Trypan Blue Dye Exclusion Method Research Articles

Synergistic Growth Inhibition by the Combination of Acyclic Retinoid Plus Vitamin K2 in HL-60 Cells

Introduction: All-trans retinoic acid (ATRA) inhibits the proliferation and induces terminal differentiation in APL cells by binding to retinoic acid receptor-a (RARa). The activation of retinoid X receptor (RXR) also plays an essential role. These receptors form heterodimeric complex (RAR/RXR) and thereby regulate the expression of target genes. We have recently reported that the accumulation of phosphorylated forms of RXRa (p-RXRa), which loses heterodimeric activity with RAR, is associated with the abnormal cell growth and the resistance to retinoic acid in APL cells (Kanemura N. et al. Leuk Res. 2008). Acyclic retinoid (ACR) inhibits cell growth and induces apoptosis in liver cancer cells by inhibiting the phosphorylation of RXRa. ACR acted synergistically to inhibit growth of these cancer cells when combined with vitamin K2 (VK2). In order to develop a new effective regimen for APL treatment, we investigated whether the combination of ACR plus VK2 exerts synergistic growth-inhibitory effects on HL-60 cells.Materials and Methods: HL-60 cells were treated with the indicated concentrations of ACR (0.01 – 50 mM) or ATRA (0.01 – 50 mM) in the presence or absence of VK2 (1 mM) for 72 hours. The cells were also treated with the indicated concentrations of VK2 (0.01 – 50 mM) in the presence or absence of ACR (1 mM) for same period and the cell numbers were then determined by using the Trypan Blue dye exclusion method. To determine whether the combined effects of ACR plus VK2 were synergistic, the combination index-isobologram was calculated and used in the combination assays. The induction of apoptosis by the combination of ACR plus VK2 was examined using a fluorescent annexin V/propidium iodide staining. Cell cycle analysis was performed by determinng the BrdU-labeled cells using flow cytometry. The effects of the combination of ACR plus VK2 on the expression levels of phosphorylated (i.e. activated) form of ERK (p-ERK) and p-RXRa proteins were examined by Western blot analysis.Result: ACR and ATRA dose-dependently inhibited proliferation in HL-60 cells. Although VK2 (1 mM) alone could not inhibit the growth of HL-60 cells, the combination of various concentrations of ACR plus 1 mM VK2 synergistically inhibited the growth of these leukemic cells. On the other hand, no synergistic growth inhibition was observed when the cells were treated with the combination of ATRA plus VK2. The induction of apoptosis was significantly increased by the combined treatment with ACR plus VK2 (38.0% to 46.6%, p<0.05). Cell cycle analysis indicated that the percentage of G0/G1 phase cells was significantly increased by the treatment with ACR alone (32.5% to 62.3%, p<0.05) or VK2 alone (27.8% to 52.8%, p<0.05), respectively. The expression of p-ERK protein was decreased when the cells were treated with VK2 alone or the combination of ACR plus VK2. Treatment of these leukemic cells with ACR alone or combination of ACR plus VK2 also caused a marked decreased in the expression level of p-RXRa. This decrease was most apparent when the cells were treated with a combination of ACR plus VK2.Discussion: The present study indicated that the combination of ACR plus VK2 caused the synergistic growth inhibition and induction of apoptosis in HL-60 cells, whereas the combination of ATRA plus VK2 did not exert such beneficial effects. The synergism might be associated with the induction of G0/G1 phase of cell cycle arrest and the inhibition of the expression of p-ERK and p-RXRa proteins caused by the combination of these agents. Among these findings, restoration of the function of RXRa by inhibiting its aberrant phosphorylation plays a critical role to exert synergistic anti-cancer effects.Conclusion: The combination of ACR plus VK2 might hold promise as a clinical modality for the treatment of APL due to their synergism. Targeting the RXRa phosphorylation might be one of the effective strategies for the therapy of certain types of human leukemia.

