Acquired drug resistance interferes with the susceptibility of prostate cancer cells to metabolic stress.

BACKGROUND: Metformin is an inhibitor of oxidative phosphorylation that displays an array of anticancer activities. The interference of metformin with the activity of multi-drug resistance systems in cancer cells has been reported. However, the consequences of the acquired chemoresistance for the adaptative responses of cancer cells to metformin-induced stress and for their phenotypic evolution remain unaddressed. METHODS: Using a range of phenotypic and metabolic assays, we assessed the sensitivity of human prostate cancer PC-3 and DU145 cells, and their drug-resistant lineages (PC-3_DCX20 and DU145_DCX20), to combined docetaxel/metformin stress. Their adaptation responses have been assessed, in particular the shifts in their metabolic profile and invasiveness. RESULTS: Metformin increased the sensitivity of PC-3 wild-type (WT) cells to docetaxel, as illustrated by the attenuation of their motility, proliferation, and viability after the combined drug application. These effects correlated with the accumulation of energy carriers (NAD(P)H and ATP) and with the inactivation of ABC drug transporters in docetaxel/metformin-treated PC-3 WT cells. Both PC-3 WT and PC-3_DCX20 reacted to metformin with the Warburg effect; however, PC-3_DCX20 cells were considerably less susceptible to the cytostatic/misbalancing effects of metformin. Concomitantly, an epithelial-mesenchymal transition and Cx43 upregulation was seen in these cells, but not in other more docetaxel/metformin-sensitive DU145_DCX20 populations. Stronger cytostatic effects of the combined fenofibrate/docetaxel treatment confirmed that the fine-tuning of the balance between energy supply and expenditure determines cellular welfare under metabolic stress. CONCLUSIONS: Collectively, our data identify the mechanisms that underlie the limited potential of metformin for the chemotherapy of drug-resistant tumors. Metformin can enhance the sensitivity of cancer cells to chemotherapy by inducing their metabolic decoupling/imbalance. However, the acquired chemoresistance of cancer cells impairs this effect, facilitates cellular adaptation to metabolic stress, and prompts the invasive front formation.

Long-Term Fenofibrate Treatment Stimulates the Phenotypic Microevolution of Prostate Cancer Cells In Vitro.

Fenofibrate is a widely used anti-hyperlipidemic agonist of peroxisome proliferator-activated receptor alpha (PPARα). As a metabolic blocker, fenofibrate interferes with cancer promotion/progression via its misbalancing effects on cellular metabolism. However, the consequences of its long-term application for patients with diagnosed drug-resistant cancers are unknown. We addressed this point by tracing the phenotypic microevolution of naïve and drug-resistant prostate cancer PC3_DCX20 cells that underwent a long-term exposition to 10 µM and 50 µM fenofibrate. Their resistance to fenofibrate, metabolic profile and invasive phenotype were estimated in the control conditions and under fenofibrate-induced stress. Apparently, drug efflux systems are not effective against the cytostatic FF action. However, wtPC3 and PC3_DCX20 cells that survived the long-term 50 µM fenofibrate treatment gave rise to lineages that displayed an increased proliferation rate, lower motility in the control conditions and enhanced fenofibrate resistance. Attenuated fenofibrate bioavailability modified the pattern of PC3 microevolution, as illustrated by phenotypic differences between wtPC3/PC3_DCX20 lineages propagated in the presence of 50 µM and 10 µM fenofibrate. Collectively, our observations indicate that fenofibrate acts as a selective factor that affects prostate cancer microevolution. We also pinpoint potential consequences of long-term exposition of prostate cancer patients to metabolic blockers.

Monocyte exposure to fine particulate matter results in miRNA release: A link between air pollution and potential clinical complication.

Chronic exposure to PM2.5 contributes to the pathogenesis of numerous disorders, although the underlying mechanisms remain unknown. The study investigated whether exposure of human monocytes to PM2.5 is associated with alterations in miRNAs. Monocytes were exposed in vitro to PM2.5 collected during winter and summer, followed by miRNA isolation from monocytes. Additionally, in 140 persons chronically exposed to air pollution, some miRNA patterns were isolated from serum seasonally. Between-season differences in chemical PM2.5 composition were observed. Some miRNAs were expressed both in monocytes and in human serum. MiR-34c-5p and miR-223-5p expression was more pronounced in winter. Bioinformatics analyses showed that selected miRNAs were involved in the regulation of several pathways. The expression of the same miRNA species in monocytes and serum suggests that these cells are involved in the production of miRNAs implicated in the development of disorders mediated by inflammation, oxidative stress, proliferation, and apoptosis after exposure to PM2.5.

Bioactive compounds from Lactarius deterrimus interfere with the invasive potential of gastric cancer cells.

Stomach cancer is the 4th most common cancer diagnosed worldwide. Despite intensive research on its etiopathology, its treatment strategies have not changed in the last 50 years. Mushrooms have recently attracted much attention as the source of bioactive compounds that can potentially complement cancer therapies. Here, we extracted a phenolic fraction from Lactarius deterrimus and analyzed its composition and bioactivity against the gastric cancer (AGS) cells. The complexity of L. deterrimus compounds was revealed by an HPLC assay, and was accompanied by cytostatic, cytotoxic and anti-invasive effects of the L. deterrimus extract (LDE). These are illustrated by inhibition of the AGS cells' proliferation, metabolic activity and motility, and by induction of the cytoskeleton rearrangements. Apparently, these effects are exerted via activation of intracellular oxidative stress and decreased ATP production in AGS cells that could not be compensated by induction of autophagy. Less severe LDE effects were seen on physiology of normal gastric fibroblasts; however, inhibition of their motility indicates that LDE can interfere with gastric cancer development via an effect on stromal cells. Along with the observed synergy of LDE and cisplatin/5-fluorouracil effects on AGS cells, our data show the potential of LDE for supplementation of the gastric cancer therapy.

The influence of IONPs core size on their biocompatibility and activity in in vitro cellular models.

Although the key factor affecting the biocompatibility of IONPs is the core size, there is a lack of regular investigation concerning the impact of the parameter on the toxicity of these nanomaterials. Therefore, such studies were carried out in this paper. Their purpose was to compare the influence of PEG-coated-magnetite NPs with the core of 5, 10 and 30 nm on six carefully selected cell lines. The proliferation rate, viability, metabolic activity, migration activity, ROS levels and cytoskeleton architecture of cells have been evaluated for specified incubation periods. These were 24 and 72-h long incubations with IONPs administered in two doses: 5 and 25 µg Fe/ml. A decrease in viability was observed after exposure to the tested NPs for all the analyzed cell lines. This effect was not connected with core diameter but depended on the exposure time to the nanomaterials. IONPs increased not only the proliferation rate of macrophages-being phagocytic cells-but also, under certain conditions stimulated tumor cell divisions. Most likely, the increase in proliferation rate of macrophages contributed to the changes in the architecture of their cytoskeleton. The growth in the level of ROS in cells had been induced mainly by the smallest NPs. This effect was observed for HEK293T cells and two cancerous lines: U87MG (at both doses tested) and T98G (only for the higher dose). This requires further study concerning both potential toxicity of such IONPs to the kidneys and assessing their therapeutic potential in the treatment of glioblastoma multiforme.

Stiffening of DU145 prostate cancer cells driven by actin filaments - microtubule crosstalk conferring resistance to microtubule-targeting drugs.

The crucial role of microtubules in the mitotic-related segregation of chromosomes makes them an excellent target for anticancer microtubule targeting drugs (MTDs) such as vinflunine (VFL), colchicine (COL), and docetaxel (DTX). MTDs affect mitosis by directly perturbing the structural organisation of microtubules. By a direct assessment of the biomechanical properties of prostate cancer DU145 cells exposed to different MTDs using atomic force microscopy, we show that cell stiffening is a response to the application of all the studied MTDs (VFL, COL, DTX). Changes in cellular rigidity are typically attributed to remodelling of the actin filaments in the cytoskeleton. Here, we demonstrate that cell stiffening can be driven by crosstalk between actin filaments and microtubules in MTD-treated cells. Our findings improve the interpretation of biomechanical data obtained for living cells in studies of various physiological and pathological processes.

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner.

Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMTlow (U87) and MGMThigh (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMThigh T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMTlow and MGMThigh cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMThigh T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.

Bioinspired Bola-Type Peptide Dendrimers Inhibit Proliferation and Invasiveness of Glioblastoma Cells in a Manner Dependent on Their Structure and Amphipathic Properties.

(1) Background: Natural peptides supporting the innate immune system studied at the functional and mechanistic level are a rich source of innovative compounds for application in human therapy. Increasing evidence indicates that apart from antimicrobial activity, some of them exhibit selective cytotoxicity towards tumor cells. Their cationic, amphipathic structure enables interactions with the negatively-charged membranes of microbial or malignant cells. It can be modeled in 3D by application of dendrimer chemistry. (2) Methods: Here we presented design principles, synthesis and bioactivity of branched peptides constructed from ornithine (Orn) assembled as proline (Pro)- or histidine (His)-rich dendrons and dendrimers of the bola structure. The impact of the structure and amphipathic properties of dendrons/dendrimers on two glioblastoma cell lines U87 and T98G was studied with the application of proliferation, apoptosis and cell migration assays. Cell morphology/cytoskeleton architecture was visualized by immunofluorescence microscopy. (3) Results: Dimerization of dendrons into bola dendrimers enhanced their bioactivity. Pro- and His-functionalized bola dendrimers displayed cytostatic activity, even though differences in the responsiveness of U87 and T98G cells to these compounds indicate that their bioactivity depends not only on multiple positive charge and amphipathic structure but also on cellular phenotype. (4) Conclusion: Ornithine dendrons/dendrimers represent a group of promising anti-tumor agents and the potential tools to study interrelations between drug bioactivity, its chemical properties and tumor cells' phenotype.

Comparison of ultrasmall IONPs and Fe salts biocompatibility and activity in multi-cellular in vitro models.

In the paper, the results of the first regular studies of ultra-small iron oxide nanoparticles (IONPs) toxicity in vitro were presented. The influence of PEG-coated NPs with 5 nm magnetite core on six different cell lines was examined. These were: human bronchial fibroblasts, human embryonic kidney cells (HEK293T), two glioblastoma multiforme (GBM) cell lines as well as GBM cells isolated from a brain tumor of patient. Additionally, mouse macrophages were included in the study. The influence of IONPs in three different doses (1, 5 and 25 µg Fe/ml) on the viability, proliferation and migration activity of cells was assessed. Moreover, quantifying the intracellular ROS production, we determined the level of oxidative stress in cells exposed to IONPs. In the paper, for the first time, the effect of Fe in the form of IONPs was compared with the analogical data obtained for iron salts solutions containing the same amount of Fe, on the similar oxidation state. Our results clearly showed that the influence of iron on the living cells strongly depends not only on the used cell line, dose and exposure time but also on the form in which this element was administered to the culture. Notably, nanoparticles can stimulate the proliferation of some cell lines, including glioblastoma multiforme. Compared to Fe salts, they have a stronger negative impact on the viability of the cells tested. Ultra-small NPs, also, more often positively affect cell motility which seem to differ them from the NPs with larger core diameters.

Epidermal Growth Factor (EGF) Augments the Invasive Potential of Human Glioblastoma Multiforme Cells via the Activation of Collaborative EGFR/ROS-Dependent Signaling.

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear-front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

High bisphenol A concentrations augment the invasiveness of tumor cells through Snail-1/Cx43/ERRγ-dependent epithelial-mesenchymal transition.

Bisphenol A (BPA) is commonly present in plastics used for food storage and preservation. The release of BPA from these products results in a permanent human exposition to BPA; however, the quality and quantity of BPA adverse effects remain a matter of controversy. The common presence of BPA in the human environment and the controversies concerning the relations of human exposition to BPA and cancer incidence justify the research on the interactions between BPA and pro-metastatic signaling in cancer cells. Here, we describe a novel BPA-reactive signaling axis that induces the epithelial-mesenchymal transition (EMT) in lung adenocarcinoma A549 cells. BPA exerted negligible effects on their properties in a wide range of concentrations (10 nM - 100 nM), whereas it considerably induced A549 invasiveness at high concentrations (10 µM). The BPA-induced EMT was illustrated by morphologic changes, E/N-cadherin switch and vimentin/Snail-1/connexin(Cx)43 up-regulation in A549 populations. It was followed by enhancement of A549 drug-resistance. Corresponding effects of BPA were observed in prostate cancer cell populations. Concomitantly, we observed increased levels and perinuclear accumulation of estrogen-related receptor gamma (ERRγ) in BPA-treated cells, its interactions with Cx43/Snail-1, and the corresponding effects of phenol red on A549 cells. Collectively, these data identify a novel, pro-metastatic Snail-1/Cx43/ERRγ signaling pathway. Its reactivity to BPA underlies the induction of cancer cells' invasiveness in the presence of high BPA concentrations in vitro. Thus, the chronic exposition of cancer cells to extrinsic and intrinsic BPA should be considered as a potential obstacle in a cancer therapy.

High doses of sodium ascorbate interfere with the expansion of glioblastoma multiforme cells in vitro and in vivo.

AIMS: Constant development of chemotherapeutic strategies has considerably improved the efficiency of tumor treatment. However, adverse effects of chemotherapeutics enforce premature treatment cessation, which leads to the tumor recurrence and accelerated death of oncologic patients. Recently, sodium ascorbate (ASC) has been suggested as a promising drug for the adjunctive chemotherapy of glioblastoma multiforme (GBM) and prostate cancer (PC). To estimate whether ASC can interfere with tumor recurrence between the first and second-line chemotherapy, we analyzed the effect of high ASC doses on the expansion of cells in vitro and in vivo. MAIN METHODS: Brightfield microscopy-assisted approaches were used to estimate the effect of ASC (1-14 mM) on the morphology and invasiveness of human GBM, rat PC and normal mouse 3T3 cells, whereas cytostatic/pro-apoptotic activity of ASC was estimated with flow cytometry. These assays were complemented by the in vitro CellROX-assisted analyses of intracellular oxidative stress and in vivo estimation of GBM tumor invasion. KEY FINDINGS: ASC considerably decreased the proliferation and motility of GBM and PC cells. This effect was accompanied by intracellular ROS over-production and necrotic death of tumor cells, apparently resulting from their "autoschizis". In vivo studies demonstrated the retardation of GBM tumor growth and invasion in the rats undergone intravenous ASC administration, in the absence of detectable systemic adverse effects of ASC. SIGNIFICANCE: Our data support previous notions on anti-tumor activity of high ASC doses. However, autoschizis-related cell responses to ASC indicate that its application in human adjunctive tumor therapy should be considered with caution.

Therapeutic potential of monoterpene α-thujone, the main compound of Thuja occidentalis L. essential oil, against malignant glioblastoma multiforme cells in vitro.

Thuja occidentalis L. is indigenous for Northern America and commonly cultivated in Europe. Raw materials obtained from this tree are widely applied in the ethnomedicine and phytotherapy of numerous ailments, incl. scurvy, cystitis, rheumatism and cancer. Despite wide medicinal applications of Thuja occidentalis, still little is known on its therapeutic potential in tumor treatment. α-thujone is the main component of Thuja occidentalis essential oil, which has been suggested to possess anti-tumor activities. This monoterpene easily penetrates the blood-brain barrier. Therefore, we examined its effects on the malignancy of glioblastoma multiforme (GBM) cells, with the special emphasis on the mechanisms of its effect on cell viability and invasiveness. α-thujone exerted the attenuating effect on the viability and proliferation of GBM cells when administered at the concentrations between 100 and 500 µg/ml (660 µM - 3.2 mM). This effect was correlated with the induction of apoptosis in GBM cell populations and with considerable inhibition of GBM cells motility. Mechanistic analyses demonstrated the induction of oxidative stress and autophagy in α-thujone-treated tumor cells, whereas normal astrocytes displayed considerably lower sensitivity to α-thujone. Our observations demonstrate that α-thujone exerts pro-apoptotic and anti-invasive effects on GBM cells. They confirm the potential of α-thujone for the treatment of glioblastoma multiforme.