A 50 Hz magnetic field influences the viability of breast cancer cells 96 h after exposure.

BACKGROUND: The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line. METHODS AND RESULTS: The breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4-8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines. Cancer and normal breast cells exhibited different responses to ELF-MF. Mitochondrial membrane potential and reactive oxygen species (ROS) production were altered after ELF-MF exposure, suggesting that the mitochondria are a probable target of ELF-MF in breast cells. CONCLUSIONS: The viability of breast cells in vitro is influenced by ELF-MF exposure at magnetic flux densities compatible with the limits for the general population and for workplace exposures. The effects are apparent after 96 h and are related to the ELF-MF exposure time.

Effects of extremely low-frequency magnetic fields on human MDA-MB-231 breast cancer cells: proteomic characterization.

Extremely low-frequency electromagnetic fields (ELF-MF) can modify the cell viability and regulatory processes of some cell types, including breast cancer cells. Breast cancer is a multifactorial disease where a role for ELF-MF cannot be excluded. ELF-MF may influence the biological properties of breast cells through molecular mechanisms and signaling pathways that are still unclear. This study analyzed the changes in the cell viability, cellular morphology, oxidative stress response and alteration of proteomic profile in breast cancer cells (MDA-MB-231) exposed to ELF-MF (50 Hz, 1 mT for 4 h). Non-tumorigenic human breast cells (MCF-10A) were used as control cells. Exposed MDA-MB-231 breast cancer cells increased their viability and live cell number and showed a higher density and length of filopodia compared with the unexposed cells. In addition, ELF-MF induced an increase of the mitochondrial ROS levels and an alteration of mitochondrial morphology. Proteomic data analysis showed that ELF-MF altered the expression of 328 proteins in MDA-MB-231 cells and of 242 proteins in MCF-10A cells. Gene Ontology term enrichment analysis demonstrated that in both cell lines ELF-MF exposure up-regulated the genes enriched in "focal adhesion" and "mitochondrion". The ELF-MF exposure decreased the adhesive properties of MDA-MB-231 cells and increased the migration and invasion cell abilities. At the same time, proteomic analysis, confirmed by Real Time PCR, revealed that transcription factors associated with cellular reprogramming were upregulated in MDA-MB-231 cells and downregulated in MCF-10A cells after ELF-MF exposure. MDA-MB-231 breast cancer cells exposed to 1 mT 50 Hz ELF-MF showed modifications in proteomic profile together with changes in cell viability, cellular morphology, oxidative stress response, adhesion, migration and invasion cell abilities. The main signaling pathways involved were relative to focal adhesion, mitochondrion and cellular reprogramming.

Keratinocytes Exposed to Blue or Red Light: Proteomic Characterization Showed Cytoplasmic Thioredoxin Reductase 1 and Aldo-Keto Reductase Family 1 Member C3 Triggered Expression.

Several cell-signaling mechanisms are activated by visible light radiation in human keratinocytes, but the key regulatory proteins involved in this specific cellular response have not yet been identified. Human keratinocytes (HaCaT cells) were exposed to blue or red light at low or high irradiance for 3 days in cycles of 12 h of light and 12 h of dark. The cell viability, apoptotic rate and cell cycle progression were analyzed in all experimental conditions. The proteomic profile, oxidative stress and mitochondrial morphology were additionally evaluated in the HaCaT cells following exposure to high-irradiance blue or red light. Low-irradiance blue or red light exposure did not show an alteration in the cell viability, cell death or cell cycle progression. High-irradiance blue or red light reduced the cell viability, induced cell death and cell cycle G2/M arrest, increased the reactive oxygen species (ROS) and altered the mitochondrial density and morphology. The proteomic profile revealed a pivotal role of Cytoplasmic thioredoxin reductase 1 (TXNRD1) and Aldo-keto reductase family 1 member C3 (AKR1C3) in the response of the HaCaT cells to high-irradiance blue or red light exposure. Blue or red light exposure affected the viability of keratinocytes, activating a specific oxidative stress response and inducing mitochondrial dysfunction. Our results can help to address the targets for the therapeutic use of light and to develop adequate preventive strategies for skin damage. This in vitro study supports further in vivo investigations of the biological effects of light on human keratinocytes.

