Inhibition Of Thymidine Incorporation Research Articles

Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells.

The active form of vitamin D (1α,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 μM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.

Statins Modulate Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 in Human Hepatic Myofibroblasts.

Statins have been shown to exert anti-inflammatory and anti-fibrogenic properties in the liver. In the present study, we explored the mechanisms underlying anti-fibrogenic effects of statins in isolated hepatic myofibroblasts and focused on cyclooxyegnase-2, a major anti-proliferative pathway in these cells. We show that simvastatin and fluvastatin inhibit thymidine incorporation in hMF in a dose-dependent manner. Pretreatment of cells with NS398, a COX-2 inhibitor, partially blunted this effect. cAMP levels, essential to the inhibition of hMF proliferation, were increased by statins and inhibited by non-steroidal anti-inflammatory drugs. Since statins modify prenylation of some important proteins in gene expression, we investigated the targets involved using selective inhibitors of prenyltransferases. Inhibition of geranylgeranylation resulted in the induction of COX-2 and mPGES-1. Using gel retardation assays, we further demonstrated that statins potentially activated the NFκB and CRE/E-box binding for COX-2 promoter and the binding of GC-rich regions and GATA for mPGES-1. Together these data demonstrate that statin limit hepatic myofibroblasts proliferation via a COX-2 and mPGES-1 dependent pathway. These data suggest that statin-dependent increase of prostaglandin in hMF contributes to its anti-fibrogenic effect.

Anti‐c‐MET Nanobody® – a new potential drug in multiple myeloma treatment

c-MET is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-MET signaling is involved in many human malignancies, including multiple myeloma (MM). Recently, multiple agents have been developed directed to interfere at different levels in HGF-c-MET signaling pathway. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy-chain-only antibodies. In this study, we wanted to determine the anticancer effect of a novel anti-c-MET Nanobody in MM. We examined the effects of an anti-c-MET Nanobody on thymidine incorporation, migration, adhesion of MM cells, and osteoblastogenesis in vitro. Furthermore, we investigated the effects of the Nanobody on HGF-dependent c-MET signaling by Western blotting. We show that the anti-c-MET Nanobody effectively inhibited thymidine incorporation of ANBL-6 MM cells via inhibition of an HGF autocrine growth loop and thymidine incorporation in INA-6 MM cells induced by exogenous HGF. HGF-induced migration and adhesion of INA-6 were completely and specifically blocked by the Nanobody. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced alkaline phosphatase activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced phosphorylation of tyrosine residues in c-MET, MAPK, and Akt. We also compared the Nanobody with anti-c-MET monoclonal antibodies and revealed the similar or better effect. The anti-c-MET Nanobody inhibited MM cell migration, thymidine incorporation, and adhesion, and blocked the HGF-mediated inhibition of osteoblastogenesis. The anti-c-MET Nanobody might represent a novel therapeutic agent in the treatment of MM and other cancers driven by HGF-c-MET signaling.

Antibody–drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin

A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

Evaluation of potential antirheumatic drugs in vitro using lymphocytes and epithelial cells. The selective action of indoxole, methyl glyoxal and chloroquine.

Abstract Anti-inflammatory steroids and non-steroid drugs rapidly inhibit the incorporation of radioactivity from tritiated uridine and thymidine into the nucleic acid fraction of cultured epithelial cells and lymphocytes obtained from sheep, rats, rabbits and chickens. Acidic (uncoupling) drugs were also found to inhibit protein synthesis in these particular cells, and protein and nucleic acid synthesis in other cells which were steroid-insensitive, DNA synthesis in epithelial cells and lymphocytes was inhibited by methyl glyoxal (pyruvaldehyde). Chloroquine selectively inhibited thymidine incorporation by the epithelial cells. Indoxole was a potent inhibitor of the metabolism of circulating lymphocytes incubated in protein-free media. These findings may indicate why some of these drugs suppress adjuvant arthritis in rats.