Safety and Efficacy of Hepatic Progenitor Cell Transplantation Through Hepatic Artery for the Treatment of Chronic Liver Failure

Purpose: Orthotopic liver transplantation (OLT) is the ultimate treatment for end-stage liver failure and for numerous liver based inborn errors of metabolism. However, several limitations such as shortage of organ donors, high cost and several complications/risks of OLT. Liver cell transplantation (LCT) is emerging as successful treatment. Stem cells from different sources are eligible for liver cell therapy according to their hepatic potential, for instance mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and adult liver stem/progenitor cells. However, the current research on the hepatic fate of the stem cells is still facing difficulties to demonstrate the acquisition of a functional hepatocyte, both in vitro and in vivo. In experimental studies heaptic progenitors from human fetal is shown promising results in the engraftment and proliferation. Recently Hisaya azuma et al. demonstrated the transplantation of serial human hepatic progenitors completely repopulated the whole liver. In this study we are reporting transplantation of hepatic progenitors in 5 patients with cirrhosis of liver (Child Pugh B, C) with 6 months follow-up. Methods: This clinical study was approved by Institutional Ethics Committee. Informed consent was taken from patient for carrying out the study. Briefly, liver cells were isolated by two step collagenase digestion method, from the aborted fetus of 14–20 weeks gestation. Total liver cells were subjected to magnetic activated cell sorting (MACS) with CD326 antibody directly labeled to microbeads (MACS, Miltenyi Biotec) and collected hepatic progenitor cells. Cell viability and cell activity were assessed by trypan blue dye exclusion method and MTT assay respectively and reported 90% of viability with higher cellular activity. Cells were infused by trans femoral catheterization technique, Hepatic artery was selectively cannulated with 8 F guage catheter. 0.1 × 108 fetal liver progenitor cells in 10–12 ml of DNS solution were infused at the rate of 1 ml/per minute to achieve reconstitution of the liver with transplanted cells. Results: All the procedures were performed safely without any post infusion complications. Follow up results after 6 months showed oveall improvement in geneal condition. All the patients showed improvement in serum albumin, bilirubin, ALT after first month of cell infusion. Ultrasonography findings showed decrease in ascitis. Conclusion: This study demonstrates the efficacy of hepatic progenitor cell in management of cirrhosis liver. As the procedure is very simple, infusion of hepatic progenitors can be repeated to improve the liver functions.

Citotoxicidad del glifosato en células mononucleares de sangre periférica humana

Glyphosate is a broad-spectrum, non-selective herbicide and commonly used to eliminate weeds in agricultural and forest settings. Studies evaluating glyphosate toxicity in animals and environment show that commercial formulations of glyphosate are more toxic than the active component itself. Technical grade glyphosate was compared with the commercial formulation Roundup in their respective toxicities on human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, either technical grade or in the form of Roundup for 24 h, 48 h, 72 h, and 96 h. Cytotoxicity was assayed by trypan blue dye exclusion method and reduction of (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2Htetrazolium-5-carboxyanilide inner salt)XTT reagent. Both technical grade glyphosate and Roundup formulation were toxic to human peripheral blood mononuclear cells. Cytotoxicity of Roundup was higher than cytotoxicity of glyphosate, since the LC50 (50% lethal concentration) determined by the trypan blue exclusion method at 24 h was the equivalent of 56.4 microg/ml of glyphosate in the form of Roundup and 1,640 microg/ml (1.64 mg/ml) for technical grade glyphosate. This in vitro study confirmed the toxic effects on human cells by glyphosate and its commercial preparations. Commercial formulations were more cytotoxic than the active component alone, supporting the concept that additives in commercial formulations play a role in the toxicity attributed to glyphosate-based herbicides.

A novel homospermidine conjugate inhibits growth and induces apoptosis in human hepatoma cells

To elucidate the mechanism responsible for the antiproliferative effects of a novel homospermidine conjugate, anthracenylmethyl homospermidine (ANTMHspd), in the human hepatoma BEL-7402 cell line. The viability of the cells was assessed by MTT assay and the trypan blue dye exclusion method. Morphological changes were observed by fluorescence microscopy with Hoechst 33258 staining. Cell cycle distribution, apoptosis, and mitochondrial membrane potential were measured by flow cytometry. Protein expression was detected by Western blot analysis. ANTMHspd strongly decreased BEL-7402 cell proliferation in a dose- and time-dependent manner. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release and enhance cleaved caspase-3, cleaved caspase-9, and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. ANTMHspd could induce BEL-7402 cell apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.

