Differential molecular response of larynx cancer cell lines to combined VPA/CDDP treatment.

Successful treatment of advanced larynx squamous cell carcinoma (LSCC) remains a challenge, mainly due to limited response to chemotherapy and the phenomenon of the drug resistance. Therefore, new chemotherapeutic solutions are needed. The aim of this study was to explore benefit of combined cisplatin (CDDP) and valproic acid (VPA) therapy in patients' derived LSCC cell lines. Cell viability assay was used to establish cellular response to the drug by isobolography followed by RNA sequencing (RNAseq) analysis. Danio rerio were used for in vivo studies. Depending on the cell line, we found that the combinations of drugs resulted in synergistic or antagonistic pharmacological interaction, which was accompanied by significant changes in genes expression profiles. The presented therapeutic scheme efficiently blocked tumor growth in an in vivo model, corresponding to the in vitro performed studies. Interestingly the RK5 cell line, upon the combined treatment acquired a molecular profile typically associated with epithelial to mesenchymal transition (EMT). Hence, our studies demonstrates that patient-specific personalized therapy of larynx cancer should be considered and the combination of cisplatin and valproic acid should be explored as a potential therapeutic strategy in the treatment of larynx cancer.

Imperatorin as a Promising Chemotherapeutic Agent Against Human Larynx Cancer and Rhabdomyosarcoma Cells.

Naturally occurring coumarins are bioactive compounds widely used in Asian traditional medicine. They have been shown to inhibit proliferation, induce apoptosis, and/or enhance the cytotoxicity of currently used drugs against a variety of cancer cell types. The aim of our study was to examine the antiproliferative activity of different linear furanocoumarins on human rhabdomyosarcoma, lung, and larynx cancer cell lines, and dissolve their cellular mechanism of action. The coumarins were isolated from fruits of Angelica archangelica L. or Pastinaca sativa L., and separated using high-performance counter-current chromatography (HPCCC). The identity and purity of isolated compounds were confirmed by HPLC-DAD and NMR analyses. Cell viability and toxicity assessments were performed by means of methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. Induction of apoptosis and cell cycle progression were measured using flow cytometry analysis. qPCR method was applied to detect changes in gene expression. Linear furanocoumarins in a dose-dependent manner inhibited proliferation of cancer cells with diverse activity regarding compounds and cancer cell type specificity. Imperatorin (IMP) exhibited the most potent growth inhibitory effects against human rhabdomyosarcoma and larynx cancer cell lines owing to inhibition of the cell cycle progression connected with specific changes in gene expression, including CDKN1A. As there are no specific chemotherapy treatments dedicated to laryngeal squamous cell carcinoma and rhabdomyosarcoma, and IMP seems to be non-toxic for normal cells, our results could open a new direction in the search for effective anti-cancer agents.

Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells.

Objective: Laryngeal squamous cell carcinoma is one of the most common malignant tumors in the head and neck region. Due to the poor response to chemotherapeutics in patients and low survival rate, successful treatment of larynx cancer still remains a challenge. Therefore, the identification of novel treatment options is needed. We investigated the anticancer effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on two different laryngeal cancer cell lines RK33 and RK45. We also studied the antiproliferative action of SAHA in combination with cisplatin and defined the type of pharmacological interaction between these drugs. Materials and Methods: Viability and proliferation of larynx cancer cell lines were studied by methylthiazolyldiphenyl-tetrazolium bromide method and 5-bromo-2-deoxyuridine incorporation assay, respectively. The type of interaction between SAHA and cisplatin was determined by an isobolographic analysis. Western blotting, flow cytometry and quantitative polymerase chain reaction method were used to determine acetylation of histone H3, cell cycle progression and genes expression, respectively. Apoptosis was assessed by means of nucleosomes released to cytosol. Results: SAHA alone or in combination with cisplatin inhibited larynx cancer cells proliferation, whereas displayed relatively low toxicity against normal cells - primary cultures of human skin fibroblasts. The mixture of SAHA with cisplatin exerted additive and synergistic interaction in RK33 and RK45 cells, respectively. We showed that SAHA induced hyperacetylation of histone H3 K9, K14 and K23 and triggered apoptosis. SAHA also caused cell cycle arrest by upregulation of CDKN1A and downregulation of CCND1 encoding p21WAF1/CIP1 and cyclin D1 proteins, respectively. Conclusion: Our studies demonstrated that SAHA may be considered as a potential therapeutic agent against larynx tumors.

Glutamate receptors in laryngeal cancer cells.

