Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Small Cell Carcinoma of the Urinary Bladder: A Retrospective, Multicenter Rare Cancer Network Study of 107 Patients

PURPOSE Small cell carcinomas of the bladder (SCCB) account for fewer than 1% of all urinary bladder tumors. There is no consensus regarding the optimal treatment for SCCB. METHODS AND MATERIALS Fifteen academic Rare Cancer Network medical centers contributed SCCB cases. The eligibility criteria were as follows: pure or mixed SCC; local, locoregional, and metastatic stages; and age ≥18 years. The overall survival (OS) and disease-free survival (DFS) were calculated from the date of diagnosis according to the Kaplan-Meier method. The log-rank and Wilcoxon tests were used to analyze survival as functions of clinical and therapeutic factors. RESULTS The study included 107 patients (mean [±standard deviation, SD] age, 69.6 [±10.6] years; mean follow-up time, 4.4 years) with primary bladder SCC, with 66% of these patients having pure SCC. Seventy-two percent and 12% of the patients presented with T2-4N0M0 and T2-4N1-3M0 stages, respectively, and 16% presented with synchronous metastases. The most frequent curative treatments were radical surgery and chemotherapy, sequential chemotherapy and radiation therapy, and radical surgery alone. The median (interquartile range, IQR) OS and DFS times were 12.9 months (IQR, 7-32 months) and 9 months (IQR, 5-23 months), respectively. The metastatic, T2-4N0M0, and T2-4N1-3M0 groups differed significantly (P=.001) in terms of median OS and DFS. In a multivariate analysis, impaired creatinine clearance (OS and DFS), clinical stage (OS and DFS), a Karnofsky performance status <80 (OS), and pure SCC histology (OS) were independent and significant adverse prognostic factors. In the patients with nonmetastatic disease, the type of treatment (ie radical surgery with or without adjuvant chemotherapy vs conservative treatment) did not significantly influence OS or DFS (P=.7). CONCLUSIONS The prognosis for SCCB remains poor. The finding that radical cystectomy did not influence DFS or OS in the patients with nonmetastatic disease suggests that conservative treatment is appropriate in this situation.

Retinoic receptor gamma in squamous epithelial tumorigenesis

Retinoids, important modulators of squamous epithelial differentiation and proliferation, are effective in the treatment and prevention of squamous epithelial cancers, including squamous cell carcinomas (SCCs) of the skin. However, the mechanism is not well understood. Retinoids exert their effects primarily through two nuclear receptor families, retinoic acid receptors (RARα, β and γ) and retinoid X receptors (RXR(α, β and γ), ligand-dependent DNA-binding transcription factors that are members of the steroid hormone receptor superfamily. Retinoid receptor loss has been correlated with squamous epithelial malignancy. This has lead to the hypothesis that reduced RARγ expression and the resulting suppression of retinoid signaling contributes to squamous epithelial malignancy. To test this hypothesis, I attempted to reduce or abolish expression of RARγ, the predominant RAR in squamous epithelia, in several nontumorigenic human squamous epithelial cell lines. The most useful of these cell lines has been SqCCY1, the human head and neck squamous cell carcinoma cell line, along with several subclones stably transfected with RARγ sense and antisense expression constructs. By several criteria, we observed an overall suppression of squamous differentiation in RARγ sense transfectants and an enhancement in RARγ antisense transfectants, relative to parental SqCCY1 cells. We also observed that both sense and antisense cells could form tumors in athymic mice in vivo, while parental SqCCY1 cells could not. Although these results appear contradictory, several conclusions can be drawn. First, loss of RARγ contributes to squamous epithelial tumorigenesis. Second, overexpression of RARγ leads to tumor formation, suppressing differentiation and promoting proliferation, possibly due to a competitive inhibition of limiting concentrations of RXRα, a common heterodimeric partner for many nuclear receptors in addition to RARs, representing a mechanism for RARγ to modulate squamous epithelial homeostasis. The cause for tumorigenesis in the two conditions is likely due to different mechanisms/roles of RARγ in the cell, with the former as a retinoid signaling regulator; and the latter as an RXRα concentration modulator. Finally, High level of RARγ expression sensitizes cells to environmental RA, enhancing RARγ/RXRα-mediated RA signaling. Therefore, RA should be used in skin lesions with suppressed RARγ expression levels, not in skin lesions with overexpressed RARγ levels. ^

