In vitro effects of heparin-binding epidermal growth factor on adhesion stage of implantation.

A member of the epidermal growth factor (EGF) family, the heparin-binding EGF (HB-EGF) is expressed in the uteri of both humans and mice during the implantation process. To study the effects of HB-EGF on adhesion stage, we developed an in vitro implantation model employing Ishikawa cell line and JAR cell line, which may attach to Ishikawa cells. For 1, 6, 12, and 24 hours, co-cultures of JAR spheroids grown on Ishikawa monolayers were treated with 1, 10, and 100 ng∕mL doses of HB-EGF. Using immunocytochemistry and Western blot analysis, the effects of HB-EGF on the protein expressions of E-cadherin, Erb-B2 receptor tyrosine kinase 4 (ErbB4), and integrin ανß3 in Ishikawa and JAR cells were examined semi-quantitatively and quantitatively. Ultrastructural changes of in vitro implantation model were investigated by transmission electron microscopy. We revealed that HB-EGF influenced trophoblast cell adhesion to endometrial cells by upregulating the expression of the proteins ErbB4 and trophoblastic integrin ανß3. Decrease in trophoblastic E-cadherin expression and increase in endometrial E-cadherin expression were demonstrated accompanying morphological variations in cells required for the invasion. We discovered ultrastructurally that Ishikawa cells acquired uterodome-like appearance, including the organelles, when 10 and 100 ng∕mL dosages of HB-EGF were administered for 12 and 24 hours. However, following additional hours of adhesion and invasion, their intercellular spaces enlarged. The trafficking of vesicular transport was enhanced by JAR spheroids. We therefore discovered that in this implantation paradigm, HB-EGF may enhance the receptivity of Ishikawa cells and the adherence of JAR cells.

Midkine silencing enhances the anti-prostate cancer stem cell activity of the flavone apigenin: cooperation on signaling pathways regulated by ERK, p38, PTEN, PARP, and NF-κB.

Prostate cancer (PCa) is the most common cancer in men worldwide. Midkine (MK) is overexpressed in PCa, as well as in tumor-initiating cells termed cancer stem cells (CSCs). Apigenin is a dietary flavone with considerable anti-tumor activities. In this study, we explored the possible therapeutic use of MK silencing, apigenin treatment, and a combination of both on human PCa and prostate cancer stem cells (PCSCs). CD44+CD133+ PC3 and CD44+ LNCaP CSCs were isolated from their parent cell lines. Both MK knockdown and apigenin treatment resulted in loss of cell viability in PCSCs, and these effects were significantly elevated when apigenin was applied with MK silencing. Combined treatment of CD44+CD133+ PC3 cells with apigenin and MK siRNA was also more effective in inducing apoptotic and non-apoptotic cell death when compared with individual applications. Treatment of CD44+ LNCaP cells with apigenin significantly decreased viability, although the combination treatment did not markedly alter the individual therapy. Molecular events underlying cell cycle arrest and inhibition of the survival, proliferation, and migration of CD44+CD133+ PC3 cells were found to be associated with upregulated p21, p27, Bax, Bid, caspase-3, and caspase-8 expression, as well as downregulated p-p38, p-ERK, NF-κB, and PARP. In addition, the combination of apigenin treatment and MK silencing showed better outcomes on the anticancer efficacy of docetaxel in CD44+CD133+ PC3 cells. In conclusion, MK-regulated events are different between PCSCs, and when combined with apigenin plus MK silencing, docetaxel treatment may be a valuable approach for the eradication of PCSCs.

Inhibition of midkine by metformin can contribute to its anticancer effects in malignancies: A proposal mechanism of action of metformin in context of endometrial cancer prevention and therapy.

