Cirka 300 000 individer drabbas årligen i världen av oral cancer och mer än nittio procent av alla orala cancerformer utgörs av skivepitelcancer. Den femåriga prognosen är generellt 50 % och den 5-åriga relativa överlevnaden har under en tioårsperiod förblivit densamma. Detta motiverar utvecklingen av bättre prognostiska markörer och diagnostiska metoder för att tidigt identifiera de patienter som har risk att utveckla oral skivepitelcancer för att förbättra prognosen och minska lidandet genom tidig insatt behandling. β-catenin är en adhesionsmolekyl som är viktig för bibehållandet av cellulär integration och avvikelser i celladhesionsmolekyler tros spela en central roll när tumörceller invaderar närliggande vävnad det vill säga metastaserar till andra organ.Syftet med studien är att med hjälp av immunohistokemi undersöka om β-catenin kan fungera som en prognostisk markör för utvecklingen av oral skivepitelcancer. Detta görs genom att jämföra förekomsten av β-catenin med hjälp av monoklonala antikroppar i normalt skivepitel, dysplasi och cancer från 18 patienter som har diagnostiserats med oral skivepitelcancer. Infärgningen av Beta catenin jämfördes i normalt oralt skivepitel med cancer och dysplasi för alla biopsier för att undersöka om det förekommer någon skillnad av infärgningen. Förutom detta skedde även en jämförelse av normalt skivepitel med dysplasi och cancer inom varje enskild biopsi.Resultaten visade att det finns en skillnad i uttrycket av β-catenin i normalt skivepitel jämfört med dysplasi och cancer i denna patientgrupp. I denna studie visade mer än 70 % av biopsierna en stark eller måttlig och stark infärgning av β-catenin i normalt skivepitel, mer än 60 % av biopsierna visade en måttlig eller måttlig och svag infärgning av dysplasi och 58,8 % av alla biopsier visade svag infärgning eller ingen och svag infärgning av skivepitelcancer. Då studien visar att mängden av β-catenin är starkast i normalt oralt skivepitel, måttligt i dysplasi och svagast i cancer tyder detta på att β-catenin skulle kunna vara en viktig faktor i utvecklingen av skivepitelcancer i munhålan vilket stämmer väl överens med resultat från andra studier. / Approximately 300,000 individuals are affected every year in the world of oral cancer and more than ninety percent of all oral cancers consists of squamous cell carcinoma. The five-year prognosis is generally 50 % and the 5-year relative survival has over ten years remained the same. This motivates the development of better prognostic markers and diagnostic methods for the early identification of patients at risk of developing oral squamous cell carcinoma to improve prognosis and reduce the suffering of these patients with early treatment.β-catenin is an adhesion molecule that is important for the maintenance of cellular integration and abnormalities of cell adhesion molecules is thought to play a central role in tumorigenesis. The abnormalites is though to enhance tumour cells to break loose from neighbouring cells and invade nearby tissues and organs, however the exact mechanisms are unknown. The purpose of the study is that using immunohistochemistry to examine whether β-catenin may serve as a prognostic marker for the development of oral squamous cell carcinoma. This is done by examining the presence of β-catenin with monoclonal antibodies in 18 biopsies with normal squamous epithelia, dysplasia and cancer from 18 patients diagnosed with oral squamous cell carcinoma from the department of Oral Pathology at Malmö Högskola, Malmö. The staining of beta catenin was compared in normal oral squamous cancer and dysplasia for all biopsies to see whether there is any difference of dyeing. Besides this, there was also a comparison of normal squamous epithelium with dysplasia and cancer in each biopsy.The results showed that there is a difference in the expression of β-catenin in normal squamous epithelium, dysplasia and cancer in this population. In this study, more than 70 % of the biopsies expressed a strong or moderate and strong staining of β -catenin in normal oral squamous epithelium, more than 60 % of the biopsies showed a moderate or moderate and weak staining of dysplasia and 58.8 % of all biopsies showed weak or no staining and weak staining of squamous cell carcinoma.As the study shows that the amount of β -catenin is strongest in normal oral squamous epithelium, moderate in dysplasia and weakest in cancer, this suggests that β -catenin could be an important factor in the development of squamous cell carcinoma of the oral cavity which is in line with results from other studies.