Inhibition of Cancer Cell Replication by Inorganic Nitrite

Nitrite has been implicated in carcinogenesis, especially under acidic conditions such as in the stomach or in urine, where it forms nitrosating species that can react with secondary amines to form nitrosamines. Recent studies have shown that nitrite and acid form a variety of other nitrogen oxides in vivo including nitric oxide—a compound with documented antitumor activity. Here we tested the effects of nitrite on bladder tumor cells incubated in mildly acidified urine. Nitrite (50 μM) inhibited thymidine incorporation in human T24 bladder cancer cells. This inhibition required slight acidification (pH 5.5–6), and no effect of nitrite could be observed at pH 7. Nitrite effects were further augmented in the presence of ascorbic acid, whereas ascorbic acid alone had no effect. The effects were paralleled by formation of nitric oxide gas. We here demonstrate an inhibitory effect of nitrite on cancer cell replication at concentrations and acidity commonly found in urine and gastric juice. The inhibitory effect is likely caused by nitric oxide and possibly other reactive nitrogen oxides formed from acidified nitrite.

Abstract 3654: Clofarabine: factors influencing cytotoxicity and resistance in hematologic and solid malignancies

Abstract Clofarabine is a new-generation nucleoside analog that was synthesized to combine the most favorable pharmacological properties of its congener fludarabine and cladribine. It acts as a strong inhibitor of DNA polymerase, DNA synthesis and ribonucletide reductase (R1/R2). Like other nucleoside analogues, clofarabine must be activated within cells to the 5′-triphodphate form initially by deoxycytidine kinase (dCK). In this study, the anticancer effect of clofarabine was investigated in three solid tumor cell lines, HCT116 (a colon carcinoma cell line), U1810 (a human non-small cell lung carcinoma cell line), and MCF7 (a mammary carcinoma cell line) and compared to four leukemic cell lines, CCRF-CEM, HL60, K562 and MOLT4. Clofarabine was investigated to enter into HCT116, U1810, and MCF7 in a clearly detectable manner by LigandTracer® White. At 10 µM a high signal is achieved and approaches equilibrium after 1∼2 hrs. Pre-incubation with 10 µM clofarabine resulted in rapid inhibition of thymidine incorporation and DNA synthesis. Induction of apoptosis by clofarabine was cell line dependent, but the average apoptotic level was higher in leukemic cell lines than solid tumor cell lines; however it seemed the sensitivity for apoptosis of cell line is correlated with the activity of dCK and thymidine kinase 1 (TK1) in the solid tumor cell lines. Upon treatment with 10 μM clofarabine for 24 hrs, apoptosis induction increased compared to untreated cells 3.1-, 2.9- and 2.1-fold; the activity of dCK 7.3-, 3.7- and 3.1-fold and the activity of TK1 4.2-, 1.4- and 1.2-fold in U1810, HCT116 and MCF7 cells, respectively. In general, the expression of R1, dCK and TK1 in leukemic cells was higher than in solid tumor cells, but the expression of R2, p53R2 and p53 varied among the cell lines. However, there was an inverse correlation between induction of apoptosis at 100nM clofarabine (upon 24 hours incubation) and the expression of p53R2 (spearman rank test −0.67, p=0.1). In addition, clofarabine treatment causes retardation in S-phase, which seems to be related to the activity of TK1 and the protein expression of R2 in U1810, HCT116 and MCF7 cells. This study is a first step to extend the application of clofarabine toward the solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3654.

The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (10(4) cells/mm(2)) and found that RA (10(-6) M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (10(2) cells/mm(2)), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10(-8) M-10(-5) M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10(-7) (422% of control) and 10(-6) (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.

Effect of retinoic acid on the proliferation and alkaline phosphatase activity of osteoblastic MC3T3-E1 cells by modulating the release of local regulators from monocytes.

There has been some evidence suggesting an important role of mononuclear cells at bone remodeling sites in the coupling of bone formation to bone resorption. Since cells of the monocyte-macrophage lineage produce important local regulators of bone remodeling, we examined effects of human monocytes-conditioned medium (CM) treated with retinoic acid on [3H] thymidine incorporation (TdR) and alkaline phosphatase (ALP) activity of osteoblastic MC3T3-E1 cells. Treatment of MC3T3-E1 cells with retinoic acid (10(-8) to 10(-6) M) caused an inhibition of TdR in a dose-dependent manner and an inhibition of ALP activity at 10(-6) M. Conditioned medium from monocytes untreated with retinoic acid caused a stimulation of TdR and an inhibition of ALP activity in these cells. In contrast, treatment of monocytes with retinoic acid (10(-8) or 10(-6) M) abolished both stimulation of DNA synthesis and inhibition of ALP activity induced by CM. The present study suggested that retinoic acid modulated osteoblast proliferation and ALP activity not only directly but also indirectly, presumably through modulating the release of local regulators as to bone remodeling from monocytes.

Insulin stimulates fibroblast proliferation through calcium-calmodulin-dependent kinase II

Insulin effects are mediated by multiple integrated signals generated by the insulin receptor. Fibroblasts, as most of mammalian cells, are a target of insulin action and are important actors in the vascular pathogenesis of hyperinsulinemia. A role for calcium-calmodulin-dependent kinases (CaMK) in insulin signalling has been proposed but has been under investigated. We investigated the role of the CaMK isoform II in insulin signalling in human fibroblasts. A rapid and transient increase of intracellular calcium concentration was induced by insulin stimulation, followed by increase of CaMKII activity, via L type calcium channels. Concomitantly, insulin stimulation induced Raf-1 and ERK activation, followed by thymidine uptake. Inhibition of CaMKII abrogated the insulin-induced Raf-1 and ERK activation, resulting also in the inhibition of thymidine incorporation. These results demonstrate that in fibroblasts, insulin-activated CaMKII is necessary, together with Raf-1, for ERK activation and cell proliferation. This represents a novel mechanism in the control of insulin signals leading to fibroblast proliferation, as well as a putative site for pharmacological intervention.

Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity

Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity in vitro, but its clinical activity is compromised by binding to the plasma protein alpha(1)-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the in vivo activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 in vitro and in vivo. Plasma NU3153 concentrations commensurate with rescue inhibition in vitro were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity in vivo. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (> or =90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved in vivo using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver.

Extracellular organelles (prostasomes) are immunosuppressive components of human semen

Numerous reports have ascribed immunosuppressive activity to human seminal plasma and there is growing agreement that much of this activity can be accounted for by the very high levels of E series prostaglandins present (up to 300 microM 19-hydroxy prostaglandin E). However not all suppressive activity is due to prostaglandin since several reports have appeared of high molecular weight active substances and we have found that stripped seminal plasma is still effective in inhibiting the mitogen-induced proliferation of lymphocytes. In this study such immunosuppressive activity has been separated by molecular size fractionation and the activity has been found to be particulate and corresponded to the previously reported prostasomes. These are trilaminar to multilaminar vesicles (150 nm diameter) which are secreted by the prostate. Pure preparations of prostasomes inhibited mitogen-induced lymphoproliferation in a dose-dependent manner with a concentration of prostasomes equivalent to 40% of that seen in seminal fluid giving 69% suppression of thymidine incorporation. The suppressive activity survived boiling and therefore was unlikely to be due to enzymatic action associated with these organelles. Interaction with the accessory cells, involved in full development of the lymphoproliferation induced by mitogen, was indicated and this possibility was supported by the demonstration of a direct effect of prostasomes on macrophage function using a mouse macrophage cell line. The prostasomes in semen may play a complementary role to the prostaglandins in neutralizing the immune defences of the female reproductive tract. This combination would allow the alloantigenic spermatozoa the best chance of achieving fertilization, but at the same time leave the recipient open to any infection present in the semen.

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.

Serum amyloid A induces WISH cell apoptosis

Serum amyloid A (SAA) is an important mammalian acute reactant. Here, we aim to investigate the effect of SAA on apoptosis and its mechanism of action in human amniotic WISH cells. The expression of formyl peptide receptor (FPRL1), which is reported as a SAA receptor, was tested using RT-PCR and ligand binding assay with radio-labeled FPRL1 ligand. The effect of SAA on proliferating cell population was evaluated by thymidine incorporation assay. The protein phosphorylation levels and caspase-3 activity were detected by Western blot assay. SAA inhibits thymidine incorporation in human amniotic WISH cells. A SAA-induced decrease of proliferating cell population was accompanied with nuclear condensation and caspase-3 activation in WISH cells, suggesting that SAA induces WISH cell apoptosis. Since FPRL1 has been reported as a SAA receptor, we investigated the effects of several FRPL1 agonists on a proliferating cell population in WISH cells. Among the tested FPRL1 agonists, only SAA induced a decrease of proliferating cell population in WISH cells. On the downstream signaling of SAA, we found that SAA stimulated extracellular signal-regulated kinase and p38 kinase, which were not inhibited by pertussis toxin (PTX), ruling out the role of PTX-sensitive G-proteins. Furthermore a SAAinduced decrease of proliferating cell population was not affected by PTX, suggesting that SAA inhibits WISH cell apoptosis in a PTX-sensitive G-proteinindependent manner. A SAA-induced decrease of a proliferating cell population was completely blocked by PD98059 and SB203580, suggesting that mitogenactivated protein kinase activities are essentially required for the process. SAA is a novel inducer for WISH cell apoptosis, and the PTX-insensitive pathway is involved in the process.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

