Sensitivity of different phases of the cell cycle to selected hepatotoxins in cultured liver-derived (BL9L) cells

1. Many toxins are active against dividing cells and cytofluorometric analysis of synchronized dividing liver-derived (BL9L) cells has been employed to study the relative sensitivity of the G1(G0), S and G2/M phases of the cell cycle to selected hepatotoxins. 2. The cytotoxic metal beryllium, which inhibits cell division, caused a specific block at the G1 phase of the cell cycle. 3. Dehydroretronecine, an antimitotic metabolite of the hepatotoxic plant pyrrolizidine alkaloids, retarded progression of cells through the cell cycle with a consistent accumulation at the late S to G2 phase. 4. Exposure of cells to aflatoxin B1-8,9-epoxide, the putative carcinogenic metabolite of the hepatocarcinogen aflatoxin B1, particularly during the early period of S phase, produced morphologically transformed cells.     

Sensitivity of different phases of the cell cycle to selected hepatotoxins in cultured liver-derived (BL9L) cells

1. Many toxins are active against dividing cells and cytofluorometric analysis of synchronized dividing liver-derived (BL9L) cells has been employed to study the relative sensitivity of the G₁(G₀), S and G₂/M phases of the cell cycle to selected hepatotoxins.

2. The cytotoxic metal beryllium, which inhibits cell division, caused a specific block at the G₁ phase of the cell cycle.

3. Dehydroretronecine, an antimitotic metabolite of the hepatotoxic plant pyrrolizidine alkaloids, retarded progression of cells through the cell cycle with a consistent accumulation at the late S to G₂ phase.

4. Exposure of cells to aflatoxin B₁-8,9-epoxide, the putative carcinogenic metabolite of the hepatocarcinogen aflatoxin B₁, particularly during the early period of S phase, produced morphologically transformed cells.     

Inhibition of cell cycle progression through specific phase by pancratistatin derivatives

Pancratistatin derivatives, 1-O-(3-hydroxybutyryl)pancratistatin (HBP) and 1-O-(3-O-beta-D-glucopyranosylbutyryl)pancratistatin (GBP), showed strong cytostatic activity against rat embryo fibroblast 3Y1 at concentrations less than 1 microM. When the effect on cell cycle progression was examined in 3Y1 fibroblasts arrested at G0/G1 phase by serum deprivation, HBP, GBP, and pancratistatin inhibited the progression of 3Y1 fibroblasts from G0/G1 to S phase. In addition, when the effect on cell cycle progression was studied in 3Y1 fibroblasts synchronized at late G1/early S phases by treating with hydroxyurea, HBP blocked further progression through S phase, while GBP and pancratistatin did not affect the progression, but retarded it. On the other hand, when the effect of HBP and GBP on the progression was evaluated in promyelocytic leukemia HL-60RG cells synchronized at G0/G1 phase, the cells did not progress into S phase and accumulated in sub G0/G1 phase, which indicated apoptotic cells. These findings suggest that of Amaryllidaceae alkaloids, HBP blocks the progression of cell cycle at least at G0/G1 and S phases and GBP does at least at G0/G1 phase, resulting in apoptosis induction in tumor cells.     

Assessment of the role of DNA damage and repair in the survival of primary cultures of rat cutaneous keratinocytes exposed to bis(2-chloroethyl)sulfide

Toxicity manifests itself as vesication in human skin exposed topically to bis(2-chloroethyl)sulfide (BCES). The destruction of the proliferating population of epidermal cells is a major component of the pathogenic process. Available data strongly suggest that damage to cellular DNA is a critical factor in the loss of these cells. However, the influence of DNA repair on this toxic response has not been adequately studied. Therefore, a study was undertaken to ascertain the influence of DNA repair on the survival of primary monolayer cultures of rat cutaneous keratinocytes exposed to BCES. The sensitive nucleoid sedimentation assay was employed for the determination of DNA damage in cultures exposed to very low levels of BCES. Initial experiments demonstrated that within 1 hr of exposure to as little as 0.1 microM BCES the structural integrity of cellular DNA was compromised, presumably resulting from the appearance of single-strand breaks in the nucleic acid. This same effect was demonstrated in basal cells derived from a stratified, cornified culture grown at the air-liquid interface and exposed topically to the vesicant. Further studies with the monolayer culture demonstrated that the gross structural integrity of the DNA in cells exposed to as much as 5 microM BCES was completely restored within the first 22 hr following the exposure. However, this repair process appeared to be inefficient since a depression of thymidine incorporation into DNA and a significant loss of DNA were exhibited in exposed cultures as long as 72 hr after the initial exposure.     

Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole

UCN-01 (7-hydroxystaurosporine) and CGP 41 251 (4'-N-benzoyl staurosporine), both of which were discovered as protein kinase C selective inhibitors, have entered in phase 1 clinical trials as anti-cancer drugs. In this study, we have directly compared the effects of these drugs as well as staurosporine (STP) on cell cycle progression of A431 human epidermoid carcinoma cells synchronized at M phase by treatment with nocodazole. The nocodazole-synchronized cells progressed from M to G1 phase in the absence of the drug, which was accompanied by a decrease of cyclin B1 protein expression, disappearance of the complex formation of CDC2 with cyclin B1 and reduction of the kinase activity. Treatments of the M phase cells with UCN-01, STP and CGP 41 251 at 80% growth-inhibitory concentrations (IC80S) resulted in specific G1 block, G2M block and polyploidy, respectively. Decreases of cyclin B1 protein expression was partially prevented by treatments with STP and CGP 41 251 but not with UCN-01 at IC80S. Reductions of active complex and kinase activity of CDC2/cyclin B1 were also observed in the presence of the three drugs. In addition, augmentation of CDC2 protein tyrosine phosphorylation was induced only when the cells were treated with STP. These observations demonstrated that higher concentrations of UCN-01, STP and CGP 41 251 showed different effects on cell cycle progression as well as CDC2/cyclin B1 regulation in A431 cells synchronized at M phase. The data suggest that UCN-01 and CGP 41 251 may act at quite different points on the cell cycle.     

The effects of cell cycle position on the cytotoxicity and mutagenicity of benzo(a)pyrene in cultured Chinese hamster cells

The induction of cytotoxicity and mutation to 6-thioguanine resistance (6TGr) by S9-activated benzo(a)pyrene (B(a)P) was studied in asynchronized and synchronized Chinese hamster V79 cells. After treatment of asynchronized populations with B(a)P (0.25-2 micrograms/ml) in the presence of S9 for 3 h, the number of 6TGr cells increased. The increase was concentration-dependent up to 2 micrograms/ml, and was accompanied by a concomitant concentration-dependent decrease in cell survival. Synchronized cells were treated with B(a)P for 2 h at 2-h intervals after release from the G1/S block by hydroxyurea (HU). The cytotoxicity of 2 micrograms/ml of B(a)P was maximal at 0 h after HU release, i.e., G1/S phase, and also at 2 h after HU release, i.e., early S phase. Thereafter, it decreased with the progression of the cell cycle. Similarly, treatment with B(a)P at 0 h and 2 h after HU release resulted in the maximum incidence of 6TGr mutants, after which the incidence showed a decrease from 4-10 h after HU release. These results indicate that the cells in G1/S and early S phase are highly susceptible to cytotoxic and mutagenic damage induced by B(a)P and suggest the presence of a specific hot spot in the cell cycle for mutagenesis by the carcinogen B(a)P in cultured hamster cells.     

The cytokinetic and cytotoxic effects of ICRF-159 and ICRF-187 in vitro and ICRF-187 in human bone marrow in vivo

The cytotoxic and cytokinetic effects of ICRF-159 and its d-enantiomer ICRF-187 have been examined in vitro. The effects of both agents were identical. Cytotoxicity is dependent on both the drug concentration and the duration of drug exposure. Drug exposure for twice the cell cycle time is necessary for maximum effect. Cytotoxicity is also dependent upon the rate of cell proliferation. A rapidly growing cell population is more sensitive to brief drug exposure than a slowly growing population. The cytokinetic effects were studied using flow cytometry, determination of [3H]-thymidine incorporation and mitotic index. ICRF-159/187 appears to act only during the G2 phase of the cell cycle. There is no detectable delay in cell passage through the G1/S boundary or in transit through S phase. Inhibition of DNA synthesis occurs only after the G2 block prevents subsequent entry of cells in S phase. A fraction of the cells, depending upon drug concentration, undergo further DNA synthesis without cell division, resulting in a tetrapoid cell population. The cytokinetic effects were determined in the bone marrow of patients receiving ICRF-187. All dose-rates produced G2/M accumulation in the marrow with depletion of S phase cells. One patient was given a single injection of 1.0 gm/M2 . G2/M accumulation was observed 24 h after treatment, with recovery to a pretreatment DNA cycle distribution 24 h later. These studies suggest that a continuous drug infusion, or intermittent infusions timed to allow the normal cell population to recover, may produce superior clinical activity with this agent. A Phase I study of such an intermittent schedule is indicated.     