Background. Head and neck cancers (HNC) are among the most common malignancies worldwide, and about 90–92% of oral neoplasias are oral squamous cell carcinomas (OSCC). Alcohol and tobacco consumption have been recognized as the main risk factors for OSCC development. Oncogenic viruses, such as human papillomavirus (HPV) or Epstein-Barr virus (EBV), as well as genetic alterations may also contribute to tumour formation.  Aims. To study the prevalence of HPV, EBV, Herpes simplex type-1 (HSV-1), and HPV-16 and their integration status as well as the molecular mechanisms that can serve as a basis for the development of OSCC. Results. In Paper I we reported a statistically significant increase in the prevalence of HPV-16 in oral epithelial dysplasia (OED) and OSCC samples compared to controls. A statistically significant increase was also seen in integrated HPV-16 compared to episomal viral forms when comparing OED and OSCC samples. Paper II reported the detection of HSV-1 in 54% of healthy samples, in 36% of oral leukoplakia samples, and 52% of OSCC samples. However, these differences were not statistically significant. In Paper III we reported a statistically significant increase in the detection of HPV-positive samples when comparing nested polymerase chain reaction (PCR) with single-PCR results in OSCC and fresh oral mucosa. Paper IV reported that the highest prevalence of HPV (65%) was seen in Sudan, while an HSV-1 prevalence of 55% and an EBV prevalence of 80% were seen in the UK. Finally, Paper V reported that the mRNA levels of Bcl-2, keratin 1, keratin 13, and p53 were significantly lower and that the level of survivin was significantly higher in the OSCC samples of the toombak users than in their paired control samples. Significant downregulation in keratin 1 and keratin 13 expression levels was found in the OSCC samples of the non-toombak users relative to their normal control samples. Conclusion. HPV-16 integration was increased in oral epithelial dysplasia and OSCC compared to normal oral mucosa. Nested PCR is a more accurate method of establishing HPV prevalence in samples containing low copy numbers of HPV DNA. HPV and EBV may be a risk factor in OSCC development. Our findings confirmed the role of survivin in OSCC carcinogenesis and survivin might be interesting as a biomarker to be monitored. The results presented here provide both clinical and biological insights that will bring us closer to the goal of managing this disease and improving treatment and outcomes for future patients.

Photodynamic therapy, PDT, is a method to diagnose and treat cancer. In PDT a sensitizer is administered to the patient and this sensitizer is accumulated in tumors. If the sensitizer-containing tumor is subjected to a laser of a specific wavelength the tumor is fluorescing allowing diagnostics. If other wavelengths are used a process involving reactive oxygen species and singlet oxygen is started and the tumor cells are killed. This process thus requires oxygen as well. This thesis investigates how UV-induced damage of the skin and different physiological factors of the skin influences the uptake of 5- aminolevulinic acid, ALA, and its conversion to the active sensitizer protoporphyrin IX, PpIX. It shows that UV-induced damage affects both the uptake and production of PpIX. UV-induced damage lowers the PpIX produced after ALA application both if the damage is acute and in chronically UV-affected skin. The PpIX production differs inter and intra individually. When looking how different physiological factors affect the PpIX production after topically applied ALA the thesis shows that an increase of temperature increases the production. No correlation between the formation of PpIX and the density of hair follicles was found and a weak correlation was seen comparing the epidermal and total dermal thickness and PpIX production The thesis also shows how PDT is used in treating laryngeal malignancies. It shows that it is possible to cure laryngeal tumors (both squamous cell carcinomas and sarcomas) using PDT primarily, and that the cure rate as well as outcome of voice and patient safety is comparable to the conventional treatment modalities. PDT can also be used as a function and organ sparing treatment for recurring laryngeal cancers, both squamous cell carcinomas and sarcomas.