Oxidative stress in retinal pigment epithelium impairs stem cells: a vicious cycle in age-related macular degeneration.

Aging, chronic oxidative stress, and inflammation are major pathogenic factors in the development and progression of age-related macular degeneration (AMD) with the loss of retinal pigment epithelium (RPE). The human RPE contains a subpopulation of progenitors (i.e., RPE stem cells-RPESCs) whose role in the RPE homeostasis is under investigation. We evaluated the paracrine effects of mature RPE cells exposed to oxidative stress (H2O2) on RPESCs behavior through co-cultural, morphofunctional, and bioinformatic approaches. RPESCs showed a decline in proliferation, an increase of the senescence-associated ß-galactosidase activity, the acquisition of a senescent-like secretory phenotype (SASP), and the reduction of their stemness and differentiation competencies. IL-6 and Superoxide Dismutase 2 (SOD2) seem to be key molecules in RPESCs response to oxidative stress. Our results get insight into stress-induced senescent-associated molecular mechanisms implicated in AMD pathogenesis. The presence of chronic oxidative stress in the microenvironment reduces the RPESCs abilities, inducing and/or maintaining a pro-inflammatory retinal milieu that in turn could affect AMD onset and progression.

Pro-inflammatory cytokines and microRNAs in male infertility.

BACKGROUND: Male infertility is a problem that affects 10-15% of men of reproductive age. In particular, gametogenesis is a complex process in which inflammation may play a central role through the secretion of cytokines and the expression of microRNAs. We assessed the potential role of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and microRNAs (miR-146a-5p, miR-34a-5p and miR-23a-3p) in the seminal plasma of infertile men compared to controls, evaluating their correlation with seminal and biochemical parameters. METHODS AND RESULTS: Expression of cytokines and microRNAs was analyzed by ELISA and q-PCR. Our data shows that IL-1α was significantly increased in the azoospermic group compared to controls, TNF-α mRNA was more expressed in the oligozoospermic group than controls. There were no significant differences in miRNAs expression among the three groups. The correlations between sperm parameters and inflammatory markers were evaluated, however no significance was highlighted. CONCLUSIONS: The determination of each inflammatory marker separately in the seminal plasma of subfertile men, despite some significant differences, does not have a diagnostic value in male infertility even if an assay of selective pro-inflammatory cytokines and microRNAs in the semen may improve the diagnosis of male infertility.

New Evidence and Insights on Dalbavancin and Wound Healing in a Mouse Model of Skin Infection.

Dalbavancin is an effective antibiotic that is widely used to treat skin infection. Our aim was to determine the effect of dalbavancin administration on wound healing compared to that of vancomycin and to elucidate if epidermal growth factor receptor (EGFR), matrix metalloproteinase 1 (MMP-1), MMP-9, and vascular endothelial growth factor (VEGF) could be involved in its therapeutic mechanism. A mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection was established. Mice were treated daily with vancomycin (10 mg/kg) and weekly with dalbavancin at day 1 (20 mg/kg) and day 8 (10 mg/kg). After 14 days, wounds were excised, and bacterial counts were performed. Wound healing was assessed by histological and immunohistochemical staining, followed by protein extraction and immunoblotting. Our microbiological results confirmed that both dalbavancin and vancomycin are effective in reducing the bacterial load in wounds. The dalbavancin group showed a strong effect compared with infected untreated animals and the vancomycin-treated group. The wounds treated with dalbavancin showed robust epidermal coverage with reconstitution of the regular and keratinized epidermal lining and well-organized granulation tissue with numerous blood vessels, although slightly less than that in the uninfected group. While in the vancomycin-treated group the epithelium appeared, in general, still hypertrophic, the granulation tissue appeared even less organized. We observed elevated EGFR and VEGF expression in both treated groups, although it was higher in dalbavancin-treated mice. MMP-1 and MMP-9 were decreased in uninfected tissue and in both treated tissues compared with untreated infected wounds. This study showed faster healing with dalbavancin treatment that might be associated with higher EGFR and VEGF levels.

