Effects of different pulp-capping materials on cell death signaling pathways of lipoteichoic acid-stimulated human dental pulp stem cells.

PURPOSE: This study aimed to evaluate the response of dental pulp stem cells (DPSCs) cultured with and without lipoteichoic acid (LTA) to different pulp-capping materials. METHODS: The cells were cultured and seeded in 6-well plates and exposed to 1% LTA solution. Dycal, ProRoot MTA and Biodentine materials were applied on cells and all groups were evaluated by cell proliferation, viability, cell cycle and cell death signaling pathways for 24 and 72 h. RESULTS: LTA + Dycal treatment significantly inhibited the proliferation of DPSCs and increased the apoptosis rate of cells more than the other groups at 72 h. Compared to other groups, LTA + Dycal treatment significantly increased the levels of Caspase-3 and AKT and decreased the levels of p-AKT. CONCLUSIONS: The results of this study revealed that all tested materials caused apoptosis in DPSCs via an extrinsic apoptotic pathway. The DPSCs showed an early apoptosis response to the Dycal and a late apoptosis response to the ProRoot MTA and Biodentine treatments. LTA led autophagy and inhibited the proliferation of DPSCs. ProRoot MTA and Biodentin eliminated the LTA's bioactivity with higher efficiency than Dycal.

1,25-Dihydroxyvitamin D3 stimulates odontoblastic differentiation of human dental pulp-stem cells in vitro.

BACKGROUND: 1,25-Dihydroxyvitamin D3 (1,25-OH D3) plays an important role in mineralized tissue metabolism, including teeth. However, few studies have addressed its role in odontoblastic differentiation of human dental pulp-stem cells (hDPSCs). AIM: This study aimed to understand the influence of various concentrations of 1,25-OH D3 on the proliferation capacity and early dentinogenesis responses of hDPSCs. MATERIALS AND METHODS: hDPSCs were obtained from the impacted third molar teeth. Monolayer cultured cells were incubated with a differentiation medium containing different concentrations of 1,25-OH D3 (0.001, 0.01, and 0.1 µM). All groups were evaluated by S-phase rate [immunohistochemical (IHC) bromodeoxyuridine (BrdU) staining], STRO-1 and dentin sialoprotein (DSP)+ levels (IHC), and alkaline phosphatase (ALP, enzyme-linked immunosorbent assay (ELISA)) levels. RESULTS: The number of cells that entered the S-phase was determined to be the highest and lowest in the control and 0.001 µM 1,25-OH D3 groups, respectively. The 0.1 µM vitamin D3 group had the highest increase in DSP+ levels. The highest Stro-1 levels were detected in the control and 0.1 µM 1,25-OH D3 groups, respectively. The 0.1 µM 1,25-OH D3 induced a mild increase in ALP activity. CONCLUSIONS: This study demonstrated that 1,25-OH D3 stimulated odontoblastic differentiation of hDPSCs in vitro in a dose-dependent manner. The high DSP + cell number and a mild increase in ALP activity suggest that DPSCs treated with 0.1 µM 1,25-OH D3 are in the later stage of odontoblastic differentiation. The results confirm that 1,25-OH D3-added cocktail medium provides a sufficient microenvironment for the odontoblastic differentiation of hDPSCs in vitro.

Antibacterial effect of Kenger gum on mutans streptococci and its cytotoxic effect on the 3T3 fibroblast cell line.

PURPOSE: To evaluate the antibacterial effect of Kenger gum on mutans streptococci (in vivo) and Streptococcus mutans (in vitro) and its cytotoxic effect on the 3T3 fibroblast cell line. MATERIALS AND METHODS: In vitro antibacterial activity of Kenger gum extracts against S.mutans was determined by the disk-diffusion method. The broth dilution method was used to determine the minimum inhibitory concentration (MIC). The cytotoxic effect on 3T3 fibroblast cells at different time intervals was determined using cell culture and viability assays. Clinical studies were then performed on 20 healthy adult subjects, where a sugar-free chewing gum was used as a control. To determine the MS counts, oral rinse samples were taken before chewing as well as 30 and 60 min after 15 min of chewing. Repeated-measures ANOVA was used to compare the bacteria level in the oral rinse samples between the two chewing gums. The Least Significant Difference test was used for adjustment for multiple comparisons. RESULTS: The MIC of the acetone extract of Kenger gum was 30 µg/ml. The acetone extract of Kenger gum possessed moderate antiproliferative properties against the non-tumorigenic cell line 3T3. A statistically significant decrease was observed for both chewing gums at 30 and 60 min. The decrease continued at 60 min after chewing Kenger gum, while the values for control gum tended to approach the baseline after 60 min. CONCLUSION: This preliminary study showed that Kenger gum had particular and prolonged antibacterial activity against S. mutans and salivary mutans streptococci.