Extracellular adenosine triphosphate protects oxidative stress‐induced increase of p21WAF1/Cip1 and p27Kip1 expression in primary cultured renal proximal tubule cells: Role of PI3K and Akt signaling

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in causing many renal diseases. Adenosine triphosphate (ATP) is an important extracellular signal in the regulation of many intracellular processes in normal tubular cells as well as in the pathogenesis of cell injury. This study investigated the effect of ATP on H(2)O(2)-induced increase of cyclin kinase inhibitors (CKI) expression and its related signal molecules in primary cultured renal proximal tubule cells (PTCs). H(2)O(2) inhibited DNA synthesis in a concentration- (>50 microM) and time-dependent manner (>2 h), as determined by thymidine and BrdU incorporation, and by increase in the p21(WAF/Cip1) and p27(Kip1) expression levels. In contrast, ATP increased the level of thymidine, BrdU incorporation (>10(-5) M), and decreased the p21(WAF/Cip1) and p27(Kip1) expression levels, suggesting that ATP has a protective effect against H(2)O(2)-induced oxidative damage. Suramin, reactive blue 2 (RB-2), MRS 2159, and MRS 2179 did block the reversing effect of ATP. In addition, AMP-CPP or 2-methylthio-ATP blocked H(2)O(2)-induced inhibition of DNA synthesis, suggesting all these P2 purinoceptors may be potentially involved. ATP-induced stimulation of DNA synthesis was blocked by phosphatidylinositol 3-kinase (PI3K) and Akt inhibitors. These results suggest the involvement of P2 purinoceptors-mediated PI3K/Akt signal pathway in the protective effect of ATP against H(2)O(2)-induced oxidative damage. Indeed, pre-treatment with PI3K or Akt inhibitors did not protect H(2)O(2)-induced lipid peroxide (LPO) production and inhibition of thymidine incorporation. In conclusion, ATP, in part, blocked H(2)O(2)-induced increase of p21(WAF1/Cip1) and p27(Kip1) expression through PI3K and Akt signal pathway in renal PTCs.

Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats

Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2α; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10 −8 to 10 −4 M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G 0/G 1 phase. Upon a 24 h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10 −4 M). Replenishments of PGE1, PGE2 or PGF2α could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG-independent mechanisms. COX-2 selective NSAIDs showed less deleterious effects on chondrocytic proliferation and death.

On the Mechanisms of 12-Otetradecanoylphorbol-13-acetate-induced Growth Arrest in Pancreatic Cancer Cells

Protein kinase C (PKC) is involved in cell growth, differentiation, and apoptosis. We investigated the effects of the PKC activator, the tetradecanylphorbol acetate (TPA), in human pancreatic cancer cells. Cell proliferation was measured by thymidine incorporation. Expression of cell cycle proteins was investigated by Western blot. Real-time reverse transcriptase-polymerase chain reaction was used to measure p21 messenger RNA expression, whereas knockdown of its expression was accomplished with a specific small interferring RNA. Cell cycle phases were determined by flow cytometry. TPA time and concentration dependently inhibited thymidine incorporation in Panc-1 and CD18 cells and induced G2/M cell cycle arrest. The TPA decreased cyclin A and B expression, increased cyclin E, and markedly increased the expression of p21 at both the messenger RNA and protein levels. TPA-induced p21 expression and growth inhibition were blocked by the PKC inhibitor, bisindoylmaleimide. TPA induced extracellular signal-regulated kinase1/2 phosphorylation, whereas the MEK inhibitor, PD98059, blocked the TPA-induced p21 expression. Small interferring RNA targeted to p21 blocked TPA-induced p21 protein expression but not TPA-induced cell growth arrest. TPA-induced p21 expression is mediated by the MEK/ERK pathway but is not involved in TPA-induced growth inhibition. In contrast, cyclin A and cyclin B are likely involved in TPA-induced G2/M arrest because both proteins are involved in S phase and G2/M transition during cell proliferation.

Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line

Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2-4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.

Extracts From a Traditional Chinese Herbal Remedy Inhibit Cell Proliferation and Induce Apoptosis in Human Endometrial and Endometriosis Cells

Extracts From a Traditional Chinese Herbal Remedy Inhibit Cell Proliferation and Induce Apoptosis in Human Endometrial and Endometriosis Cells