Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest

AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry.RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a doseand time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/G1 phase, cells treated with high concentrations of PBA were arrested at the S phase. CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.

EFFECTS OF S86019, AN ACTIVE COMPONENT FROM PURALIA LOB AT A, ON CELL DIFFERENTIATION AND CELL CYCLE TRAVERSE OF HL-60 CELLS

The effects of S86019, an active component from Puralia lobata, on the induction of cell differentiation and cell cycle traverse of HL-60 cells were described. It was shown that cell proliferation of HL-60 cells was inhibited by S86019 in vitro. Under the action of S86019 the HL-60 cells were induced to differentiate into metamyelocytes, myelocytes and much matured cells with banded or segmented nucleus. Flow cytometry demonstrated that the cell population of HL-60 cells was blocked at G1 phase which resulted in the elevation of percentage of G1 cells and decrease of percentage of cells in S phase. Experimental results demonstrated that S86019 is an active inducer of cell differentiation in HL-60 cells.

Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity

Background:The cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms,among which transcriptional regulation is one of the most important components.Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle.However,the patterns of transcript isoform expression in the cell cycle are unclear.Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies,but none of them have been designed or evaluated at the alternative splicing transcript level.The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown,and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare.Methods:To explore alternative splicing patterns during cell cycle progression,we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines,using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples,and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle.Genomics of Drug Sensitivity in Cancer(GDSC)drug sensitivity datasets and Cancer Cell Line Encyclopedia(CCLE)transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity.We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients.Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6(CDK4/6)inhibitors.Finally,alternative splicing transcripts associated with mitotic(M)phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis.Results:We established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells.The results of the cell cycle assessment showed that 43,326,41,578 and 29,244 transcripts were found to be periodically expressed in HeLa,HCT116 and MDA-MB-231 cells,respectively,among which 1280 transcripts showed this expression pattern in all three cancer cell lines.Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes.Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904.The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts.Combined with the cell cycle-related drug screening,the results also showed that a set of periodic transcripts,for example,ENST00000314392(a dolichylphosphate mannosyltransferase polypeptide 2 isoform transcript),was more associated with drug sensitivity than the levels of their corresponding gene transcripts.Conclusions:In summary,we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity,providing novel insights into alternative splicing-related drug development and evaluation.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Effects of estradiol on cell cycle and cyclin proteins of vascular smooth muscle cells in rats

To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0～100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Experimental study on antitumor effect of arsenic trioxide in combination with cisplatin or doxorubicin on hepatocellular carcinoma

INTRODUCTIONThe main component of a traditional Chinese drug 'Pishuang'. arsenic trioxide (As2O3), has obviously selective anti-tumor effect on human hepatocellular carcinoma (HCC)in both in vitro and in vivo studies[1-5]. Due to limited effectiveness when any anti-carcinogen is used alone and obviously increased toxicity when the dose is raised, there is no exception for As2O3. Furthermore, combined chemotherapy contributes to improve therapeutic effectiveness, disperse toxicity and surmount drug-resistance,in which the combination of traditional Chinese and modern medicine has more advantages and characteristics. As a result,we made an experimental study on anti-tumor effect of As2O3in combination with cisplantin (PDD) or doxorubicin (ADM)on HCC. to investigate the possibility of AS2O3 in combination with PDD or ADM and nature of interaction between them,and to provide experimental basis for clinical application.

Efficacy of Bee Products(Anzer Honey,Pollen and Propolis)in Detection and Healing of Damage Induced by Antidiabetic Drug Vildagliptin/Metformin Hydrochloride in Healthy Human Pancreatic Cells:Cytotoxic,Genotoxic and Biochemical Studies

Background and Objective Although drugs are powerful therapeutic agents,they have a range of side effects.These side effects are sometimes cellular and not clinically noticeable.Vildagliptin/metformin hydrochloride is one of the most widely used oral antidiabetic drugs with two active ingredients.In this study,we investigated its harmful effects on the metabolic activation system in healthy human pancreatic cells“hTERT-HPNE”,and we aimed to improve these harmful effects by natural products.To benefit from the healing effect,we used the unique natural products produced by the bees of the Anzer Plateau in the Eastern Black Sea Region of Turkey.Methods Cytotoxic and genotoxic effects of the drug were investigated by different tests,such as MTT,flow cytometry-apoptosis and comet assays.Anzer honey,pollen and propolis were analyzed by gas chromatography/mass spectrometry(G/C-MS).A total of 19 compounds were detected,constituting 99.9%of the samples.Results The decrease in cell viability at all drug concentrations was statistically significant compared to the negative control(P<0.05).A statistically significant decrease was detected in the apoptosis caused by vildagliptin/metformin hydrochloride with the supplementation of Anzer honey,pollen and propolis in hTERT-HPNE cells(P<0.05).Conclusion This study can contribute to other studies testing the healing properties of natural products against the side effects of oral antidiabetics in human cells.In particular,Anzer honey,pollen and propolis can be used as additional foods to maintain cell viability and improve heal damage and can be evaluated against side effects in other drug studies.

Effect of polo-like kinase 1 gene silence on cell cycle and drug resistance in K562/A02 cell

Polo-like kinase 1（PLK1） plays an important role in many cell-cycle-related events. At G2/Mtransition, PLK1 contributes to the activation of cyclinB/Cdc by phosphorylation of Cdc25C, centrosome functional maturation, bipolar spindle formation. In later stage of mitosis, PLK1 is involved in regulating components of the anaphase-promoting complex （APC） for mitotic exit and in the execution of cytokinesis. Moreover, recent reports have shown that PLK1 is involved in both G2 and mitotic DNA damage checkpoints. When G2/M DNA damage occurs, PLK1 activity is suppressed and cell cycle arrests to repair the damaged DNA. So far, the deregulated expression of PLK1 has been detected in many types of human tumors and PLK1 is considered as a novel prognostic marker for several tumors.

吲哚美辛诱导肝癌HepG2细胞凋亡及其对cyclin E蛋白表达的影响

目的 观察吲哚美辛诱导肝癌 Hep G2细胞凋亡 ,及其对 cyclin E蛋白的影响。方法 采用 MTT比色法观察吲哚美辛对肝癌 Hep G2细胞增殖的影响 ;采用流式细胞仪检测吲哚美辛对 Hep G2细胞周期分布的影响 ,同时结合透射电子显微镜观察吲哚美辛诱导肝癌 Hep G2细胞凋亡的作用 ;免疫细胞化学方法观察吲哚美辛对Hep G2细胞周期调控蛋白 cyclin E的影响。结果 吲哚美辛可抑制肝癌 Hep G2细胞的增殖 ,诱导其凋亡 ,改变细胞周期分布 ,使 G0 /G1 期细胞比例增高 ,S期比例降低 ,同时还可使 cyclin E蛋白表达下降。上述作用具有剂量和时间依赖性 (P<0 .0 5 )。结论 吲哚美辛可诱导肝癌 Hep G2细胞凋亡 ,改变细胞周期分布 ,影响细胞周期调控蛋白表达 ,从而抑制细胞增殖。

胃肠道恶性肿瘤Cyclins表达分型与MDR1及TNM分期的关系

目的:研究胃肠道恶性肿瘤的4种主要Cyclins (Cyclin D1,E,A,B1)的表达规律,以此为依据对恶性肿瘤进行分型,并结合肿瘤标本中 MDR1(多药耐药基因1)的表达情况,与肿瘤 TNM分期进行相关性分析．方法:采用流式细胞术分析新鲜手术获取的 62例肿瘤标本(胃癌32例,结直肠癌30例)的 Cyclin D1,E,A,B1及MDR1表达情况,再记录术后病理报告进行TNM分期．结果:根据4种主要Cyclins的表达情况对恶性消化道肿瘤分为Ⅰ-Ⅳ型Cyclin非时相性表达类型,此Ⅰ-Ⅳ型细胞周期类型与TNM分期具有一致性,Kappa系数等于0．599(P<0．01)．Ⅰ- Ⅳ型细胞周期类型中MDR1的表达水平逐渐增高,等级相关系数rs为0．495(P<0．01)．结论:根据Cyclins的表达情况对消化道恶性肿瘤进行的分型具有与TNM分期相同的意义．在消化道肿瘤中,MDR1与肿瘤细胞周期素表达类型有关,对治疗具有指导意义．

