Markéta Sztalmachová

Clinical significance of head and neck squamous cell cancer biomarkers

Head and neck tumors belong among the six leading causes of cancer death worldwide. The predominant type of head and neck tumors consists of squamous cell carcinomas (HNSCC). Early detection of primary tumor and relapse is a key factor for enhancing the survival rate of HNSCC patients, because high rates of cases are recognized at advanced stages. Accordingly, biomarkers suitable for the early detection of HNSCC are sorely needed to improve patient outcomes. HNSCC evolve through a multistep process by the accumulation of genetic and phenotypic changes. Searching for specific biomarkers capable of characterizing each degree is therefore really essential. In this review, genomic and gene expression alterations of HNSCC are summarized and associated with HPV status, clinicopathological conditions, and patient history from the perspective of potential biomarker utilization. The emphasis is placed on non-invasive markers detectable from saliva and blood and clinically relevant studies are mentioned in particular. These include analyses of tumorous tissues, saliva, and blood from patients with histologically defined tumors; cell culture- and other in vitro-based studies with no clinical correlations are rather excluded.

Modulation of Induced Cytotoxicity of Doxorubicin by Using Apoferritin and Liposomal Cages

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin (“Apodox” and “lip-8-dox”) and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.

Haloperidol cytotoxicity and its relation to oxidative stress.

Haloperidol (HP) is used for the symptomatic treatment of psychosis, manic phases, hyperactivity, aggressiveness, and acute delirium. Long-term use leads to various adverse side effects, especially to severe impairment of extrapyramidal nerve tracts and in particular, altered QT interval and increased incidence of arrhytmias. It is believed that cytotoxicity of HP and its metabolites is responsible for both neurotoxicity and cardiotoxicity. Extrapyramidal and cardiac adverse side effects may be explained by the HP-induced oxidative stress, as implicated by many studies. HP was reported to induce lipid peroxidation with subsequent membrane changes, responsible for cell death. Vice versa, cells resistant to oxidative stress are also resistant to the toxic effects of HP. Similarly, high percentage of patients suffering from extrapyramidal symptoms treated by vitamin E and other lipid-soluble antioxidants demonstrates diminishing of these adverse side effects. HPs ability to induce oxidative stress by multi-modal action (increased metabolism of dopamine, decrease of glutathione content, induction of NF-κB transcription factor, and inhibition of complex I of respiratory chain) has been established just recently. This review brings summarizing view on the cytotoxicity of haloperidol and involvement of reactive oxygen species and oxidative stress HP-induced cytotoxicity.

Monitoring of the prostate tumour cells redox state and real-time proliferation by novel biophysical techniques and fluorescent staining

The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are >1.3-fold higher. Impedance-based viability correlates with viable count (r > 0.92; p < 0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment >2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio > 1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis.

Prognostic significance of the tumour-adjacent tissue in head and neck cancers.

Even with significant advances in operative skills and adjuvant therapies, the overall survival of patients suffering with head and neck squamous cancers (HNSCC) is unsatisfactory. Accordingly, no clinically useful prognostic biomarkers have been found yet for HNSCC. Many studies analysed the expression of potential markers in tumour tissues compared to adjacent tissues. Nevertheless, due to the sharing of the same microenvironment, adjacent tissues show molecular similarity to tumour tissues. Thus, gene expression patterns of 94 HNSCC tumorous tissues were compared with 31 adjacent tissues and with 10 tonsillectomy specimens of non-cancer individuals. The genes analysed at RNA level using quantitative RT-PCR and correlated with clinico-pathological conditions were as follows: EGF, EGFR, MKI67, BCL2, BAX, FOS, JUN, TP53, VEGF, FLT1, MMP2, MMP9, MT1A and MT2A. The elevated MT2A, BAX, EGF and JUN expression was associated with the influence of tumour cells on the rearrangement of healthy tissues, as well as a significant shift in the BAX/BCL2 ratio. Our investigation also indicated that adjacent tissues play an important role in cancerogenesis by releasing several tumour-supporting factors such as EGF. A gradual increase in the metallothionein expression, from the lowest one in tonsillectomy samples to the highest ones in tumour samples, suggests that MT expression might be tissue reaction to the presence of tumour cells. The results of this study confirmed the significance of metallothionein in tumori-genesis and gave evidences for its use as a potential HNSCC biomarker. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in prediction of HNSCC progress.

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis†

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal‐binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS‐PAGE. Optimal conditions: antigen‐binding time – 60 min, temperature − 70°C, and buffer composition and pH – acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS‐PAGE analysis.

3D-printed biosensor with poly(dimethylsiloxane) reservoir for magnetic separation and quantum dots-based immunolabeling of metallothionein.

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti‐MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P‐N) and fibroblasts (122P‐F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability.

Effect of zinc(II) ions on the expression of pro- and anti-apoptotic factors in high-grade prostate carcinoma cells

Several typical characteristics of prostate tissue have been identified including the ability to accumulate zinc(II). However, this feature of prostate cells is lost during carcinogenesis and, thus, prostate cells are unable to accumulate zinc(II) ions in high levels. Therefore, we can expect that zinc(II) ions can significantly contribute to the progression of tumour disease and to the ability of prostate cell lines to metastasize. In this study, we aimed our attention on determining the expression of Bcl-2, c-Fos, c-Jun, Ki-67, NF-κB and p53 genes in two prostate cell lines, as the 22Rv1 cell line, a model of aggressive partially androgen-sensitive prostate cancer and the PNT1A cell line, a normal prostate cell line model. Moreover, we were interested in the mechanisms through which exposure of these cell lines to zinc(II) ions could influence expression of the above-mentioned genes. We found that zinc(II) ions caused elevated expression of Ki-67, a marker of proliferation, extremely low expression of p53, high expression of Bcl-2 and no changes in the expression of p53. Our experimental data show different effect of zinc(II) ions on expression of the above-mentioned regulatory genes, which may give us more information on their impact on cancer development and progression with possible using for cancer therapy.

Determination of oxidative stress and activities of antioxidant enzymes in guinea pigs treated with haloperidol

Guinea pigs (Cavia porcellus) were treated with haloperidol (HP), and free radical (FR) and ferric reducing antioxidant power (FRAP) assays were used to determine oxidative stress levels. Furthermore, the superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST) activity levels were detected and glucose levels and the reduced and oxidized glutathione (GSH/GSSG) ratio were measured in HP-treated and untreated guinea pigs. The present study demonstrated that the administration of HP causes significant oxidative stress in guinea pigs (P=0.022). In animals treated with HP, the activity of GST was significantly increased compared with a placebo (P= 0.007). The elevation of SOD and GR activity levels and increase in the levels of glutathione (GSH) in HP-treated animals were not statistically significant. In the HP-untreated animals, a significant positive correlation was observed between oxidative stress detected by the FR method and GST (r=0.88, P=0.008) and SOD (r=0.86, P= 0.01) activity levels, respectively. A significant negative correlation between the levels of plasma glucose and oxidative stress detected by the FRAP method was observed (r=−0.78, P=0.04). Notably, no significant correlations were observed in the treated animals. In the HP-treated group, two subgroups of animals were identified according to their responses to oxidative stress. The group with higher levels of plasma HP had higher enzyme activity and reactive oxygen species production compared with the group with lower plasma levels of HP. The greatest difference in activity (U/μl) between the two groups of animals was for GR.

Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin‐modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20, serum vs. oligo(dT)20, serum and SMPs). RNA content was measured, and the expression of metallothionein‐2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT‐PCR detection. Isolation is possible on serum volume range (10–200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10–30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1–0.4 μL. This optimized protocol was also modified to fit needs of automated one‐step single‐tube analysis with identical efficiency compared to conventional setup. One‐step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.