Short-term autophagy inhibition by autophinib or SAR405 does not alter the effect of cisplatin on ATP production in prostate cancer cells.

OBJECTIVES: Cisplatin is a widely used anticancer drug for the treatment of many solid cancers. DNA damage is thought to be the key mechanism of cisplatin's anticancer activity. However, cisplatin may also affect cellular metabolism. The aim of this study was to determine the effect of cisplatin on the types of ATP production (OXPHOS versus glycolysis) and their rate in prostate cancer cells and to determine the potentially protective effect of autophagy and amino acids during cisplatin treatment. We also wanted to investigate the potential synergy between the metabolic effects of cisplatin on ATP production and the inhibition of autophagy. METHODS: Cisplatin treatment can significantly affect the metabolism of cancer cells. Important metabolic pathways can be altered, leading to changes in energy production and nutrient utilization. Autophagy and amino acid pool modulations can serve as protective mechanisms significantly affecting tumor cell survival under metabolic stress caused by anticancer treatment. By enabling the recycling of amino acids, autophagy helps cancer cells maintain cellular homeostasis and overcome nutrient limitations. Thus, inhibition of autophagy could have a supportive effect on the metabolic effects of cisplatin. RESULTS: After cisplatin treatment, ATP production by way of OXPHOS was significantly decreased in 22Rv1 and PC-3 cells. On the other hand, ATP production by glycolysis was not significantly affected in 22Rv1 cells. DU145 cells with dysfunctional autophagy were the most sensitive to cisplatin treatment and showed the lowest ATP production. However, short-term autophagy inhibition (24h) by autophinib or SAR405 in 22Rv1 and PC-3 cells did not alter the effect of cisplatin on ATP production. Levels of some amino acids (arginine, methionine) significantly affected the fitness of cancer cells. CONCLUSION: Persistent defects of autophagy can affect the metabolic sensitivity of cancer cells due to interference with arginine metabolism. Amino acids contained in the culture medium had an impact on the overall effect of cisplatin (Fig. 3, Ref. 38).

Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism.

The combination of in ovo and ex ovo chorioallantoic membrane (CAM) assay provides an excellent platform which extends its relevance in studying carcinogenesis to the field of screening of anticancer activity of platinum nanoparticles (PtNPs) and further study of the amino acids' fluctuations in liver and brain. PtNPs are promising candidates for replacing cisplatin (CDDP); however, insufficient data of their antitumor efficiency and activity on the cancer-related amino acid metabolism are available, and the assessment of the in vivo performance has barely scratched the surface. Herein, we used CAM assay as in vivo model for screening of novel therapeutic modalities, and we conducted a comparative study of the effects of CDDP and polyvinylpyrrolidone coated PtNPs on MDA-MB-231 breast cancer xenograft. PtNPs showed a higher efficiency to inhibit the tumor growth and metastasis compared to CDDP. The amino acids profiling in the MDA-MB-231 â€‹cells revealed that the PtNPs had an overall depleting effect on the amino acids content. Noteworthy, more side effects to amino acid metabolism were deduced from the depletion of the amino acids in tumor, brain, and liver upon CDDP treatment. Different sets of enzymes of the tricarboxylic acid (TCA) cycle were targeted by PtNPs and CDDP, and while mRNA encoding multiple enzymes was downregulated by PtNPs, the treatment with CDDP affected only two TCA enzymes, indicating a different mechanism of action. Taken together, CAM assay represents and invaluable model, demonstrating the PtNPs capability of repressing angiogenesis, decrease amino acid contents and disrupt the TCA cycle.

Platinum-based drug-induced depletion of amino acids in the kidneys and liver.