Notch signaling-related therapeutic strategies with novel drugs in neuroblastoma spheroids.

Neuroblastoma is a severe pediatric tumor characterized by poor prognosis. Identification of novel molecular targets and diversion of investigations on new drug trials is mandatory for cancer therapy. In this study, vinorelbine tartrate, lithium chloride, clomipramine, and medroxyprogesterone acetate are used for the possible new treatment modalities in neuroblastoma cells. Notch and c-kit are novel molecules in cancer research, and Notch pathway is one of the emerging molecules in the neuroblastoma pathogenesis. Cytotoxic effects of these drugs at different time points, with different doses were studied in the SH-SY5Y human neuroblastoma cell line. Analysis of Notch and c-kit signaling with immunohistochemistry were constituted in multicellular tumor spheroids, and morphologic investigation was performed for digital imaging of cancer stem cells (CSCs) with electron microscopy. Size kinetics of spheroids was also determined after drug treatment. Results showed that all drugs were cytotoxic for neuroblastoma cells. Yet, this cytotoxic action did not correlate with the inhibitory effects in cell signaling. Neuroblastoma spheroids showed increased immunoreactivity of Notch signaling and c-kit. Altered ultrastructural CSCs morphology was observed after clomipramine and medroxyprogesterone acetate treatment compared with other drugs. Lithium chloride showed cellular membrane destruction for both CSCs and the remaining population. In this study, independent effects of cytotoxicity in tumor cells with respect to CSCs were determined. Redundant cells, which are the bulk population in tumor a compound, destroyed with therapy, were neither a target for treatment nor a remarkable investigation of cancer.

Low-level laser therapy vs. pulsed electromagnetic field on neonatal rat calvarial osteoblast-like cells.

To compare the effects of pulsed electromagnetic field (PEMF) and low-level laser therapy (LLLT) on osteoblast cells in a cell culture model. Fifty thousand neonatal rat calvarial osteoblast-like cells per milliliter were seeded and 0.06 mT PEMF, 0.2 mT PEMF, and LLLT at 808 nm were applied for 24 and 96 h on the cells. To evaluate cellular proliferation and differentiation, specimens were examined for DNA synthesis, alkaline phosphatase (ALP) activity, cell numbers, and viability of the cells. Morphological appearances of the cells were observed using scanning electron microcopy after 24 and 96 h of incubation. At 24 and 96 h, the control group had a higher cell proliferation than 0.06 and 0.2 mT PEMF groups (p=0.001). At 96 h, 0.2 mT PEMF group had higher cell proliferation rate than 0.06 mT PEMF and LLLT groups (p=0.001). The cell count and cell viability in 0.2 mT PEMF group were higher than the 0.06-mT PEMF and LLLT groups, although these differences were not statistically significant at 96 h (p>0.05). At 24 and 96 h, cell viability in the control group was higher than the test groups. Alkaline phosphatase levels of the groups were comparable in both time intervals (p>0.05). 0.2 mT PEMF application on osteoblast-like cells led to cell proliferation and differentiation better than 0.06 mT PEMF and LLLT at 808 nm, although a remarkable effect of both PEMF and LLLT could not be detected. The ALP activity of 0.2 and 0.06 mT PEMF and LLLT were comparable.

The effects of pulsed electromagnetic field (PEMF) on osteoblast-like cells cultured on titanium and titanium-zirconium surfaces.

