.7%. In contract we see HPV 16 entry results for exposure to two Actinomycete derived extracts; one not significantly different from control; Extract B. (11.5%+/-8.1%) (p<0.1687, NS) and the second Extract E, which was significantly different from control: E. (31.4%+/-7.0%) (p<0.0001). This results parallels results from 293TT cells showed increases in HPV 16 entry but Extract B: 15.63% was lower than Extract E. 42.51% results.

Figure7Capture

Fig. 7: Exposure to marine Actinomycetes and HPV 16 entry into hTERT HOK cells (72h)

A. Untreated hTERT HOK cells: Bright field (grey-scale) and comparative dark-field (fluorescence). Cells containing replication of plasmid contained in HPV 16 PsV (GFP). A count (% of fluorescent cells/total number of cells present in at least three fields (10X) is obtained from fluorimeter and visual counting. (8.5% +/-5.7%)

B. Extract B. Counted as stated above. Increase compared to control (A.) (11.5%+/-8.1%). This result demonstrated a variation in HPV 16 entry in relation to cell type and time of incubation.

C. Extract E. Counted as stated above. Increase compared to control (A.) (28.4%+/-7.0%). This result demonstrated a variation in HPV 16 entry in relation to cell type and time of incubation.

In the following assay we attempted to link extract results to ROS production such as, anthraquinones20,22,25 that are suggested mediators of EGFR expression22,29,35 and influence HPV 16 entry.

To test this relationship we used anthraquinone; two poly-cyclic aromatic hydrocarbons: B[a]P; DBP; TSNAs, NNK; NNAL and nicotine which are also sources for ROS and possible synthesis of quinones and anthraquione.1,24,39

HPV 16 Entry following Treatment with B[a]P; DBP, or NNK.

In Table. 01, we show that the vehicle DMSO did not produce a significant difference from untreated 293TT cells. However, B[a]P dissolved in DMSO in three different concentrations (dilutions) significantly increased HPV 16 entry. In contrast, only the highest concentration of 50 microliters (36.7 nanoMolar) DBP produced significant increased HPV 16 entry compared to controls.

Another comparison was made with the TSNAs, NNK, and NNAL solubilized in methanol and derived from metabolism of nicotine. Methanol treatment did not increase HPV 16, but we did record a significant increase for NNK; although close to significance at the highest concentration (50ul) (28.9 nanoMolar), and recorded increase in HPV 16. However, NNAL treatment was significant for treatment that used 20 (138 nanoMolar) and 50 microliters (345 nanoMolar). Furthermore, anthraquinone our suggested ROS secreted chemical from Antinomycete extracts was sufficient to produce a significant HPV 16 entry at treatments of 50 microliters (4.9 nanoMolar).

Furthermore, a 12mg. concentration of nicotine compared to a 6mg. concentration did show a significant increase in HPV 16 entry.

Discussion

This laboratory study showed a variation in capacity for HPV 16 entry following treatment from an initial 100 marine derived Actinomycete extracts and this trend persisted with a reduced number of 12 extracts (Figure .01 and 03). An increase in HPV 16 entry was obtained for human epithelial cells using a standard assay (293TT) and oral focus; human oral keratinocytes. (hTERT HOK) (Figure. 07) We also found HPV 16 entry was linked to FC and EGFR expression (Figure .03-06). This finding may have implications for better understanding HPV 16 related OPC presentations; impact on risk, treatment selection, and expected clinical responses.

Previous reports established marine and non-marine Actinomycetes spp. synthesized anthraquinones12,1721,23,24 and as stated Actinomycetes spp. are a common commensal microbe with membes of the genera including Rhodococcus and Streptomyces spp. that under a variety of conditions can become a virulent pathogenic microorganism associated with inflammatory sites and increased release of ROS.911 Moreover treatment responses (Table. 01) from anthraquinone; two PAHs derived from tobacco products, B[a]P and DBP; two TSNAs, NNK and NNAL and nicotine can also generate ROS to increase HPV 16 entry in a dose dependent manner for specific agents. This association although requiring extensive clinical assessment with populations of tobacco product users may indicate a plausible reason for epidemiologic variability between tobacco use and HPV16 risk for infection and presence in OPC versus OSCC. Moreover, these assays are unclear as to the extent of ROS activity independent of quinone metabolism by any specific Actinomycete spp. extract. Although extract effects on oxidative capability was discerned by the degree and immediacy of toxicity with loss of viability encountered with treatment of epithelial cells with these extracts. Moreover, Actinomycete derived products such as salinosporamide A, which is a potent 20S proteasome inhibitor is documented to produce apoptosis with treatment for multiple myeloma.

Stated before some Actinomycete spp. are capable of metabolic oxidation (e.g., cytochrome P450: 1A1, 1A2, 1B1, 3A/4) and degradation of PAH carcinogens; such as, B[a]P and DBP and TSNAs; such as, NNK and NNAL.1,2 The latter nitrosamines are notably degraded using a NAD(P)H:quinone oxidoreductase 1 (NQO1); a cytosolic enzyme that uses a two-electron reduction of quinoid compounds into hydroquinones.2226

Comparison of HPV 16 Entry Following Exposure to anthraquinone, B[a]P, DBP, NNK, NNAL and Nicotine.