AIM: Despite recent improvements in treatment strategies, the results of chemotherapy in patients with advanced squamous cell carcinoma of the larynx are not satisfactory. Thus, the development of new approaches which influence specific metabolic pathways are needed. In the last decade, evidence has emerged implicating a role for glutamate as a signal mediator in tumors. MATERIALS AND METHODS: The presence of glutamate receptor subunits in two laryngeal cancer cell lines (RK33 and RK45) was evaluated by means of end-point PCR, real-time PCR, and immunocytochemistry. RESULTS: Glutamate receptor subunits are differentially expressed in laryngeal cancer cell lines. In addition, we show that selected ionotropic glutamate receptor antagonists and metabotropic glutamate receptor 5 antagonist inhibit proliferation of laryngeal cancer cells. Glutamate antagonists also affected activity of extracellular signal-regulated kinases 1/2 in tumor cells. CONCLUSION: Signaling through glutamate receptors may influence growth of laryngeal cancer cells and may constitute an adjunctive therapeutic target.

[Evaluation of cytochrome concentration in peripheral blood lymphocytes of patients with laryngeal cancer]. / Ocena stezenia cytochromu c w limfocytach krwi obwodowej chorych na raka krtani.

The aim of the study was evaluation of cytochrome concentration in the peripheral blood lymphocytes of patients with laryngeal cancer. The study was conducted in a group of 62 patients presenting different clinical advancement of the disease. The study material consisted of the studied population's peripheral blood from which T lymphocytes were isolated and incubated with monoclonal antibodies. To evaluate antigens' expression, a FACS Galibur flow cytometre was used; the evaluated cells were labeled with fluorochrome-conjugated monoclonal antibodies. Next, mononuclear cells were rinsed with cold PBS and suspended in the lysis buffer. In the obtained cell lysate the c cytochrome concentration was determined with the use of an immunoenzymatic test, HumanCytochrome cELISA Kit (BenderMed Systems, Austria). Obtained results were compared with the measurements taken in 20 healthy individuals who constituted the control group. On the basis of conducted study it was found that the level of c cytochrome concentration was significantly increased in the T CD3+ lymphocytes in the peripheral blood of patients with stage IV of laryngeal cancer. A positive correlatio was also found between the cytochrome level in lymphocytes and the advancing stage of the disease. Our own observation give grounds to conjecture that together with the progress of laryngeal cancer the energetic potential of lymphocytes increases and so does the readiness of the lymphatic cells to undergo the redox processes, therefore the amount of c cytochrome in the cells increases.

Preliminary results of prognostic significance of proliferating cell nuclear antigen expression in advanced primary larynx carcinomas and lymph node metastases.

INTRODUCTION: The aim of this study was to investigate the prognostic significance of proliferating cell nuclear antigen (PCNA) expression in laryngeal carcinoma in relation to clinicopathological features. Special emphasis was placed on examining the relationship of PCNA expression in the primary tumour and PCNA expression in corresponding lymph node metastases obtained from the same patients. MATERIAL AND METHODS: The study included 60 patients with advanced larynx carcinoma who had received treatment and follow-up for at least 5 years. Sixty laryngeal carcinoma specimens and metastatic lymph nodes from 24 patients were examined for immunohistochemical PCNA expression. RESULTS: The percentages of PCNA positive cells were significantly higher in the primary tumours which developed lymph node metastases than in those without metastases. The fraction of PCNA immunolabelled cells in metastatic lymph nodes increased significantly when compared with the PCNA positive cell score in their corresponding primary tumours obtained from the same patient. There was a significant difference in PCNA index score in primary tumours between the group of patients who survived a 5-year period and those who died within 5 years after treatment. CONCLUSIONS: Our data demonstrate that a high proliferation index in primary larynx tumours is retained and increased in corresponding lymph node metastases. Measurement of the fraction of cancer cells stained for PCNA in primary larynx carcinomas can be helpful in selecting tumours with high aggressiveness potential that are more likely to develop neck metastases and thereby in identifying patients who need elective lymph node dissection or additional treatment.

[Cigarette smoking and alcohol consumption in patients with oral cancer]. / Palenie papierosów i spozywanie alkoholu w grupie chorych z nowotworami zlosliwymi jamy ustnej.

The study aim was to analyse the frequency of simultaneous smoking and alcohol consumption in 142 patients with oral cancer and in the control group. A connection between the prevalence of these habits and epidemiological traits of the patients and features of malignant lesions was evaluated. Statistical analysis revealed that simultaneous smoking and alcohol abuse were significantly more frequent in the cases group. A strong influence of these habits on the development of oral squamous cell carcinoma was noted.

Fluoxetine inhibits the extracellular signal regulated kinase pathway and suppresses growth of cancer cells.