BACKGROUND: Commercially pure Ti, together with Ti Ni, Ti-6Al-4V, and Ti-6Al-7Nb alloys, are among the materials currently being used for this purpose. Titanium-zirconium (TiZr) has been developed that allows SLActive surface modification and that has comparable or better mechanical strength and improved biocompatibility compared with existing Ti alloys. Furthermore, approaches have targeted making the implant surface more hydrophilic, as with the Straumann SLActive surface, a modification of the SLA surface. PURPOSE: The aim of this study is to evaluate the effects of pulsed electromagnetic field (PEMF) to the behavior of neonatal rat calvarial osteoblast-like cells cultured on commercially pure titanium (cpTi) and titanium-zirconium alloy (TiZr) discs with hydrophilic surface properties. MATERIALS AND METHODS: Osteoblast cells were cultured on titanium and TiZr discs, and PEMF was applied. Cell proliferation rates, cell numbers, cell viability rates, alkaline phosphatase, and midkine (MK) levels were measured at 24 and 72 hours. RESULTS: At 24 hours, the number of cells was significantly higher in the TiZr group. At 72 hours, TiZr had a significantly higher number of cells when compared to SLActive, SLActive + PEMF, and machine surface + PEMF groups. At 24 hours, cell proliferation was significantly higher in the TiZr group than SLActive and TiZr + PEMF group. At 72 hours, TiZr group had higher proliferation rate than machine surface and TiZr + PEMF. Cell proliferation in the machine surface group was lower than both SLActive + PEMF and machine surface + PEMF. MK levels of PEMF-treated groups were lower than untreated groups for 72 hours. CONCLUSIONS: Our findings conclude that TiZr surfaces are similar to cpTi surfaces in terms of biocompatibility. However, PEMF application has a higher stimulative effect on cells cultured on cpTi surfaces when compared to TiZr.

Enhanced nerve growth factor expression by mast cells does not differ significantly between idiopathic and allergic rhinitis.

BACKGROUND: The role of neurotrophins in allergic rhinitis (AR) has been well studied, but it has not been evaluated in idiopathic rhinitis (IR). OBJECTIVE: We aimed to evaluate the nasal ß-nerve growth factor (ß-NGF) expressions of mast cells in patients with AR and IR. METHODS: Seventeen patients with house dust mites-induced persistent moderate/severe allergic rhinitis (mean age: 29.7 ± 11.96), 14 patients with idiopathic rhinitis (mean age, 29.3 ± 10.62), and 16 healthy controls (29.9 ± 11.57) were included in the study. Nasal biopsy specimens were taken from the posterior part of the inferior turbinate from all of the study subjects. Nasal ß-nerve growth factor and its receptors, pan-neurotrophin receptor p75, and tyrosine kinase A (trkA) were assessed with an immunofluorescence assay. Mast cells were determined by both an immunofluorescence assay and immunohistochemistry as tryptase-positive cells. RESULTS: The ß-NGF, trkA, and p75 receptor counts were significantly higher in AR and IR patients than in the control group (P < .001, for each), but they were not different between AR and IR patients. Similarly, the ratio of ß-NGF+ mast cells/total mast cells and the ratio of ß-NGF+ mast cells/total ß-NGF+ cells in AR and IR patients was found to be elevated when compared with the control group (P < .001, P < .001, P < .001, and P = .046, respectively); furthermore, the 2 ratios were not statistically different between the 2 patient groups. CONCLUSION: The increase in ß-NGF-expressing mast cells does not differ between idiopathic and allergic rhinitis. Therefore, we propose that mast cells do play a role in the pathogenesis of IR as important as in that of AR.

Inhibition of cell survival, viability and proliferation by dentin adhesives after direct and indirect exposure in vitro.

OBJECTIVES: The influence of dentin adhesive systems (Scotchbond Multi-Purpose, XP Bond, Xeno V, Clearfil Protect Bond, AdheSE) on cell survival, viability and proliferation was characterized after direct and indirect exposure using different cell culture techniques. MATERIALS AND METHODS: The primers and cured bonding parts were directly exposed to cells using cell culture inserts, and complete materials were analyzed in a dentin barrier test. Cell responses were examined in 3T3 mouse fibroblasts after 24- and 72-h exposure periods by the estimation of total cell numbers (survival), apoptosis (viability) and cell proliferation. RESULTS: Cell numbers were effectively reduced by the primers of AdheSE, Protect Bond, and Scotchbond Multi-Purpose as well as XP bond after direct exposure in a cell culture insert test device. Likewise, Scotchbond Multi-Purpose primer induced a rate of apoptosis (93.9%) even higher than detected with Protect Bond primer (91.6%). Cell proliferation was entirely inhibited by primers and by Xp Bond as well. The Scotchbond Multi-Purpose was most cytotoxic in a dentin barrier test device after a 24-h indirect exposure. It also increased the percentage of cells in apoptosis to 15.4% compared to untreated controls. CONCLUSION: Unpolymerized primers of dentin adhesives were more cytotoxic than polymerized bonding counterparts. Moreover, total etch dentin adhesives were more cytotoxic than self-etch adhesives. CLINICAL RELEVANCE: When dentin adhesives are used in deep cavities without a protective dentin barrier the leachable hydrophobic and hydrophilic component of dentin adhesive systems can penetrate to the pulp and may induce cytotoxic responses in pulp tissues.