Analysis of prognostic factors for the development of metastases and survival in renal cell carcinoma patients

Introduction and objective. A number of prognostic factors have been reported for predicting survival in patients with renal cell carcinoma. Yet few studies have analyzed the effects of those factors at different stages of the disease process. In this study, different stages of disease progression starting from nephrectomy to metastasis, from metastasis to death, and from evaluation to death were evaluated. ^ Methods. In this retrospective follow-up study, records of 97 deceased renal cell carcinoma (RCC) patients were reviewed between September 2006 to October 2006. Patients with TNM Stage IV disease before nephrectomy or with cancer diagnoses other than RCC were excluded leaving 64 records for analysis. Patient TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were analyzed in relation to time to metastases. Time from nephrectomy to metastasis, TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were tested for significance in relation to time from metastases to death. Finally, analysis of laboratory values at time of evaluation, Eastern Cooperative Oncology Group performance status (ECOG), UCLA Integrated Staging System (UISS), time from nephrectomy to metastasis, TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were tested for significance in relation to time from evaluation to death. Linear regression and Cox Proportional Hazard (univariate and multivariate) was used for testing significance. Kaplan-Meier Log-Rank test was used to detect any significance between groups at various endpoints. ^ Results. Compared to negative lymph nodes at time of nephrectomy, a single positive lymph node had significantly shorter time to metastasis (p<0.0001). Compared to other histological types, clear cell histology had significant metastasis free survival (p=0.003). Clear cell histology compared to other types (p=0.0002 univariate, p=0.038 multivariate) and time to metastasis with log conversion (p=0.028) significantly affected time from metastasis to death. A greater than one year and greater than two year metastasis free interval, compared to patients that had metastasis before one and two years, had statistically significant survival benefit (p=0.004 and p=0.0318). Time from evaluation to death was affected by greater than one year metastasis free interval (p=0.0459), alcohol consumption (p=0.044), LDH (p=0.006), ECOG performance status (p<0.001), and hemoglobin level (p=0.0092). The UISS risk stratified the patient population in a statistically significant manner for survival (p=0.001). No other factors were found to be significant. ^ Conclusion. Clear cell histology is predictive for both time to metastasis and metastasis to death. Nodal status at time of nephrectomy may predict risk of metastasis. The time interval to metastasis significantly predicts time from metastasis to death and time from evaluation to death. ECOG performance status, and hemoglobin levels predicts survival outcome at evaluation. Finally, UISS appropriately stratifies risk in our population. ^

Role of obesity, weight gain, physical activity, and polymorphisms in the mTOR pathway and the risk of renal cell carcinoma

The interplay between obesity, physical activity, weight gain and genetic variants in mTOR pathway have not been studied in renal cell carcinoma (RCC). We examined the associations between obesity, weight gain, physical activity and RCC risk. We also analyzed whether genetic variants in the mTOR pathway could modify the association. Incident renal cell carcinoma cases and healthy controls were recruited from the University of Texas MD Anderson Cancer Center in Houston, Texas. Cases and controls were frequency-matched by age (±5 years), ethnicity, sex, and county of residence. Epidemiologic data were collected via in-person interview. A total of 577 cases and 593 healthy controls (all white) were included. One hundred ninety-two (192) SNPs from 22 genes were available and their genotyping data were extracted from previous genome-wide association studies. Logistic regression and regression spline were performed to obtain odds ratios. Obesity at age 20, 40, and 3 years prior to diagnosis/recruitment, and moderate and large weight gain from age 20 to 40 were each significantly associated with increased RCC risk. Low physical activity was associated with a 4.08-fold (95% CI: 2.92-5.70) increased risk. Five single nucleotide polymorphisms (SNPs) were significantly associated with RCC risk and their cumulative effect increased the risk by up to 72% (95% CI: 1.20-2.46). Strata specific effects for weight change and genotyping cumulative groups were observed. However, no interaction was suggested by our study. In conclusion, energy balance related risk factors and genetic variants in the mTOR pathway may jointly influence susceptibility to RCC. ^

Effects of exogeneous p53 on growth suppression, apoptosis, and differentiation in oral cancer cell lines