Effects of valproic acid and levetiracetam on viability and cell cycle regulatory genes expression in the OVCAR-3 cell line

Concentration- and time-dependent effects of two antiepileptic drugs (AEDs), levetiracetam (LEV) and valproic acid (VPA), on proliferation, cytotoxicity and expression of cell cycle regulatory genes were investigated in a human ovarian cancer cell line, OVCAR-3. Cells were cultured with VPA or LEV, at concentrations between 100 μM and 10 mM. Cell proliferation was determined by alamarBlue and BrdU incorporation assays; cytotoxic effects by tetrazolium hydroxide (XTT), acid phosphatase (AP) and lactate dehydrogenase (LDH) assays. Expression of cell cycle regulatory genes was determined by real-time PCR. Exposure to VPA caused a concentration- and time-dependent decrease in cell proliferation (alamarBlue and BrdU incorporation assays), cytotoxic effects above 2.5 mM (XTT and AP assays) and modulated expression of genes primarily responsible for cell cycle arrest in G(1) phase. Cell proliferation was unaffected by exposure to LEV for 24 h and 120 h (alamarBlue assay), but increased when exposed to LEV for 72 h and 168 h, at concentrations from 250 μM to 1 mM. The BrdU incorporation assay showed no effect of LEV on cell proliferation. LEV was cytotoxic at higher concentrations (AP assay), but modulation in expression of cell cycle regulatory genes was not observed. Changes in LDH release were not observed with either AED. In summary, VPA apparently decreased cell proliferation by down-regulating genes responsible for transition from G(1) to S phase and up-regulating genes responsible for G(1) phase arrest, which suggest its potential as an anticancer drug. LEV does not exhibit such action.     

Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies

In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular.     

Studies on antitumor activity of prumycin. III. Mode of action of prumycin on HeLa S-3 cells

The mode of action of prumycin was investigated using synchronized and asynchronous cultured HeLa S-3 cells. Prumycin inhibited significantly the growth of HeLa S-3 cells at the concentration over 5 mcg/ml. DNA synthesis as well as protein synthesis was strongly inhibited at the concentration of 10 mcg/ml of prumycin, but RNA synthesis was not inhibited by the same concentration. Prumycin did not block the transition of the cells from M phase to G1 phase, however, G2 phase cells were blocked clearly by this antibiotic.     

Schedule-dependent cytotoxicity of etoposide (VP-16) and cyclophosphamide in leukemia cell line K-562

In allogeneic bone marrow transplantation (allo-BMT) in patients with leukemia, the combination of VP-16 and cyclophosphamide (CY) is commonly used for the conditioning regimen. In the present study, we demonstrated schedule-dependent cytotoxicity of VP-16 and CY in K-562 cells. K-562 cells were pretreated with low concentrations (2.5 and 5?μg/mL) of 4-hydroperoxycyclophosphamide (40487S), which is a preactivated analog of CY. It was confirmed that these concentrations did not influence cell viability. Cells subsequently exposed to 0.5-100?μg/mL of VP-16 showed reduced the viability compared to that of control cells not treated with 40487S. In contrast, there was no change in the viability of K-562 cells pretreated with low concentrations (0.5 and 1?μg/mL) of VP-16. It was confirmed that these concentrations did not influence cell viability. Viability of subsequently exposed to 1-20?μg/mL was not different from that of control cells not treated with VP-16. VP-16 caused cell cycle arrest at G?/M phase. On the other hand, 40487S arrested the cell cycle at S phase. Thymidine-synchronized cells, VP-16 showed cell cycle specificity for cell killing from early-S to mid-S phase. On the other hand, 40487S showed cell cycle-independent cytotoxicity. Exposure of cells to VP-16 after 40487S induced a greater cytotoxic effect on K-562 cells. The findings may lead to improvements in clinical combination chemotherapy.     