Personalized targeted therapy for esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma continues to heavily burden clinicians worldwide. Researchers have discovered the genomic landscape of esophageal squamous cell carcinoma, which holds promise for an era of personalized oncology care. One of the most pressing problems facing this issue is to improve the understanding of the newly available genomic data, and identify the driver-gene mutations, pathways, and networks. The emergence of a legion of novel targeted agents has generated much hope and hype regarding more potent treatment regimens, but the accuracy of drug selection is still arguable. Other problems, such as cancer heterogeneity, drug resistance, exceptional responders, and side effects, have to be surmounted. Evolving topics in personalized oncology, such as interpretation of genomics data, issues in targeted therapy, research approaches for targeted therapy, and future perspectives, will be discussed in this editorial.

Antiproliferation and apoptosis induction of paeonol in HepG_2 cells

AIM： To investigate the antiproliferative effect of paeonol （Pae） used alone or in combination with chemotherapeutic agents [cisplatin （CDDP）, doxorubicin （DOX） and 5-fluorouracil （5-FU）] on human hepatoma cell line HepG2 and the possible mechanisms. METHODS： The cytotoxic effect of drugs on HepG2 cells was measured by 3-（4, 5-dimethylthiazol-2- yl）-2, 5-diphenyltetra-zolium bromide （MTT） assay. Morphologic changes were observed by acridine orange （AO） fuorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry （FCM）. Drug-drug interactions were analyzed by the coefficient of drug RESULTS： Pae （7.81-250 mg/L） had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of （104.77±7.28） mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae （15.63, 31.25, 62.5 rag/L） was combined with CDDP （0.31-2.5 mg/L）, DOX （0.16-1.25 mg/L）, or 5-FU （12.5-100 mg/L） at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae （P 〈 0.01）. Of the three different combinations, the sensitivity of cells to drugs was considerably different.CONCLUSION： Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2, which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.

Effects of low frequency electromagnetic fields on osteoblasts proliferation and cell cycle

After primary mouse osteoblasts and ROS osteo-like cells were exposed to 50 Hz low frequency electromagnetic fields (EMF), the MTT method and flow cytometry have been used to determine cell proliferation, cell cycle and apoptosis. The results show that, compared

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

Ruxolitinib add-on in corticosteroid-refractory graft-vs-host disease after allogeneic stem cell transplantation:Results from a retrospective study on 38 Chinese patients

BACKGROUND Graft-vs-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation.Some patients have steroid-refractory(SR) GVHD.AIM To evaluate the effect and safety of ruxolitinib add-on in the treatment of patients with SR acute (a) and chronic (c) GVHD.METHODS We retrospectively analyzed 38 patients administered ruxolitinib add-on to standard immunosuppressive therapy for SR-aGVHD or SR-cGVHD following allogeneic hematopoietic stem cell transplantation.Ruxolitinib was administered5-10 mg/d depending on disease severity,patient status,and the use of antifungal drugs.Overall response rate,time to best response,malignancy relapse rate,infection rate,and treatment-related adverse events were assessed.RESULTS The analysis included 10 patients with SR-aGVHD (gradeⅢ/Ⅳ,n=9) and 28patients with SR-cGVHD (moderate/severe,n=24).For the SR-aGVHD and SRcGVHD groups,respectively:Median number of previous GVHD therapies was 2(range:1-3) and 2 (1-4);median follow-up was 2.5 (1.5-4) and 5 (1.5-10) mo;median time to best response was 1 (0.5-2.5) and 3 (1-9.5) mo;and overall response rate was 100%(complete response:80%) and 82.1%(complete response:10.7%) with a response observed in all GVHD-affected organs.The malignancy relapse rates for the SR-aGVHD and SR-cGVHD groups were 10.0%and 10.7%,respectively.Reactivation rates for cytomegalovirus,Epstein-Barr virus,and varicella-zoster virus,respectively,were 30.0%,10.0%,and 0%for the SR-aGVHD group and 0%,14.3%,and 7.1%for the SR-cGVHD group.CONCLUSION Ruxolitinib add-on was effective and safe as salvage therapy for SR-GVHD.