Metformin, a drug widely used in the treatment of type II diabetes mellitus (T2DM), has been the focus of interest as a potential therapeutic agent for certain types of malignancies, including gynaecological cancers [i.e. endometrial cancer (EC)]. Although the exact mechanism behind the potential anticancer activity of metformin is still not completely understood, certain studies have suggested that different effects on cell functions, such as inhibition of cell migration, apoptosis and tumor cell proliferation, are involved in its preventive and therapeutic effects in certain types of malignancies, including EC. In contrast, midkine (MK), a heparin-binding growth factor and cytokine, which induces carcinogenesis and chemoresistance, promotes the development and progression of many malignant tumours by increasing diverse cell functions such as cell proliferation, cell survival and antiapoptotic activities via mainly the activation of phosphatidyl inositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The same pathways are also subject to certain therapeutic effects of metformin, although this cytokine and this drug have some different mechanism of action pathways as well. Taken together, MK and metformin appear to have opposite effects in various biological processes such as apoptosis, cell proliferation, cell survival, cell migration, and angiogenesis. On the other hand, MK activates PI3K and MAPK cell signal pathways, whereas metformin inhibits these two pathways. It seems likely that almost all the pathways and cell functions, which play important roles in malignancies, are inhibited by metformin and activated by MK. Given the opposite relationship between the actions of metformin and MK, we hypothesize that metformin may act like a novel MK inhibitor in some malignancies. We also discuss the possible relationship between metformin and MK in the context of EC, the most common gynecological cancer worldwide, which incidence is rising rapidly, in parallel with the increase in obesity, T2DM and insulin resistance. In this respect, the therapeutic use of metformin may improve the survival of EC or other cancers, via inhibiting or overcoming the unwanted effects of MK in carcinogenesis.

The Possible Interactions and Therapeutic Roles of Lithium Chloride and Midkine on Cancer Treatment.

Midkine (MK) is a heparin-binding anti-apoptotic growth factor or cytokine also known as neurite growth-promoting factor 2 (NEGF2). It is developmentally an important retinoic acid-responsive gene product strongly induced during the mid-gestation stage. Midkine promotes different cellular events such as cell growth, differentiation, survival, gene expression, and drug resistence. Midkine, the phosphatidylinositol 3-kinases (PI3-kinase, PI3K) and glycogen synthase kinase-3 beta (GSK-3ß) inhibitors together with lithium chloride may be a very effective treatment modality, especially in tumors with high expression of these two molecules. PI3 kinase and GSK-3ß, both serine threonine kinases located in the center of the signaling network, are very important regulator molecules for cell survival or death. Lithium chloride (LiCl), with its newly discovered antineoplastic effect and cytotoxicity potentiation, has become a promising agent in the application of new combination treatments. Although the LiCl mechanism of action is still not fully understood, previous studies have shown that LiCl is an inhibitör of the inositol monophosphatase (IMPase) and GSK-3ß. GSK-3ß, is a serine-threonine protein kinase involved in cell proliferation, differentiation, survival, apotosis, and tumorogenesis. The role of GSK-3ß in tumorigenesis and cancer remains controversial. It may have a function as a tumor suppressor for certain types of tumors, but it promotes cell growth and development in other tumor types.

Gemcitabine, vinorelbine and cyclooxygenase inhibitors in the treatment of glioblastoma. Ultrastructural analyses in C6 glioma in vitro.

OBJECTIVES: To define ultrastructural features accompanying to antitumor effects of gemcitabine, vinorelbine and cyclooxygenase inhibitors in C6 glioma cells in vitro. Vinorelbine is a semisynthetic vinca alkaloid and recent studies showed its antitumor activity in pediatric optic and pontine gliomas. Vinorelbine infusion induces a severe tumor site-pain in systemic cancers, but it is unknown whether algesia and inflammation contribute to its antitumor effects. Gemcitabine is a nucleoside-chemotherapeutic which was recently shown to act as a radiosensitizer in high-grade glioma. Some studies showed synergism of anti-inflammatory cyclooxygenase-inhibitors with microtubule inhibitors and gemcitabine. DMSO is a solvent and blocks both cylooxygenase and ribonucleotide reductase, another target of gemcitabine. Rofecoxib is withdrawn from the market, yet we used it for investigational purposes, since it blocks cylooxygenase-2 1000-times more potently than cylooxygenase -1 and is also a selective inhibitor of crinophagy. METHODS: Plating efficacy, 3D-spheroid S-phase analysis with BrdU labelling and transmission electron microscopical analyses were performed. RESULTS: Vinorelbine induced frequent mitotic slippage/apoptosis and autophagy. Despite both DMSO and rofecoxib induced autophagy alone and in synergy, they reduced mitotic catastrophe and autophagy triggered by vinorelbine, which was also reflected by reduced inhibition of spheroid S-phase. Gemcitabine induced karyolysis and margination of coarse chromatin towards the nuclear membrane, abundant autophagy, gutta adipis formation and decrease in mitochondria, which were enhanced by DMSO and rofecoxib. CONCLUSIONS: Detailed ultrastructural analysis of the effects of chemotherapeutic drugs may provide a broader insight about their actions and pave to develop better strategies in treatment of glioblastoma.