The p53 gene is known to be one of the most commonly mutated genes in human cancers. Many squamous cell carcinomas of the head and neck (SCCHNs) have been shown to contain nonfunctional p53 as well. The use of p53-mediated gene therapy to treat such cancers has become an intensive area of research. Although there have been varied treatment responses to p53 gene therapy, the role that endogenous p53 status plays in this response has not been thoroughly examined. Because of this, the hypothesis of this study examined the role that the endogenous p53 status of cells plays in their response to p53 gene therapy. To test this, an adenoviral vector containing p53 (p53FAd) was administered to three squamous cell carcinoma lines with varied endogenous p53. The SCC9 cell line demonstrates no p53 protein expression, the SCC4 cell line displays overexpression of a mutant p53 protein, and the 1986LN cell line displays low to no expression of wild-type p53 protein as a consequence of human papillomavirus infection. After treatment with p53FAd, the cells were examined for evidence of exogenous p53 expression, growth suppression, alterations in cellular proteins, G1 growth arrest, apoptosis, and differentiation state. Each cell line exhibited exogenous p53 protein. Growth suppression was seen most prominently in the SCC9 cells, to some extent in the 1986LN cells, and little was seen with the SCC4 cells. WAF1/p21 protein was induced in all three cell lines, while PCNA, bcl-2, and bax expression was not significantly affected in any of the lines. Apoptosis developed first in SCC9 cells, next in 1986LN cells, with little seen in the SCC4 cells. The SCC9 line was the only line to show significant GI growth arrest. No significant differences were observed in the overall expression of differentiation markers, aside from increased keratin 13 mRNA levels in all three lines indicating a possible tendency toward differentiation. This study indicates that the endogenous p53 status of squamous cell carcinomas appears to play a critical role in determining the response to p53 adenoviral gene therapy. ^

Constitutive expression of interleukin-6 in renal cell carcinoma is modulated by p53

Several studies indicate that interleukin-6 (IL-6) production is elevated in renal cell carcinoma (RCC) cells, and that IL-6 can serve as an autocrine growth factor in this malignancy. Wild type (wt) p53 represses transcription from the IL-6 promoter in an inducible system. The objective of this study was to determine the role of p53 in regulating constitutive IL-6 production in RCC cells. RCC cell lines containing mutant (mut) p53 produced significantly higher levels of IL-6 than those containing wt p53 (p < 0.05). Transfection of wt p53 into RCC cell lines resulted in significant repression of IL-6 promoter CAT activity p < 0.05). Mutant p53 was less effective at repressing IL-6 promoter activity in ACHN cells, and actually enhanced IL-6 promoter activity in the A498 cell line. A498 cells stably transfected with mutant p53 produced significantly higher levels of IL-6 than A498 cells transfected with an empty expression vector (p < 0.05). Electrophoretic mobility shift assay showed a significant decrease in binding of C/EBP, CREB, and NF-kB transcription factors to the IL-6 promoter in A498 cells transfected with wt p53. Mut p53 was unable to inhibit transcription factor binding to the IL-6 promoter in these cells. Mutant p53-expressing UOK 121LN cells showed decreased binding of C/EBP and CREB, but not NF-kB, following wt p53 transfection. These data suggest that (i) mutation of p53 contributes to the over-expression of IL-6 in RCC; and (ii) wt p53 represses IL-6 expression at least in part by interfering with the binding of C/EBP, CREB, and in some cases, NF-kB transcription factors to the IL-6 promoter. ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^

Virus del papiloma humano : presentación oral y su relación con el carcinoma espinocelular

El virus del papiloma humano (VPH) (HPV en su sigla en inglés) es un virus ADN con especial afinidad por células epiteliales, tanto cutáneas como mucosas, que incluyen el epitelio del cérvix, región anogenital y orofaríngea. Existen más de 120 subtipos de VPH subdivididos en dos grupos según su bajo o alto riesgo de inducir actividad oncogénica. En cavidad oral la manifestación clínica benigna de la infección es el papiloma plano.Sin embargo, un 12% de los carcinoma~ o rofaríngeos son causados por este virus. Se ha demostrado que la infección por VPH tiene un papel independiente como factor de riesgo para el desarrollo de carcinoma espinocelular en cavidad oral.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1–38 (20 μg/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with 125I-labeled GHRH antagonist JV-1–42, we demonstrated the presence of specific high-affinity (Kd = 0.25 ± 0.03 nM) binding sites for GHRH with a maximal binding capacity (Bmax) of 70.2 ± 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of 125I-JV-1–42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with 125I-VIP (Kd = 0.89 ± 0.14 nM; Bmax = 183.5 ± 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His1,125I-Tyr10,Nle27]hGHRH(1–32)NH2. Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of 125I-JV-1–42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

FHIT gene alterations in head and neck squamous cell carcinomas.