Decreased therapeutic effects of noscapine combined with imatinib mesylate on human glioblastoma in vitro and the effect of midkine.

BACKGROUND: Glioblastoma (GBM) develops resistance to the advances in chemotherapy leading to poor prognosis and life quality. Consequently, new treatment modalities are needed. Our aims were to investigate the effects of combined noscapine (NOS) and imatinib mesylate (IM) on human GBM in vitro and the role of midkine (MK) in this new combination treatment. METHODS: Monolayer and spheroid cultures of T98G human GBM cell line were used to evaluate the effects of IM (10 µM), Nos (10 µM) and their combination on cell proliferation and apoptotic indexes, cell cycle, the levels of antiapoptotic MK, MRP-1, p170, PFGFR-α, EGFR, bcl-2 proteins, apoptotic caspase-3 levels, morphology (SEM) and ultrastructure (TEM) for 72 hrs. Results were statistically analyzed using the Student's t-test. RESULTS: The combination group induced highest decrease in cell proliferation and apoptotic indexes, caspase-3 levels, MRP-1 and PDGFR-α levels. The decrease in p170 levels were lower than IM but higher that NOS. The highest increases were in EGFR, MK, bcl-2 and cAMP levels in the combination group. The G0+G1 cell cycle arrest at the end of 72nd hr was the lowest in the combination group. Apoptotic appearence was observed rarely both in the morphologic and ultrastructural evaluation of the combination group. In addition, autophagic vacuoles which were frequently observed in the IM group were observed rarely. CONCLUSIONS: The combination of Nos with IM showed antagonist effect in T98G human GBM cells in vitro. This antagonist effect was correlated highly with MK levels. The effects of NOS on MRP-1, MK and receptor tyrosine kinase levels were firstly demonstrated in our report. In addition, we proposed that MK is one of the modulator in the switch of autophagy to cell death or survival/resistance.

Evaluation of light-emitting diode (LED-660 nm) application over primary osteoblast-like cells on titanium surfaces: an in vitro study.

BACKGROUND: The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED). METHODS: An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM). RESULTS: Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED. CONCLUSIONS: One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.

Effects of scopolamine treatment and consequent convulsion development in c­fos expression in fed, fasted, and refed mice.

Fasting, anticholinergics, and seizures affect c­fos activation in the brain. Additionally, antimuscarinic treated fasted animals develop convulsion soon after re­feeding. Therefore, we assessed whether c­fos expression changes in fed, fasting, and refed animals and how scopolamine treatment affects these changes. We further assessed whether there is a change in c­fos expression after convulsions. For this purpose, BALB/c mice fasted for 1, 3, 6, 12, 24 and 48 h periods were used. The animals were treated with saline or scopolamine. Half\r\nof the animals treated with saline or scopolamine were given food 20 min after injection. All animals were observed for development of convulsions for 30 min. At the end of this period, the brains of all animals were removed, and the percentage of c­fos active cells in the hypothalamus was determined immunohistochemically. Convulsions occurred within 1­48 h of fasting, after scopolamine treatment and re­feeding. Compared to fed animals, c­fos expression was not significantly changed in those undergoing different fasting periods, but significantly decreased after 12 h fasting. After animals were allowed to eat, c­fos activation significantly increased in the 1, 3, 6 and 12 refed­saline groups and decreased in the 48 refed­saline group. Scopolamine treatment in 1­24 h fasted animals increased c­fos expression, but decreased in 48 h fasted animals. Whereas convulsion development in scopolamine­treated 3, 6, 12 and 24 h refed animals suppressed c­fos expression. These results demonstrate that re­feeding and scopolamine treatment induces neuronal activity in the hypothalamus, while scopolamine induced convulsions after food intake suppressed the c­fos activity.

Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y.

The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.

Design of colloidal drug carriers of celecoxib for use in treatment of breast cancer and leukemia.

Inflammation develops initiation and pathological process of cancer. Nanoscale drug carriers are required to be investigated for delivery of actives at cellular level for treatment of various cancer types. Solid lipid nanoparticles (CLX-SLN), nanostructured lipid carriers (CLX-NLC) and a nanoemulsion (CLX-NE) of celecoxib (CLX), a selective cyclooxygenase-2 inhibitor, were formulated for use in remedy of breast cancer and acute promyelocytic leukemia. The hot high pressure homogenization technique was employed to product formulations. Scanning electron micrographs were utilized for morphological characterization of formulations. Laser diffraction (LD), photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC) were used for examination of their physical stability by storing them at various temperatures. Drug release profiles of formulations were obtained. Their activity on cancer cells was investigated in the cell culture experiments. Stable formulations having homogenous size distribution were obtained below 200 nm with high drug payloads between 93.76% and 96.66%. Nanoparticles were ascertained to contribute controlled drug release. CLX-SLN induced the highest decrease in numbers of human breast cancer and human acute promyelocytic leukemia cells through the activation of the cell death cascades especially apoptosis in comparison to CLX-NLC, CLX-NE and the pure CLX application (p < 0.05). Nanoformulations of CLX optimized in this study were found to have various advantages expected from sophisticated drug delivery systems in order to achieve higher CLX efficiency at cellular level. Thus, they are able to be administered efficaciously alone and in combination therapies in remedy of breast cancer and acute promyelocytic leukemia.

Potentiation of cytotoxicity by combination of imatinib and chlorimipramine in glioma.

Rat C6 glioma is a chemo-resistant experimental brain tumor that is difficult to treat with various drug combinations. Previous studies suggested that imatinib mesylate (Gleevec) is effective in pre-clinical trials for glioblastoma. Also, chlorimipramine (Anafranil) is an anti-depressant drug in use in the clinic and shown to have anti-neoplastic activity. We hypothesized that treatment of resistant C6 glioma with combination of imatinib and chlorimipramine may potentiate cytotoxicity and reverse resistance. C6 glioma was examined both as monolayer and as spheroid cultures. Several experimental designs were examined all of which showed synergistic activity albeit at different time kinetics. Combination treatment resulted in inhibition of cell growth and enhanced cell death as determined by dye exclusion. Further, the combination treatment resulted in significant induction of apoptosis as determined by Annexin V-FITC and PI. Also, there was inhibition of DNA synthesis and cAMP. Altogether, these findings supported the anti-proliferative and cytotoxic effects of the combination treatment. Morphological studies were also performed using transmission and scanning electron microscopy. Significant synergistic apoptosis was detected by the combination treatment in both the monolayers and spheroid cultures. There was also a synergistic effect in autophagy by the combination. Several altered morphological features were noted by both the individual compound and enhanced by the combination treatment. The present findings support our hypothesis and demonstrate the potentiation of cytotoxicity by the combination of imatinib and chlorimipramine in C6 glioma. Further, the findings suggest the potential clinical application of the combination in the treatment of drug-resistant glioma.

The inhibitory effect of suramin on telomerase activity and spheroid growth of C6 glioma cells.

BACKGROUND: The significant roles of telomerase in carcinogenesis and drug resistance have attracted growing attention as potential therapies. The aim of the study was to investigate the effect of suramin on the telomerase activity of C6 glioma cells and on the growth of C6 glioma spheroids. MATERIAL/METHODS: C6 rat glioma cells were treated with suramin at doses of 175 and 250 microM, and also with an increasing dosage for 96 hours. The effect of suramin on monolayer and spheroid cultures was determined by evaluating cell proliferation, spheroid growth, bromodeoxyuridine labeling index, changes of spheroid ultrastructure, and telomerase activity. RESULTS: Suramin inhibited telomerase activity in a dose-dependent manner. Suramin at 250 microM and 175 microM reduced telomerase activity and cell proliferation, spheroid growth, and bromodeoxyuridine labeling index. These groups commonly revealed distorted nuclei (i.e., nucleolus segregation at 250 microM, many vacuoles, vacuole fusions, and smoothing of the cell surface). Contrary to the effects of individually applied suramin, the increased dose of suramin increased telomerase activity but inhibited cell proliferation and bromodeoxyuridine labeling index only at the 24th and 96th hours; it inhibited spheroid growth at the 96th hour. The increased dose of the suramin-treated group showed changes of spheroid ultrastructure similar to those of the control group except for rarely observed vacuoles or vacuole fusion and microvilli. CONCLUSIONS: To the best of our knowledge, this is the first study in the English language to show in vitro cytotoxic effect of suramin via telomerase inhibition on C6 glioma cells and spheroids.

Lithium chloride has a biphasic effect on prostate cancer stem cells and a proportional effect on midkine levels.

Prostate cancer (PCa) is the second most frequent type of cancer in men worldwide and the levels of differentiation growth factor midkine (MK) are increased in PCa. Cancer and/or the treatment process itself may lead to psychiatric disorders. Lithium chloride (LiCl) has anti-manic properties and has been used in cancer therapy; however, it has a queried safety profile. In addition, cancer stem cells are responsible for the heterogeneous phenotype of tumor cells; they are involved in progression, metastasis, recurrence and therapy resistance in various cancer types. The aims of the present study were to investigate the effect of different concentrations of LiCl on PCa stem cells (whether a shift from tumorigenic to non-tumorigenic cells occurs) and to determine if these results can be explained through changes in MK levels. Monolayer and spheroid cultures of human prostate stem cells and non-stem cells were incubated with low (1, 10 µM) and high (100, 500 µM) concentrations of LiCl for 72 h. Cell proliferation, apoptotic indices, MK levels and ultrastructure were evaluated. Cells stimulated with low concentrations showed high proliferation, low apoptotic indices, high MK levels and more healthy ultrastructure. Opposite results were obtained at high concentrations. Furthermore, stem cells were more sensitive to stimulation and more resistant to inhibition than non-stem cells. LiCl exhibited concentration-dependent effects on stem cell and non-stem cell groups. MK levels were not involved in the biphasic effect of LiCl; however, they were proportionally affected. To the best of our knowledge, the present study was the first to show the effect of LiCl on PCa stem cells through MK.

Antagonist activity of the antipsychotic drug lithium chloride and the antileukemic drug imatinib mesylate during glioblastoma treatment in vitro.

OBJECTIVES: Glioblastoma (GBM), the most common primary tumour of the central nervous system, is characterised by a high malignancy and poor prognosis. The aims of this study were to investigate whether the combination of imatinib mesylate (IM) and lithium chloride (LiCl) exhibited a synergistic effect in treatment and to determine whether midkine (MK) affected the fate of this treatment in vitro. METHODS: Monolayer and spheroid cultures of the T98G human GBM cell line were treated with an IM and LiCl combination for 72 h. The cell proliferation index, apoptotic index, cell cycle distribution, apoptotic and anti-apoptotic protein levels, and cAMP level as well as the cellular morphology and ultrastructure were evaluated. RESULTS: All applications inhibited cell proliferation and induced apoptosis. The most substantial decreases in cell proliferation and the caspase-3, epidermal growth factor receptor (EGFR), platelet derived growth factor receptor-alpha (PDGFR-α), multidrug resistance protein-1 (MRP-1), aquaporin-4 (AQP-4) and cAMP levels were induced by the LiCl treatment, which exhibited more pronounced effects compared with the combination treatment. LiCl was less effective in decreasing the MK and B cell lymphoma-2 (Bcl-2) levels compared with the combination treatment. The most substantial decrease in the p170 levels was identified following the combination treatment, whereas IM induced the second greatest decrease. LiCl alone had no effect on the p170 levels. IM induced the most substantial decrease in the phospho-glycogen synthase kinase 3-beta (p-GSK-3ß)/glycogen synthase kinase 3-beta (GSK-3ß) ratio, and LiCl induced the second most substantial decrease. Both LiCl and the combination treatment induced G2 + M arrest, whereas IM induced G0 + G1 arrest after 72 h of exposure. An apoptotic appearance and autophagic vacuoles were commonly identified in the LiCl, combination and IM groups, respectively. CONCLUSIONS: The combination of IM and LiCl exhibited an antagonist effect, and MK had a role at this antagonism.

The Effects of Adipose Tissue-Derived Mesenchymal Stem Cell Transplantation During the Acute and Subacute Phases Following Spinal Cord Injury.

