Dose-dependent Decrease In Cell Number Research Articles

P-326 - Eucalyptus globulus Labill. decoction extract inhibits the growth of human tumor cell lines

Eucalyptus globulus Labill. is a widespread evergreen plant with a plethora of medicinal properties, most notably antitumoral activity. The aims of this work were to: (i) test the cell growth inhibitory effect of the E. globulus, prepared by decoction and infusion, in three human tumor cell lines models: lung (NCI-H460), colorectal (HCT-15) and pancreatic (PANC-1) cancer; (ii) study the mechanism of action of the most potent extract in the most sensitive cell line. Both extracts inhibited the growth of all cell lines and revealed gallotannins as the main compounds. The decoction extract was the most potent in inhibiting the NCI-H460 cell growth. Thus, we further investigated the effect of the decoction extract on the NCI-H460 cell viability (Trypan blue exclusion assay), cell cycle profile, apoptosis (Flow cytometry) and protein expression (Western blot). Interestingly, the E. globulus decoction extract caused a dose-dependent decrease in cell number, which was correlated with a cell cycle arrest in the G1 phase and an increase in p53 and p21 proteins expression. However, no differences were found in the levels of programmed cell death. The relevance of identifying novel bioactive compounds with antitumoral activity in Eucalyptus globulus L. is thus highlighted in this work.

Inhibition of Proliferation of Prostate Cancer Cell Line DU-145 in vitro and in vivo Using Salvia miltiorrhiza Bunge.

To investigate the antiproliferative activity of Salvia miltiorrhiza Bunge. (SM) on the castration-resistant prostate cancer (CRPC) cell line DU-145, in vitro and in vivo. Prostate cancer cell line (DU-145) and normal prostate cell line (RWPE-1) were treated with SM at different concentrations (3.125, 12.5, 25 and 50 μg/mL) to investigate the antiproliferative effects. DNA laddering analysis was performed to investigate the apoptosis of DU-145 cells. Molecular mechanism was investigated by Western blot analysis of p53, Bcl-2, prostate specific antigen (PSA), and androgen receptor (AR). Six-week-old male BALB/c nude mice were randomly divided into normal control group (n=101) and treated group (n=101) which administered 500 mg/kg SM for 2 weeks. Tumor volumes were measured. Treatment with SM resulted in a dose-dependent decrease in cell number of DU-145 cells in comparison with RWPE-1. DNA laddering analysis indicated the apoptosis of DU-145 cells. Treatment with SM increased the expression of p53 and reduced the expression of Bcl-2 proteins. The levels of PSA were considerably reduced in SM-treated group compared to the controls, and a decrease in AR expression was observed when cells were treated with SM in the same pattern as a reduction in PSA. In the tumour xenograft study, SM given once a day for 2 weeks significantly inhibited tumour growth. SM might contribute to the anticancer actions such as induction of apoptosis and inhibition of proliferation of prostate cancer cells.

Xenoestrogens Alter Estrogen Receptor (ER) α Intracellular Levels

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

Role of anthocyanin-enriched purple-fleshed sweet potato p40 in colorectal cancer prevention.

Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention. However, the content of anthocyanins in staple foods is typically low and the mechanisms by which they exert anticancer activity is not yet fully defined. We selected an anthocyanin-enriched purple-fleshed sweet potato clone, P40, and investigated its potential anticancer effect in both in vitro cell culture and in vivo animal model. In addition to a high level of total phenolics and antioxidant capacity, P40 possesses a high content of anthocyanins at 7.5 mg/g dry matter. Treatment of human colonic SW480 cancer cells with P40 anthocyanin extracts at 0-40 μM of peonidin-3-glucoside equivalent resulted in a dose-dependent decrease in cell number due to cytostatic arrest of cell cycle at G1 phase but not cytotoxicity. Furthermore, dietary P40 at 10-30% significantly suppressed azoxymethane-induced formation of aberrant crypt foci in the colons of CF-1 mice in conjunction with, at least in part, a lesser proliferative PCNA and a greater apoptotic caspase-3 expression in the colon mucosal epithelial cells. These observations, coupled with both in vitro and in vivo studies reported here, suggest anthocyanin-enriched sweet potato P40 may protect against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptotic mechanisms.

Abstract 945: Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy.

Abstract The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis and the development of platinum resistance. T-type calcium channels have recently garnered interest as potential targets for the treatment of many cancers. We investigated the hypothesis that inhibiting T-type calcium channels would sensitize ovarian cancer cells to platinum based chemotherapy using cell culture and mouse models. Sequential treatment of cultured ovarian cancer cell lines or primary cells obtained from patients with mibefradil followed by carboplatin showed a dose-dependent decrease in cell number that was greater than observed with either drug alone. Importantly, treatment of platinum-resistant tumor cells with mibefradil followed by carboplatin also decreased cell number. Moreover, either simultaneous administration of both drugs or treatment with carboplatin followed by mibefradil did not alter cellular response to carboplatin. Based on these findings, we tested the effect of mibefradil and carboplatin in a mouse model of peritoneal ovarian cancer metastasis with platinum-resistant ovarian cancer cells. Tumor-bearing mice were subjected to 3 cycles of Interlaced TherapyTM, which consists of 5 days of mibefradil treatment followed by one dose of carboplatin and 2 days off. We found that while neither drug alone significantly affected tumor growth compared to control, the mice that received the dual treatment had a dramatic reduction in tumor burden. Together, the data provide pre-clinical evidence supporting the use of T-type calcium channel blockers with platinum-based chemotherapy to treat ovarian cancer. Citation Format: Eli V. Casarez, Lloyd Gray, Amir A. Jazaeri, Jill K. Slack-Davis. Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2013-945

Antiproliferative effects of zinc-citrate compound on hormone refractory prostate cancer

To investigate the antiproliferative effects of zinc-citrate compound on hormone refractory prostate cancer (HRPC). HRPC cell line (DU145) and normal prostate cell line (RWPE-1) were treated with zinc, citrate and zinc-citrate compound at different time intervals and concentrations to investigate the effect of zinc-citrate compound. Mitochondrial (m)-aconitase activity was determined using aconitase assay. DNA laddering analysis was performed to investigate apoptosis of DU145 cells. Molecular mechanism of apoptosis was investigated by Western blot analysis of P53, P21(waf1), Bcl-2, Bcl-xL and Bax, and also caspase-3 activity analysis. Treatment with zinc-citrate compound resulted in a time- and dose-dependent decrease in cell number of DU145 cells in comparison with RWPE-1. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of DU145 cells. Zinc-citrate compound increased the expression of P21(waf1) and P53, and reduced the expression of Bcl-2 and Bcl-xL proteins but induced the expression of Bax protein. Zinc-citrate compound induced apoptosis of DU145 cells by activation of the caspase-3 pathway. Zinc-citrate compound can induce apoptotic cell death in DU145, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.

Induction of Apoptosis of Bladder Cancer Cells by Zinc-Citrate Compound

PurposeZinc is one of the trace minerals in the body and is known to have an anticancer effect by inducing apoptosis in prostate cancer. We aimed to investigate the antiproliferative effects of a zinc-citrate compound in bladder cancer.Materials and MethodsA bladder cancer cell line (MBT-2) was treated with a zinc-citrate compound at different time intervals and concentrations. Mitochondrial (m)-aconitase activity was determined by use of the aconitase assay. DNA laddering analysis was performed to investigate apoptosis of MBT-2 cells. The molecular mechanism of apoptosis was investigated by Western blot analysis of p53, p21waf1, Bcl-2, Bcl-xL, and Bax and also by caspase-3 activity analysis.ResultsTreatment with the zinc-citrate compound resulted in a time- and dose-dependent decrease in cell number of MBT-2 cells. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of MBT-2 cells. The zinc-citrate compound increased the expression of p21waf1 and p53 and reduced the expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. The zinc-citrate compound induced apoptosis of MBT-2 cells by activation of the caspase-3 pathway.ConclusionsWe have shown that a zinc-citrate compound induces apoptotic cell death in a bladder cancer cell line, MBT-2, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.