Fluoxetine (FLX) is a widely prescribed antidepressant. Concerns were raised about the potential impact of FLX on cancer growth, because FLX was shown to promote development of breast cancer in rodents. Here we studied the effect of FLX on tumor growth in lung (A549), colon (HT29), neuroblastoma (SKNAS), medulloblastoma/rhabdomyosarcoma (TE671), astrocytoma (MOGGCCM) and breast (T47D) cancer cells and explored potential mechanisms of its action. In our study, FLX reduced growth of cancer cells in vitro in a concentration dependent manner. The antiproliferative effect of FLX was already evident after 24 hours exposure and more pronounced at 96 hours. We demonstrate that FLX inhibits phosphorylation of ERK1/2 kinases in a time and concentration-dependent manner, followed by reduced phosphorylation of transcription factor c-Myc in A549 and HT29 cells. After treatment with FLX, A549 and HT29 cells demonstrated concentration-dependent decrease in the expression of c-fos, c-jun, cyclin A, cyclin D1, and increased expression of p21(waf1) and p53 genes, which resulted in slowing of the cell cycle progression. We suggest that these changes could be responsible for observed inhibition of cancer cell proliferation during FLX treatment in vitro.

Application of primary cell cultures of laryngeal carcinoma and laser scanning cytometry in the evaluation of tumor reactivity to cisplatinum.

Unsatisfactory effects of treatment of laryngeal carcinoma patients stimulate the clinicians as well as researchers to develop new more effective treatment models and to find new reliable prognostic factors. The aim of the present study was the evaluation of the use of primary cell cultures of the laryngeal carcinoma and laser scanning cytometry (LSC) in the assessment of tumor reactivity to cisplatinum. Nineteen primary cultures of laryngeal carcinoma cells established from fragments of laryngeal carcinoma infiltrations were cultured with or without cisplatin, stained with monoclonal antibodies against P53 and BCL-2 proteins and analyzed by LSC. Cisplatin added to the culture medium leads to the significant increase of P53 expression and decrease of BCL-2 expression. Moreover, changes of P53 and BCL-2 expressions were significantly correlated. Our findings of apoptosis regulatory mechanisms could be useful in patient qualification for the chemotherapeutic follow-up treatment.

[Tobacco smoking as a risk factor for oral cancer]. / Palenie tytoniu jako czynnik ryzyka zachorowan na nowotwory zlosliwe jamy ustnej.

The study involved 254 patients with primary oral cancer. The analysis of prevalence, duration and intensity of smoking habit was carried out both in cases and control group. The prevalence of smoking was significantly more frequent in cases than in controls. Any significant differences between cases and controls in duration of the habit and in number of cigarettes smoked daily were not noted. The findings indicated significant dependence between the smoking habit and the prevalence of oral squamous cell carcinoma.

[Lack of prognostic significance of carcinoembryonic antigen (CEA) preoperative serum levels in patients with advanced larynx cancer]. / Brak wartosci diagnostycznej przedoperacyjnego oznaczania steienia antygenu rakowo-plodowego (CEA) w surowicy pacjentów z zaawansowanym rakiem krtani.

The purpose of this study was to identify the relationship between preoperative serum levels of carcinoembryonic antigen (CEA) and clinicopathological features in advanced stage of larynx cancer. The mean CEA serum concentrations were below cutoff value, which exclude the CEA as a serum marker in diagnosis of larynx cancer. However, significant correlations were found between CEA levels and tumor size, lymph node metastasis and clinical stage of the disease. The pretreatment CEA level was increased above cut-off value only in 6% of tested patients and thereby is not a prognostic factor in larynx cancer.

[Squamous cell carcinoma antigen levels in patients with laryngeal cancer]. / Antygen raka plaskonablonkowego w surowicy krwi pacjentów z rakiem krtani.

Squamous cell carcinoma antigen (SCCAg) is one of the most common markers used in diagnosis of head and neck cancer and larynx cancer. We tested correlations between level of SCC Ag and tumor size, presence of lymph node metastasis, clinical advances of tumour and histopathological diagnosis. Pretreatment level of SCC antigen was evaluated in 34 patients with squamous cell carcinoma of the larynx. Microparticle enzyme immunoassay was used to measure the SCCAg level. Elevated SCCAg serum levels were found in 41% of patients. The magnitude of the marker elevations were correlated with lymph node metastases (N0 versus N2, and N1 versus N2). Our date indicate that in patients with larynx cancer SCCAg does not appear to be a sensitive marker in the primary diagnosis. However, seem to be useful marker for monitoring nodal invasion.

[Diagnostic value of tissue polypeptide antigen in serum of larynx cancer patients]. / Wartosc diagnostyczna oznaczania tkankowego antygenu polipeptydowego w surowicy chorych na raka krtani.

UNLABELLED: Tissue polypeptide antigen (TPA) is a serological tumour marker used in the diagnosis, management and follow-up of patients with head and neck cancer. The aim of this study was to evaluate the diagnostic and prognostic significance of pretreatment TPA serum levels in patients with larynx cancer. The predicting ability of this tumour marker with respect to histological type, pathological state and lymph node metastasis was also assessed. MATERIALS AND METHOD: Concentrations of the TPA in the serum from 35 patients were measured by immunoradiometric assay. RESULTS: The results showed the sensitivity value for the group of 35 patients was 29.4%, but in clinical stage IV was 70%. TPA levels correlate with T classification and lymph node metastasis. In T4 tumors it was significantly higher than in T2 (p=0.047). TPA levels were significantly higher in patients with nodal invasion and were generally lower in patients with spinocellular carcinoma (p=0.0048). CONCLUSION: Our date indicate that TPA is of limited usefulness in the primary diagnosis in patients with larynx cancer, but is useful in detecting lymph node metastasis.

[Histone deacetylase inhibitors as a new generation of anti-cancer agents]. / Inhibitory deacetylaz histonów jako potencjalne cytostatyki nowej generacji.

The acetylation and deacetylation of histones mediated by histone acetylases and deacetylases influence DNA accessibility to factors regulating replication, repair, and transcription. Histone deacetylases inhibitors (HDI) are inducers of growth arrest, differentiation, and/or apoptosis of many tumors cells by altering the transcription of a small number of genes. The selective tumor specificity of these compounds underscores their potential as new agents for the treatment of cancer. Several HDI have shown anti-tumor activity in vitro and in vivo with remarkably low toxicity in preclinical studies and are currently in phase I and II clinical trials. This review summarizes the molecular mechanism of action of HDI and its clinical application in the treatment of cancer.

Reactive oxygen species production by blood neutrophils of patients with laryngeal carcinoma and antioxidative enzyme activity in their blood.

Squamous cell carcinoma of the head and neck is a devastating illness with a severe impact on affected individuals. Several mechanisms may lead to oxidative stress in tumor-bearing patients, among others chronic inflammation. Inflammatory cells, especially macrophages and neutrophil leukocytes, may produce reactive oxygen species (ROS) which participate in carcinogenesis and tumor-associated immunosuppression. The aim of the study presented in this paper was to compare the production of reactive oxygen species (ROS)--superoxide anion (O2-) and hydrogen peroxide (H2O2)--by neutrophils isolated from the blood of 16 patients with larynx carcinoma and 15 healthy controls. The serum activity of superoxide dismutase and catalase as well as the total peroxidase activity in serum have also been estimated. The production of ROS, especially spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced O2-, was relatively higher in the patients with larynx carcinoma than in the healthy controls and increased parallel with the tumor stage (tumor, node, metastasis-TNM staging). The serum activity of catalase and peroxidase was also highest in the patients with stage T3 and T4 larynx carcinoma. After partial or total laryngectomy, a significant decrease in ROS production and the serum activity of catalase and peroxidase was observed. In contrast, the serum level of superoxide dismutase, which had been low prior to surgery, especially in the patients with advanced tumor stages (T3-T4), increased significantly afterwards. The results indicate the existence of oxidative stress in the blood of patients with larynx carcinoma and its significant decrease after partial or total laryngectomy.

[Is syndecan-1 an independent prognostic factor in patients with laryngeal carcinoma?]. / Czy syndekan-1 jest niezaleznym czynnikiem prognostycznym u pacjentów z rakiem krtani?

Syndecan-1 is a member of the syndecan family of cell membrane heparan sulphate proteoglicans. The aim of this study was the evaluation of prognostic value of syndecan-1 expression in laryngeal cancer. The findings were correlated with the clinico-pathological parameters of the tumours, as well as with patient survival rate. Paraffin-embedded samples from 99 patients with laryngeal cancer selected from the files of the ENT-Dept. of Medical Academy in Lublin were immunostained with anti-syndecan-1 monoclonal antibody. The patients' mean age was 57 years and the over 5-year survival rate was 53.2%. Syndecan-1 immunoreactivity was observed in 99 (100%) of carcinomas. In our study, a statisti- cally significant correlation between syndecan-1 expression and patient survival rate was observed (chi2 = 9631; p = 0.008) as well as between syndecan-1 expression and various clinical stages of disease (chi2 = 6771; p = 0.034). The significant difference in the presence of syndecan-1 expression among the patients with various stage of histological differentiation of carcinoma (chi2 = 14.9; p = 0.001), and among the patients with present and absent metastatic changes in regional lymph nodes (chi2 = 16.698; p = 0.001) was observed. In a Coxs multivariate analysis syndecan-1 had an independent prognostic value (p = 0.014). Our results indicate that syndecan-1 could be used as a prognostic marker in laryngeal cancer.