AIM: To investigate the effectiveness of rat adipose tissue-derived (rAT) mesenchymal stem cell (MSC) transplantation on the functional restoration and regeneration of spinal cord injury (SCI). MATERIAL AND METHODS: Six of 48 Wistar albino rats were sacrificed to obtain MSCs, and the remaining rats were divided randomly into six groups. SCI was performed using the clip compression method. The control and transplantation groups were injected with physiological saline and a rAT-MSC suspension at the injury sites, respectively. Each animal was evaluated using the Basso, Beattie and Bresnahan (BBB) rating system and sacrificed at 28 days post-injury period (p.i.). The regeneration process was evaluated based on immunostaining against ß3-tubulin, BDNF, CNTF, and CNPase. RESULTS: rAT-MSC transplantation into the SCI site substantially improved the tissue regeneration and functional recovery (p < 0.05). However, the rAT-MSC transplantation at 9 days p.i. was not more efficient on functional recovery than the transplantation immediately after injury. The expression of ß3-tubulin, BDNF and CNTF at the injury site indicated the potential for functional regeneration. CONCLUSION: The adaptive nature of rat-MSCs enabled the remodulation and regeneration of the lesion site, decreasing the importance of transplantation time in the treatment of SCI.

Effect of simvastatin on mitochondrial enzyme activities, ghrelin, hypoxia-inducible factor 1α in hepatic tissue during early phase of sepsis.

We aimed to investigate the effects of prior treatment of simvastatin on mitochondrial enzyme, ghrelin, and hypoxia-inducible factor 1 α (HIF-1 α) on hepatic tissue in rats treated with Lipopolysaccharides (LPS) during the early phase of sepsis. Rats were divided into four groups: control, LPS (20 mg/kg, i.p.), Simvastatin (20 mg/kg, p.o.), and LPS + Simvastatin group. We measured citrate synthase, complex I, II, I-III, II-III enzymes activities, serum and tissue levels of TNF-α, IL-10 using ELISA. Liver sections underwent histopathologic examination and TNF-α, IL-10, HIF-1α and ghrelin immunoreactivity were examined using immunohistochemistry methods. There were no differences in all groups for mitochondrial enzyme activities. In terms of both ELISA and immunohistochemistry findings; the levels of serum and tissue TNF-α and IL-10 were higher in the experimental groups than controls (P < 0.05). In the LPS group, the hepatocyte cell membrane and sinusoid structure were damaged. In the Simvastatin +LPS group, hepatocytes and sinusoidal cord structure were partially improved. For HIF-1α, in all experimental groups immunoreactivity was increased (P < 0.05). In the Simvastatin group, Ghrelin levels were increased in comparison with the other groups (P < 0.01). Ghrelin levels were greatly decreased in LPS (P < 0.05). We observed that the degree of hepatocellular degeneration was partially reduced depending on the dosage and duration of prior simvastatin treatment with LPS, probably due to alterations of Ghrelin and HIF-1α levels.

Sorafenib and lithium chloride combination treatment shows promising synergistic effects in human glioblastoma multiforme cells in vitro but midkine is not implicated.

OBJECTIVES: The objectives of this study were to test the effects of the new combination treatment modality, sorafenib (SOR) and lithium chloride (LiCl) and to assess whether midkine (MK) protein has a role in any potential effects. METHODS: Monolayer and spheroid cultures of T98G human glioblastoma multiforme (GBM) cells were treated with LiCl and SOR (inhibition concentration 50 value  =  100 µM), or their combination, or were left untreated (control). Cell proliferation and apoptotic indices, the mechanism of action, and the levels of apoptotic and anti-apoptotic proteins were evaluated in monolayer cultures and ultrastructure was evaluated by transmission electron microscopy (TEM) in spheroid cultures after for 72 hours. RESULTS: All drug applications decreased cell numbers and increased the apoptotic index. The combination shows a synergistic effect. In the combination group, the decrease in cell numbers and the increase in the apoptotic index were significantly greater than with the individual drugs (P < 0.01). The combination treatment led to the greatest decreases in MRP-1 and p170 levels; but the greatest decreases in p-STAT-3, p-ERK (P < 0.05), p-AKT, p-GSK-3-beta (P < 0.01), EGFR (P < 0.01), NF-kappa-ß levels were with SOR alone, followed by the combination. The decreases in MK levels in the SOR and combination groups were similar (P  =  0.06). Severe ultrastructural damage was more frequently observed in the combination group compared with the other groups. CONCLUSIONS: These results suggest the possibility that the addition of LiCl to SOR could improve the prognosis in at least some patients who need both cancer and psychotherapy and indicate the need for further studies.