Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G(1)/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G(1)/S arrest in response to severe hypoxia. We found a general decrease in p16(INK4a) (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G(1)/S arrest. Furthermore, cell lines that underwent G(1)/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1alpha) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12-24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G(1)/S arrest, also did not show decreased HIF-1alpha, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3beta, which was the opposite pattern to that exhibited by the cell lines showing the G(1)/S arrest phenotype. Inhibition of GSK3beta by lithium chloride treatment of HeLa cells converted the HIF-1alpha, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1alpha, p16 and p27 was associated with both a hypoxia-induced G(1)/S arrest phenotype and increased GSK3beta activity.     

HIV protease inhibitor nelfinavir inhibits growth of human melanoma cells by induction of cell cycle arrest

HIV protease inhibitors (HIV PI) are a class of antiretroviral drugs that are designed to target the viral protease. Unexpectedly, this class of drugs is also reported to have antitumor activity. In this study, we have evaluated the in vitro activity of nelfinavir, a HIV PI, against human melanoma cells. Nelfinavir inhibits the growth of melanoma cell lines at low micromolar concentrations that are clinically attainable. Nelfinavir promotes apoptosis and arrests cell cycle at G(1) phase. Cell cycle arrest is attributed to inhibition of cyclin-dependent kinase 2 (CDK2) and concomitant dephosphorylation of retinoblastoma tumor suppressor. We further show that nelfinavir inhibits CDK2 through proteasome-dependent degradation of Cdc25A phosphatase. Our results suggest that nelfinavir is a promising candidate chemotherapeutic agent for advanced melanoma, for which novel and effective therapies are urgently needed.     

Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells.We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.     

土贝母皂苷诱导人宫颈癌HeLa细胞周期阻滞和细胞凋亡

背景和目的：土贝母皂苷是由传统中药土贝母块茎中分离得到的,由土贝母苷甲（79%）和土贝母苷乙（21%）组成。本研究旨在探讨土贝母皂苷抗肿瘤作用的机制。方法：MTT法检测土贝母皂苷对肿瘤细胞生长的抑制作用,流式细胞仪分析细胞周期,荧光显微镜和电子显微镜观察细胞形态,琼脂糖凝胶电泳检测DNA电泳图谱的变化。结果：土贝母皂苷对人宫颈癌HeLa细胞的生长具有强制作用,且这种作用呈时间剂量依赖关系,处理24、48和72h的IC50值分别为20.0、18.8和8.8μmol/L.流式细胞术分析显示,15、30、35μmol/L土贝母皂苷处理5h,HeLa细胞G2/M期细胞数从9.80%分别增加到21.90%、25.92%和27.00%;处理12h,G2/M期细胞数增加更显著,从8.20%分别增加到21.40%、31.15%和34.55%。40μmol/L土贝母皂苷处理一定时间,形态学检查发现细胞核明显皱缩、核染色质凝集边聚等特征,流式细胞术检测发现凋亡峰,DNA琼脂糖凝胶电泳可见明显的梯形条带。结论：土贝母皂苷使细胞周期阻滞和诱导细胞凋亡可能在其抗肿瘤作用中具有重要意义。     

Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines

Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC₅₀ (SD) for growth inhibition by the SRB assay was 200 (101) µM for a 48 h exposure with a range of 64–358 µM. Cell killing was schedule-dependent, favoring exposure times ≥48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations ≤200 µM for 48 h. When concentrations were increased to 300 µM for ≥48 h, the MiaPaCa-2 cells arrested in G₂ phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC₈₀ concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of ≥400 µM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC’s are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G₂ arrest, accumulation of ROS, and the induction of apoptosis.     

Retinoic acid-induced cell cycle arrest of human myeloid cell lines

Retinoic acid-induced terminal differentiation of myeloid cells involves the sequential regulation of cell cycle regulatory genes, coordinating the process of differentiation with arrest in the G0/G1 phase of the cell cycle. In this review we have summarized changes in expression and activity of cell cycle regulatory proteins associated with retinoic acid induced-growth arrest in human myeloid cell lines. These changes involve: (i) an early down-regulation of c-Myc; (ii) up-regulation of p21CIP1 and p27KIP1 and, in some cases, p15INK4b or p18INK4c; (iii) down-regulation of cyclin E and cyclin D1/D3, and, at later stages, cyclin A and cyclin B; and (iv) decreased CDK activity and dephosphorylation of pRb.     

大蒜素诱导人胃癌细胞M期阻滞的研究

目的探讨大蒜素对人胃癌细胞系MGC803和SGC7901细胞周期的影响及其作用机制。方法以台盼蓝拒染法检测人胃癌细胞系MGC803和SGC7901细胞的增殖抑制率;以透射电镜观察两种胃癌细胞的直接损伤改变;以流式细胞仪及瑞士姬姆萨染色光镜观察两种胃癌细胞周期的改变;以SP免疫组化及RTPCR方法检测两种胃癌细胞的p21WAF1和p16INK4基因及蛋白表达的变化。结果大蒜素可抑制MGC803细胞生长,24h药物半数抑制浓度(IC50)为6.4μg/ml;也可抑制SGC7901细胞的生长,24hIC50为7.3μg/ml。12μg/ml的大蒜素作用两种胃癌细胞24h后,细胞出现急性直接损伤,膜系统改变明显。以3μg/ml、6μg/ml、9μg/ml终浓度大蒜素分别作用于两种胃癌细胞系24h后,与对照组比较,G0和G1期细胞周期百分比(%)明显减少,而G2和M期明显增加(P<0.01);6μg/ml大蒜素作用培养两种胃癌细胞24h后,与对照组比较,细胞分裂指数明显增加,提示两种细胞系经大蒜素处理后,细胞周期阻滞于M期。经大蒜素处理后,MGC803细胞的p21WAF1和p16INK4基因及蛋白表达均明显增加;SGC7901细胞的p21WAF1基因及蛋白表达明显增加。结论大蒜素可使MGC803及SGC7901两种细胞周期阻滞于M期;大蒜素的M期阻滞作用可能与其上调细胞的p21WAF1和p16INK4基因表达有关。     

Knockdown of USP39 induces cell cycle arrest and apoptosis in melanoma

The spliceosome machinery composed of multimeric protein complexes guides precursor messenger RNAs (mRNAs) (pre-mRNAs) splicing in eukaryotic cells. Spliceosome components have been shown to be downregulated in cancer and could be a promising molecular target for anticancer therapy. The ubiquitin-specific protease 39 (USP39) is essential for pre-mRNA splicing, and upregulated USP39 expression is noted in a variety of cancers. However, the role of USP39 in the development and progression of melanoma remains unclear. In the present study, USP39 expression was found to be increased in melanoma tissues compared with that in nevus tissues. USP39 silencing via lentivirus-mediated short hairpin RNA (shRNA) significantly suppressed melanoma cell proliferation, induced G0/G1 cell cycle phase arrest, and increased apoptosis in vitro. Moreover, USP39 knockdown suppressed melanoma tumor growth in a xenograft model. In addition, USP39 silencing was associated with the increased expressions of p21, p27, and Bax. Furthermore, the inhibition of USP39 expression decreased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, indicating that ERK signaling pathways might be involved in the regulation of melanoma cell proliferation by USP39. Our findings suggest that USP39 may play crucial roles in the development and pathogenesis of melanoma, and it may serve as a potential therapeutic target for melanoma.

     

Cell growth inhibition, G2M cell cycle arrest and apoptosis induced by the imidazoacridinone C1311 in human tumour cell lines

The cytotoxic activity of the imidazoacridinone C1311 was assessed on two ovarian cancer cell lines (A2780, OAW42) and one osteogenic sarcoma cell line (U2-OS) and their sublines (A2780Cp8, OAW42-MER and U2-OS-R) with experimentally induced resistance to cisplatin. A 1-h exposure to C1311 significantly inhibited the growth of all cell lines, with IC50 values ranging from 0.50 +/-0.11 to 4.10+/-0.36 microM. No or only partial cross-resistance was found between C1311 and cisplatin in the different cell lines. Treatment with equitoxic (IC50) C1311 concentrations consistently induced accumulation of cells in the G2M phase. The cyclin B1-associated p34(cdc2) kinase activity in cells arrested in G2M was superimposable to that of control cells in the OAW42-MER and U2-OS cell lines, whereas a reduction of cdc2 catalytic activity was observed in OAW42 and U2-OS-R cells. Exposure to C1311 (IC50) induced apoptosis in the U2-OS and U2-OS-R cell lines, whereas in the OAW42 and OAW42-MER cell lines there was a negligible percentage of apoptotic cells. In U2-OS, U2-OS-R and OAW42 cells, C1311 induced an increase in p53 expression and an increase in p21waf1 protein, whereas p53 failed to transactivate p21waf1 in OAW42-MER cells. An almost complete abrogation of bcl-2 was observed in U2-OS-R cells in correspondence with the peak of apoptosis induction. Our results indicate that C1311 is active against human ovarian cancer and osteogenic sarcoma cells and is not cross-resistant with CDDP. Moreover, C1311 blocks cells in the G2M phase and induces apoptosis in a small percentage of osteogenic sarcoma cells.     