帕唑帕尼对甲状腺癌细胞和血管内皮细胞增殖的影响及其作用机制

目的 研究新型酪氨酸激酶抑制剂帕唑帕尼对甲状腺癌细胞株SW579和血管内皮细胞株HUVEC增殖的影响及作用机制.方法 将SW579和HUVEC暴露于不同浓度（1、2、4、8和16 μmoL/L）的帕唑帕尼至72 h,无药物处理的细胞作为对照.采用细胞活度测定、图像分析技术及分裂细胞荧光免疫染色观察评价帕唑帕尼对细胞增殖的影响,应用流式细胞仪进行细胞周期时相分析.结果 细胞活度测定显示,帕唑帕尼对SW579和HUVEC细胞的生长增殖均有明显的抑制作用,并呈浓度依赖性及时间依赖性,其半数抑制浓度（IC50）分别约为2和4μmol/L.图像分析显示,在IC50作用下,帕唑帕尼使两种细胞的细胞密度降低,分裂指数下降.帕唑帕尼作用48 h时,SW579细胞分裂指数（0.9％）明显低于其对照细胞（2.2％）,差异有统计学意义（P＜0.01）;HUVEC细胞分裂指数（1.9％）亦明显低于其对照细胞（4.0％）,差异有统计学意义（P＜0.01）.细胞周期时相分析表明,帕唑帕尼可使SW579和HUVEC发生细胞周期休止,分别休止于G1期和S期.帕唑帕尼作用24、48 h时,SW579在G1期的比例均明显高于其对照组细胞[（60.4±2.2）％比（49.2±1.6）％,（70.4±1.6）％比（58.6±3.3）％],差异有统计学意义（P＜0.01）;HUVEC细胞在S期的比例均明显高于其对照组细胞[（47.3±2.7）％比（39.5±0.6）％,（43.5±1.6）％比（28.8±4.2）％],差异有统计学意义（P＜0.01）.结论 帕唑帕尼对甲状腺癌细胞和血管内皮细胞的增殖均有明显的抑制效应,其作用是藉细胞周期休止作用而实现,但在两种细胞细胞周期休止时相不同.     

Effect of bis(beta-chloroethyl)sulfide (BCES) on base mismatch repair of DNA in monkey kidney cells

Sulfur mustard, bis(beta-chloroethyl)sulfide (BCES), a bifunctional alkylating agent, is a vesicant whose mode of action involves interference with the integrity of cellular DNA. Alkylation of DNA is responsible for some of the biological effects of BCES in tissue. Another possible mechanism by which BCES could exert its toxic effect is interference with high fidelity repair of damaged DNA. This study evaluated the possible effects of BCES on the repair of specific errors, i.e., mismatched bases, in the DNA. Heteroduplex (ht) DNA, formed between two temperature-sensitive mutants of SV40 virus, tsA239 and tsA255, each having a different point mutation in the gene for large T antigen, was used to study the effect of BCES on mismatched base repair in African green monkey kidney (AGMK) cells. AGMK cells were exposed to dilute solutions of BCES in methylene chloride (MC) prior to cationic lipofection with ht DNA. In order for the cells to produce wild type (wt) SV40 DNA at a nonpermissive temperature (41 degrees C), repair of at least one of the two mismatches in the DNA had to occur. It was observed that (a) as the concentration of BCES was increased, a proportionally longer delay in the appearance of wt DNA at 41 degrees C was observed in treated cells transfected with ht DNA as compared with cultures exposed to MC alone and then transfected with ht DNA, (b) there was no such effect in exposed AGMK cells transfected with wt DNA, (c) wt and ht DNA were transfected at similar rates in unexposed cells, and (d) BCES did not affect the rate of transfection of wt cells. These observations are consistent with the hypothesis that BCES affects mismatched base repair.     

bis-(beta-chloroethyl)sulfide (BCES)-induced changes in epidermal cell homeostasis in vitro