The mean and standard variation (S.D.) and the P value for statistical significance were ascertained. Results show anthraquinone; tobacco derived carcinogens and nicotine at the highest concentration produces a general increase in HPV 16 entry. This result is a product of metabolic oxidation that produces quinones and/or an oxidative state in the epithelial cell. 1= Students T test comparison for significance to untreated

HPV 16 entry in 293TT Cells (%)
Treatment Mean% S.D. P Value1
Untreated 37.6 2.8 NA
DMSO 33.4 10.0 <0.442 NS
Methanol
10 ul 30.8 17.2 <0.591 NS
20 ul 25.3 15.8 <0.237 NS
B[a]P
10 ul 52.9 8.5 <0.0045
20 ul 72.8 14.2 <0.0002
50 ul 61.3 13.0 <0.0019
DBP
10 ul 45.6 9.8 <0.058 NS
20 ul 42.9 10.6 <0.141 NS
50ul 53.2 10.4 <0.0072
Nicotine
6 mg 33.7 18.0 <0.410 NS
12 mg 62.5 11.4 <0.0009
NNK
10 ul 27.7 8.0 <0.746 NS
20 ul 34.5 9.6 <0.250 NS
50 ul 44.7 14.0 <0.048 NS
NNAL
10 ul 30.7 9.9 <0.257 NS
20 ul 51.2 4.7 <0.013
50 ul 84.7 9.0 <0.001
Anthraquinone
10 ul 41.9 5.3 <0.374 NS
20 ul 46.8 7.0 <0.094 NS
50 ul 56.4 8.9 <0.006

This reaction can be viewed as protective because metabolic reduction can lead to less toxic products to be excreted by cells. In eukaryotes, this is observed by EGFR activation and genes such as the Nrf2/Keap1- mediated stress complex.3941 Several Actinomycete species use a two-electron reduction to the hydroquinone form to avoid generation of one-electron reduced semiquinone which is known to cause oxidative stress. These enzymes utilize a flavin cofactor, either FMN or FAD, to transfer a hydride from an electron donor, such as NAD(P)H, to a quinone substrate.4143Arthrobacter spp. is an Actinomycete spp. found in the soil which uses a nicotinic form of this enzyme.44

Nicotine is also linked to production of quinones through TSNA synthesis,39 or cyclic organic compounds that are present in pro- and eukaryotic cellular environments.

Our results suggest a common relationship is present between synthesis of quinones from Actinomycetes extracts, anthraquinone, PAHs, TSNA, and nicotine (>12mg) because they all generate a ROS and induce an oxidation state to regulate EGFR.1,2,24,26,35,36,39 Furthermore our recent publication shows oral commensal bacteria mediate HPV 16 entry through phosphorylation of EGFR, and enhanced function of membrane furin convertase in support of the concept that specific microbe genera participate in regulation of HPV 16 entry because they are capable of induction of ROS physiology alterations in HOK.

Expression of phosphorylated-L-tyrosine active form of EGFR is tied to expression and function of the PI3K/ AKT/PTEN /nTOR pathway.37,4547 This pathway mediates oncogenes, tumor suppressors and oxidation /cell cycle regulatory factors such as c-myc, p53, Hypoxia-inducible factor-1, and cyclin D1.46,47

Ultimately differentiation of epithelial targets is related to HPV 16 entry which is shown to occur with differentiated oral keratinocytes and influences protein synthesis of E6 and E7 derived from HPV 16 genome integration to regulate a complex containing several factors (e.g., p300/Cyclin D/p16/p53etc).5

Our results suggest that Actinomycetes spp. which are composed of a variety of species should be considered in evaluation of risk for HPV 16 related OPC.6 In addition, FC and EGFR expressions are possible biomarkers for this association.

Summary

Significant highlights documented in this study are:

Marine Actinomcyte synthesize ROS chemicals such as anthraquinones.

• Marine Actinomycetes extracts contain chemical mediators for HPV16 entry into epithelial cells (293TT and hTert HOK cells).

• HPV 16 activity is through EGFR phosphorylation and furin convertase activity.

• Environmental derived chemicals (B[a]P, DBP, NNK, NNAL, Nicotine, and anthraquinone) parallel oral Actinomycetes effect HPV 16 entry.

Abbreviations

HPV 16: human papilloma virus subtype 16

EGFR: epidermal growth factor receptor);

FC: furin convertase

PAH: poly-cyclic aromatic-hydrocarbon

(B[a]P): benzo[a]pyrene

DBP: dibenzo[a,l]pyrene

NNK: Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

HNSCC: head and neck squamous cell carcinoma

DMSO: dimethylsulfoxide

CMK: chloromethyl ketone

TSNA: tobacco specific nitrosamines