From 2646 to 15: differentially regulated microRNAs between progenitors from normal myometrium and leiomyoma.

BACKGROUND: Uterine leiomyomas (fibroids) are smooth muscle neoplasms of the myometrial layer of the uterus and are the most common benign tumors in women. Although their etiology is still unclear, progenitor cells seem to be implicated. OBJECTIVE: To identify the dysregulated pathways involved in leiomyoma onset by microRNA profiling of progenitor cells isolated from normal myometrium and leiomyoma tissue. MATERIALS AND METHODS: Pairs of normal myometrium and uterine fibroid specimens were collected from 12 myomectomy patients. Myometrial progenitor cells and leiomyoma progenitor cells were isolated and characterized for stemness. After total RNA extraction and profiling of their 2646 microRNAs, DIANA-miRPath analysis was applied to find any dysregulated pathways. RESULTS: Only 30 microRNAs showed a significant differential regulation between myometrial progenitor cells and leiomyoma progenitor cells. Removal of those that had values close to the cut-off or that were not consistent among triplicates left 15 microRNAs, of which 7 were downregulated and 8 were upregulated in leiomyoma progenitor cells compared to myometrial progenitor cells. According to DIANA-miRPath analysis, the 7 downregulated microRNAs (hsa-miR-146b-5p; hsa-miR-335-3p; hsa-miR-335-5p; hsa-miR-135b-5p; hsa-miR-10a-3p; hsa-miR-10a-5p; hsa-miR-200a-3p) are all related to 3 pathways, "ECM-receptor interaction" (33 targeted genes), "Adherens junction" (33 targeted genes), and "Hippo signaling" (69 targeted genes), whereas the 8 upregulated miRNAs (hsa-miR-146a-5p; hsa-miR-576-3p; hsa-miR-122-5p; hsa-miR-1246; hsa-miR-595; hsa-miR-658; hsa-miR-4284; hsa-miR-924) are related to 4 pathways, "PI3K-Akt signaling pathway" (71 targeted genes), "Pathways in Cancer" (80 targeted genes), "Cell Cycle" (37 targeted genes), and "Regulation of actin cytoskeleton" (41 targeted genes). CONCLUSION: The findings that only 15 of 2646 microRNAs are differentially regulated in normal myometrium and leiomyoma and that they are involved in 7 dysregulated pathways provides interesting insights into the development of uterine fibroids, and lends support to the hypothesis that leiomyoma onset is the result of alterations affecting progenitor cells.

TNF-α inhibitors reduce the pathological Th1 -Th17 /Th2 imbalance in cutaneous mesenchymal stem cells of psoriasis patients.

Psoriasis is a disease characterized by an imbalance between Th1 and Th17 and Th2 inflammatory axes, in which cutaneous mesenchymal stem cells (MSCs) are early involved, as they show a greater relative expression of several genes encoding for Th1 and Th17 cytokines. Therapeutic implications of TNF-α inhibitors on differentiated skin cells have been largely described in psoriasis; however, their effects on MSCs derived from patients with psoriasis have been only partially described. The aim of this work was to evaluate the effect of TNF-α inhibitors on cytokine milieu expressed by MSCs isolated from the skin of patients with psoriasis. Resident MSCs from skin of patients with psoriasis and healthy subjects have been isolated, characterized and profiled by PCR and ELISA for the expression of 22 cytokines involved in Th1 , Th2 and Th17 pathways, both before and after 12 weeks therapy with TNF-α inhibitors. The administration of TNF-α inhibitors for 12-weeks acts on MSCs as follows: it reduces the expression of several Th1 -Th17 cytokines whose levels are elevated at baseline (IL-6, IL-8, IL-12, IL-23A, IFN-γ, TNF-α, CCL2, CCL20, CXCL2, CXCL5, IL-17A, IL-17C, IL-17F, IL-21, G-CSF). Similarly, it enhances the expression of several Th2 cytokines which are underexpressed at baseline (IL-2, IL-4, IL-5), reducing the expression of those overexpressed at baseline (TGF-ß and IL-13). TNF-α inhibitors could contribute to reduce the pathological imbalance between the Th1 -Th17 vs Th2 axis in MSCs of patients with psoriasis.