Methylation Changes of Primary Tumors, Monolayer, and Spheroid Tissue Culture Environments in Malignant Melanoma and Breast Carcinoma.

Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for PAX5, TMPRSS2, and SBDS. These findings will play an important role in developing in vitro model system to understand the effect of methylation inhibitors on primary tumor tissue. In a previous study PAX5, TMPRSS2, and SBDS genes that we are investigating were reported to be methylated more than 60% in breast cancer and malignant melanoma cell lines. However, these genes have never been studied in primary tumor tissues. Thus, primary tumor tissues of breast cancer and malignant melanoma were first grown in 2D and 3D cultures. Then these two types of tumor tissues and their 2D and 3D cultures were investigated for changes considering methylation levels in PAX5, TMPRSS2, and SBDS genes using real-time polymerase chain reaction. No differences were observed in the primary tissues and culture systems for both PAX5 and TMPRSS2 in malignant melanoma tissues. We found that PAX5 gene was an efficient marker to measure the effects of methylation inhibitors for in vitro systems for malignant melanoma tissue.

Medroxyprogesterone effects on colony growth, autophagy and mitochondria of C6 glioma cells are augmented with tibolone and temozolomide: Cell kinetic and electron microscopical studies with a broad review of the literature.

OBJECTIVE: Risk of high grade gliomas is lower in young females and its incidence enhances after menopause suggesting likely protective roles of female hormones. Hormone replacement therapy (HRT) was widely employed to treat osteoporosis and some epidemiological studies showed that HRT regimes including progesterone analogs such as medroxyprogesterone acetate (MPA) decreased risk of glial tumors. Tibolone is a unique progesterone analog employed in HRT with tissue specific estrogenic effects and stimulates gene expressions very similar to those induced by MPA. Tibolone's pro-estrogenic effects occur particularly in bone and brain and both MPA and tibolone inhibit AKR1C enzymes, which involve in temozolomide chemoresistance. Hence, we aimed to investigate interactions between MPA, tibolone and temozolomide in modification of glioma cell growth and fine structure. PATIENTS AND METHODS: For our studies, we have particularly chosen C6 rat glioma cell line due to several reasons: i) We previously showed that MPA reduced growth and induced procarbazine-sensitization in C6 cells; ii) temozolomide has a triazene-type molecular structure like procarbazine; iii) other groups previously showed that C6 glioma cell line is more resistant to temozolomide than human glioma cells; hence it may provide a native model of chemoresistance. Monolayer plating efficacy, soft agar colony growth, 3D-spheroid S-phase (as determined by BrdU-labeling) and electron microscopical analyses were performed to assess mutual interactions between MPA, tibolone and temozolomide. RESULTS: MPA inhibited clonogenic growth of C6 glioma and this effect is augmented by both tibolone and temozolomide. MPA and tibolone inhibited DNA synthesis in C6 glioma spheroids to similar levels which can be achieved with temozolomide. Electron microscopical analyses revealed synergisms between MPA, tibolone and temozolomide involved mitochondrial proliferation, condensation, mitophagy and autophagy. CONCLUSIONS: MPA and tibolone shall be studied in further experimental models of glioblastoma in vitro and in vivo.

Involvement of midkine in autoimmune and autoinflammatory diseases.