HNSCC accounts for 7 percent of all new cancer occurrences. Despite currently available treatments, there continues to be a high mortality and recurrence rate in HNSCC. Well over 50 percent of all cancer patients receive chemotherapy as a standard treatment. However, only 5 percent of these cases have been shown to help with treatment of the disease. Formerly, two options were available for drug testing: in vivo animal models, and in vitro two-dimensional models. While in vivo models remain the most representative, their use is burdened by high costs, time constraints, and ethical concerns. 2D models are simple to use and cost effective, although they have been shown to produce inaccurate data regarding chemotherapeutic drug resistance due to their 2D arrangement and altered gene expression. Researchers for the past decade have been working to create 3D models that more accurately represent in vivo systems in order to evaluate chemotherapeutic efficacy and improve clinical outcomes. In line with this agenda, novel 3D head and neck cancer models were created out of electrospun synthetic polymers seeded with either HN6 or HN12 cancer cells. The models were then treated with chemotherapeutic drugs (either paclitaxel or cisplatin), and, after 72 hours, subjected to a live-dead assay in order to determine the cytotoxic effects of the drugs. 2D cultures of HN6 and HN12 were also and subject to a WST-1 assay after 72 hours. The results of the treated-scaffold assays were then compared to the results of the 2D culture assays, and, as predicted, the cancer cells in a 3D culture system proved to be more resistant to chemotherapeutic drugs. The underlying assumption for this study being that a 3D culture system based on precisely defined structural parameters would provide a practical environment to screen therapeutics for anti-cancer efficacy. To prove this, 3D scaffolds of three different fiber sizes were developed by electrospinning different concentrations of Poly(L-lactic acid) (“PLLA”) (55mg/ml, 115mg/ml, and 180mg/ml) onto a mandrel that was perforated to allow for increased porosity. The resultant small, medium, and large scaffolds were then subjected to concentrated hydrochloric acid (HCl) pretreatment in order to make them less hydrophobic. Different fiber diameters represented different ECM environments for both HN6 and HN12. It was proven that both cell types thrived best in small fibers (55mg/ml-115mg/ml) than in large fibers. It was also reaffirmed through live-dead anlaysis of cells seeded on 3D scaffolds and treated with IC90 values of cisplatin that the head and neck cancer cells were more resistant which is more representative to the 3D environment of cancer cells in vivo.

The primary focus of this research is the mechanisms of cell death in head and neck squamous cell carcinoma (HNSCC) treatment. These cancers typically originate in squamous cells that line the moist mucosal surfaces of head and neck. HNSCC is commonly treated with a platinum based agent, cisplatin. While the drug does offer strong antitumor effects, its prolonged use often results in tumor-acquired resistance, which limits treatment effectiveness. We have shown that cisplatin treatment induces the expression of a pro-apoptotic BCL-2 family member Noxa, which then initiates caspase- dependent apoptosis through its binding and sequestration of pro-survival protein MCL-1 for its inactivation. Without Noxa induction, cell death is significantly reduced when treating HNSCCs with cisplatin. The objectives of this study are (1) to determine the molecular mechanisms by which Noxa induces cell death in HNSCC cells; (2) to determine the molecular mechanisms of cisplatin-resistance in isogenic HNSCC cell lines. We observed an increase of apoptosis by ectopic expression of Noxa in all HNSCC cell lines tested, but not in immortalized human normal oral keratinocytes (NOK), suggesting that Noxa overexpression is sufficient to induce tumor-specific cell death. Noxa-induced cell death was mediated by BAX and BAK activation. BAK activation was mediated through Noxa binding to MCL-1, but not BCL-XL. Cisplatin- resistant cells induced less Noxa and apoptosis, supporting that Noxa induction is prerequisite for apoptosis induced by cisplatin. Taken together, Noxa induces tumor- specific cell death in HNSCC cells primarily through BAX and BAK activation, which suggests the therapeutic potential of this protein.

No description available.