Protective effects of a hibernation-inducer on hepatocyte injury induced by hypothermic preservation

For hepatocyte-based cell therapy to be realistic, the method chosen for cryopreservation or hypothermic preservation is critical. The aim of the present study was to clarify whether D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, has protective effects on hepatocytes with regard to hypothermic preservation injury. A suspension of rat hepatocytes was stored at 4 degrees C for 24 h with or without DADLE. Their viability was measured by the trypan blue dye exclusion method, and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in the preservation solution were measured. After 24 h of cold storage, viable hepatocytes were cultured at 37 degrees C for another 24 h. Then albumin production and lidocaine clearance were measured. DADLE significantly improved the survival rate of hepatocytes. The levels of ALT and LDH in the preservation solution with DADLE were significantly lower than those in the preservation solution without DADLE. The treated viable hepatocytes maintained both albumin synthesis and lidocaine clearance. DADLE appears to have protective effects on hepatocytes with regard to hypothermic preservation injury in vitro. This hibernation-inducer is useful in prolonged hypothermic preservation for hepatocyte-based therapy.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Bcl2 Suppresses DNA Repair by Enhancing c-Myc Transcriptional Activity

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

The CLN9 Protein, a Regulator of Dihydroceramide Synthase

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Antiproliferation effects of oridonin on HPB-ALL cells and its mechanisms of action

Oridonin, an ent-kaurane diterpenoid derived from the herbal Rabdosia rubescens, has been recently reported to have antitumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of oridonin on HPB-ALL cell lines and its mechanisms of action. HPB-ALL cells in culture medium in vitro were treated with different concentrations of oridonin (16-56 micromol/L). MTT assay was used to detect the cell growth inhibitory rate, and the cell viability was assessed by the trypan blue dye-exclusion method. Cell apoptosis and the mitochondrial membrane potential (delta psi m) were investigated by flow cytometry (FCM), Hoechst 33258 staining, and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators, including Fas and Bcl-2 family members, was analyzed by Western blotting. The results revealed that oridonin could significantly inhibit the growth of HPB-ALL cells and cause apoptosis, and the suppression was both time- and dose-dependent. After treatment with oridonin for 48 hr, the percentage of disruption of delta psi m gradually increased in a dose-dependent manner along with marked changes of cell apoptosis, and necrotic cells increased remarkably after the cells were treated with oridonin for 72 hr; Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 20-kDa subunit when apoptosis occurred; expression of Bcl-2 and Bcl-XL was downregulated remarkably while expression of Bax and Bid was upregulated concurrently after the cells were treated with oridonin for 24 hr. Of note, the expressions of Fas and other Bcl-2 family members including Bak and Bad remained constant before and after apoptosis occurred. We therefore conclude that oridonin has significant antiproliferation effects on HPB-ALL cells by induction of apoptosis as well as directly causing cell necrosis and that oridonin-induced apoptosis on HPB-ALL cells is mainly related to the disruption of delta psi m and activation of caspase-3 as well as downregulation of anti-apoptotic protein Bcl-2, Bcl-XL, and upregulation of pro-apoptotic proteins Bax and Bid. The results indicate that oridonin may serve as a potential antileukemia reagent.

Cytotoxicity and Genotoxicity Induced by the Pesticide Profenofos on Cultured Human Peripheral Blood Lymphocytes

Profenofos is a broad-spectrum organophosphate insecticide and acaricide used widely for agricultural and household purposes. The aim of this investigation was to determine its toxicity profile in vitro, using lymphocytes from peripheral blood samples of healthy human donors. We found the IC50 of profenofos to be 3.5 μM as measured by Trypan blue dye exclusion method. Chromosomal analyses of the metaphase plates of the samples treated with sublethal concentrations of profenofos revealed satellite associations and chromatid breaks and gaps, indicating its effect on chromosomes. The results were further supported by comet assay, where single-strand breaks in DNA were observed as comet tail lengths. The results were statistically significant (p < 0.01, ANOVA). Hence, it may be proposed that in vitro assays like the comet assay and chromosomal aberrations test, which indicate genetic damage, could be used to study the effect of organophosphorus pesticide poisoning in humans.

Proapoptotic and antitumor activities of the HMG-CoA reductase inhibitor, lovastatin, against Dalton's Lymphoma Ascites tumor in mice