Midkine (MK) is a heparin-binding growth factor that markedly expressed during embryogenesis but downregulated to inconsiderable levels in healthy adults. However, MK is upregulated during tissue repair and in many pathologic conditions, mostly malignancies and inflammatory diseases. MK promotes a number of functions in target cells such as migration, proliferation, survival, growth, reproduction and repair, angiogenesis, and gene expression. It acts as a pro-inflammatory cytokine and contributes to chronic inflammation via promoting chemotaxis and tissue infiltration of neutrophils and macrophages. Furthermore, MK upregulated the production of various inflammatory mediators (i.e. interleukin (IL) 6 and IL8). Recent studies have demonstrated strong evidence that MK is involved in the onset and progression of autoimmune rheumatic diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren's syndrome (SS) and other autoimmune conditions such as multiple sclerosis (MS). Additionally, it has been shown that MK is overexpressed in two major clinically defined forms of inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), which are classified as autoinflammatory diseases. Taken together, MK is involved in the pathogenesis of autoimmune and autoinflammatory diseases and may serve as an indicator and biomarker in these conditions. Furthermore, MK inhibitors are expected to contribute in the management of these diseases.

Erucic acid, a component of Lorenzo's oil and PPAR-δ ligand modifies C6 glioma growth and toxicity of doxorubicin. Experimental data and a comprehensive literature analysis.

BACKGROUND: PPAR-δ is a transcription factor which has crucial roles in stimulating oligodendroglial differentiation and myelination and its activation was also shown to differentiate malignant C6 glioma cells into oligodendrocytes. OBJECTIVE: One of the ligands of PPAR-δ is erucic acid (EA), an edible omega-9 fatty acid consumed more by Asian populations and exists highly in Chinese womens milk. There exist epidemiological evidence that pediatric brain tumor incidence is among the lowest in the Chinese population. EA is also an ingredient of Lorenzo's oil used against adrenoleukodystrophy, a pediatric demyelinating disease. EA was inappropriately assumed as a strong cardiotoxin based on Spanish oil syndrome, caused by toxic-aniline dye refined rapeseed oil. In this study, we studied whether EA is capable to block growth of C6 glioma cells and modify cardiotoxicity of doxorubicin. MATERIALS AND METHODS: We studied effects of EA on the 3-dimensional appearance of the adherent cells, soft agar colony formation and S-phase in the 3-dimensional spheroids in C6 glioma cell cultures. We also investigated the effects of EA on hepatic and cardiac toxicity of doxorubicin. RESULTS: EA decreased in vitro growth of C6 glioma cells at therapeutically achievable concentrations. EA effects were more prominent in 3D-assays (soft agar colonies and spheroids) and induced cell fusions in monolayer cultures. EA decreased S-phase inhibitory potency of doxorubicin (DOX), yet augmented its efficacy to induce a senescent morphology (as assessed by scanning electron microscopy) in monolayer and to increase iNOS and eNOS expression in spheroids. In our study, EA reduced DOX-induced necrosis in mice heart and liver and induced healthier morphology of heart mitochondria (as assessed by transmission electron microscopy); yet intercalated disks (ID) were more disturbed with DOX + EA. CONCLUSIONS: Both the antitumor and cardiac effects of EA may associate with the cell-to-cell contact mechanisms. Combining systemic EA with intrathecal DOX-chemotherapy via Ommaya reservoirs may reduce DOX concentrations in systemic circulation, hinder toxic interactions with EA and induce selective kill of glioma cells.

Midkine downregulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ERK pathway.

AIMS: To examine the functions of growth factor midkine (MK) and a flavonoid quercetin on survival, apoptosis and migration of prostate cancer (PCa) stem cells (CSCs). MAIN METHODS: CD44+/CD133+ and CD44+ stem cells were isolated from PC3 and LNCaP cells, respectively by magnetic-activated cell sorting system. 3D cell culture was used to evaluate the ability of quercetin, MK siRNA, and the combination of both to inhibit spheroid formation, apoptosis and cell cycle arrest. Image-based cytometer, RT-qPCR, Western blotting and transwell migration assays were performed. KEY FINDINGS: Quercetin treatment for 24-72 h inhibited PC3 and CD44+/CD133+ stem cell proliferation in a time- and dose-dependent manner. Knockdown of endogenous MK expression significantly suppressed proliferation of CD44+/CD133+ and CD44+ cells as well as their parent cells. Co-administration of MK siRNA and quercetin reduced the cell survival, induced apoptosis and caused G1 phase cell cycle arrest more effectively than the individual therapy. Knockdown of MK significantly enhanced the inhibitory effect of quercetin on CD44+/CD133+ migration and spheroid formation. In addition, the combined therapy inhibited the phosphorylation of PI3K, AKT and ERK1/2, and reduced the protein expression of p38, ABCG2 and NF-κB. SIGNIFICANCE: Quercetin alone exhibited significant cytotoxic effects on CD44+/CD133+. MK plays an important role in the proliferation of CD44+/CD133+ and CD44+ cells in particular, and quercetin and MK-silencing therapy may be an important strategy in targeting CSCs that play a role in relapse, migration and drug resistance.

