Evaluation of EGFR as a prognostic and diagnostic marker for head and neck squamous cell carcinoma patients.

Approximately 90% of all head and neck tumors are squamous cell carcinomas. The overall survival of patients with head and neck squamous cell carcinoma (HNSCC) is low (≤50%). A non-invasive marker of disease progression is sorely required. The present study focused on the plasmatic levels of epidermal growth factor receptor (EGFR) in HNSCC patients (N=92) compared with healthy (N=29) and diabetic [type 2 diabetes mellitus (T2DM); N=26] controls. Enzyme-linked immunosorbent assay using antibodies against the extracellular region of EGFR (L25-S645) was performed. No significant changes were observed between diabetic and healthy controls. However, there were significantly higher EGFR plasma levels in HNSCC patients compared with both control groups (P=0.001 and 0.005, respectively). Receiver operating characteristic curve analysis identified a sensitivity of 76.09%, a specificity of 67.27% and an area under curve of 0.727 for this comparison. No significant association was observed between EGFR plasma levels and tumor stage, tumor grade, lymph node or distant metastasis occurrence, smoking habit or hypertension. However, the presence of human papillomavirus infection and T2DM in HNSCC patients had borderline effect on the plasma EGFR levels. Survival analysis revealed no significant influence of plasmatic EGFR levels on the overall and disease-specific survival of HNSCC patients. In conclusion, EGFR plasma levels appear to be a relatively promising diagnostic, but poor prognostic, HNSCC marker.

Expression profiles of miR-29c, miR-200b and miR-375 in tumour and tumour-adjacent tissues of head and neck cancers.

Altered expression of microRNAs (miRNAs) has been shown in many types of malignancies including the head and neck squamous cell carcinoma (HNSCC). Although there are many new and innovative approaches in the treatment of HNSCC, a clear marker of this disease is still missing. Three candidate miRNAs (miR-29c-3p, miR-200b-5p and miR-375-3p) were studied in connection with HNSCC using quantitative real-time PCR expression levels in 42 tissue samples of HNSCC patients and histologically normal tumour-adjacent tissue samples of these patients. Primary HNSCC carcinoma tissues can be distinguished from histologically normal-matched noncancerous tumour-adjacent tissues based on hsa-miR-375-3p expression (sensitivity 87.5 %, specificity 65 %). Additionally, a significant decrease of hsa-miR-200b-5p expression was revealed in tumour-adjacent tissue samples of patients with node positivity. Lower expression of hsa-miR-200b-5p and hsa-miR-29c-3p in HNSCC tumour tissue was associated with higher tumour grade. Consequently, survival analysis was performed. Lower expression of hsa-miR-29c-3p in tumour-adjacent tissue was associated with worse overall and disease-specific survivals. Lower expression of miR-29c-3p in tumourous tissue was associated with worse relapse-free survival. hsa-miR-375-3p seems to be a relatively promising diagnostic marker in HNSCC but is not suitable for prognosis of patients. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in HNSCC progress (significant decrease of hsa-miR-200b-5p expression in tumour-adjacent tissue of patients with node positivity and low expression of hsa-miR-29c-3p in HNSCC tumour-adjacent tissue associated with worse prognosis).

Novel biophysical determination of miRNAs related to prostate and head and neck cancers.

In this study we have chosen a new approach and characterized three miRNAs (miR-23a, miR-34a and miR-320a) related to prostate cancer and head and neck cancer by spectral (circular dichroic and UV-absorption spectra) and electrochemical (voltammetry at graphite and mercury electrodes) methods. The spectral and voltammetric results, reflecting different nucleotide sequences of miRNAs, were complemented by the results of DNAs(U) having the same oligonucleotide sequences as miRNAs. The effect of the substitution of ribose for deoxyribose was shown and structural diversity was confirmed. The stability of RNA and DNA(U) was studied using CD and UV-absorption spectroscopy and melting points were calculated. MiRNA-320a with the highest content of guanine provided the highest melting point. With respect to the rapid progress of miRNA electrochemical sensors, our results will be useful for the research and development of sensitive, portable and time-efficient miRNA sensors, which will be able to diagnose cancer and other diseases.

Molecular response of 4T1-induced mouse mammary tumours and healthy tissues to zinc treatment.

Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their documented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes (p<0.003). Significant differences were also revealed in the gene expression between the tissues (p<0.001). The highest expression levels were observed in the liver. As compared to brain, lung and liver, significantly lower concentrations of MT protein were found in the primary tumour; an inverse trend was observed in the concentration of Zinc(II). In non-tumour mice, the amount of hepatic hydrosulphuryl groups significantly increased by the exposure to Zn(II), but the animals with tumour induction showed no similar trend. The primary tumour size of zinc-treated animals was 20% smaller (p=0.002); however, no significant effect on metastasis progression due to the zinc treatment was discovered. In conclusion, Zn(II) itself may mute the growth of primary breast tumours especially at their early stages.

Label-free and amplification-free miR-124 detection in human cells.

MicroRNAs (miRNAs) are becoming a very important group of molecules especially since their connection to numerous diseases has been revealed. The potential in gene therapy as well as in diagnostics is being widely investigated leading to the demand of sensitive, selective and simple methods of isolation and detection. The combined advantages of magnetic particle-based separation with sensitive electrochemical detection may offer a very valuable tool for these purposes. In this study, the miR­124 was targeted as an example analyte for development and optimization of the isolation procedure coupled to the electrochemical detection. The sensitivity of the method was demonstrated by the limit of detection at the level of nanomolar concentration (4 nM). To verify the applicability of the procedure to the real samples, miR­124 was isolated from the human embryonic kidney cells naturally expressing this miRNA molecule and the results were compared to the amount of miR­124 isolated from the cells transfected by the pENTR-miR­124 plasmid leading to the overexpression of miR­124.

Cisplatin-resistant prostate cancer model: Differences in antioxidant system, apoptosis and cell cycle.

Differences in the antioxidant system, apoptotic mechanism and in cell cycle between prostatic cell lines could partially elucidate the development of cisplatin resistance. The aim of this study was to identify the most characteristic parameter for a particular cell line and/or a particular cisplatin treatment using a general regression model and to assess whether it is possible to use measured parameters as markers of cisplatin resistance. This study integrates the results of viability, antioxidant, flow cytometric and quantitative PCR assays in order to characterize the resistance of prostate cancer to cisplatin. Cell growth using metabolic- (MTT) and impedance-based assays, the expression of key cell death signaling proteins (p53, Bax and Bcl-2), cell cycle, activity of antioxidant system-related proteins (superoxide dismutase, glutathione peroxidase, glutathione reductase and metallothionein) and free radical scavenging capacity assays [free radicals (FR), ferric reducing antioxidant power (FRAP), ABTS] were analyzed in the cell lines 22Rv1, PC-3 and PNT1A with respect to rising concentrations (0-150 µM) and different length of cisplatin treatment (12-72 h). The non-functional-p53 PC-3 cell line showed decreased BAX (p<0.05) and, in contrast to PNT1A and 22Rv1, no cisplatin-induced effects on cell cycle. All cell lines showed increasing levels of free radical scavenging activity by ABTS, FRAP and FR assays in a time- and dose-dependent manner (r>0.76 at p<0.001 for ABTS, FRAP and FR at p<0.001). PC-3 showed increased (p<0.05) levels of free radical scavenging activity by ABTS and FR methods. These findings, together with significantly elevated MT, decreased p53 and Bax indicate PC-3 to be cisplatin-resistant. The differences in the antioxidant system and apoptotic mechanisms in PC-3 cells may elucidate the development of cisplatin resistance and indicate that this cell line may be further studied as a model of cytostatic resistance.

MicroRNAs and zinc metabolism-related gene expression in prostate cancer cell lines treated with zinc(II) ions.

MicroRNAs (miRNAs) are a large class of single-stranded RNA molecules involved in post-transcriptional gene silencing. miRNAs not only regulate various developmental and physiological processes but also are involved in cancer development. Additionally, they can be considered as biomarkers of some pathological processes. The aim of this study was to determine the expression levels of selected miRNA and zinc(II)-related genes (ZIP-1, BAX, MT2A and MT1A) in the non-tumor PNT1A prostate cell line in comparison with the prostate cancer cell lines 22Rv1, PC-3 and LNCaP after zinc(II) treatment. Using bioinformatic approaches we selected miRNAs with putative binding sites in the 3'UTR regions in Metallothionein 1A and 2A as miRNA 23a, 141, 224, 296-3p, 320, 375 and 376. We observed significantly higher expression of miRNA 23a in all tumor lines compared to non-tumor PNT1A (13.6-fold in 22Rv1, 7.3-fold in PC-3, 8.3-fold in LNCaP, p<0.01). We also observed that the 22Rv1 cell line has significantly higher expression of miRNA 224 in comparison to other cell lines. In addition, all tumor cell lines expressed significantly higher levels of miRNA 375 in comparison to non-tumor PNT1A (87.1­fold in 22Rv1, nearly 2,000-fold in PC-3, 56.3­fold in LNCaP, p<0.01). Nevertheless, miRNA 375 and 23a expression levels strongly suggest their potential to contribute to the diagnosis of prostate cancer and miRNA 224 eventually may be suitable for classification of primary tumors. The expression of miRNA 224 in 22Rv1 cell line was negatively correlated with increasing zinc(II) concentration only. Our experiments revealed significant negative correlation of miRNA 376 and MT2A in 22Rv1 and a negative correlation between miRNA 224 and MT1A in PC-3 cells which may denote possible direct regulation of MT genes by specific miRNAs in prostate cancer.