To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.

Validation of a new easy score to predict outcome in early stage renal cell carcinoma

Background e scopi dello studio. Il carcinoma renale rappresenta circa il 3% delle neoplasie e la sua incidenza è in aumento nel mondo. Il principale approccio terapeutico alla malattia in stadio precoce è rappresentato dalla chirurgia (nefrectomia parziale o radicale), sebbene circa il 30-40% dei pazienti vada incontro a recidiva di malattia dopo tale trattamento. La probabilità di recidivare può essere stimata per mezzo di alcuni noti modelli prognostici sviluppati integrando sia parametri clinici che anatomo-patologici. Il limite principale all’impiego nella pratica clinica di questi modelli è legata alla loro complessità di calcolo che li rende di difficile fruizione. Inoltre la stratificazione prognostica dei pazienti in questo ambito ha un ruolo rilevante nella pianificazione ed interpretazione dei risultati degli studi di terapia adiuvante dopo il trattamento chirurgico del carcinoma renale in stadio iniziale. Da un' analisi non pre-pianificata condotta nell’ambito di uno studio prospettico e randomizzato multicentrico italiano di recente pubblicazione, è stato sviluppato un nuovo modello predittivo e prognostico (“score”) che utilizza quattro semplici parametri: l’età del paziente, il grading istologico, lo stadio patologico del tumore (pT) e della componente linfonodale (pN). Lo scopo del presente studio era quello di validare esternamente tale score. Pazienti e Metodi. La validazione è stata condotta su due coorti retrospettive italiane (141 e 246 pazienti) e su una prospettica americana (1943 pazienti). Lo score testato prevedeva il confronto tra due gruppi di pazienti, uno a prognosi favorevole (pazienti con almeno due parametri positivi tra i seguenti: età < 60 anni, pT1-T3a, pN0, grading 1-2) e uno a prognosi sfavorevole (pazienti con meno di due fattori positivi). La statistica descrittiva è stata utilizzata per mostrare la distribuzione dei diversi parametri. Le analisi di sopravvivenza [recurrence free survival (RFS) e overall survival (OS)] sono state eseguite il metodo di Kaplan-Meier e le comparazioni tra i vari gruppi di pazienti sono state condotte utilizzando il Mantel-Haenszel log-rank test e il modello di regressione di Cox. Il metodo di Greenwood è stato utilizzato per stimare la varianza e la costruzione degli intervalli di confidenza al 95% (95% CI), la “C-statistic” è stata utilizzata per descrivere l’ accuratezza dello score. Risultati. I risultati della validazione dello score condotta sulle due casistiche retrospettive italiane, seppur non mostrando una differenza statisticamente significativa tra i due gruppi di pazienti (gruppo favorevole versus sfavorevole), sono stati ritenuti incoraggianti e meritevoli di ulteriore validazione sulla casistica prospettica americana. Lo score ha dimostrato di performare bene sia nel determinare la prognosi in termini di RFS [hazard ratio (HR) 1.85, 95% CI 1.57-2.17, p < 0.001] che di OS [HR 2.58, 95% CI 1.98-3.35, p < 0.001]. Inoltre in questa casistica lo score ha realizzato risultati sovrapponibili a quelli dello University of California Los Angeles Integrated Staging System. Conclusioni. Questo nuovo e semplice score ha dimostrato la sua validità in altre casistiche, sia retrospettive che prospettiche, in termini di impatto prognostico su RFS e OS. Ulteriori validazioni su casistiche internazionali sono in corso per confermare i risultati qui presentati e per testare l’eventuale ruolo predittivo di questo nuovo score.

Resistência de células de carcinoma epidermóide bucal à terapia fotodinâmica mediada pelo ácido 5-aminolevulínico

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais.

Expressão dos receptores Toll-like em carcinoma espinocelular de orofaringe

Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe.