The effect of cannabinoids on wound healing: A review.

Background and Aims: Cannabis and its various derivatives are commonly used for both recreational and medicinal purposes. Cannabinoids have been shown to have anti-inflammatory properties. Inflammation is an important component of wound healing and the effect of cannabinoids on wound healing has become a recent topic of investigation. The objective of this article is to perform a comprehensive review of the literature to summarize the effects of cannabinoids on wound healing of the skin and to guide future avenues of research. Methods: A comprehensive literature review was performed to evaluate the effects of cannabinoids on cutaneous wound healing. Results: Cannabinoids appear to improve skin wound healing through a variety of mechanisms. This is supported through a variety of in vitro and animal studies. Animal studies suggest application of cannabinoids may improve the healing of postsurgical and chronic wounds. There are few human studies which evaluate the effects of cannabinoids on wound healing and many of these are case series and observational studies. They do suggest cannabinoids may have some benefit. However, definitive conclusions cannot be drawn from them. Conclusion: While further human studies are needed, topical application of cannabinoids may be a potential therapeutic option for postsurgical and chronic wounds.

Exposure to Secondhand Smoke Extract Increases Cisplatin Resistance in Head and Neck Cancer Cells.

Chemotherapy and radiotherapy resistance are major obstacles in the long-term efficacy of head and neck squamous cell carcinoma (HNSCC) treatment. Secondhand smoke (SHS) exposure is common and has been proposed as an independent predictor of HNSCC recurrence and disease-free survival. However, the underlying mechanisms responsible for these negative patient outcomes are unknown. To assess the effects of SHS exposure on cisplatin efficacy in cancer cells, three distinct HNSCC cell lines were exposed to sidestream (SS) smoke, the main component of SHS, at concentrations mimicking the nicotine level seen in passive smokers' saliva and treated with cisplatin (0.01-100 µM) for 48 h. Compared to cisplatin treatment alone, cancer cells exposed to both cisplatin and SS smoke extract showed significantly lower cisplatin-induced cell death and higher cell viability, IC50, and indefinite survival capacity. However, SS smoke extract exposure alone did not change cancer cell viability, cell death, or cell proliferation compared to unexposed control cancer cells. Mechanistically, exposure to SS smoke extract significantly reduced the expression of cisplatin influx transporter CTR1, and increased the expression of multidrug-resistant proteins ABCG2 and ATP7A. Our study is the first to document that exposure to SHS can increase cisplatin resistance by altering the expression of several proteins involved in multidrug resistance, thus increasing the cells' capability to evade cisplatin-induced cell death. These findings emphasize the urgent need for clinicians to consider the potential role of SHS on treatment outcomes and to advise cancer patients and caregivers on the potential benefits of avoiding SHS exposure.

Tobacco Cessation Following Laryngeal Cancer Diagnosis Predicts Response to Treatment and Laryngectomy-Free Survival.

OBJECTIVE: To determine the effect of tobacco cessation following laryngeal cancer diagnosis on response to first-line therapy, laryngectomy-free survival, and overall survival in patients who were current smokers at the time of diagnosis. STUDY DESIGN: Retrospective, case-control study. SETTING: OU Stephenson Cancer Center, National Cancer Institute-Designated Cancer Center. METHODS: We included 140 patients diagnosed with laryngeal squamous cell carcinoma, who were current smokers at the time of diagnosis, and were treated with first-line definitive radiation or chemo/radiation with the intent to cure. The association between patient characteristics and treatment response was assessed using the χ2 test and logistic regression analysis. Survival outcomes were analyzed using Kaplan-Meier methods and Cox proportional-hazards models. RESULTS: Of the 140 current smokers, 61 patients (45%) quit smoking prior to treatment initiation. In adjusted logistic regression analysis, quitters had 3.7 times higher odds of achieving a complete response to first-line therapy than active smokers (odds ratio: 3.694 [1.575-8.661]; P = .003). In the adjusted Cox proportional-hazards model, quitters were 54% less likely to require salvage laryngectomy within 7 years of diagnosis than active smokers (hazard ratio: 0.456 [0.246-0.848]; P = .013). Quitters had a statistically significant increase in 7-year overall survival compared to active smokers (P = .02). CONCLUSION: This is the first study to show that in newly diagnosed laryngeal cancer patients who are current smokers at the time of diagnosis, tobacco cessation significantly increases therapy response, laryngectomy-free survival, and overall survival. These data stress the importance of systematically incorporating tobacco cessation programs into laryngeal cancer treatment plans.