Background Diet rich in fat have a clear effect on the tumor incidence in humans. Increased level of lipid peroxidation were found in colon, liver, breast and kidney carcinogenesis. Although the beneficial effects of statins for cardiovascular diseases are well established, their importance in the area of cancer therapeutics has recently gained recognition. Many studies of lovastatin in in vitro systems and experimental animals have been reported as an effective antitumor agent. However, phase I/II clinical trials in cancer patients demonstrated a minor to non-significant responses. Hence more studies in different tumor models using doses corresponding to that used to reduce lipid in human are required to support the antitumor activity. Methods The antitumor activity was evaluated using Daltons' Lymphoma Ascites (DLA) cell line-induced ascites tumor model in mice. Proapoptotic activity was evaluated in DLA cell line induced ascites animals after the treatment of lovastatin. Apoptosis was analyzed morphologically by staining with Giemsa and biochemically by observing the laddering of DNA in agarose gel electrophoresis. In vitro cytotoxic activity of lovastatin was studied by trypan blue dye exclusion method. Lipid peroxidation inhibiting activity was demonstrated in Fe 2+-ascorbate induced rat whole liver homogenate. Results Lovastatin dose dependently inhibited the ascites tumor growth at 4 and 16 mg/kg body wt (p.o). The percentage increase in life span (%ILS) in the 16 mg/kg treated group was 61.8% ( P < 0.01). Single dose of lovastatin (16 mg/kg body wt, p.o) was also effective to accelerate the apoptosis in the ascites tumor bearing mice that was evident from the multiple fragmentation of DNA in gel electrophoresis. Further the morphological analysis of DLA cells aspirated from the lovastatin treated animals showed a significant ( P < 0.01) increase of apoptotic cells (15.5 ± 3%) than the control animals (6.5 ± 1%). Concentration of lovastatin required for the 50% of the cytotoxicity was 37 ± 5 μg/ml. Lovastatin at its low concentrations were effective to inhibit lipid peroxidation. Conclusions The antitumor activity of lovastatin against the ascites tumor is due to its proapoptotic and cytotoxic activities. Lovastatin at low concentrations inhibited Fe 2+ induced lipid peroxidation in in vitro system. The proapoptotic and lipid peroxidation inhibiting activities of the lipid lowering drug lovastatin may further suggest its possible therapeutic use as a cancer chemopreventive agent.

Inhibition of Cellular Proliferation and Induction of Apoptosis by Curcumin in Human Malignant Astrocytoma Cell Lines

Nuclear factor (NF)-kappaB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-kappaB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-kappaB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-kappaB activity. In this study, we investigated inhibitory effects of curcumin on NF-kappaB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-kappaB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [(3)H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-kappaB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-kappaB activity by curcumin.

Dual regulatory effects of resveratrol on activation of NF-κB and cell proliferation in human embryonal kidney 293 cells

Resveratrol (3,4′,5-trihydroxystilbene, Res), a naturally occurring polyphenol, exhibits antioxidant, anti-inflammatory, potential chemopreventive and chemotherapeutic properties in preclinical studies. To further understand its potential clinical efficacy and safety, effect of Res at 10−9–10−4 mol/L on human embryonal kidney (HEK293) cell proliferation and its potential mechanism were investigated in present study. Cell viability was detected by using trypan blue dye exclusion method. Cell cycle and apoptosis were analyzed by flow cytometry with propidium iodide stain. Activation of nuclear factor-kB (NF-kB) was determined by luciferase reporter gene assay using stably transfected HEK293/kB-1uc cells. Secretion of human interleukin-8 (hIL-8) was measured by ELISA. Our results show that HEK293 cell proliferation was significantly stimulated by 10−7 mol/L Res after treatment for 48 hours, or by 10−8-10−7 mol/L Res combinated with 10 ng/mL TNFα for 24 h, but was suppressed by 10−4 mol/L Res with or without TNFα. Both endogenous and TNFα-induced NF-kB activation were downregulated by Res at 10−7 mol/L, but were upregulated at 10−4 mol/L. With 10−4 mol/L Res, the content of secreted IL-8 was increased, and apoptosis rate was increased from less than 5% to 10%, together with significant cell-cycle arrest in S phase. TNFα has coordinative effects with Res on HEK293 cell apoptosis, cell-cycle arrest and IL-8 secretion. These results indicate that Res promotes cell proliferation at low concentration through down-regulation of NF-kB activation in HEK293, but suppresses its growth at high concentration through up-regulation of NF-kB activation, increasing IL-8 and cell-cycle arrest. As resveratrol has dual regulatory effect on cell proliferationin vitro, comprehensive evaluation of its potential clinical utility is needed.

Apoptotic effect of oridonin on NB4 cells and its mechanism

The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.

The Chinese Herbal Medicine Tien-Hsien Liquid Inhibits Cell Growth and Induces Apoptosis in a Wide Variety of Human Cancer Cells

Tien-Hsien liquid (THL) is a commercially available Chinese herbal mixture that has been used as an anticancer dietary supplement for more than 10 years. We recently showed that THL has strong immunomodulatory effects on peripheral blood mononuclear cells (PBMC) and T cells. To investigate the antitumor activity of THL further, we sought to test whether THL could induce apoptosis in various human cancer cell lines based on the fact that THL contains several components with tumor killing functions. The growth inhibitory effect of THL on human cervical carcinoma C-33A cells, human lung carcinoma H1299 cells, and human PBMC was assessed by counting viable cells using the trypan blue dye exclusion method. The apoptosis-inducing activity of THL in H1299 cells was assessed by analyzing the cells with four assays: (1) Hoechst 33258 nuclear DNA staining; (2) the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay; (3) the nucleosomal DNA fragment ladder assay; and (4) the sub-G1 cell analysis. The activities of caspase-8, -9, and -3 in H1299 cells treated with or without THL were also measured to elucidate the possible mechanism underlying THL apoptosis-inducing effect. Finally, the apoptotic effect of THL on fifteen human cancer cell lines and normal human cells were analyzed by the TUNEL assay. THL could induce apoptosis in all human cancer cell lines tested but not in normal human cells. THL treatment of H1299 cancer cells resulted in activation of caspase-8, -9, and -3 and the inhibitors of these caspases could partially block THL-induced apoptosis. THL has been used by numerous patients with cancer for many years with no known adverse effect. Our present study showing that THL had a broad-range tumor killing function has provided a molecular basis underlying THL therapeutic activity. Furthermore, because THL had apoptotic effects only on cancer cells but not on normal cells, this selectivity suggests that THL could be a potential cancer therapeutic agent.

Brine shrimp lethality and cytotoxicity assay of Araucaria bidwillii Hook in human carcinoma cell lines

The leaf extracts of Araucaria bidwillii Hook. (Araucariaceae) were evaluated for their cytotoxic effect in various human cancer cell lines. Preliminary investigation by brine shrimp lethality assay indicated that <TEX>$LC_{50}$</TEX> value of various successive extracts were found to be less than <TEX>$1000\;{\mu}g/ml$</TEX>, where the ethyl acetate extract showed maximum activity of less than <TEX>$100\;{\mu}g/ml$</TEX>. Further cytotoxic evaluation of various leaf extracts of Araucaria bidwilli Hook was carried out in four different human cancer cell lines-acute myeloblastic leukemia (HL-60), chronic myelogenic leukemia (K-562), breast adenocarcinoma (MCF-7) and cervical epithelial carcinoma (HeLa). Cytotoxicity was assessed by trypan blue dye exclusion method and 3-(4,5-dimethyl thiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay. From the present investigation it was found that the ethyl acetate and methanol extract of Araucaria bidwilli Hook was found to be more effective in leukemic cell lines and was less effective in MCF-7 and HeLa. The <TEX>$IC_{50}$</TEX> value of the ethyl acetate extract in leukemic cell lines was found to be <TEX>$28.18\;and\;34.64\;{\mu}g/ml$</TEX> and methanol extract was found to be <TEX>$33.11\;&\;39.81\;{\mu}g/ml$</TEX>. It can be concluded that various extract from the leaves of Araucaria bidwillii Hook. posses cytotoxic activity tested in brine shrimps and various human carcinoma cell lines.

Cytogenetic Biomarkers of Carbofuran Toxicity Utilizing Human Lymphocyte Cultures In Vitro

Short-term lymphocyte cultures from human peripheral blood samples were incubated with various aliquots of the carbofuran pesticide. After 48 h of initiation and 24 h of exposure to the carbofuran pesticide aliquots, it was seen that carbofuran caused an increase in the frequency of chromosomal aberrations, and the increase was significant (p < 0.05) in treated samples compared to controls. Karyotype analysis revealed more satellite associations, gaps, and breaks in treated samples. Single-strand breaks in the DNA assessed by comet assay revealed that the pesticide caused increase in the comet tail length implicating genotoxicity in somatic cells. The LD50 of carbofuran was found to be 18 µM as calculated by probit analysis and determined by trypan blue dye exclusion method. The results presented here indicate that in vitro assays could be used as indicators of cyto- and genotoxicity of the pesticide, and their end points could be used as biomarkers.