Cisplatin (cis-diamminedichloroplatinum II; CDDP) is a widely used cytostatic agent; however, it tends to promote kidney and liver disease, which are a major signs of drug-induced toxicity. Platinum compounds are often presented as alternative therapeutics and subsequently easily dispersed in the environment as contaminants. Due to the major roles of the liver and kidneys in removing toxic materials from the human body, we performed a comparative study of the amino acid profiles in chicken liver and kidneys before and after the application of CDDP and platinum nanoparticles (PtNPs-10 and PtNPs-40). The treatment of the liver with the selected drugs affected different amino acids; however, Leu and Arg were decreased after all treatments. The treatment of the kidneys with CDDP mostly affected Val; PtNPs-10 decreased Val, Ile and Thr; and PtNPs-40 affected only Pro. In addition, we tested the same drugs on two healthy cell lines, HaCaT and HEK-293, and ultimately explored the amino acid profiles in relation to the tricarboxylic acid cycle (TCA) and methionine cycle, which revealed that in both cell lines, there was a general increase in amino acid concentrations associated with changes in the concentrations of the metabolites of these cycles.

Lycorine and UV-C stimulate phenolic secondary metabolites production and miRNA expression in Chlamydomonas reinhardtii.

Studying stress pathways on the level of secondary metabolites that are found in very small concentration in the cells is complicated. In the algae, the role of individual metabolites (such as carotenoids, phenolic compounds, organic acids, and vitamins) and miRNAs that participate in plant's defence are very poorly understood during stressful conditions. Therefore, in the present experiment, the model organism Chlamydomonas reinhardtii was exposed to stress conditions (Lyc and UV-C irradiation) to detect these substances, even at very low concentrations. The purpose was to monitored changes at each response level with a future view to identifying their specific roles under different stress factors. In stress-treated cultures, numerous transcriptomic and metabolomic pathways were triggered in C. reinhardtii. Although Lyc significantly decreased the concentration of AA, suggesting that Lyc has a similar function in C. reinhardtii as in plants. The negative effect of UV-C radiation was based on the production of ROS and enhancement of antioxidant responses, resulting in increased levels of polyphenols and simple phenolic compounds. Both treatments did lead to extensive changes in transcript levels and miRNA expression patterns.

Prognostic Significance of Serum Free Amino Acids in Head and Neck Cancers.

Despite distinctive advances in the field of head and neck squamous cell cancer (HNSCC) biomarker discovery, the spectrum of clinically useful prognostic serum biomarkers is limited. As metabolic activities in highly proliferative transformed cells are fundamentally different from those in non-transformed cells, specific shifts in concentration of different metabolites may serve as diagnostic or prognostic markers. Blood amino acids have been identified as promising biomarkers in different cancers before, but little is known about this field in HNSCC. Blood amino acid profiles of 140 HNSCC patients were examined using high-performance liquid chromatography. Cox proportional hazards regression model was used to assess the prognostic value of amino acid concentrations in serum. Colony forming assay was used to identify the effect of amino acids that were significant in Cox proportional hazards regression models on colony forming ability of FaDu and Detroit 562 cell lines. In the multivariable Cox regression model for overall survival (OS), palliative treatment was associated with an unfavourable prognosis while high serum levels of methionine have had a positive prognostic impact. In the relapse-free survival (RFS) multivariable model, methionine was similarly identified as a positive prognostic factor, along with tumor localization in the oropharynx. Oral cavity localization and primary radio(chemo)therapy treatment strategy have been linked to poorer RFS. 1mM serine was shown to support the forming of colonies in both tested HNSCC cell lines. Effect of methionine was exactly the opposite.

The effects of 5-azacytidine and cadmium on global 5-methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda.

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S-adenosyl-L-methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5-azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S-adenosylhomocysteine, 5'-methylthioadenosine and global 5-methylcytosine (5-mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5-mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5-azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5-mC content of C. reinhardtii was 1%-1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S-adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.

Post-treatment urinary sarcosine as a predictor of recurrent relapses in patients with prostate cancer.

To date, there has been no evidence regarding the association between urinary sarcosine content and prostate cancer survival. Our main objective was to investigate whether levels of post-treatment urinary sarcosine are associated with relapse. The inclusion criteria were (in accordance with EAU 2017) as follows: histopathologically verified adenocarcinoma in prostate biopsy cores or specimens from transurethral resection of the prostate (TURP) or prostatectomy for benign prostatic enlargement (BPE) with retained ability to urinate. The median follow-up was 53 months. In the study, we retrospectively evaluated a cohort of 511 patients with prostate cancer with various risk factors and treatment strategies. Post-treatment sarcosine levels were elevated in 266 (52%) patients and highly elevated (≥200 nmol/L) in 71 (13%) patients. Urinary sarcosine content was significantly associated with number of relapses that patients experienced, P = 0.002 for sarcosine ≥200 vs ≤30 nmol/L. Multivariate analysis revealed that sarcosine was an independent predictor of recurrent relapses (≥2 relapses with an intermediate period of remission), HR = 3.89 (95% CI 1.29-11.7) for sarcosine >200 vs <30 nmol/L. This trend was even more pronounced in a subgroup of patients who underwent radical prostatectomy, HR = 3.29 (95% CI 1.06-10.18), where (single) relapse-free survival could also be predicted by sarcosine levels, HR = 1.96 (1.05-3.66). Urinary sarcosine may become a possible predictor for patients' outcomes, because patients with elevated post-treatment sarcosine could be predicted to have recurrent relapses of the disease.

Antioxidant status of rats' blood and liver affected by sodium selenite and selenium nanoparticles.

BACKGROUND: Selenium is an essential element; however, at higher doses, it can be toxic. Therefore, alternative nanotechnological solutions are required to overcome toxicological issues, rather than conventional alternatives. Nanoparticles show new and promising properties that may be able to suppress toxicity while maintaining the positive effects of selenium on an organism. The aim of the experiment was to determine the influence of sodium selenite and selenium nanoparticles (SeNPs) on the antioxidant status of rats. METHODS: The males of the outbreed rat strain Wistar albino were selected as a model organism. Animals were fed different forms of selenium. The control group was given a mixture without selenium addition, whereas other groups were fed a mixture containing sodium selenite, Se-49, and Se-100 SeNPs respectively. The duration of the trial was 30 days. RESULTS: Analysis of blood and liver was performed where the concentration of reduced (GSH) and oxidised (GSSG) glutathione, and total selenium content were measured. In the liver, a significant reduction in GSSG was found for all experiment groups. Blood samples showed a significant reduction in GSH and an increase in GSSG. DISCUSSION: These results show that SeNPs may be an alternative to dietary selenium for animal organisms.

Differences in Urinary Amino Acid Patterns in Individuals with Different Types of Urological Tumor Urinary Amino Acid Patterns as Markers of Urological Tumors.

BACKGROUND: Insufficient specificity and invasiveness of currently used diagnostic methods raises the need for new markers of urological tumors. The aim of this study was to find a link between the urinary excretion of amino acids and the presence of urological tumors. MATERIALS AND METHODS: Using ion-exchange chromatography, we tested urine samples of patients with prostate cancer (n=30), urinary bladder cancer (n=28), renal cell carcinoma (n=16) and healthy volunteers (control group; n=21). RESULTS: In each category, we found a group of amino acids which differed in concentration compared to the control group. These differences were most significant in sarcosine in patients with prostate cancer; leucine, phenylalanine and arginine in those with bladder cancer; and sarcosine, glutamic acid, glycine, tyrosine and arginine in the those with renal cell carcinoma. CONCLUSION: Results of our research imply a possible connection between the occurrence of specific types of amino acids in the urine and the presence of urological tumors.

Amino Acid Profiling of Zinc Resistant Prostate Cancer Cell Lines: Associations With Cancer Progression.

BACKGROUND: Failure in intracellular zinc accumulation is a key process in prostate carcinogenesis. Nevertheless, epidemiological studies of zinc administration have provided contradicting results. In order to examine the impact of the artificial intracellular increase of zinc(II) ions on prostate cancer metabolism, PNT1A, 22Rv1, and PC-3 prostatic cell lines-depicting different stages of cancer progression-and their zinc-resistant counterparts were used. To determine "benign" and "malignant" metabolic profiles, amino acid patterns, gene expression, and antioxidant capacity of these cell lines were assessed. METHODS: Amino acid profiles were examined using an ion-exchange liquid chromatography. Intracellular zinc content was measured by atomic absorption spectrometry. Metallothionein was quantified using differential pulse voltammetry. The content of reduced glutathione was determined using high performance liquid chromatography coupled with an electrochemical detector. Cellular antioxidant capacity was determined by the ABTS test and gene expression analysis was performed by qRT-PCR. RESULTS AND CONCLUSIONS: Long-term zinc treatment was shown to reroute cell metabolism from benign to more malignant type. Long-term application of high concentration of zinc(II) significantly enhanced cisplatin resistance, invasiveness, cellular antioxidant capacity, synthesis of glutathione, and expression of treatment resistance- and stemness-associated genes (SOX2, POU5F1, BIRC5). Tumorous cell lines universally displayed high accumulation of aspartate and sarcosine and depletion of essential amino acids. Increased aspartate/threonine, aspartate/methionine, and sarcosine/serine ratios were associated with cancer phenotype with high levels of sensitivity and specificity. Prostate 77: 604-616, 2017. © 2017 Wiley Periodicals, Inc.

Fully automated two-step assay for detection of metallothionein through magnetic isolation using functionalized γ-Fe2O3 particles.

Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.

Influence of Long-Distance Bicycle Riding on Serum/Urinary Biomarkers of Prostate Cancer.

Herein, we present a study focused on the determination of the influence of long-distance (53 km) bicycle riding on levels of chosen biochemical urinary and serum prostate cancer (PCa) biomarkers total prostate-specific antigen (tPSA), free PSA (fPSA) and sarcosine. Fourteen healthy participants with no evidence of prostate diseases, in the age range from 49-57 years with a median of 52 years, underwent physical exercise (mean race time of 150 ± 20 min, elevation increase of 472 m) and pre- and post-ride blood/urine sampling. It was found that bicycle riding resulted in elevated serum uric acid (p = 0.001, median 271.76 vs. 308.44 µmol/L pre- and post-ride, respectively), lactate (p = 0.01, median 2.98 vs. 4.8 mmol/L) and C-reactive protein (p = 0.01, 0.0-0.01 mg/L). It is noteworthy that our work supports the studies demonstrating an increased PSA after mechanical manipulation of the prostate. The subjects exhibited either significantly higher post-ride tPSA (p = 0.002, median 0.69 vs. 1.1 ng/mL pre- and post-ride, respectively) and fPSA (p = 0.028, median 0.25 vs. 0.35 ng/mL). Contrary to that, sarcosine levels were not significantly affected by physical exercise (p = 0.20, median 1.64 vs. 1.92 µmol/mL for serum sarcosine, and p = 0.15, median 0.02 µmol/mmol of creatinine vs. 0.01 µmol/mmol of creatinine for urinary sarcosine). Taken together, our pilot study provides the first evidence that the potential biomarker of PCa-sarcosine does not have a drawback by means of a bicycle riding-induced false positivity, as was shown in the case of PSA.

Relation of exposure to amino acids involved in sarcosine metabolic pathway on behavior of non-tumor and malignant prostatic cell lines.

BACKGROUND: Sarcosine (N-methylglycine) was previously delineated as a substantial oncometabolite of prostate cancer (PCa) and its metabolism seems to be significantly involved in PCa development and behavior. METHODS: We focused on investigation whether the exposure of prostate cells (PNT1A, 22Rv1, and PC-3) to sarcosine-related amino acids (glycine, dimethylglycine, and sarcosine) affects their aggressiveness (cell mobility and division rates, using real-time cell based assay). The effect of supplementation on expression of glycine-N-methyltransferase (GNMT) mRNA was examined using qRT-PCR. Finally, post-treatment amino acids patterns were determined with consequent statistical processing using the Ward's method, factorial ANOVA and principal component analysis (P < 0.05). RESULTS: The highest migration induced sarcosine and glycine in metastatic PC-3 cells (a decrease in relative free area about 53% and 73%). The highest cell division was achieved after treatment of 22Rv1 and PC-3 cells with sarcosine (time required for division decreased by 65% or 45%, when compared to untreated cells). qRT-PCR revealed also significant effects on expression of GNMT. Finally, amino acid profiling shown specific amino acid patterns for each cell line. In both, treated and untreated PC-3 cells significantly higher levels of serine, glutamic acid, and aspartate, linked with prostate cancer progression were found. CONCLUSIONS: Sarcosine-related amino acids can exceptionally affect the behavior of benign and malignant prostate cells.

Electrochemical sensing of etoposide using carbon quantum dot modified glassy carbon electrode.

In this study, enhancement of the electrochemical signals of etoposide (ETO) measured by differential pulse voltammetry (DPV) by modifying a glassy carbon electrode (GCE) with carbon quantum dots (CQDs) is demonstrated. In comparison with a bare GCE, the modified GCE exhibited a higher sensitivity towards electrochemical detection of ETO. The lowest limit of detection was observed to be 5 nM ETO. Furthermore, scanning electron microscopy (SEM), fluorescence microscopy (FM), and electrochemical impedance spectroscopy (EIS) were employed for the further study of the working electrode surface after the modification with CQDs. Finally, the GCE modified with CQDs under optimized conditions was used to analyse real samples of ETO in the prostate cancer cell line PC3. After different incubation times (1, 3, 6, 9, 12, 18 and 24 h), these samples were then prepared prior to electrochemical detection by the GCE modified with CQDs. High performance liquid chromatography with an electrochemical detection method was employed to verify the results from the GCE modified with CQDs.

Exposure to 17ß-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida.

BACKGROUND: The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17ß-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida. METHODOLOGY/PRINCIPAL FINDINGS: Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 µg/kg of E2). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17ß-oestradiol (E2), significantly affected the molecules involved in antioxidant defence. Exposure to E2 results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner. CONCLUSIONS/SIGNIFICANCE: The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels.

Differences in urinary proteins related to surgical margin status after radical prostatectomy.

Presented exploratory pilot study was aimed at evaluation of proteins present in urinary specimens collected from prostate cancer suffering subjects after radical prostatectomy, divided into two experimental cohorts: positive (n=15) and negative (n=15) surgical margins (PSM/NSM). The presence of PSM suggests inadequate cancer clearance and the possible need for additional treatment. Proper identification of these risk-patients is therefore of a paramount importance. Total protein profiles were firstly identified by using SDS-PAGE and compared by using partial least square discrimination analysis (PLS-DA), which revealed differences in molecular weights of 80-99 and 150-235 kDa between the experimental groups. For further identification of proteins, comparative proteomic technologies were employed. Two-dimensional gel electrophoresis with subsequent identification of protein spots by using MALDI-TOF mass fingerprinting revealed differential expression of proteins between NSM/PSM cohorts. Moreover, in PSM group, three uniquely identified proteins (cyclin-dependent kinase 6, galectin-3-binding protein and L-lactate dehydrogenase C chain) were found, which show tight connection with prostate cancer and presence of all of them was previously linked to certain aspects of prostate cancer. These proteins may be associated with the molecular mechanisms of prostate cancer development; hence, their identification may be helpful for the assessment of disease progression risk after radical prostatectomy, but also for possible early diagnosis.

Study of linkage between glutathione pathway and the antibiotic resistance of Escherichia coli from patients' swabs.

In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase-GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of "more resistant" E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.

Paramagnetic nanoparticles as a platform for FRET-based sarcosine picomolar detection.

Herein, we describe an ultrasensitive specific biosensing system for detection of sarcosine as a potential biomarker of prostate carcinoma based on Förster resonance energy transfer (FRET). The FRET biosensor employs anti-sarcosine antibodies immobilized on paramagnetic nanoparticles surface for specific antigen binding. Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity. The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm). The saturation curve of sarcosine based on FRET efficiency (F604/F510 ratio) was tested within linear dynamic range from 5 to 50 nM with detection limit down to 50 pM. Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients.

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.

17ß-estradiol-containing liposomes as a novel delivery system for the antisense therapy of ER-positive breast cancer: An in vitro study on the MCF-7 cell line.

The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and ß. The delivery system is composed of a cationic liposome envelope containing 17ß-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carcinoma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and ß exhibited an antiproliferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/GSSG redox ratio), metallothionein (MT) and malondialdehyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.