Comparative study between amniotic-fluid mesenchymal stem cells and retinal pigmented epithelium (RPE) stem cells ability to differentiate towards RPE cells.

Dysfunction of the retinal pigmented epithelium (RPE) is one of the first effects of dry age-related macular degeneration (AMD) with consequent blindness. Hence, patients affected by this retinal disorder could benefit from a cell-based transplantation strategy for RPE. Actually, an effective protocol to approach this problem is lacking, though recently, it has been postulated the existence of a subpopulation of RPE stem cells (RPESCs) derived from adult RPE and able to reconstitute a functional RPE. On the other hand, the evidence related to the differentiative potential of human mesenchymal stem cells (MSCs) is continuously increasing. Among others, amniotic fluid-derived MSCs (AF-MSCs) may be a promising candidate, since these cells are characterized by high proliferation and differentiative potential. In this study, AF-MSCs and RPESCs were isolated, characterized to assay their stemness and induced to neuronal/retinal differentiation; specific RPE markers were then analyzed. Our results indicate that RPESCs are more suitable candidates for RPE replacement than AF-MSCs.

mRNAs and miRNAs profiling of mesenchymal stem cells derived from amniotic fluid and skin: the double face of the coin.

Mesenchymal stem cells (MSCs) can be isolated from different adult sources and, even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still open. In particular, it is debated whether MSCs of different origin have the same grade of stemness or whether the source affects their undifferentiated status. Here, we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) and skin (S-MSCs) but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that, even if both AF- and S-MSCs are mostly regulated by the same pathways (such as Wnt, MAPK and TGF-ß), there are some important differences at the molecular level that directly affect important cellular features, such as the ability to differentiate into adipocytes. In conclusion, even if further studies are necessary to improve the knowledge about the role of each dysregulated miRNAs gene, these differences may actually strengthen the question about the importance of tissue origin.

Night shift work and serum markers of bone turnover in male shift workers.

Night shift work is related to sleep disorders, disruption of circadian rhythm and low serum levels of vitamin D. It is known that all these conditions can adversely affect bone mass. The rate of bone turnover can be assessed through the measurement of molecules called bone turnover markers, including C-terminal telopeptide fragment of type I collagen (CTX) and procollagen type I N-terminal propeptide (P1NP). In this study, we evaluated the serum levels of CTX, P1NP and 25-Hydroxy Vitamin D in 82 male subjects (42 daytime workers and 40 night shift workers) to assess the possible risk of osteoporosis in male shift workers. Serum levels of CTX and P1NP were found to be higher in night shift workers than in daytime workers. No significant difference was found in vitamin D levels between night shift and daytime workers. The increased CTX and P1NP levels reveal a higher rate of bone turnover in night shift workers and thus a possible increased risk of osteoporosis in this category of workers compared with daytime workers. In view of this, our results highlight the importance of further studies investigating the bone health in male night shift workers.

Can extremely low frequency magnetic field affect human sperm parameters and male fertility?

Exposure to extremely low frequency magnetic fields (ELF-MF) may have different effects on spermatozoa depending on the waveform, magnetic flux density, frequency of ELF-MF, and duration of exposure. In this study, we investigated the possible role of ELF-MF (50 Hz; 1 mT) exposure in altering sperm parameters. In this study we found that exposure to ELF-MF at the frequency of 50 Hz (1 mT) for two hours induces statistically significant alterations in progressive motility, morphology and reactive oxygen species (ROS) production of human spermatozoa, suggesting a role of ELF-MF in altering reproductive function of spermatozoa. Our results represent an important discovery in the field since occupational exposure to the sine waveform 1 mT 50 Hz ELF-MF used in our study is possible in workplace. Moreover, these electromagnetic fields are product by many electronic devices and household appliances. Thus, alterations of progressive motility and morphology of spermatozoa would be important consequences of human exposures to ELF-MF.