Tobacco cessation after head and neck cancer diagnosis is an independent predictor of treatment response and long-term survival.

Head and neck squamous cell carcinoma (HNSCC) patients who are current smokers when diagnosed have inferior clinical outcomes compared to never-smokers or previous smokers. However, the impact of quitting after HNSCC diagnosis has not been quantified. In this retrospective, case-control study (n = 134), the odds of complete response to first-line therapy were 3.7 times higher among smokers at diagnosis who quit before treatment initiation (quitters; n = 55) than among those continuing to smoke (p = 0.03). Disease-free survival was also higher among quitters (aHR, 0.33; 95 % CI, 0.12-0.90; p = 0.029). Quitters were 67 % less likely to die of all causes than active smokers (aHR, 0.33; 95 % CI, 0.15-0.71; p = 0.004). These data show for the first time that, smoking cessation after HNSCC diagnosis is predictive of higher therapy efficacy and long-term survival.

The Effect of Flow Rate on a Third-Generation Sub-Ohm Tank Electronic Nicotine Delivery System-Comparison of CORESTA Flow Rates to More Realistic Flow Rates.

Many types of electronic cigarettes (ECs) are currently in use, but the default flow rate used to simulate puffing is centered on tobacco cigarette flow rates. CORESTA offers several methods and technical guides for evaluation of ECs but there are few puffing topography studies focusing on sub-ohm ECs; differences between real-world usage and that found in the literature appear large. This study focuses on how power and flow rate affect the nicotine yield of a sub-ohm EC. A puffing system (Puff3rd) has been designed and used to produce and collect EC aerosol. Nicotine yield was measured by GC-MS at three power levels and four flow rates. Data analysis was conducted in SAS using the MIXED procedure. Power, flow rate, and their interaction were all significant predictors of nicotine yield. Nicotine yield increased with both the vaping power and the puff flow rate with significant interaction of the two. Findings indicate that using the current CORESTA flow rate (1100 mL/min) to evaluate third-generation ECs underestimates nicotine yield and likely overestimates pyrolysis products. Real users are expected to have 2-3× the nicotine dose measured at 1100 mL/min, which could confound epidemiological studies seeking to link nicotine delivery to product satisfaction and acceptability.

Electronic cigarette aerosols alter the expression of cisplatin transporters and increase drug resistance in oral cancer cells.

Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies.

Regulation of MicroRNAs in Inflammation-Associated Colorectal Cancer: A Mechanistic Approach.

The development of colorectal cancer (CRC) is a multistage process. The inflammation of the colon as in inflammatory bowel disease (IBD) such as ulcerative colitis (UC) or Crohn's disease (CD) is often regarded as the initial trigger for the development of inflammation-associated CRC. Many cytokines such as tumor necrosis factor alpha (TNF-α) and interleukins (ILs) are known to exert proinflammatory actions, and inflammation initiates or promotes tumorigenesis of various cancers, including CRC, through differential regulation of microRNAs (miRNAs/miRs). miRNAs can be oncogenic miRNAs (oncomiRs) or anti-oncomiRs/tumor suppressor miRNAs, and they play key roles during colorectal carcinogenesis. However, the functions and molecular mechanisms of regulation of miRNAs involved in inflammation-associated CRC are still anecdotal and largely unknown. Consolidating the published results and offering perspective solutions to circumvent CRC, the current review is focused on the role of miRNAs and their regulation in the development of CRC. We have also discussed the model systems adapted by researchers to delineate the role of miRNAs in inflammation-associated CRC.

Role of Inflammation in the Development of Colorectal Cancer.

Chronic inflammation can lead to the development of many diseases, including cancer. Inflammatory bowel disease (IBD) that includes both ulcerative colitis (UC) and Crohnmp's disease (CD) are risk factors for the development of colorectal cancer (CRC). Many cytokines produced primarily by the gut immune cells either during or in response to localized inflammation in the colon and rectum are known to stimulate the complex interactions between the different cell types in the gut environment resulting in acute inflammation. Subsequently, chronic inflammation, together with genetic and epigenetic changes, have been shown to lead to the development and progression of CRC. Various cell types present in the colon, such as enterocytes, Paneth cells, goblet cells, and macrophages, express receptors for inflammatory cytokines and respond to tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, and other cytokines. Among the several cytokines produced, TNF-α and IL-1ß are the key pro-inflammatory molecules that play critical roles in the development of CRC. The current review is intended to consolidate the published findings to focus on the role of pro-inflammatory cytokines, namely TNF-α and IL-1ß, on inflammation (and the altered immune response) in the gut, to better understand the development of CRC in IBD, using various experimental model systems, preclinical and clinical studies. Moreover, this review also highlights the current therapeutic strategies available (monotherapy and combination therapy) to alleviate the symptoms or treat inflammation-associated CRC by using monoclonal antibodies or aptamers to block pro-inflammatory molecules, inhibitors of tyrosine kinases in the inflammatory signaling cascade, competitive inhibitors of pro-inflammatory molecules, and the nucleic acid drugs like small activating RNAs (saRNAs) or microRNA (miRNA) mimics to activate tumor suppressor or repress oncogene/pro-inflammatory cytokine gene expression.

Evaluation of Respiratory Symptoms Among Youth e-Cigarette Users.

Importance: Use of e-cigarettes (ECs) among youths has increased in recent years. e-Cigarette aerosol contains chemical constituents, such as diacetyl or benzaldehyde, which are known to affect the respiratory system. Objective: To examine the association between EC use and self-reported wheezing in a cohort of US adolescents. Design, Setting, and Participants: This cohort study used data from waves 3 and 4 (October 19, 2015, to January 3, 2018) of the Population Assessment of Tobacco and Health (PATH) study, a longitudinal, nationally representative cohort survey. Adolescent respondents aged 12 to 17 years who did not have asthma were included. Exposures: e-Cigarette use during the previous year. Main Outcomes and Measures: Self-reported wheezing in the past 12 months (yes or no) and EC use (no use in past year or never use, use in past year, use in past 30 days, and use in past 7 days). Survey-weighted logistic regression models adjusted for demographic characteristics and other risk factors. Results: Among 7049 adolescents without asthma from waves 3 and 4 of the PATH study, 49.9% were female and 54.4% were non-Hispanic White. In unadjusted models, the odds of wheezing in the past 12 months were higher for youths who had used ECs in the past year compared with those who had not (odds ratio, 1.74; 95% CI, 1.22-2.48; P = .003). In the adjusted model, after controlling for the variables of race/ethnicity, household rules about the use of tobacco, contact with a smoker in the previous 7 days, and current use of combustible tobacco products, the association of EC use with wheezing was not significant (adjusted odds ratio for EC use in the past year, 1.37 [95% CI, 0.91-2.05]; in the past 30 days, 1.35 [95% CI, 0.63-2.88]; in the past 7 days, 0.74 [95% CI, 0.28-1.97]; P = .33). Conclusions and Relevance: In this cohort study, use of ECs alone was not associated with increased odds of experiencing wheezing episodes. Future studies incorporating the use of objective data appear to be needed to more accurately understand the potential respiratory harms associated with vaping among adolescents.

Electronic cigarette power affects count concentration and particle size distribution of vaping aerosol.

INTRODUCTION: Electronic cigarettes (EC) have evolved rapidly toward higher powered devices that produce more vaping aerosol and a more satisfying vaping experience. This research characterized the particle size distribution and estimated the mass concentration of vaping aerosols produced at power outputs spanning the operating range typical of second generation variable voltage EC devices. METHODS: EC aerosol was characterized from a single coil atomizer powered by a variable voltage EC battery at the minimum and maximum dial settings (3.3, 11.2 Watts, W), and a lab controlled power supply (3-11.9 W). Aerosol particle size distribution was measured by a Scanning Mobility Particle Sizer and Aerodynamic Particle Sizer, spanning 16 nm to 19.8 µm. A mouth puff was simulated using a 100 mL glass syringe. RESULTS: Consistent with prior studies, sub-micron EC aerosol size distributions were bimodal, with peaks at 40 and 200 nm, however a previously unreported third mode was observed at approximately 1000 nm. The ~1000 nm mode accounted for 7-20x the aerosol mass of the smaller modes. Increasing atomizer power decreased count concentration of particles <600 nm but increased particle count >600 nm. Particle mass distribution shifted toward micron sized particles with increasing power and increased the respirable fraction of aerosol, likely due to increased coagulation and condensation around nano-sized particles. CONCLUSIONS: Vaping power greatly affects EC aerosol count and mass distribution. Mouth puffed EC aerosol spans a much wider particle size range than previously reported, although the major portion of the mass is still well within the alveolar size range the larger particles will deposit within the oro-pharyngeal cavity at 2-3x greater efficiency than in alveoli. These observations have major clinical implications, as aerosol particle size distribution determines deposition sites along the respiratory tract. The results of this experiment stress the need for further research to inform the design, regulation and use of e-cigarette products.

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage.

BACKGROUND: Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. OBJECTIVE: The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. METHODS: Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. RESULTS: EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. CONCLUSIONS: Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public.

Have combustible cigarettes met their match? The nicotine delivery profiles and harmful constituent exposures of second-generation and third-generation electronic cigarette users.

INTRODUCTION: Electronic cigarettes' (e-cigarettes) viability as a public health strategy to end smoking will likely be determined by their ability to mimic the pharmacokinetic profile of a cigarette while also exposing users to significantly lower levels of harmful/potentially harmful constituents (HPHCs). The present study examined the nicotine delivery profile of third- (G3) versus second-generation (G2) e-cigarette devices and their users' exposure to nicotine and select HPHCs compared with cigarette smokers. METHODS: 30 participants (10 smokers, 9 G2 and 11 G3 users) completed baseline questionnaires and provided exhaled carbon monoxide (eCO), saliva and urine samples. Following a 12-hour nicotine abstinence, G2 and G3 users completed a 2-hour vaping session (ie, 5 min, 10-puff bout followed by ad libitum puffing for 115 min). Blood samples, subjective effects, device characteristics and e-liquid consumption were assessed. RESULTS: Smokers, G2 and G3 users had similar baseline levels of cotinine, but smokers had 4 and 7 times higher levels of eCO (p<0.0001) and total 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (i.e., NNAL, p<0.01), respectively, than G2 or G3 users. Compared with G2s, G3 devices delivered significantly higher power to the atomiser, but G3 users vaped e-cigarette liquids with significantly lower nicotine concentrations. During the vaping session, G3 users achieved significantly higher plasma nicotine concentrations than G2 users following the first 10 puffs (17.5 vs 7.3 ng/mL, respectively) and at 25 and 40 min of ad libitum use. G3 users consumed significantly more e-liquid than G2 users. Vaping urges/withdrawal were reduced following 10 puffs, with no significant differences between device groups. DISCUSSION: Under normal use conditions, both G2 and G3 devices deliver cigarette-like amounts of nicotine, but G3 devices matched the amount and speed of nicotine delivery of a conventional cigarette. Compared with cigarettes, G2 and G3 e-cigarettes resulted in significantly lower levels of exposure to a potent lung carcinogen and cardiovascular toxicant. These findings have significant implications for understanding the addiction potential of these devices and their viability/suitability as aids to smoking cessation.

An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma.

Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.

Detection of in vivo DNA damage induced by very low doses of mainstream and sidestream smoke extracts using a novel assay.

BACKGROUND: Mainstream (MS) smoke, the main smoke inhaled by active smokers, and sidestream (SS) smoke, the main component of secondhand smoke, induce a wide range of DNA lesions. Owing to technical limitations, the in vivo levels of tobacco-induced DNA damage are unknown. Recently, the authors developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to quantify endogenous and induced DNA damage. PURPOSE: To quantify the in vivo levels of DNA damage induced by MS and SS smoke extracts in human cells using PADDA and define the strand-specific patterns of DNA damage and repair following exposure to diverse doses of MS and SS smoke. METHODS: Human epithelial cells were exposed to escalating doses of hydrogen peroxide (H2O2), MS, or SS smoke. TP53 gene DNA damage was quantified using PADDA at various time points. DNA double-strand breaks were detected by immunofluorescence analysis of phosphorylated histone H2AX (γ-H2AX). Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Data were collected and analyzed by t-test in 2012-2014. RESULTS: A dose-dependent increase in DNA damage was detected in vivo with increasing doses of H2O2, MS, and SS smoke. Even 1 hour of exposure to very low doses of MS or SS smoke resulted in significant DNA damage (p<0.01). MS and SS smoke induced distinctive strand-specific patterns of DNA damage and DNA repair kinetics. CONCLUSIONS: Very low concentrations of MS and SS smoke induce significant DNA damage in human cells. Application of PADDA to population studies has major potential to establish biomarkers of susceptibility to tobacco-induced diseases.

Detection of In Vivo DNA Damage Induced by Very Low Doses of Mainstream and Sidestream Smoke Extracts Using a Novel Assay.