Effect of contrast media on vascular smooth muscle cells

In order to alleviate the adverse effects of contrast media (CMs) on the vascular system, the role of Ca2+ in the viability of vascular smooth muscle cells (VSMCs) was investigated. VSMCs were obtained from swine thoracic aorta. The number of VSMCs was counted under a microscope using the trypan blue dye-exclusion method 24 h after culture in RPMI containing physiological saline (SAL) as control, iothalamate (IOT), or iohexol (IOH) at 10% by volume with CaCl2 added at 0 to 2.0 mmol/l. Free Ca2+ in the above media was measured using an ion-selective electrode. Free Ca2+ was 0.4 to 1.5 mmol/l with ionic IOT and 0.4 to 1.8 mmol/l with non-ionic IOH as well as with control. The ratio of viable cells grown in the presence of CMs to those grown in the control was optimal at approximately 0.60 near 1 mmol/l Ca2+ and decreased markedly to 0.00 at 1.5 mmol/l Ca2+ in the presence of IOT and to 0.39 at 1.8 mmol/l Ca2+ in the presence of IOH, while the ratios decreased gradually to 0.28 in the presence of IOT and 0.53 in the presence of IOH at 0.4 mmol/l Ca2+. Ionic IOT is more cytotoxic to VSMCs than non-ionic IOH. However, the cytotoxicity was minimal and similar between both CMs at 1 mmol/l Ca2+ in accordance with the sodium-calcium balance.

Synergistic antiproliferative and apoptotic effects induced by mixed epidermal growth factor receptor inhibitor ZD1839 and nitric oxide donor in human prostatic cancer cell lines.

The specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, ZD1839 induces potent antitumoral effects on several advanced cancer types. The present study was undertaken to determine whether the combination of ZD1839 with an agent donating nitric oxide (NO(*)), sodium nitroprusside (SNP) results in a synergy of anticarcinogenic responses on metastatic prostate cancer (PC) cells. The antiproliferative and apoptotic/necrotic effects of ZD1839 and SNP alone or in combination were estimated on EGF- and serum-stimulated LNCaP, DU145, and PC3 cells by MTT growth tests, trypan blue dye exclusion method, and flow cytometric analyses. Moreover, the cellular ceramide levels were evaluated by the diacylglycerol kinase enzymatic method and the amounts of cytosolic cytochrome c by ELISA assays. ZD1839 and SNP alone or in combination at lower concentrations induced an inhibition of EGF- and serum-stimulated growth of LNCaP, DU145, and PC3 concomitant with an arrest in the G1 phase of cellular cycle. Interestingly, the mixed ZD1839 and SNP also caused a more substantial apoptotic/necrotic death of these PC cells as compared to drugs alone. Moreover, we have observed that an inhibition of acidic sphingomyelinase, hydrogen peroxide (H(2)O(2)) accumulation and caspase cascades results in a significant reduction of apoptotic/necrotic death induced by mixed ZD1839 and SNP in EGF-stimulated PC3 cells. In addition, the combined ZD1839 plus SNP also induced a higher cellular ceramide and reactive oxygen species (ROS) production, mitochondrial transmembrane potential decrease, and cytochrome c amount released into cytosol as compared to drugs alone. The simultaneous use of EGFR inhibitor and compound releasing NO(*) might lead to a synergy in the ceramide and ROS production which might cause cellular membrane damages resulting in a massive apoptotic/necrotic death of metastatic PC cells.

Cerivastatin-induced apoptosis of human aortic smooth muscle cells through partial inhibition of basal activation of extracellular signal-regulated kinases

Background The beneficial effects of cerivastatin including hypolipidemic properties have been demonstrated to involve nonlipid as well as lipid mechanisms. In the present study, we examined the mechanisms underlying cerivastatin-induced growth inhibition of human aortic smooth muscle (ASM) cells. Methods Human ASM cells were cultured in 96-well plates with or without cerivastatin in the presence or absence of mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase MEK1/MEK 2 inhibitor PD98059. Cell growth was assessed by colorimetric quantitation of NADH, and cell viability was determined by trypan blue dye exclusion method. The induction of apoptosis was determined by propidium iodide (PI) staining method using flow cytometer. The activation of ERKs or c-Jun N-terminal kinases (JNKs) was determined by Western blotting using antibodies (Abs) specific for phospho-ERKs or phospho-JNKs. Results Treatment of the ASM cells with cerivastatin prevented cell growth in a concentration-dependent manner through at least induction of apoptosis. The cerivastatin-induced apoptosis was reversed by coincubation with isoprenoid [mevalonate, geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP)] suggesting a role for isoprenoid in the cerivastatin-induced apoptosis. The cerivastatin cooperated with a MEK1/MEK2 inhibitor PD98059 to induce apoptosis, which appeared to correlate with down-regulation of ERK activation (phospho-ERKs expression) induced by the combination. Conclusion Cerivastatin-induced blockade of ERK activation in ASM cells might result in growth inhibition including apoptosis, which might explain some aspects of the beneficial effects of cerivastatin on coronary artery disease.