Alterations in Pregnenolone and Testosterone Levels in Male Shift Workers.

Steroid hormone levels are closely related to the endogenous circadian rhythm induced by sleep-wake and dark-light cycles. Shift work that disrupts the circadian rhythm may influence the levels of steroid hormones. The association between shift work and alterations in female sex steroid hormone levels has been studied, but little is known about testosterone and its precursor pregnenolone levels in male shift workers. The present study investigated serum pregnenolone and testosterone levels in a group of shift and daytime male workers. All participants were sampled at the beginning of the morning shift. Lower levels of serum pregnenolone and total testosterone were found in the shift workers compared to the daytime workers. Variations in pregnenolone levels may have consequences for well-being, and they might produce consequences for the levels of hormones downstream of the steroid hormone cascade, such as testosterone. The low levels of testosterone found in shift workers demonstrate the perturbative effect of shift work on testosterone serum levels, which may be independent and/or related to pregnenolone synthesis.

A Possible Cause for the Differential Expression of a Subset of miRNAs in Mesenchymal Stem Cells Derived from Myometrium and Leiomyoma.

The aetiology of leiomyoma is debated; however, dysregulated progenitor cells or miRNAs appear to be involved. Previous profiling analysis of miRNA in healthy myometrium- (M-MSCs) and leiomyoma- (L-MSCs) derived mesenchymal stem cells (MSCs) identified 15 miRNAs differentially expressed between M-MSCs and L-MSCs. Here, we try to elucidate whether these differentially regulated 15 miRNAs arise as a conversion of M-MSCs along the differentiation process or whether they may originate from divergent cell commitment. To trace the origin of the dysregulation, a comparison was made of the expression of miRNAs previously identified as differentially regulated in M-MSCs and L-MSCs with that detected in MSCs from amniotic fluid (considered as a substitute for embryonic cells). The results do not allow for a foregone conclusion: the miRNAs converging to the adherens junction pathway showed a gradual change along the differentiation process, and the miRNAs which coincided with the other three pathways (ECM-receptor interaction, TGFß and cell cycle) showed a complex, not linear, regulation and, therefore, a trend along the hypothetical differentiation process was not deduced. However, the role of miRNAs appears to be predominant in the onset of leiomyoma and may follow two different mechanisms (early commitment; exacerbation); furthermore, miRNAs can support the observed (epigenetic) predisposition.

Predicting microRNA modulation in human prostate cancer using a simple String IDentifier (SID1.0).

To make faster and efficient the identification of mRNA targets common to more than one miRNA, and to identify new miRNAs modulated in specific pathways, a computer program identified as SID1.0 (simple String IDentifier) was developed and successfully applied in the identification of deregulated miRNAs in prostate cancer cells. This computationally inexpensive Fortran program is based on the strategy of exhaustive search and specifically designed to screen shared data (target genes, miRNAs and pathways) available from PicTar and DIANA-MicroT 3.0 databases. As far as we know this is the first software designed to filter data retrieved from available miRNA databases. SID1.0 takes advantage of the standard Fortran intrinsic functions for manipulating text strings and requires ASCII input files. In order to demonstrate SID1.0 applicability, some miRNAs expected from the literature to associate with cancerogenesis (miR-125b, miR-148a and miR-141), were randomly identified as main entries for SID1.0 to explore matching sequences of mRNA targets and also to explore KEGG pathways for the presence of ID codes of targeted genes. Besides genes and pathways already described in the literature, SID1.0 has proven to useful for predicting other genes involved in prostate carcinoma. These latter were used to identify new deregulated miRNAs: miR-141, miR-148a, miR-19a and miR-19b. Prediction data were preliminary confirmed by expression analysis of the identified miRNAs in androgen-dependent (LNCaP) and independent (PC3) prostate carcinoma cell lines and in normal prostatic epithelial cells (PrEC).

MicroRNA Profiling in Mesenchymal Stromal Cells: the Tissue Source as the Missing Piece in the Puzzle of Ageing.

Ageing is among the main risk factors for human disease onset and the identification of the hallmarks of senescence remains a challenge for the development of appropriate therapeutic target in the elderly. Here, we compare senescence-related changes in two cell populations of mesenchymal stromal cells by analysing their miRNA profiling: Human Dental Pulp Stromal Cells (hDPSCs) and human Periosteum-Derived Progenitor Cells (hPDPCs). After these cells were harvested, total RNA extraction and whole genome miRNA profiling was performed, and DIANA-miRPath analysis was applied to find the target/pathways. Only 69 microRNAs showed a significant differential expression between dental pulp and periosteum progenitor cells. Among these, 24 were up regulated, and 45 were downregulated in hDPSCs compared to hPDPCs. Our attention was centered on miRNAs (22 upregulated and 34 downregulated) involved in common pathways for cell senescence (i.e. p53, mTOR pathways), autophagy (i.e. mTOR and MAPK pathways) and cell cycle (i.e. MAPK pathway). The p53, mTOR and MAPK signaling pathways comprised 43, 37 and 112 genes targeted by all selected miRNAs, respectively. Our finding is consistent with the idea that the embryological origin influences cell behavior and the ageing process. Our study strengthens the hypothesis that ageing is driven by numerous mediators interacting through an intricate molecular network, which affects adult stem cells self-renewal capability. Graphical abstract.

Apoptotic mechanism activated by blue light and cisplatinum in cutaneous squamous cell carcinoma cells.

New approaches are being studied for the treatment of skin cancer. It has been reported that light combined with cisplatinum may be effective against skin cancer. In the present study, the effects of specific light radiations and cisplatinum on A431 cutaneous squamous cell carcinoma (cSCC) and HaCaT non­tumorigenic cell lines were investigated. Both cell lines were exposed to blue and red light sources for 3 days prior to cisplatinum treatment. Viability, apoptosis, cell cycle progression and apoptotic­related protein expression levels were investigated. The present results highlighted that combined treatment with blue light and cisplatinum was more effective in reducing cell viability compared with single treatments. Specifically, an increase in the apoptotic rate was observed when the cells were treated with blue light and cisplatinum, as compared to treatment with blue light or cisplatinum alone. Combined treatment with blue light and cisplatinum also caused cell cycle arrest at the S phase. Treatment with cisplatinum following light exposure induced the expression of apoptotic proteins in the A431 and HaCaT cell lines, which tended to follow different apoptotic mechanisms. On the whole, these data indicate that blue light combined with cisplatinum may be a promising treatment for cSCC.

Vascular endothelial growth factor/semaphorin-3A ratio and SEMA3A expression in cutaneous malignant melanoma.

Breslow thickness and Clark level are still important factors for cutaneous melanoma, but do not provide a precise prognosis in all cases. It is necessary to find new factors capable of a more accurate prediction of the tumor course. Angiogenesis is essential for tumor development and progression and is regulated by vascular endothelial growth factor A (VEGF-A) and semaphorins (SEMA), in particular, SEMA3A inhibits angiogenesis by affecting VEGF signaling. However, the prognostic role of angiogenetic factors remains unclear. To date, no information is available on SEMA3A in human melanoma. Microvessel density, immunohistochemical and mRNA VEGF and SEMA3A expression level in 60 thin (Breslow thickness ≤ 1.0 mm), 60 intermediate (1.1-4.0 mm) and 50 thick (>4.0 mm) primary human cutaneous melanomas were investigated and related to clinical/pathological parameters and disease-specific survival. No positive association between Breslow thickness, Clark level, metastasis presence and survival was identified; Clark level was poorly related to survival. VEGF and microvessel density were significantly higher in intermediate and thick melanomas and related to Breslow thickness and Clark level but not to metastasis status and survival. On the contrary, SEMA3A was significantly reduced in intermediate and thick melanomas and associated to metastasis and poor survival. VEGF/SEMA3A ratio was higher in the worst prognosis, resulting the most closely related factor with metastasis and survival. SEMA3A expression and VEGF/SEMA3A ratio turned out to be valuable prognostic biomarkers in patients affected by cutaneous melanoma, in particular with Breslow thickness >1 mm. SEMA3A might serve as a candidate tumor suppressor in cutaneous melanoma therapy.