A rat cutaneous keratinocyte culture system was developed to study the effects of the vesicant bis-(beta-chloroethyl)sulfide (BCES) on the homeostasis of cell proliferation and differentiation. Lectins were used to reveal cell surface carbohydrate changes as the keratinocytes differentiate. In the newborn rat epidermis, the isolectin, Griffonia simplicifolia I-B4 (GS I-B4), binds to basal cell surfaces. Ulex europeus agglutinin I (UEA) binds to the surfaces of spinous and lower granular cells and is therefore considered an indicator of keratinocyte differentiation. A fluorometric assay was developed which determines the ratio of bound UEA to bound GS I-B4 (the UEA/B4 ratio) in primary monolayer cultures of rat cutaneous keratinocytes maintained in low Ca2+ medium. The UEA/B4 ratio was found to be a representation of the relative sizes of the differentiating and proliferating cell compartments in the monolayer cultures, respectively (W.W. Ku and I.A. Bernstein, 1988, Exp. Cell Res., 175, 298-316). Monolayer cultures exposed for 1 hr to BCES at Day 1 exhibited a dose-related increase in the UEA/B4 ratio at Day 7 when compared to solvent controls. The results from the analysis of lectin binding sites showed a decrease in GS I-B4 binding with little or no change in UEA binding as a result of BCES exposure, contributing to the increase in the UEA/B4 ratio. BCES-exposed monolayers also showed early perturbations in replicative DNA synthesis as revealed by autoradiography. Subsequent to the perturbations in replicative DNA synthesis was an inability of BCES-exposed cultures to produce cells into the monolayer through mitosis. In addition to an increase in the UEA/B4 ratio, BCES-exposed monolayers also showed a dose-related loss of DNA, with the appearance of enlarged cells at Day 7. These enlarged cells failed to show evidence of DNA synthesis, with groups of these cells showing intense UEA staining with only faint GS I-B4 staining. Overall, exposure to low concentrations of BCES appeared to disrupt the normal homeostasis of cell proliferation and differentiation in this monolayer culture system. This disruption was primarily through a reduction in the fraction of germinative (basal) cells with concomitant retention of some early differentiated cells, presumably early spinous or spinous cells.     

半边旗抗肿瘤有效成分对HL—60细胞DNA拓扑异构酶活性及其细胞 …

目的；研究半边旗有效成分５Ｆ，６Ｆ，Ａ对ＨＬ－６０细胞ＤＮＡ拓扑异构酶活性和细胞周期的影响。方法：应用ｐＢＲ３２２质粒ＤＮＡ作为底物测定酶的活性；细胞周期用流式细胞仪测定；应用噻唑蓝法测定药物对细胞生长的抑制率。结果：５Ｆ，６Ｆ，Ａ均能够抑制ＤＮＡ拓扑异构酶Ⅰ，Ⅱ的活性。     

Cytotoxic effect of inositol hexaphosphate and its Ni(II) complex on human acute leukemia Jurkat T cells

Inositol hexaphosphate (InsP₆) is present in cereals, legumes, nuts and seed oils and is biologically active against some tumor and cancer cells. Herein, this study aimed at evaluating the cellular toxicity, antiproliferative activity and effects on cell cycle progression of free InsP₆ and InsP₆–Ni(II) of leukemic T (Jurkat) and normal human cells. Treatments with InsP₆ at concentrations between 1.0 and 4.0 mM significantly decreased the viability of Jurkat cells, but showed no cytotoxic effect on normal human lymphocytes. Treatment with InsP₆–Ni(II) complex at concentrations between 0.05 and 0.30 mM showed an anti-proliferative dose and a time-dependent effect, with significantly reduced cell viability of Jurkat cells but showed no cytotoxic effect on normal human lymphocytes as compared to the control. Ni(II) free ion was toxic to normal cells while InsP₆–Ni(II) had no cytotoxic effect. The InsP₆–Ni(II) complex potentiated (up to 10 ×) the antiproliferative effect of free InsP₆ on Jurkat cells. The cytometric flow assay showed that InsP₆ led to an accumulation of cells in the G0/G1 phase of the cell cycle, accompanied by a decrease in the number of cells in S and G2/M phases, whereas InsP₆–Ni(II) has led to an accumulation of cells in the S and G2/M phases. Our findings showed that InsP₆–Ni(II) potentiates cytotoxic effects of InsP₆ on Jurkat cells and may be a potential adjuvant in the treatment of cancer.     