BACKGROUND: Mainstream (MS) smoke, the main smoke inhaled by active smokers, and sidestream (SS) smoke, the main component of secondhand smoke, induce a wide range of DNA lesions. Owing to technical limitations, the in vivo levels of tobacco-induced DNA damage are unknown. Recently, the authors developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to quantify endogenous and induced DNA damage. PURPOSE: To quantify the in vivo levels of DNA damage induced by MS and SS smoke extracts in human cells using PADDA and define the strand-specific patterns of DNA damage and repair following exposure to diverse doses of MS and SS smoke. METHODS: Human epithelial cells were exposed to escalating doses of hydrogen peroxide (H2O2), MS; or SS smoke. TP53 gene DNA damage was quantified using PADDA at various time points. DNA double-strand breaks were detected by immunofluorescence analysis of phosphorylated histone H2AX (γ-H2AX). Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Data were collected and analyzed by t-test in 2012-2014. RESULTS: A dose-dependent increase in DNA damage was detected in vivo with increasing doses of H2O2, MS, and SS smoke. Even 1 hour of exposure to very low doses of MS or SS smoke resulted in significant DNA damage (p < 0.01). MS and SS smoke induced distinctive strand-specific patterns of DNA damage and DNA repair kinetics. CONCLUSIONS: Very low concentrations of MS and SS smoke induce significant DNA damage in human cells. Application of PADDA to population studies has major potential to establish biomarkers of susceptibility to tobacco-induced diseases.

Characterization of tumorigenic cell lines from the recurrence and lymph node metastasis of a human salivary mucoepidermoid carcinoma.

UNLABELLED: The long-term outcome of patients with mucoepidermoid carcinoma is poor. Limited availability of cell lines and lack of xenograft models is considered a major barrier to improved mechanistic understanding of this disease and development of effective therapies. OBJECTIVE: To generate and characterize human mucoepidermoid carcinoma cell lines and xenograft models suitable for mechanistic and translational studies. METHODS: Five human mucoepidermoid carcinoma specimens were available for generation of cell lines. Cell line tumorigenic potential was assessed by transplantation and serial in vivo passaging in immunodeficient mice, and cell line authenticity verified by short tandem repeat (STR) profiling. RESULTS: A unique pair of mucoepidermoid carcinoma cell lines was established from a local recurrence (UM-HMC-3A) and from the metastatic lymph node (UM-HMC-3B) of the same patient, 4 years after surgical removal of the primary tumor. These cell lines retained epithelial-like morphology through 100 passages in vitro, contain the Crtc1-Maml2 fusion oncogene (characteristic of mucoepidermoid carcinomas), and express the prototypic target of this fusion (NR4A2). Both cell lines generated xenograft tumors when transplanted into immunodeficient mice. Notably, the xenografts exhibited histological features and Periodic Acid Schiff (PAS) staining patterns that closely resembled those found in human tumors. STR profiling confirmed the origin and authenticity of these cell lines. CONCLUSION: These data demonstrate the generation and characterization of a pair of tumorigenic salivary mucoepidermoid carcinoma cell lines representative of recurrence and lymph node metastasis. Such models are useful for mechanistic and translational studies that might contribute to the discovery of new therapies for mucoepidermoid carcinoma.

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

Pharmacodynamic modeling of anti-cancer activity of tetraiodothyroacetic acid in a perfused cell culture system.

Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvß3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.

Hyaluronic acid hydrogel sustains the delivery of dexamethasone across the round window membrane.

BACKGROUND: The use of intratympanic (IT) steroids for the treatment of inner ear disorders is promising, but the clinical challenges of prolonged middle ear drug application have proven burdensome, and a sustainable delivery system is yet to be developed. METHOD: In this study, a guinea pig model was used to determine if dexamethasone in combination with a hyaluronic-acid (HA)-based hydrogel is an efficient, stable and sustainable dexamethasone delivery system to the inner ear. For each animal, right and left middle ear bullae were randomly selected to be filled with dexamethasone alone or dexamethasone-HA (Dex-HA) gel. Perilymph samples were collected at different time points and dexamethasone levels were determined using an ELISA. RESULTS: Dexamethasone was measurable in the perilymph samples up to 72 h after treatment. At 24 h after treatment, the perilymph dexamethasone concentrations were significantly higher (p = 0.01) in the ears treated with Dex-HA gel than in those treated with dexamethasone alone. While the perilymph dexamethasone concentration had decreased at 48 h after treatment with Dex-HA gel, the levels were still higher than those observed at 24 h in ears treated with dexamethasone alone. A high variability in dexamethasone concentration was observed between the samples, and the variability between matched ears receiving different treatments was remarkably lower than the variability within each treatment group, suggesting that individual parameters might play a major role in perilymph dexamethasone concentration. There was no statistically significant correlation between dexamethasone concentration and sex, weight or laterality. CONCLUSIONS: Our results show that the Dex-HA gel used in this study provides an effective and sustained dexamethasone release mechanism that might be utilized to treat conditions such as sudden sensorineural hearing loss. This could potentially reduce the morbidity and costs associated with IT treatment.