Anthocyanin‐enriched Purple Sweetpotato for Cancer Prevention

Natural pigment of dietary anthocyanins has been epidemiologically associated with reduced chronic diseases including cancer risk. We previously selected a purple-fleshed sweetpotato clone, P40, from seeds obtained from the International Potato Center in Lima, Peru. In addition to a high content of total phenolics and antioxidants, P40 possesses anthocyanins up to 5 mg/g dry matter. To provide scientific evidences and understand the underling mechanisms, this study is to test a potential anticancer activity of P40 cultivar in both in vitro cell culture and in vivo animal models. Treatment with the sweetpotato extracts in human colonic SW480 cancer cells resulted in a dose-dependent decrease in cell number, which was not due to cytotoxicity, but cytostatic mechanism with an increase of cell cycle arrest at G0/G1 phase. Both cell growth inhibition and cell cycle arrest were significantly correlated with the anthocyanin contents in P40 cultivar when compared with the white (O'Henry) and yellow (NC Japanese) sweet potato controls. An on-going animal study is to assess both bioavailability of anthocyanins and chemopreventive potential in azoxymethane-induced aberrant crypt foci in rodent colons. Better understanding of the anticancer activity and underlying mechanisms may promote the health benefits of the functional purple sweetpotato grown in Kansas.

Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.

Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose- and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation.

Comparison of Ethanol and Acetaldehyde Toxicity in Rat Astrocytes in Primary Culture

This study compared the effects of toxicity of ethanol and its first metabolite acetaldehyde in rat astrocytes through cell viability and cell proliferation. The cells were treated with different concentrations of ethanol in the presence or absence of a catalase inhibitor 2-amino-1,2,4 triazole (AMT) or with different concentrations of acetaldehyde. Cell viability was assessed using the trypan blue test. Cell proliferation was assessed after 24 hours and after seven days of exposure to either ethanol or acetaldehyde.We showed that both ethanol and acetaldehyde decreased cell viability in a dose-dependent manner. In proliferation studies, after seven days of exposure to either ethanol or acetaldehyde, we observed a significant dose-dependent decrease in cell number. The protein content study showed biphasic dose-response curves, after 24 hours and seven days of exposure to either ethanol or acetaldehyde. Co-incubation in the presence of AMT significantly reduced the inhibitory effect of ethanol on cell proliferation.We concluded that long-term exposure of astrocytes to ethanol is more toxic than acute exposure. Acetaldehyde is a much more potent toxin than ethanol, and at least a part of ethanol toxicity is due to ethanol's first metabolite acetaldehyde.

Glutathione- and thioredoxin-related enzymes are modulated by sulfur-containing chemopreventive agents

We studied the effects of sulfur-containing chemopreventive agents, including allyl sulfides and isothiocyanates, on human redox networks. Isothiocyanates inhibited isolated redox-active enzymes in a time- and dose-dependent manner. As shown for the most active compound, benzyl isothiocyanate (BITC), on thioredoxin reductase, the inhibition has an initial competitive part (Ki=6.1+/-1.0 microM) followed by a time-dependent irreversible inhibition (k2=72.8+/-25.5 M(-1) s(-1)). Also, glutathione reductase and glutathione S-transferase were irreversibly modified by BITC. Sulforaphane led to irreversible inhibition of the studied redox enzymes, but with 5-10 times lower k2 values. In contrast, allyl sulfides had only moderate effects on the tested enzymes. However, diallyl disulfide was found to react directly with reduced glutathione (k2=100 M(-2) s(-1)). This reaction might contribute to enhanced oxidative stress and the induction of the selenoprotein glutathione peroxidase as determined on activity and transcript levels. All chemopreventive agents tested induced transcript levels of genes associated with cell cycle arrest and apoptosis. This upregulation was accompanied by a dose-dependent decrease in cell number. Our data indicate that modulation of cellular redox networks is likely to contribute to the effects of sulfur-containing chemopreventive agents.

Epigallocatechin‐3‐gallate and bicalutamide cause growth arrest and apoptosis in NRP‐152 and NRP‐154 prostate epithelial cells

A number of epidemiological studies have suggested that consumption of green tea reduces the risk of prostate cancer. The aim of this study was to elucidate the effects of epigallocatechin-3-gallate (EGCG), one of the major constituents of green tea, on growth inhibition and apoptosis in prostate epithelial cell lines with and without bicalutamide. The effects of EGCG and bicalutamide alone and in combination were examined on NRP-152 and NRP-154 cells derived from the dorso-lateral prostate of the Lobund-Wistar rat. Following treatments, cell number and levels of apoptosis were assessed. After treatment with EGCG, both cell lines displayed a dose-dependent decrease in cell number; this effect was more pronounced in NRP-154 cells. This decrease in cell number was caused by growth arrest in NRP-152 cells and apoptosis in NRP-154 cells. The apoptotic events in the NRP-154 cells were concurrent with a loss of manganese superoxide dismutase expression. Androgen ablation was achieved by androgen withdrawal using charcoal stripped serum or treatment with bicalutamide. Bicalutamide decreased cell number and induced apoptosis in a dose-dependent manner in both cell lines; however, androgen withdrawal did not. There was a loss of androgen receptor expression in NRP-152 cells with bicalutamide treatment. However, as the NRP-154 cells are androgen receptor negative, the loss in cell number and increased apoptotic events in these cells cannot be attributed to the anti-androgenic activity of bicalutamide. Cells treated with a combination of bicalutamide and EGCG also demonstrated a dose-dependent decrease in cell number that was significantly greater than bicalutamide alone. This study demonstrates the potential use of EGCG and other antioxidants as therapeutic candidates for prostate cancer.

Effects of IFN-αonα-Fetoprotein Expressions in Hepatocellular Carcinoma Cells

We investigated the effects of pegylated (PEG)-IFN-alpha2b on alpha-fetoprotein (AFP) expression as demonstrated by protein and mRNA levels in six human hepatocellular carcinoma (HCC) cell lines. The number of KIM-1 cells in culture with PEG-IFN-alpha2b decreased between 24 amd 240 h, whereas the levels of intracellular and secreted AFP per cellular protein increased (except at 192 h), with levels 1.9-fold and 2.9-fold higher at maximum, respectively, than cells without PEG-IFN-alpha2b (control). The mRNA level increased between 72 and 192 h, when the level was 3-fold higher than that of the control. In the 72-h culture with 40-5000 IU/mL PEG-IFN-alpha2b, there were dose-dependent increases in AFP protein and mRNA expression and dose-dependent decrease in cell number resulting from apoptosis and blockage of the cell cycle at the S-phase. The rate of fucosylated AFP in the cell lysate decreased in a dose-dependent and time-dependent manner. In the PEG-IFN-alpha2b culture of the other five HCC cell lines, cell proliferation was suppressed, but the expressions of AFP protein and mRNA increased in only two cell lines, and suppression of cell proliferation was not related to the increase in AFP expressions. Our findings demonstrated that PEG-IFN-alpha2b induces an increase in AFP expression at both the protein and mRNA levels.

Effects of propofol on intracellular Ca 2+ homeostasis in human astrocytoma cells

The effects of propofol, a short-acting general anesthetic, upon cell growth and Ca 2+ signaling in a human astrocytic cell line were examined. Exposure of cells to graded concentrations of propofol resulted in a dose-dependent decrease in cell number with an inhibitory concentration of cell viability (IC50) of 31.7 ± 1.2 μM. To evaluate the changes in intracellular Ca 2+ homeostasis induced by propofol, cytoplasmic and mitochondrial Ca 2+ were measured by fluorescence imaging. Mitochondrial Ca 2+ increased while cytoplasmic Ca 2+ decreased significantly at a propofol concentration lower than the IC50 (10 μM for 24 h, 1 μM for 72 h). In addition, propofol diminished the Ca 2+ response induced by fetal bovine serum (FBS). To determine the source of Ca 2+ alterations induced by propofol, pharmacologic agents targeting intracellular Ca 2+ homeostasis mechanisms were used. Nifedipine, an L-type Ca 2+ channel blocker, decreased FBS-induced Ca 2+ response of control cells to a level similar to propofol treated cells. However, diazoxide (a K +-ATP channel opener) administered 1 h before FBS addition restored the FBS response in propofol treated cells to a level similar to control. In addition, diazoxide increased mitochondrial Ca 2+ in control cells to a level comparable to propofol treated cells suggesting activation of these channels by propofol treatment. Addition of 1 μM RU-360 (a selective blocker of the mitochondrial Ca 2+ uniporter) for 30 min prior to propofol treatment restored mitochondrial and cytoplasmic Ca 2+ to control levels. These data suggest that voltage operated Ca 2+ channels, mitochondrial Ca 2+ and K +-ATP channels may be targets of propofol action in astrocytes.

Antiproliferative and apoptotic effects of zinc–citrate compound (CIZAR®) on human epithelial ovarian cancer cell line, OVCAR-3

Objective. Zinc inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. The intracellular concentration of zinc and its dynamic changes are critically important in cell biology. We investigated the effects of zinc–citrate compound (CIZAR®) on normal human ovarian epithelial cells (NOSE) and human epithelial ovarian cancer cell line, OVCAR-3. Methods. To investigate the potential effect of CIZAR® on cell growth and survival, cells were treated with different doses and exposed to different times. Intracellular concentration of zinc was measured by colorimetric assay. Mitochondrial aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, and morphological analysis were done to investigate cytotoxic effects of CIZAR®. Molecular mechanism of cell death was investigated by p53, Bcl-xL, Bcl-2, Bax protein, activity of caspase-3 and -12, and activity of telomerase. Results. CIZAR®-induced zinc accumulation in OVCAR-3 cells was higher than that in NOSE cells. CIZAR® treatment resulted in a time- and dose-dependent decrease in cell number in OVCAR-3 cells in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells within 4 h exposure to CIZAR® but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering, and morphological analysis indicated apoptosis in OVCAR-3 cells but not in NOSE cells. CIZAR® increased the expression of p21 waf1 which is a part of p53-independent pathway and induced reduction of telomerase activity. CIZAR® reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR® induced apoptosis of OVCAR-3 cells by activation of caspase-12 and caspase-3 pathway. Conclusions. Exposure to CIZAR® induces apoptosis in OVCAR-3 cells which accumulate high intracellular levels of zinc, but not in NOSE cells, which do not accumulate high levels of zinc. CIZAR® prevents the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induces apoptosis by induction of proapoptotic gene (Bax), repression of antiapoptotic genes (Bcl-2, Bcl-xL), and consequently activation of caspase-3. CIZAR® also induced activation of caspase-12. The CIZAR® will offer new window in prevention and treatment of epithelial ovarian cancer.

Inhibition of human neuroblastoma cell growth by CAY10404, a highly selective Cox-2 inhibitor

Neuroblastomas constitute about 10% of childhood cancers and are responsible for 15% of pediatric cancer mortality. We evaluated the efficacy and the mechanism of cell death induced by CAY10404, a selective cyclooxygenase-2 (Cox-2) inhibitor in four human neuroblastoma cell lines (SH-EP, SH-SY5Y, SK-N-MC and MSN). Treatment with CAY10404 in the range of 15-115 microM revealed a dose-dependent decrease in cell number and an average IC50 (inhibitory concentration 50%) of 60 microM. About 20-30% of the cells were terminal deoxynucleotidyltransferase-mediated UTP nick-end-labeling (TUNEL) positive 48 h after treatment. Western blot analysis of CAY10404-treated cells showed poly(ADP-ribose) polymerase (PARP) cleavage and cleaved caspase-3 signifying caspase activity and apoptotic cell death. Inhibitor-of-apoptosis proteins including X-linked inhibitor-of-apoptosis protein (XIAP) and survivin did not change significantly after CAY10404 treatment. Fluorescence activated cell sorter (FACS) analysis performed in two different cell lines 48 h following CAY10404 treatment showed a reduction in the number of cells in the G1 phase of the cell cycle and an increase in the number of cells in the G2 phase. When radioresistant SH-EP cells were treated with CAY10404, a 49% decrease in cell viability was observed relative to DMSO-treated cells; pretreatment with CAY10404 followed by ortho-voltage irradiation further enhanced cell death (58%) suggesting radiosensitization by CAY10404.

Initial Effects of Partially Purified Bone Morphogenetic Protein on the Expression of Glycosaminoglycan, Collagen, and Alkaline Phosphatase in Nonunion Cell Cultures

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.

Cell Cycle Changes and Cytotoxicity in Irradiated Cultures of Bovine Aortic Endothelial Cells

The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type.