São freqüentes alterações em vias de sinalização de genes de desenvolvimento, no que diz respeito ao carcinoma epidermóide de boca (CEB). Vinte e nove casos de CEB e tecido não tumoral adjacente à neoplasia foram investigados através da técnica de hibridização “in situ". Os transcritos dos genes WNT5A e HOXB5 foram observados em todos os casos de tumor. A hibridização “in situ" revelou que o WNT5A estava mais expresso em tumores bem diferenciados. Adicionalmente, observou-se transcritos do WNT5A em glândulas salivares menores, estroma glandular, estroma tumoral e alguns vasos sanguíneos Entretanto, com respeito ao HOXB5 não foi possível estabelecer mudança do padrão do transcrito nos diferentes graus histológicos nas amostras de CEB. O HOXB5 também pôde ser identificado em alguns fragmentos de glândulas salivares menores, tecido muscular e em endotélio. Os resultados do presente estudo sugerem que a expressão dos genes WNT5A e HOXB5 podem estar relacionadas com a diferenciação e progressão do câncer de boca. / Disruption in developmental genes pathway are common in oral squamous cell carcinoma (OSCC). This study investigated the pattern of expression of two developmental genes, WNT5A and HOXB5, in 29 cases of OSCC and adjacent non tumoural tissue using in situ hybridization technique. Transcripts for WNT5A and HOXB5 were detected in all tumoral samples. In situ hybridization technique demonstrated that WNT5A transcripts were mainly detected in well differentiated tumors when compared with moderately and undifferentiated OSCC. WNT5A transcripts were also observed in accessory salivary glands, glandular stroma, and vessels. Therefore, for the HOXB5 transcript it was not possible to stability a relationship with the tumoral histological grade. The expression of HOXB5 transcripts in non tumoral samples was detected in salivary glands, glandular stroma, endothelium, and muscle. Results suggest that WNT5A and HOXB5 genes play a possible role in tumor differentiation and cancer progression.

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. / The incidence of HPV- associated oropharynx squamous cell carcinoma (OPSCC) has increased in recent years. HPV- associated OPSCC has a better prognosis than OPSCC not infected with HPV. In cervical carcinoma, HPV oncoproteins E6 and E7 influence the expression of Toll-like receptors (TLR). To date, 10 TLRs have been identified in humans and many are important for the detection of bacteria, fungi and viruses, as well as regression and tumor promotion. This influence of TLR in carcinogenesis has been the subject of numerous studies, due to the connection between inflammation and cancer. This study aimed to determine differences in the expression and function of Toll-like receptor in OPSCC. Thirty-seven tumours were selected and immunohistochemistry for p16 and TLR4 was performed. Two HPV16-associated OPSCC and two OPSCC not associated to HPVcell lines were used. qPCR was performed to analyze the expression of TLR1-10, IL-6 and IL-8. The detection of the main TLRs (TLR1, TLR2, TLR6 and TLR4) was performed by flow cytometry. For activation of the TLR2-signaling pathway and subsequent analysis of IL6 and IL8 expression, cells were stimulated with peptidoglycan. To verify the expression and function TLR4 cells were stimulated with LPS and LPS UP and subsequent analysis of IL-6 and IL-8 by ELISA. The results showed differences in the expression of TLR1 and TLR6 between the HPV- group and the HPV+ group and differences in protein expression of TLR9. TLR2 has increased protein expression in all cell lines and demonstrates the trigger of the immune response, with the secretion of IL6 and IL8 in HPV- cell lines (SCC72 and SCC89) and one HPV+ cell line (SCC2). Interestingly, TLR4 showed no significant differences in gene and protein expression. However, HPV+ cell lines showed no pro-inflammatory response even when stimulated with LPS and LPS UP, a specific agonist of TLR4. This work helps to establish the profile of the expression of Toll-like receptors in OPSCC (SCC72 and SCC89) and HPV- associated OPSCC (SCC2 and SCC90), in vitro, and points to changes in the signaling pathway mediated by TLR4. In addition, our results open new avenues for future studies to assess changes in the innate immune system caused by HPV in oropharynx carcinomas.