Guanine nucleotide depletion triggers cell cycle arrest and apoptosis in human neuroblastoma cell lines

Mycophenolic acid (MPA) specifically inhibits inosine-5'-monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester pro-drug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1-10 microM) caused a decrease of intracellular levels of guanine nucleotides, a G(1) arrest and a time- and dose-dependent death by apoptosis. These effects, associated with an up-regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down-regulation of bcl-2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G(1) arrest and apoptosis through p53-mediated pathways, indicating a potential role of its morpholinoethyl ester pro-drug in the management of patients with neuroectodermal tumors.     

三氧化二砷对人肺癌细胞株增殖和凋亡的影响

目的 探讨三氧化二砷（Ａｓ２Ｏ３）对人肺癌的潜在性治疗作用及可能的机制。方法 选用人肺癌细胞株ＧＬＣ－８３,运用细胞培养法、ＭＴＴ法、流式细胞术（ＦＣＭ）检测细胞生长曲线、细胞增殖、细胞周期和细胞凋亡。结果 三氧化二砷可明显抑制ＧＬＣ－８２细胞的增殖,其抑制作用具有时－效和量－效关系。当４．０μｍｏｌ／Ｌ三氧化二砷处理ＧＬＣ－８２细胞９６小时,增殖抑制率达８１．０５％,ＦＣＭ检测肿瘤细胞ＤＮＡ含量,观察到三氧化二砷ＧＬＣ－８２细胞周期阻滞于Ｇ２／Ｍ期,细胞周期进程变慢,同时出现剂量依赖性Ｇ１峰。结论 三氧化二砷能有效地抑制人肺癌细胞株ＧＬＣ－８２的增殖,其可能的机制与三氧化二砷使细胞阻滞于Ｇ２／Ｍ期并诱导其调亡有关。     

Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation

The incidence of cutaneous malignant melanoma is undergoing a dramatic increase in persons with light-color skin in all parts of the world. The prognosis for individuals with advanced disease is dismal due to the lack of effective treatment options. Thus, there is a need for new approaches to control tumor progression. Epidemiological, experimental, and mechanistic data implicate omega-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chain omega-3 PUFAs as inhibitors of development and progression of a range of human cancers, including melanoma. The aim of this study was to assess the mechanisms by which docosahexaenoic acid (DHA), an omega-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposed in vitro to DHA and then assessed for (a) inhibition of cell growth; (b) expression of cyclins and cyclin-dependent kinase inhibitors in individual cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to cyclin D1, cyclin E, p21WAF1/CIP1, or p27(KIP1); and (c) expression of total pRb(T) independent of phosphorylation state and hypophosphorylated pRb(P-) in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb(T) or pRb(P-), respectively. After treatment with increasing concentrations of DHA, cell growth in a majority of melanoma cell lines (7 of 12) was inhibited, whereas in 5 of 12 cell lines, cell growth was minimally affected. Two melanoma cell lines were examined in detail, one resistant (SK-Mel-29) and one sensitive (SK-Mel-110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 microg/ml DHA whether grown in the absence or presence of 1% fetal bovine serum (FBS). No appreciable change was observed in cell growth, cell cycle distribution, the status of pRb phosphorylation, cyclin D1 expression, or the levels of the cyclin-dependent kinase inhibitors p21 and p27. In contrast, SK-Mel-110 cell growth was inhibited by DHA with the cells accumulating either in G1 or S phase: 0% in SK-Mel-29 versus 13.3 or 41.2% in SK-Mel-110 in the absence or presence of FBS, respectively. In the absence of serum, considerable death occurred by apoptosis. In addition, DHA treatment resulted in increasing numbers of SK-Mel-110 cells (from 12 to >40%) expressing hypophosphorylated pRb, whereas the levels of cyclin D1 and p21 changed little. Expression of p27 in these cells increased >2.5 times when grown in the absence of FBS but not in the presence of 1% FBS. Thus, we show for the first time that DHA inhibits the growth of cultured metastatic melanoma cells. Furthermore, growth inhibition correlates with a quantitative increase in hypophosphorylated pRb in the representative sensitive melanoma cell line SK-Mel-110. Although multiple factors influence pRb phosphorylation, it appears that both cyclin D1 and p21 expression do not change in the presence of DHA, although p27 was strikingly increased in SK-Mel-110 cells in the absence of FBS. The fact that pRb became hypophosphorylated after exposure to DHA suggests a cross-talk mechanism between fatty acid metabolism and the pRb pathway. Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents.     

Exogenous morphine inhibits human gastric cancer MGC- 803 cell growth by cell cycle arrest and apoptosis induction

Morphine is not only an analgesic treating pain for patients with cancer but also a potential anticancer drug inhibiting tumor growth and proliferation. To gain better insight into the involvement of morphine in the biological characteristics of gastric cancer, we investigated effects on progression of gastric carcinoma cells and the expression of some apoptosis-related genes including caspase-9, caspase-3, survivin and NF-κB using the MGC-803 human gastric cancer cell line. The viability of cells was assessed by MTT assay, proliferation by colony formation assay, cell cycle progression and apoptosis by flow cytometry and ultrastructural alteration by transmission electron microscopy. The influences of morphine on caspase-9, caspase-3, survivin and NF-κB were evaluated by semi-quantitative RT-PCR and Western blot. Our data showed that morphine could significantly inhibit cell growth and proliferation and cause cell cycle arrest in the G2/M phase. MGC-803 cells which were incubated with morphine also had a higher apoptotic rate than control cells. Morphine also led to morphological changes of gastric cancer cells. The mechanism of morphine inhibiting gastric cancer progression in vitro might be associated with activation of caspase-9 and caspase-3 and inhibition of survivin and NF-κB.     

HIV-1Vpr体外诱导肿瘤细胞凋亡与细胞周期阻滞的研究

目的 研究HIV-1 Vpr诱导Hela、Lovo、HepG2细胞凋亡和G2期阻滞效果.方法 构建含有HIV-1 Vpr基因的pcDNA4-Vpr重组载体,体外转染Hela、Lovo、HepG2细胞,同时设pcDNA4-EGFP质粒转染对照组、FUGENE组和空白对照组.转染后24、48、72 h,通过甲臜化合物法检测...     

Radiation-induced cell cycle delays and p53 status of early passage melanoma cell lines

Cell cultures exposed to DNA-damaging agents such as gamma radiation respond by arresting at cell cycle checkpoints, and the p53 tumor suppressor protein is strongly implicated in this behavior. We have investigated the TP53 status and cell cycle response to ionizing radiation of a series of early passage cell lines (designated NZM1 to NZM15) previously developed from patients with metastatic melanoma. The TP53 status of each of the cell lines was determined by single-strand conformation polymorphism and DNA sequence analysis. The majority of the lines appeared to have a wild-type TP53 gene sequence, consistent with published studies. Two lines (NZM4 and NZM7.2) were found to have an identical T-->C transition mutation in nucleotide 721 (exon 7) of the coding region. NZM7.2 (mutant) and NZM7.4 (wild-type) were clonally derived from the same line (NZM7). The existence of radiation-induced cell cycle arrest in G and/or G2M phase was determined 16 h after irradiation (6.3 Gy) by DNA staining and flow cytometric analysis. The mitotic inhibitor paclitaxel was used as a reference compound, with or without irradiation, to assess the efficiency of radiation-induced cell cycle arrest. G1 phase arrest was associated only with the presence of the wild-type TP53 gene, but the efficiency of induced arrest varied among the cell lines and the period of G phase arrest appeared to be short. A significant difference (P < 0.002) was also found between the efficiency of induction of G2 phase arrest and the presence of wild-type TP53 gene. The results provide evidence that although the melanoma cell lines generally had an intact TP53 gene, the efficiency of p53-mediated cycle arrest might be deficient and contribute to the resistance of this tumor to treatment.     

Derivate isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis

Tumor Biology - We have previously demonstrated that isocorydine (ICD) can be served as a potential antitumor agent in hepatocellular carcinoma (HCC). A novel derivate of isocorydine (d-ICD) could...     

Involvement of p53, nuclear factor kappaB and Fas/Fas ligand in induction of apoptosis and cell cycle arrest by saikosaponin d in human hepatoma cell lines

In this study, we report the proapoptotic effect of saikosaponin d in two liver cancer cell lines, Hep G2 and Hep 3B cells. Treatment with saikosaponin d decreased the cell proliferation of Hep G2 and Hep 3B cells in a dose dependent manner. In Hep G2, saikosaponin d blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up-regulating p21/WAF1 expression. In addition, an enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by saikosaponin d. Furthermore, saikosaponin d also inhibited the cell survival signaling by enhancing the amount of IkappaBalpha in cytoplasm and reducing the level and activity of NF-kappaB in the nucleus, and subsequently attenuated the expression of Bcl-XL in Hep G2 and Hep 3B cells. Saikosaponin d therefore decreased the cell proliferation and inducted apoptosis both in p53-positive Hep G2 and p53-negative Hep 3B cells.