Enhanced antitumor therapy by inhibition of p21waf1 in human malignant mesothelioma.

PURPOSE: The p21 cyclin-dependent kinase inhibitor was frequently expressed in human malignant pleural mesothelioma (MPM) tissues as well as cell lines. Recent data indicate that p21 keeps tumor cells alive after DNA damage, favoring a survival advantage. In this study, we assessed the possibility of p21 suppression as a therapeutic target for MPM. EXPERIMENTAL DESIGN: We established two different MPM-derived (from H28 and H2052 cells) subclones using vector-based short hairpin RNA (shRNA). Then, chemosensitivity against low doses of antineoplastic DNA-damaging agents was investigated by colony formation assays, and furthermore, the type of cell response induced by these drugs was analyzed. To examine the effect of p21 shRNA on chemosensitivity in vivo, tumor formation assays in nude mice were done. RESULTS: In colony formation assay, the IC50 of doxorubicin was 33 +/- 3.0 nmol/L in p21 shRNA-transfected cells with respect to 125 +/- 10 nmol/L of control vector-transfected cells. This enhancement of growth inhibition was achieved by converting a senescence-like growth arrest to apoptosis in response to doxorubicin, etoposide, and CPT11. In the in vivo assays, CPT11 and loss-of-expression of p21 in combination led to considerable suppression of tumor growth associated with a substantially enhanced apoptotic response, whereas CPT11 alone was ineffective at inducing these responses. CONCLUSIONS: These results indicated that p21 might play an important role in chemosensitivity to anticancer agents, and the suppression of its expression might be a potential therapeutic target for MPM.

The senescent status of endothelial cells affects proliferation, inflammatory profile and SOX2 expression in bone marrow-derived mesenchymal stem cells.

Human aging is a physiological process characterized by a chronic low-grade inflammation. Senescence may affect endothelial cells, subsequently involved in the most common age-related diseases (ARDs), as well as mesenchymal stem cells (MSCs) with an impairment of their properties in tissues regeneration. Endothelial cells seem to be able to exert a paracrine effect on BM-MSCs through the secretion of pro-inflammatory factors. This work is aimed to evaluate if the senescent status of human umbilical vein endothelial cells (HUVECs) could affect bone marrow derived MSCs (BM-MSCs) proliferative ability and stemness. HUVECs were cultured until the senescence status. Young (passage 3) and senescent HUVECs (passage 13) were indirectly co-cultured with BM-MSCs for 8 days in order to evaluate the effect of their senescence status on proliferative ability and stemness of MSCs. The co-culture of senescent HUVECs with BM-MSCs was associated with a reduced proliferative ability of BM-MSCs, an enforced pro-inflammatory phenotype of BM-MSCs (increased synthesis of proinflammatory cytokines such as IL-6 and TNF-α) and an increased expression of miR-126a-3p, in association with a significant decrease of SOX2, a stemmness- associated gene, targeted by miR-126a-3p. A more general IPA analysis, revealed as miR-126a-3p also modulates the expression of IRS1, IRS2, IL6ST and PIK3R2, all targets that enforce the hypothesis that senescent endothelial cells may reduce the proliferative ability and the stemness phenotype of bone marrow-derived mesenchymal stem cells.