Effects of different drug treatments on the proliferation of human ovarian carcinoma cell line MDAH-2774

Background/aim: In this study, the effects of resveratrol as a natural polyphenol compound, gemcitabine as an antimetabolite that has nucleoside structure analogous to deoxycytidine, and para-aminophenol-derived paracetamol were investigated with single and combined applications in monolayers of the MDAH-2774 human ovarian cancer cell line. Materials and methods: Drugs were evaluated in cell culture with respect to cell proliferation, cell cytotoxicity (trypan blue dye exclusion test), synthesis phase of cell cycle, and cell structure in 24, 48, 72, and 96 h. Result: Resveratrol and gemcitabine diminished both cell proliferation and cell cycle synthesis phase indication in monolayer cell cultures (P < 0.05). All combination groups showed similar effects that were mainly more effective in respect to single usage of resveratrol and gemcitabine in monolayer cell cultures. Conclusion: The effects of gemcitabine, resveratrol, and paracetamol were investigated in monolayers of the MDAH-2774 human ovarian cancer cell line and a decrease in cell number in cell cycle synthesis phase, prevention of cell proliferation, and destruction of cell structure were observed.

Dual inhibition of P-glycoprotein and midkine may increase therapeutic effects of anticancer drugs.

Multidrug resistance (MDR) to chemotherapy may significantly affect the outcome of cancer treatment. ATP-dependent drug efflux pumps, including P-glycoprotein (P-gp), contribute to the resistance of various chemotherapeutic agents. Overexpression of P-gp in tumor cells induces chemoresistance via pumping the anticancer drugs out of the cells. In addition to taking part in many biological processes such as development, reproduction and repair, midkine (MK) also plays important roles in the pathogenesis of malignant diseases as well as in the regulation of MDR. Although, the mechanisms of action of P-gp and MK are different, overexpression of both proteins prevents the accumulation of many chemotherapeutics in tumor cells, leading to decreased therapeutic effects of anticancer drugs. Therefore, identification of the result of dual inhibition of P-gp and MK in overcoming chemoresistance may enhance the likelihood for a more efficient chemotherapy.

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.

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.

The flavonoid apigenin reduces prostate cancer CD44(+) stem cell survival and migration through PI3K/Akt/NF-κB signaling.

AIMS: Cancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. MAIN METHODS: Prostate CSCs (CD44(+)) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48h. KEY FINDINGS: Apigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8, -3 and TNF-α, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-α and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2, -9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-κB p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-κB signaling. SIGNIFICANCE: Our data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs.

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.

Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts.

BACKGROUND: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. AIMS: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. STUDY DESIGN: In vitro study. METHODS: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group). Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2). RESULTS: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10(th) day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5(th) and 10(th) days. For the second method, the membranous form on the 10(th) day and solution form on the 5(th) day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5(th) day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5(th) day. CONCLUSION: The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches.

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.

Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

Effects of hemostatic agents on fibroblast cells.

PURPOSE: Hemostatic agents may be used topically to control hemorrhage, especially in patients with bleeding disorders. The agent used may have a negative effect on the tissue prolonging the healing time. The aim of this study was to compare the effects of 3 different hemostatic agents on fibroblast cells on a rat primary fibroblast cell culture model. MATERIALS AND METHODS: Ankaferd Blood Stopper (ABD) (Ankaferd Pharmaceuticals Cosmetics Production and Marketing Co.), fibrin glue, and tranexamic acid were the agents to be evaluated for their effects on cell proliferation, cell numbers, cell viability, and cell morphology. Also lactate dehydrogenase, basic fibroblast growth factor, and vascular endothelial growth factor C levels were measured. RESULTS: It was found that all of the agents used in the study have negative effects on fibroblasts, with ABD having the lowest values of cell proliferation, cell number, and cell viability. CONCLUSION: The results of this study indicate that ABD, fibrin glue, and tranexamic acid may negatively affect tissue healing.