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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.

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.

The core size of iron oxide nanoparticles (IONPs) is a crucial factor defining not only their magnetic properties but also toxicological profile and biocompatibility. On the other hand, particular IONPs may induce different biological response depending on the dose, exposure time, but mainly depending on the examined system. New light on this problem may be shed by the information concerning biomolecular anomalies appearing in various cell lines in response to the action of IONPs with different core diameters and this was accomplished in the present study. Using Raman microscopy we studied the abnormalities in the accumulation of proteins, lipids and organic matter within the nucleus, cytoplasm and cellular membrane of macrophages, HEK293T and U87MG cell line occurring as a result of 24-hour long exposure to PEG-coated magnetite IONPs. The examined nanoparticles had 5, 10 and 30 nm cores and were administered in doses 5 and 25 μg Fe/ml. The obtained results showed significant anomalies in biochemical composition of macrophages and the U87MG cells, but not the HEK293T cells, occurring as a result of exposure to all of the examined nanoparticles. However, IONPs with 10 nm core diminished the accumulation of biomolecules in cells only when they were administered at a larger dose. The Raman spectra recorded for the macrophages subjected to 30 nm IONPs and for the U87MG cells exposed to 5 and 10 nm showed the presence of additional bands in the wavenumber range 1700-2400 cm

The experiments presented in this paper show how growing cells on portable substrats can be useful to facilitate and accelerate the passaging (subculture) of anchorage-dependent cells. Experiments have shown that portable substrats are cheap, commercially available, and transparent. They are easily cut into various shapes and sizes, and are easy to sterilize. Portable substrats are also friendly to cells and permit faster than usual cell passaging procedures. Anchorage-dependent cells growing on the bottom of a culture vessel made of glass or polystyrene can be quickly passaged with previously-cut small fragments of glass fiber or nylon meshes or small fragments of polyester foil as well as nylon fishing lines and biodegradable surgeon threads that have been inserted into the vessel. The surfaces of such fragments of portable supports are quickly overgrown with cells and can be easily transferred to a new culture vessel. As with tissue explants, cells migrate and grow over the bottom of the new culture vessels. Using cell viability tests, analyses of proliferation and fluorescence microscopy, we confirmed the utility of the investigated substrats for cell culture. In addition, the passaging cells, together with a portable support (like explants), eliminate the need for an application of proteolytic enzymes which modify numerous cell properties and activities and would keep the cell from detaching from the substratum which would lead to the cell rounding and changes in the cell's cytoskeleton architecture.

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-$1\beta$ level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-$\kappa$ signaling in a RIPK4-dependent ($RIPK4^{high}$) or independent ($RIPK4^{low}$) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

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 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.

A patient’s body accepting a bone implant depends not only on the biomaterial used, but also on its surface, which allows it to properly interact with bone cells. Therefore, research has focused on improving the bioactive and tribological properties of titanium and its alloys. Commercially pure titanium (cp-Ti) is widely used as a biomedical material. However, it is characterized by unsuitable tribological properties. In this work, yttria-stabilized zirconia (YSZ) was deposited on a cp-Ti substrate via plasma spray–physical vapor deposition (PS-PVD). The structural characteristics were determined using X-ray analysis (XRD). Additionally, the lattice parameters of each phase were determined using Rietveld’s method. High-resolution scanning microscopy (HR-SEM) showed a typical column structure of coatings that can be used with PS-PVD. Depending on the process parameters, the coatings differed in thickness in the range of 2.4–9.0 µm. The surface roughness also varied. The samples were subjected to nano-indenter testing. A slight change in hardness after deposition of the coating was observed, in addition to a significant decrease in the Young’s modulus. The Young’s modulus in relation to the metallic substrate was reduced to 58 or 78 GPa depending on the parameters of the spray-coating process.

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.

(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&rsquo, phenotype.

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.

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, colchicine, and docetaxel. MTDs affect mitosis by directly perturbing the structural organization of microtubules. By a direct assessment of the biomechanical properties of prostate cancer cells exposed to different MTDs using atomic force microscopy, we show that cell stiffening is a candidate mechanism through which cancer cells preserve the original phenotype in response to the application of MTDs. While changes in cellular rigidity are typically mainly attributed to remodeling of the actin filaments in the cytoskeleton, here we provide evidence that cell stiffening can be driven by a crosstalk between actin filaments and microtubules in drug-treated cells. Our findings improve the interpretation of biomechanical data obtained for living cells in studies of various physiological and pathological processes.

