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).

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles.

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFß1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.

Magnetically Driven Self-Degrading Zinc-Containing Cystine Microrobots for Treatment of Prostate Cancer.

Prostate cancer is the most commonly diagnosed tumor disease in men, and its treatment is still a big challenge in standard oncology therapy. Magnetically actuated microrobots represent the most promising technology in modern nanomedicine, offering the advantage of wireless guidance, effective cell penetration, and non-invasive actuation. Here, new biodegradable magnetically actuated zinc/cystine-based microrobots for in situ treatment of prostate cancer cells are reported. The microrobots are fabricated via metal-ion-mediated self-assembly of the amino acid cystine encapsulating superparamagnetic Fe3 O4 nanoparticles (NPs) during the synthesis, which allows their precise manipulation by a rotating magnetic field. Inside the cells, the typical enzymatic reducing environment favors the disassembly of the aminoacidic chemical structure due to the cleavage of cystine disulfide bonds and disruption of non-covalent interactions with the metal ions, as demonstrated by in vitro experiments with reduced nicotinamide adenine dinucleotide (NADH). In this way, the cystine microrobots served for site-specific delivery of Zn2+ ions responsible for tumor cell killing via a "Trojan horse effect". This work presents a new concept of cell internalization exploiting robotic systems' self-degradation, proposing a step forward in non-invasive cancer therapy.

Determination of Renal Distribution of Zinc, Copper, Iron, and Platinum in Mouse Kidney Using LA-ICP-MS.

The main dose-limiting side effect of cisplatin is nephrotoxicity. The utilization of cisplatin is an issue of balancing tumour toxicity versus platinum-induced nephrotoxicity. In this study, we focused on intraorgan distribution of common essential trace elements zinc, copper, and iron in healthy mouse kidneys and distribution of platinum after cisplatin treatment. Renal distribution in 12 nontreated Nu-Nu mice (males) was assessed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Furthermore, 9 Nu-Nu mice were treated with cisplatin. The order of elements concentration in kidneys was as follows: Fe > Zn > Cu. All three metals showed the higher concentrations at the cortex and medulla (28.60, 3.35, and 93.83 µg/g for Zn, Cu, and Fe, respectively) and lower concentration at the pelvis and the urinary tract (20.20, 1.93, and 62.48 µg/g for Zn, Cu, and Fe, respectively). No statistically significant difference between cortex and medulla was observed for these elements. After platinum treatment, the concentration of platinum in kidneys was enhanced more than 60-times, p < 0.001. Platinum significantly showed the highest accumulation in cortex (2.11 µg/g) with a gradient distribution. Platinum was less accumulated in medulla and pelvis than in cortex, and the lowest accumulation occurred in the urinary tract (1.13 µg/g). Image processing has been successfully utilized to colocalize metal distribution using LA-ICP-MS and histological samples images.

Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation.

We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository ( https://zenodo.org/ , Digital Object Identifiers:10.5281/zenodo.1494935).

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90-, CD44-/CD90-, CD44+/CD90+, CD44-/CD90-). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90-) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44- subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44- subpopulations. Furthermore, co-cultivation experiments revealed that CD44-/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90-). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44-/CD90+, but this subpopulation had a low migratory capacity.

VPA does not enhance platinum binding to DNA in cisplatin-resistant neuroblastoma cancer cells.

Neuroblastoma represents a malignancy of the sympathetic nervous system characteristic by biological heterogeneity. Thus, chemotherapy exhibits only low effectivity in curing high-risk forms. Previous studies revealed the cytotoxic potential of valproate on neuroblastoma cells. Nevertheless, these studies omitted effects of hypoxia, despite its undeniable tumorigenic role. In this study, we addressed the question whether valproate promotes binding of platinum-based anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to DNA and role of hypoxia, cellular antioxidant capacity and cisplatin resistance in this process. Following parameters differed significantly when cells were exposed to treatment with platinum-based drugs: elevation of platinum content bound to DNA, elevation of total thiol content, GSH/GSSG ratio, glutathione reductase and peroxidase, superoxide dismutase and elevation of antioxidant capacity. Hypoxia caused a decrease in cytosine/adenine peak, and no changes in platinum-DNA binding properties were observed. After valproate co-treatment, oxidative stress-related parameters and cytosine/adenine peak were only elevated. The amount of platinum bound to DNA was not changed significantly. Valproate is not able to enhance platinum binding to DNA in neuroblastoma cells, neither in case of intrinsic resistance (UKF-NB-4) nor in case of acquired resistance (UKF-NB-4CDDP). Therefore, another mechanism different from increase in platinum binding to DNA should be considered as a synergistic effect of valproate by cisplatin treatment.

Levels of heavy metals and their binding protein metallothionein in type 2 diabetics with kidney disease.

Hyperglycemia, a major metabolic disturbance present in diabetes, promotes oxidative stress. Activation of antioxidant defense is an important mechanism to prevent cell damage. Levels of heavy metals and their binding proteins can contribute to oxidative stress. Antiradical capacity and levels of metallothionein (MT), metals (zinc and copper), and selected antioxidants (bilirubin, cysteine, and glutathione) were determined in 70 type 2 diabetes mellitus (T2DM) subjects and 80 healthy subjects of Caucasian origin. Single nucleotide polymorphism (rs28366003) in MT gene was detected. Antiradical capacity, conjugated bilirubin, and copper were significantly increased in diabetics, whereas MT and glutathione were decreased. Genotype AA of rs28366003 was associated with higher zinc levels in the diabetic group. The studied parameters were not influenced by renal function. This is the first study comprehensively investigating differences in MT and metals relevant to oxidative stress in T2DM. Ascertained differences indicate increased oxidative stress in T2DM accompanied by abnormalities in non-enzymatic antioxidant defense systems.

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.