Cell cycle specific fluctuations of adenosine 3',5' -monophosphate and prostaglandin binding in synchronized mastocytoma P-815 cells

Endogenous adenosine 3',5' -monophosphate (cAMP) levels in mastocytoma P-815 cells, synchronized either at the G1/S transition by amethopterin- or double thymidine-block or in mitosis by colcemid block, were highest during late S and early G2 phases and lowest during mitosis. These cell cycle-dependent changes in cAMP levels were largely accounted for by changes in adenylate cyclase and phosphodiesterase activities. Similar fluctuations occurred simultaneously with specific prostaglandin E1 (PGE1) binding, histidine decarboxylase activity, histamine content, and [35S]SO-2(4) incorporation into glycosaminoglycans of the cells. In addition, endogenous levels of the E group of prostaglandins (PGEs) and "14C]carachiodonic acid incorporations into PGE, phosphatidylcholine and phosphatidylinositol also exhibited fluctuation patterns similar to that of cAMP levels. Since cAMP levels still fluctuated in a serum-depleted medium where DNA synthesis and cell division were inhibited, endogeneous levels of prostaglandin and cAMP appeared not to be regulated solely by serum factor(s). Exposure of cells at G1/S transition to 1-methyl-3-isobutylxanthine (MIX) resulted in 10-fold elevation of cAMP levels throughout the cell cycle without affecting DNA synthesis. On the other hand, PGE1 and/or MIX added at late S phase elevated cAMP levels, prolonged C2 phase and retarded the cell division, but these agents added at the beginning of mitosis elevated cAMP levels without affecting the cell division. These results suggest that prostaglandin newly synthesized by the increased metabolism of phospholipids promote the cAMP synthesis via their binding to the receptors and thereby control the division and phenotypic expression of mastocytoma P-815 cells.     

海南哥纳香醇甲(GHM-10)对体外L1210细胞的抗肿瘤活性

用体外培养法研究GHM-10对L1210细胞生长的抑制作用和作用机制。结果表明,L1210细胞在用GHM-10处理1h,24h和7d后,IC₅₀依次为6.85,3.32和1.59μg·ml^-1,提示GHM-10有非时相特异性细胞毒药物的特性。在用GHM-10 1～2μg·ml^-1作用24h后,L1210细胞的增长率和有丝分裂指数下降,细胞核形态发生变化,但活细胞率仍保持在96%以上,提示GHM-10主要抑制细胞的增殖。用流式细胞计对L1210细胞进行细胞周期动力学的分析表明,GHM-10可在一定程度上阻断G₁期细胞向S期移行,还可增加L1210细胞的膜流动性。     

吡格列酮对HepG2细胞增殖和凋亡的影响及机制研究

目的观察吡格列酮对体外培养的HepG2细胞增殖和凋亡的影响,并探讨其是否通过PPARγ依赖途径发挥上述药理作用。方法将不同浓度的吡格列酮作用于体外培养HepG2细胞,以MTT比色法检测HepG2细胞增殖情况,以3H-TdR参入实验检测细胞DNA合成速率,采用RT-PCR和Western blot检测PPARγmRNA和蛋白的表达,以流式细胞术检测细胞凋亡和细胞周期;同时观察PPARγ特异性拮抗剂GW9662和(或)瞬时转染pSG5-PPARγ真核表达质粒对吡格列酮细胞增殖作用的影响;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒稳定转染HepG2细胞,观察PPARγ沉默后吡格列酮对HepG2细胞增殖作用的影响。结果吡格列酮作用于HepG2细胞后,导致HepG2细胞的增殖受到抑制、DNA合成速率减慢,并诱导细胞凋亡,呈一定的剂量依赖关系;在此过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达没有变化;GW9662部分拮抗吡格列酮的增殖抑制作用,但转染pSG5-PPARγ真核表达质粒可以逆转GW9662的作用;吡格列酮在高浓度(20μmol.L-1)时对pGCsi-PPARγ表达质粒稳定转染的HepG2细胞仍表现出增殖抑制作用。结论吡格列酮能够抑制HepG2细胞的增殖并诱导凋亡,具有潜在的抗瘤作用,这种作用与其诱导细胞G0/G1期的停滞有关,PPARγ依赖和非依赖途径参与上述过程。