Combinations of metabolic blockers (including fenofibrate) with chemotherapeutic drugs interfere with the drug‐resistance of prostate cancer cells. However, their effect on cancer stem cells‐dependent microevolution of prostate cancer malignancy remains unaddressed. Here, we hypothesize that the combined docetaxel/fenofibrate treatment prompts the selective expansion of cancer stem cells that affects the microevolution of their progenies. Accordingly, we adapted a combined in vitro/in vivo approach to identify biological and therapeutic consequences of this process. Minute subpopulations of docetaxel‐resistant CD133high and/or CD44high cancer stem cell‐like (SCL) cells were found in prostate cancer DU145 and PC3 cell populations. When pretreated with docetaxel, they readily differentiated into docetaxel‐resistant CD44negative “bulk” cells, thus accounting for the microevolution of drug‐resistant cell lineages. Combined docetaxel/fenofibrate treatment induced the generation of poly(morpho)nuclear giant cells and drug‐resistant CD44high SCL cells. However, the CD44negative offspring of docetaxel‐ and docetaxel/fenofibrate‐treated SCLs remained relatively sensitive to the combined treatment, while retaining enhanced resistance to docetaxel. Long‐term propagation of drug‐resistant SCL‐derived lineages in the absence of docetaxel/fenofibrate resulted in their reverse microevolution toward the drug‐sensitivity and invasive phenotype. Consequently, prostate tumors were able to recover from the combined docetaxel/fenofibrate stress after the initial arrest of their expansion in vivo. In conclusion, we have confirmed the potential of fenofibrate for the metronomic treatment of drug‐resistant prostate tumors. However, docetaxel/fenofibrate‐induced selective expansion of hyper‐resistant CD44high SCL prostate cells and their “bulk” progenies prompts the microevolution of prostate tumor drug‐resistance. This process can limit the implementation of metabolic chemotherapy in prostate cancer treatment., CD44+ stem cell‐related microevolution of prostate cancer drug‐resistance under the combined chemotherapeutic/metabolic stress. Combined (DCX)/fenofibrate (FF) treatment induces the formation of polymorphonuclear cells (upper insert) and DCX/FF‐resistant CD44+/CD133+ SCL cells (lower insert) that give rise to DCX‐ (but not DCX/FF‐) “super‐resistant” cell populations (right).

Glioblastoma multiforme (GBM) is a primary brain tumor with a very high degree of malignancy and is classified by WHO as a glioma IV. At present, the treatment of patients suffering from GBM is based on surgical resection of the tumor with maximal protection of surrounding tissues followed by radio- and pharmacological therapy using temozolomide as the most frequently recommended drug. This strategy, however, does not guarantee success and has devastating consequences. Testing of new substances or therapies having potential in the treatment of GBM as well as detection of their side effects cannot be done on humans. Animal models of the disease are usually used for these purposes, and one possibility is the implantation of human tumor cells into rodent brains. Such a solution was used in the present study the purpose of which was comparison of elemental anomalies appearing in the brain as a result of implantation of different glioblastoma cell lines. These were two commercially available cell lines (U87MG and T98G), as well as tumor cells taken directly from a patient diagnosed with GBM. Using total reflection X-ray fluorescence we determined the contents of P, S, K, Ca, Fe, Cu, Zn, and Se in implanted-left and intact-right brain hemispheres. The number of elemental anomalies registered for both hemispheres was positively correlated with the invasiveness of GBM cells and was the highest for animals subjected to U87MG cell implantation, which presented significant decrease of P, K, and Cu levels and an increase of Se concentration within the left hemisphere. The abnormality common for all three groups of animals subjected to glioma cell implantation was increased Fe level in the brain, which may result from higher blood supply or the presence of hemorrhaging regions. In the case of the intact hemisphere, elevated Fe concentration may also indicate higher neuronal activity caused by taking over some functions of the left hemisphere impaired as a result of tumor growth.

Epidemiological data suggests that there are functional links between bronchial asthma and lung carcinogenesis. Bronchial fibroblasts serve a prominent role in the asthmatic process; however, their involvement in lung cancer progression remains unaddressed. To estimate the effect of the asthmatic microenvironment on the invasiveness of lung cancer cells, the present study compared the behavior of human non-small cell lung cancer A549 cells exposed to the signals from human bronchial fibroblasts (HBFs) derived from non-asthmatic donors (NA HBFs) and from asthmatic patients (AS HBFs). NA HBFs did not significantly affect A549 motility, whereas AS HBFs and the media conditioned with AS HBF/A549 co-cultures increased Snail-1/connexin43 expression and motility of A549 cells. In contrast to NA HBFs, which formed A549-impenetrable lateral barriers, α-SMA+ AS HBFs actively infiltrated A549 monolayers and secreted chemotactic factors that arrested A549 cells within AS HBF/A549 contact zone. However, small sub-populations of A549 cells could release from this arrest and colonize distant regions of AS HBF monolayers. These data indicated that the interactions between lung cancer cells and HBFs in asthmatic bronchi may facilitate the colonization of lung tumors by fibroblasts. It further stabilizes the tumor microenvironment and potentially facilitates collective colonization of novel bronchial loci by cancer cells. Potential mechanistic links between the asthmatic process and lung cancer progression suggest that bronchial asthma should be included in the list of potential prognostic markers for lung cancer therapy.

Heme oxygenase-1 (HO-1, HMOX1) degrades pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe2+) and biliverdin. The enzyme exerts multiple cytoprotective functions associated with the promotion of angiogenesis and counteraction of the detrimental effects of cellular stress which are crucial for the survival of both normal and tumor cells. Accordingly, in many tumor types, high expression of HO-1 correlates with poor prognosis and resistance to treatment, i.e. chemotherapy, suggesting inhibition of HO-1 as a possible antitumor approach. At the same time, the lack of selective and well-profiled inhibitors of HO-1 determines the unmet need for new modulators of this enzyme, with the potential to be used in either adjuvant therapy or as the stand-alone targeted therapeutics. In the current study, we provided novel inhibitors of HO-1 and validated the effect of pharmacological inhibition of HO activity by the imidazole-based inhibitor (SLV-11199) in human pancreatic (PANC-1) and prostate (DU-145) cancer cell lines. We demonstrated potent inhibition of HO activity in vitro and showed associated anticancer effectiveness of SLV-11199. Treatment with the tested compound led to decreased cancer cell viability and clonogenic potential. It has also sensitized the cancer cells to chemotherapy. In PANC-1 cells, diminished HO activity resulted in down-regulation of pro-angiogenic factors like IL-8. Mechanistic investigations revealed that the treatment with SLV-11199 decreased cell migration and inhibited MMP-1 and MMP-9 expression. Moreover, it affected mesenchymal phenotype by regulating key modulators of the epithelial to mesenchymal transition (EMT) signalling axis. Finally, F-actin cytoskeleton and focal contacts were destabilized by the reported compound. Overall, the current study suggests a possible relevance of the tested novel inhibitor of HO activity as a potential anticancer compound. To support such utility, further investigation is still needed, especially in in vivo conditions.

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.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of na&iuml, ve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPAR&alpha, and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

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.

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.

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.

Doxorubicin (DOX) therapy is limited by both cancer cells resistance and cardiotoxicity. DOX biotransformation to doxorubicinol (DOXol) by reductases enzymes (mainly by CBR1; carbonyl reductase 1) is a key process responsible for DOX adverse effects development. Thus, inhibition of CBR1 can increase the therapeutic effect of DOX. In the present study, we used a group of new synthetized cinnamic acid (CA) derivatives to improve the effectiveness and safety profile of DOX therapy against cancer cells in vitro. The possible mechanism of CBR1 inhibition was simulated by molecular modelling studies. The kinetics of DOX reduction in the presence of active CA derivatives were measured in cytosols. The chemosensitising activity of CA derivatives including proapoptotic, anti-invasiveness activity were investigated in A549 lung cancer cell line. In our research 7 from 16 tested CA derivatives binded to the active site of CBR1 enzyme and improved DOX stability by inhibition of DOXol formation. Co-treatment of A549 cells with active CA derivatives and DOX induced cells apoptosis by activation of caspase cascade. At the same time we observed decrease of invasive properties (cell migration and transmigration assays) and the rearangments of F-actin cytoskeleton in CA derivatves + DOX treated cells. Meanwhile, control, human lung fibroblasts stay realtivelly unvulnerable and viable. New synthetized CA derivatives may inhibit the activity of CBR1 leading to the stabilization of DOX therapeutic levels in cancer cells and to protect the myocardium against DOXol cytotoxic effect. Favourable physicochemical properties supported by a safety profile and multidirectional chemosensitising activity render CA derivatives a promising group for the development of agent useful in combined therapy.

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&reg, 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&ndash, 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.

The local anesthetics procaine, lidocaine and tetracaine permit the reversible detachment of viable cells and their passaging or preservation in a non-adherent state in the absence of proteolytic enzymes. The effects of these anesthetics, dissolved in various media, on cell viability, cell detachment from substrata and preservation of cells in a non-adherent state, were compared using the AT-2 line of rat prostate carcinoma cells of moderate malignancy and the 3T3 mouse fibroblast cell line. It was found that all three local anesthetics can induce cell rounding followed by detachment of over 95% of viable cells in both lines in Ca2+/Mg(2+)-free PBS. Tetracaine in 1 mM concentration was the most effective in induction of fast cell detachment. However, procaine and lidocaine in 16 mM concentrations were found to be optimal for preservation of cells in a non-adherent state and for the maintenance of cell viability for at least 2 h. The tested anesthetics also cause cell rounding and detachment when present in various cell culture media but these processes occurred much more slowly and less efficiently than in Ca2+/Mg(2+)-free PBS. Normal 3T3 mouse fibroblasts after detachment and passaging undertake growth reaching the same saturation density in cultures after detachment with procaine or lidocaine as after passaging using trypsin solution. The results suggest that the application of local anesthetics can be a very simple and effective technique for cell passaging in tissue cultures. This technique might decrease side-effects and cell injury caused by trypsinization or cell scraping. The preservation of cells in suspension in a non-adherent state may facilitate analysis of cell surface properties and fractionation of cell mixtures. Avoiding the use of trypsin allows for the preservation of cell surface proteins ICAM, CXCR4, and HCAM analyzed with FlowSight image flow cytometry.

Connexin(Cx)43

Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-β (TGF-β). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-β–induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF-β1–induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-β1 efficiently up-regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43low) fibroblasts increased their predilection to TGF-β1–induced Smad2 activation and fibroblast–myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43high) fibroblasts ...

Cellular stress responses determine tissue development, homeostasis and pathogenesis. Paracrine signaling, exchange of mechanical stimuli and intercellular transfer of small metabolites via connexin-built gap junctional channels are involved in the cellular stress detection and propagation of stress stimuli in multicellular networks. Cellular stress responses are also regulated through the activity of unpaired connexons (hemichannels) and via the intracellular interference of connexins with the cell cycle and pro-apoptotic machinery. Therefore, connexins are considered as multidirectional transmitters of the "outside-in" and "inside-out" stress signaling that are crucial for tissue homeostasis, regeneration and pathology. In particular, the disturbance of connexin function during the multi-stage process of tumor development leads to abnormal reactions of tumor cells to stress stimuli. In this review, we outline the current knowledge on the multidirectional role of connexins in the detection of stress signals. We also discuss the role of connexin-mediated intercellular transmittance of stress signals in tumour promotion, progression and metastatic cascade.1. Connexins and gap junctions protect cells from the microenvironmental stress and are involved in propagation and intracellular processing of stress signals. 2. The quality and quantity of stress stimuli, which may lead to cell adaptation or death by apoptosis, is determined by intrinsic properties of connexins and the cell phenotype. 3. Connexin deficiency increases the resistance of tumor cells to the "outside-in" stress signaling. 4. The connexin-mediated "inside-out" stress signaling participates in tumor cell invasion during the metastatic cascade.

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43high (DU-145 and MAT-LyLu) and Cx43low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43low AT-2 cells, Cx43low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43low and Cx43high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43high prostate cancer and endothelial cells.

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43

Background Multipotent cancer stem cells (CSC) are involved in micro-evolutionary tumor adaptation to therapeutic regimens. Due to their drug-resistance, they represent a promising target for treatment strategies of advanced prostate tumors. Fenofibrate (FF) has recently been pinpointed as a potential metronomic agent that augments the sensitivity of cancer cells to chemotherapeutic drugs. Here, we hypothesized that FF interferes with the drug-resistance of prostate cancer cell populations through the interference with the functions of cancer stem cell-like (SCL) cells. Methods Naive and drug-resistant DU145 cells cultivated in control conditions and in the presence of DCX and/or FF were immunostained against pluripotency markers and analyzed with fluorescent microscopy, fluorescent activated cell sorting (FACS) and image stream. Results FACS analyses of naive and docetaxel (DCX)-resistant DU145 cells revealed minute sub-populations of CD133+/CD44-, CD133+/CD44+ and CD133-/CD44+ SCL cells. SCL fractions were elevated within DCX-treated naive and DCX-resistant DU145 cell line populations. More prominent increase of SCL-fraction was also observed in naive, and to lower extent, in drug-resistant DU145 lineages upon combined DCX/FF treatment, which confirms their drug-resistance. CD44+ cells formed the aggregates of round and loosely attached cells, surrounded by morphologically diverse cellular clusters. These SCL cells offspring displayed neoplastic and invasive phenotype similar to that of parental cells; however, they showed increased, P-gp-dependent DCX- but not DCX/FF resistance. Loss of drug-resistance over time of SCL cells offspring was accompanied by induction of their EMT and invasive potential, and combined DCX/FF treatment interfered with this process. Conclusions These observations confirm the phenotypic plasticity of SCL cells and its role in the maintenance of prostate cancer cell heterogeneity and drug-resistance. FF can enhance the efficiency of palliative prostate cancer therapies through the interference with the function of cancer stem cells. Legal entity responsible for the study Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology. Funding National Science Centre Poland. Disclosure All authors have declared no conflicts of interest.

The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

Intrinsically disordered proteins (IDPs) are biologically active and crucial for cell function although they do not possess defined three-dimensional architecture. IDPs are especially prevalent in eukaryotic proteomes, and large-scale experiments have shown that many IDPs are nuclear proteins. Bioinformatic analyses have also demonstrated that the vast majority of transcription factors contain extended regions of intrinsic disorder. In the current study, we isolated and functionally analyzed IDPs expressed in the nuclei of HEK293 human cells. According to the results of MS analysis followed by subsequent analysis with the bioinformatic tools IUPred and RAPID (regression-based accurate predictor of intrinsic disorder), a heat-treatment method was able to enrich the nuclear lysate in IDPs. For approximately 85% of the proteins obtained, IUPred predicted a sequence of 30 or more consecutive disordered residues (DRs), and for approximately 83% of the proteins RAPID reported a content of at least 25% DRs (compared to ~ 66% and 49%, respectively, for the nuclear lysate). Gene Ontology analysis in terms of molecular function revealed that the obtained fraction was generally enriched in proteins involved in the process of transcription and especially in transcription factors. We also showed experimentally that IDPs are overrepresented in the cell nucleus. Significance Intrinsically disordered proteins (IDPs) are crucial cellular molecules and are especially numerous in eukaryotes. In particular, IDPs act as signaling and regulatory proteins, and impairment in their functioning may lead to serious diseases. Large-scale bioinformatic studies of IDPs have provided essential knowledge about this group of proteins. However, experimental data reflect the actual situation in living cells. Our study is the first large-scale proteomic analysis of nuclear IDPs. We showed experimentally that IDPs are overrepresented in the nucleus in comparison to the whole cell. Analysis of molecular function indicated that the nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors. The IDP isolation method from human cell nuclei presented in this article could be further applied in differential proteomic studies.

Introduction Microevolution of drug-resistant cancer cell populations is a serious obstacle for currently available cancer therapies. Reports on the inhibitory effects of fenofibrate (FF) on the growth, survival and invasiveness of prostate cancer cells suggest its potential for metronomic strategies of prostate cancer therapy. Therefore, we assessed the interference of FF with the drug-resistance of prostate cancer cells. Material and methods Additive effects of DCX/FF on the propagation, invasiveness and drug-resistance of native prostate cancer cells and of their invasive DCX-resistant variants (DU145_DCX20 and DU145_DCX50) were estimated with time-lapse and fluorescence microscopy. Flow-cytometric and cytofluorimetric tests were performed to assess the interference of FF with ATP production, pro-apoptotic and pro-autophagic pathways; and with the activity of ABC transporters. Results and discussions When administered alone, 2.5 nM DCX significantly attenuated the proliferation of native DU145 cells, but exerted no effect on the viability of DU145_DCX20 and DU145_DCX50 cells. FF (25 mM) sensitised these cells to DCX through PPARa/ROS-independent interference with intracellular ATP production and P-gp activity, as demonstrated by control assays with elacridar. Concomitantly, DCX/FF treatment considerably reduced neoplastic and invasive potential of drug-resistant DU145 cells via the activation of mTOR-sensitive suicidal autophagy signalling(s). Conclusion Our observations suggest that FF can be applied to reduce the effective doses of chemotherapeutic drugs, to attenuate their adverse effects and to inhibit the microevolution of drug-resistant cells induced by chemotherapy. Thus, it can be considered as an metronomic agent that can enhance the efficiency of long-term palliative prostate cancer treatment.

This study is focused on antioxidant and anticancer capacity of bread enriched with broccoli sprouts (BS) in the light of their potential bioaccessibility and bioavailability. Generally, bread supplementation elevated antioxidant potential of product (both nonenzymatic and enzymatic antioxidant capacities); however, the increase was not correlated with the percent of BS. A replacement up to 2% of BS gives satisfactory overall consumers acceptability and desirable elevation of antioxidant potential. High activity was especially found for extracts obtained after simulated digestion, which allows assuming their protective effect for upper gastrointestinal tract; thus, the anticancer activity against human stomach cancer cells (AGS) was evaluated. A prominent cytostatic response paralleled by the inhibition of AGS motility in the presence of potentially mastication-extractable phytochemicals indicates that phenolic compounds of BS retain their biological activity in bread. Importantly, the efficient phenolics concentration was about 12 μM for buffer extract, 13 μM for extracts after digestionin vitro, and 7 μM for extract after absorptionin vitro. Our data confirm chemopreventive potential of bread enriched with BS and indicate that BS comprise valuable food supplement for stomach cancer chemoprevention.

Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-β and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.

Analysis of the heterogeneity of clones of single cells from two established lines of the Dunning series of rat prostate carcinoma differing in the degree of malignancy, AT-2 (moderately malignant) and MAT-LyLu (highly malignant), was conducted. The results showed that not only the original tumors and primary cell cultures were heterogeneous but also the established cell lines of tumor origin. In the MAT-LyLu cell line, the clones of cells with morpho-physiological features characteristic of malignant cells dominated, whereas diverse types of clones were present in the AT-2 cell line. Differences in cell morphology (EMT), multi-layering and release from contact inhibition followed by active migration were observed and found to be correlated with increased expression of proteins involved in cancer cell invasiveness: connexin 43 and transcription factor Snail. Caffeine, theophylline and papaverine, reported to show anticancer activity in vivo, were found to decrease the proportion of clones displaying malignant cell features in the AT-2 cell line. At the tested concentrations, these compounds reversibly retarded cell growth but did not inhibit it. The results showed that the heterogeneity of cell populations within the cell lines should be taken into account in experiments carried out in vitro on established model cancer cell lines.

Neither androgen ablation nor chemotherapeutic agents are effective in reducing the risk of prostate cancer progression. On the other hand, multifaceted effects of phytochemicals, such as triterpene saponins, on cancer cells have been suggested. A promising safety and tolerability profile indicate their possible application in the treatment of advanced prostate cancers. We analyzed the specificity, selectivity and versatility of desglucoanagalloside B effects on human prostate cancer cells derived from prostate cancer metastases to brain (DU-145 cells) and bone (PC-3 cells). Prominent growth arrest and apoptotic response of both cell types was observed in the presence of sub-micromolar desglucoanagalloside B concentrations. This was accompanied by cytochrome c release and caspase 3/7 activation. A relatively low cytostatic and pro-apoptotic response of cancer cells to a desglucoanagalloside B analog, anagallosaponin IV, illustrated the specificity of the effects of desglucoanagalloside B, whereas the low sensitivity of normal prostate PNT2 cells to desglucoanagalloside B showed the selectivity of its action. Inhibition of cancer cell motility was observed in the presence of both saponins, however only desglucoanagalloside B attenuated cancer cell invasive potential, predominantly through an effect on cell elastic properties. These data demonstrate the versatility of its effects on prostate cancer cells. In contrast to PNT2 cells, cancer cells tested in this study were relatively resistant to mitoxantrone. The multifaceted action of desglucoanagalloside B on basic cellular traits, crucial for prostate cancer progression, opens perspectives for elaboration of combined palliative therapies and new prostate cancer prophylaxis regimens.