Diversos mecanismos celulares estão associados à patogênese do Carcinoma Epidermoide de Cabeça e Pescoço (CECP). Algumas dessas alterações envolvem proteínas pertencentes à via de sinalização do Akt, e o fator de transcrição NF-kB, o qual têm importante papel na fisiologia normal e no câncer. A proteína COX-2, descrita em processos inflamatórios, também participa da carcinogênese e está associada com a via de sinalização do Akt e com o NF-kB. Dendrímeros são uma forma única de nanotecnologia, surgindo como nanotransportadores com a capacidade de penetrar na célula tumoral liberando drogas quimioterápicas em seu interior. Os benefícios desta tecnologia são o aumento da eficicácia do princípio ativo utilizado e a redução dos seus efeitos secundários tóxicos. O Celecoxibe, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, no entanto seu mecanismo de ação no CECP não é totalmente compreendido. Neste trabalho, um Dendrímero de Poliglicerol associado ao Celecoxibe (PGLD-celecoxibe) foi sintetizado e caracterizado por técnicas de espectroscopia ¹H-RMN, ¹³C-RMN, Maldi-Tof, TLC e DSC. Além disso, o conjugado foi testado in vitro em três linhagens celulares de CECP. O PGLD-Celecoxibe foi sintetizado com sucesso e promoveu a redução da dose capaz de inibir a proliferação celular, reduzindo o IC 50 do Celecoxibe de forma significativa em todas as linhagens celulares, se aproximando da dose sérica alcançada por este medicamento, resultado corroborado pelo Ensaio de Migração Celular. O mecanismo de morte celular observado foi a apoptose, associada a diminuição significativa da expressão de COX-2 ou por uma via alternativa independente. Alguns dos grupos tratados apresentaram alteração na expressão das proteínas pAkt e NF-kB. / Several cellular mechanisms are associated with the pathogenesis of Head and Neck Squamous Cell Carcinoma (HNSCC). Some of these alterations involve proteins in the Akt signaling pathway and the transcription factor NF-kB, which plays an important role in normal physiology and in cancer. COX-2 protein, described in inflammatory processes, and also involved in the carcinogenesis is associated with the Akt signaling pathway and the NF-kB. Dendrimers are a unique form of nanotechnology, emerging as nanocarriers with the ability to penetrate the tumor cell releasing chemotherapeutic. This technology increases the active substance efficiency and reduces its toxic side effects. Celecoxib, a nonsteroidal anti-inflammatory, selective inhibitor of COX-2 has been shown to be an important anticancer agent, but its action mechanism in HNSCC is not fully understood. A polyglycerol dendrimer linked to celecoxib (PGLD-Celecoxibe) was synthesized and characterized by NMR spectroscopy ¹H-NMR, ¹³C-NMR,TLD, DSC and Maldi-Tof techniques. In addition, in vitro assays were performed in three HNSCC cell lines The PGLD-Celecoxibe was successfully synthesized and provided a decrease in the dose able to inhibit cell proliferation reducing the IC 50 index of Celecoxib significantly in all cell lines, approaching to the serum dose achieved for this product, result supported by Wound Healing Assay. The cell death mechanism observed was apoptosis, which can be associated with significant reduction of expression of COX-2 also may be occurring by a COX-2 independent pathway. Some of the treated groups showed alterations in pAkt and NF-kB proteins expression.

A laminina contém peptídeos que podem ser liberados por proteólise. Nosso laboratório estuda os efeitos de peptídeos da laminina em biologia tumoral. Neste trabalho, verificamos se C16 (cadeia g1) estimularia invasão e atividade de invadopódios em células de carcinoma epidermóide (CAL27) e fibrossarcoma (HT1080). C16 promoveu aumento na taxa de invasão e atividade de invadopódios em ambas às linhagens celulares, comparado ao peptídeo controle C16SX. Microscopia em time-lapse demonstrou que C16 induz aumento na atividade de invadopódios em função do tempo. C16 estimula fosforilação de Src e ERK 1/2, e inibição da via ERK reduz invasão e atividade de invadopódios relacionados ao peptídeo. C16 conjugado à rodamina foi encontrando decorando a membrana de células CAL27, sugerindo possível interação com receptores. Diminuição dos níveis de integrina b1 reduzem atividade de invadopódios em amostras tratadas com C16. Nossos dados sugerem que C16 regula invasão e atividade de invadopódios em células CAL27 e HT1080, provavelmente por meio de Src, ERK e integrina b1. / Laminin harbors bioactive peptides released upon tumor-induced proteolysis. Our Laboratory has been studying laminin peptides effects in tumor biology. Here we addressed whether C16 (g1 chain) would regulate invasion and invadopodia activity in cell lines from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). C16 increased invasion rate and invadopodia activity compared to control peptide (C16SX). Through time-lapse microscopy, we observed that C16 stimulated invadopodia activity overtime. We searched for signaling pathways related to peptide effects. C16 stimulated Src and ERK 1/2 phosphorylation, and ERK signaling cascade inhibition decreased C16-induced invasion and invadopodia. Next, we addressed how C16 would interact with tumor cells. Rhodamine-conjugated C16 was found decorating CAL27 cell membrane, suggesting an interaction with receptors. Knockdown of b1 integrin reduced invadopodia activity of C16-treated cells. We propose that C16 regulates invasion and invadopodia activity of CAL27 and HT1080 cells through Src, ERK and b1 integrin.