Taxotere resistance in SUIT Taxotere resistance in pancreatic carcinoma cell line SUIT 2 and its sublines

AIM: To investigate the specific mechanisms of intrinsic and acquired resistance to taxotere (TXT) in pancreatic adenocarcinoma (PAC). METHODS: MTT assay was used to detect the sensitivity of PAC cell line SUIT-2 and its sublines (S-007, S-013, S-020, S-028 and TXT selected SUIT-2 cell line, S2/TXT) to TXT. Mdr1 (P-gp), multidrug resistance associated protein (MRP), lung resistance protein (LRP) and beta-tubulin isotype gene expressions were detected by RT-PCR. The functionality of P-gp and MRP was tested using their specific blocker verapamil (Ver) and indomethacin (IMC), respectively. The transporter activity of P-gp was also confirmed by Rhodamine 123 accumulation assay. RESULTS: S-020 and S2/TXT were found to be significantly resistant to TXT(19 and 9.5-fold to their parental cell line SUIT-2, respectively). RT-PCR demonstrated strong expression of Mdr1 in these two cell lines, but weaker expression or no expression in other cells lines. MRP and LRP expressions were found in most of these cell lines. The TXT-resistance in S2-020 and S2/TXT could be reversed almost completely by Ver, but not by IMC. Flow cytometry showed that Ver increased the accumulation of Rhodamine-123 in these two cell lines. Compared with S-020 and SUIT-2, the levels of beta-tubulin isotype II, III expressions in S-2/TXT were increased remarkably. CONCLUSION: The both intrinsic and acquired TXT-related drug resistance in these PAC cell lines is mainly mediated by P-gp, but had no relationship to MRP and LRP expressions. The increases of beta-tubulin isotype II, III might be collateral changes that occur when the SUIT-2 cells are treated with TXT.

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Effects of arotinoid acid on induction of apoptosis and differentiation and telomerase activity and cell cycle in the HL 60 cell line

Objective To investigate the effects of arotinoid acid (Ro13 7410) on the morphological and functional alterations of leukemia HL 60 cell line and compared with those of RA Methods Differentiation of HL 60 cells was assessed by morphology and by NBT reduction Trypan blue exclusion was used to determine viability Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD assay kit Telomerase PCR ELISA kit tested telomerase activity The cell cycle was analyzed by flow cytometry Results Incubation of the HL 60 cells with 10 -6 10 -8 ?mol/L Ro13 7410 resulted in suppression of cell growth Apoptotic cells were detected following exposure to 10 -6 ?mol/LRo13 7410 for 3 hours by measurement of the “in situ” enzymatic labeling of DNA breaks with biotinylated dUTP Ultrastructural examination of Ro13 7410 treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of “apoptotic bodies” Cells induced into apoptosis were accompanied by Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ Percentage of HL 60 cells reduced NBT following incubation with Ro13 7410 was lower than with all trans retinoic acid (RA) (27% vas 85%) Telomerase PCR ELISA assay showed that HL 60 cells cultured in the absence of inducing agents had significant telomerase activity Telomerase activity declined gradually after 10 -6 ?mol/L Ro13 7410 treatment, and changes becoming evident at 1 day The inhibition of telomerase activity at day 5 of treatment with Ro13 7410 was less effective than with RA DNA flow cytofluorimetric analysis revealed that Ro13 7410 caused partial cell arrest in the G 2/M phase after a 2 day treatment and the percentage of cells arrested in the G 2/M phase increased after 4 days treatment With RA treated cells, a reduction in the percentage of cells in the G 2/M phase was observed after 2 day of treatment Conclusion Our study shows that Ro13 7410 suppresses HL 60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation Ro13 7410 dramatically inhibits telomerase activity during the course of induction and results in G 2/M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells