Cell cycle disturbances and apoptosis induced by topotecan and gemcitabine on human lung cancer cell lines

Apoptosis is a major mode of cell death in response to cytotoxic drug treatment. A correlation between induction of apoptosis and chemosensitivity has been documented in some preclinical models. Topotecan (a topoisomerase I inhibitor) and gemcitabine (a deoxycytidine analogue), two active new drugs for the treatment of lung cancer, were evaluated for their growth inhibitory effect on human lung cancer cell lines and their effect on cell cycle perturbation, apoptosis and apoptosis-related genes. The cytotoxicities of topotecan and gemcitabine on the human lung cancer cell lines H460 (wild-type-p53) and H322 (mutant p53) were determined after 72 h drug exposure employing the MTT assay. The apoptotic index (AI) was assessed by three methods: analysis of morphological changes using May–Grünwald–Giemsa (MGG) staining, the TUNEL assay and FACS analysis. Cell cycle disturbances were studied by FACS and the number of cells expressing p53 and p21 were determined by immunohistochemistry. Both gemcitabine and topotecan had potent growth inhibitory effects in human lung cancer cell lines; combination treatment with these two drugs showed some additivity but no synergism. Induction of apoptosis after treatment was concentration- and time-dependent with both drugs and ic₈₀ concentrations induced the highest values. The DNA histograms at 4, 24, 48 and 72 h indicate that topotecan at ic₈₀ concentrations causes accumulation of cells in S and G2/M phases, whereas gemcitabine at ic₈₀ concentrations causes, accumulation of cells in G1 phase. Both compounds induced p53 and p21 expression in the H460 cell line but not in the H322 cell line; the percentage of cells expressing p53 was highest at ic₈₀ values, whereas the highest percentage of p21 positive cells could be induced with ic₅₀ values. This could suggest that p53 induces cell cycle arrest at low drug concentrations, whereas p53 induces apoptosis at higher concentrations. In conclusion, p53-dependent and independent pathways of apoptosis exist in lung cancer cell lines. Activation of the p53 pathway depends on the induced cellular damage. Understanding the cell cycle disturbances induced by these drugs may help in the design of more rational treatment schedules.     

Apoptosis induced by etoposide in small-cell lung cancer cell lines

The DNA fragmentation, a parameter of apoptosis, in non-small (NSCLC) and small (SCLC) cell lung cancer cell lines (N231 and PC-9) was evaluated. The DNA fragmentation in SCLC lines, but not in NSCLC lines, was observed in overgrown cells without exposure to anticancer drugs. In etoposide (VP-16)-treated N231 but not PC-9 cells, DNA fragmentation continued to increase up to 42 h, and the increase was dependent on the concentration of VP-16. The endonuclease activity of VP-16-treated N231, but not PC-9, cells required both Ca²⁺ and Mg²⁺ for full activity. It was elevated in a time- and concentration-dependent manner. As this activity was not affected by addition of cycloheximide, the activation of the endonuclease activity without protein synthesis may be involved in VP-16-induced cytotoxicity in N231.This work was supported, in part, by grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, the Comprehensive Ten-year Strategy for Cancer Control and the Ministry of Education, Science and Culture, Support from the Bristol Myers Squibb Foundation is also appreciated     

X射线诱导人星形细胞瘤细胞系的凋亡

目的：为了观察辐射诱导胶质瘤细胞凋亡的特征，进一步探讨辐射诱导肿瘤细胞凋亡与辐射剂量、剂量率和照射方式之间的关系。方法：将ＳＷＯ细胞分为对照组、不同剂量组、不同剂量率组、单次照射组和分次照射组，以医用直线加速器Ｘ射线照射。采用光镜、电镜及ＤＮＡ琼脂糖凝胶电泳法观察Ｘ射线诱导星形细胞瘤细胞系凋亡的特征。结果：（１）低剂量、低剂理率和分次照射组（１０Ｇｙ）诱导出典型细胞凋亡的特殊（如凋亡小体，ＤＮＡ梯     

高能电磁脉冲诱导肺癌细胞株GLC-82凋亡的研究

背景与目的:电磁脉冲(electromagneticpulse,EMP)辐射已用于饮食行业的灭菌,且较2450MHz连续微波辐射消毒更加有效。本研究探讨高能电磁脉冲对肺癌细胞GLC-82凋亡的影响,以发现治疗肿瘤的新手段。方法:以场强为6×104V/m的EMP2min内辐照5次,然后采用细胞计数、MTT、流式细胞术及SP免疫组化法检测bcl-2和p53蛋白的表达,并对表达强度用CMIAS-Ⅱ图像分析仪在放大400倍条件下进行分析,通过上述方法观察EMP对肺癌细胞GLC-82的损伤作用,所有数据经SPSS8.0软件进行分析。结果:EMP可明显抑制肺癌细胞GLC-82的增殖与活力。照射后细胞的MTT光吸收值(A570)与对照组相比,在辐照后0h,1h和6h明显降低。流式细胞术证明,GLC-82细胞在辐照后6h发生明显的凋亡,凋亡率达13.38%。免疫组化的图像分析表明,照射后GLC-82细胞有不同程度的bcl-2蛋白表达的下调及p53蛋白表达的上调。结论:EMP可诱导肺癌细胞GLC-82的凋亡。bcl-2及p53蛋白参与了GLC-82细胞的凋亡过程。     

Apoptosis induced by novel aldehyde calpain inhibitors in human tumor cell lines

Calpain is a class of Ca(2+)-dependent cysteine proteases and has been suggested to be involved in several important signaling cascades. A series of novel aldehyde calpain inhibitors identified in our laboratory were more potent and specific than commercially available calpain inhibitors, and were used to assess the involvement of calpain in cancer. Our inhibitors demonstrated potent anti-proliferative activity in four cancer cell lines (PC-3, HeLa, Jurkat and Daudi) with IC(50)'s ranging from 2 to >30 microM. A non-cancer cell line (CV-1) was 4-7-fold less sensitive than the cancer cell lines. Apoptotic activity was determined and appeared to be inversely correlated to calpain expression levels in the different cell types. Leukemia cell lines (i.e., Daudi and Jurkat) with undetectable m-calpain were more susceptible to the apoptotic effects in response to calpain inhibition, while apoptosis was not detected in PC-3 prostate cancer cells, which highly express m-calpain. The extent of apoptosis in HeLa cells was moderate under identical conditions. Apoptosis induced by calpain inhibition was accompanied by caspase-3 activation. Furthermore, cell cycle analysis showed that aldehyde calpain inhibitors arrested cells at the G2/M boundary in a concentration-dependent manner. These results indicate that aldehyde calpain inhibitors exhibit their cytotoxic effects via induction of G2/M arrest and apoptosis. Importantly, the compounds failed to exert any inhibitory effects toward 20S proteasome. Collectively, our results suggest that calpain is a novel target for the treatment of a variety of cancer diseases and provide leads for further discovery and development of calpain inhibitors.     

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。     

Monoclonal antibodies against two human lung carcinoma cell lines

Monoclonal antibodies against 2 human lung carcinoma cell lines (E14 and BEN) were prepared by production and cloning of somatic cell hybrids between the murine myeloma NS1, and spleens from E14- and BEN-immune BALB/c mice. Approximately 2000 hybrid culture supernatants were screened for antibody simultaneously against the immunizing cell line and lung fibroblasts (573 Lu) using a radiolabelled Protein A binding assay. Although the vast majority secreted antibodies which recognized species-specific antigens, a few supernatants showed marked differential reactivity against E14 or BEN. These were cloned and subsequently tested against a panel of up to 25 human cell lines originating from different neoplastic and non-neoplastic tissues. Two anti-E14 clones (3E19.8 and 4EAB3.7) displayed preferential activity against lung cancer cell lines, but a low level of reactivity was also detectable with cell lines of different tissue provenance. The antibodies of 3 anti-BEN clones (7B3.5, 7B5.4, 7B17.7) likewise recognized antigens present to a higher density on lung cancer cell lines but were also reactive (to a variable extent for the different clones) with a diversity of other tumour cell lines. The antibodies of 2 further clones were exceptional in so far as one (7BC9.1) reacted only with BEN and WIDR (colorectal cancer) cells, while another (7B24.4) reacted, with apparent exclusivity, against BEN cells. With the exception of the latter, the distinction in antigen expression between many of the cell lines was quantitative rather than qualitative and the emergent picture is one of random expression of individual determinants on several disparate types of cancer cells, rather than restriction to cells of a given morphological type or histogenic derivation.     

川芎嗪对人小细胞肺癌H446细胞的增殖抑制作用

目的　观察中药川芎的提取物川芎嗪对人小细胞肺癌H4 4 6细胞的增殖抑制作用。方法　用MTT法、吖啶橙 /溴乙啶 (AO/EB)双荧光染色法、流式细胞术及扫描电镜观察川芎嗪对体外培养的人小细胞肺癌H4 4 6细胞存活率的影响、检测凋亡、分析其对细胞形态学及细胞周期的影响。结果　川芎嗪对小细胞肺癌H4 4 6细胞有增殖抑制作用 ,细胞形态学发生变化 ,如细胞体积缩小、细胞质空泡化、细胞核碎裂 ;川芎嗪可诱导H4 4 6细胞凋亡并使细胞生长停滞在S期 ,抑制细胞有丝分裂及DNA合成。结论川芎嗪对小细胞肺癌H4 4 6细胞具有增殖抑制作用 ,呈浓度相关性 ,川芎嗪可诱导H4 4 6细胞凋亡 ,其机制可能与其导致细胞S期阻滞有关。     

Selenium modulation of cell proliferation and cell cycle biomarkers in human prostate carcinoma cell lines

Prostate cancer (PCA) is the most common histological malignancy and the second leading cause of cancer deaths among North American men. There has been considerable interest in the chemopreventative properties of selenium. In this study, we assessed whether selenium inhibits cell growth and associated cell cycle regulatory proteins. Human PCA cells (LNCaP, PC3, PC3-AR2, and PC3-M) were incubated with and without selenium (Seleno-DL-methionine, 150 microM) for 24, 48, and 72 h. Cells were fixed and stained with propidium iodide for flow cytometry analysis. In parallel experiments, total protein was extracted, immunoprecipitated with cyclin E antibody, and analyzed by Western blot for the expression of cell cycle markers. Treatment with selenium caused G1 arrest and an 80% reduction in the S phase of LNCaP with no effect on PC3. However, PC3 cells transfected with the androgen receptor (PC3-AR2) exhibited a G2/M arrest and a marked reduction (57%) in the S phase during cell cycle progression. In the analysis of cell cycle regulatory molecules, selenium-treated cells demonstrated a significant induction of cyclin-dependent kinase inhibitors Cip1/p21 and Kip1/p27. These data suggest that selenium possesses strong antiproliferative properties in regard to human PCA. This effect appears to be dependent on the presence of a functioning androgen receptor. This provides a theoretical basis for Phase III studies of selenium in PCA prevention.     

Nuclear matrix protein composition of human lung carcinoma cell lines

The nuclear matrix is the RNA-protein skeleton within the nucleus that contributes to the structural and functional organization of DNA. Differences in the nuclear matrix protein composition between cancer and normal cells have been reported in various cell lines and tissues, suggesting altered gene expression. This study examined the nuclear matrix protein composition of various human lung cell lines. Using high resolution two-dimensional electrophoresis, at least ten common proteins, as well as specific differences, were identified in each category of lung cell lines. These protein differences may be responsible, at least in part, for the different phenotypes of human lung cancer.     

氨氯吡咪诱导卵巢癌细胞凋亡

目的 观察利尿药氨氯吡咪诱导卵巢癌细胞凋亡及其可能的机理。方法 用光镜及荧光显微镜从形态上了解凋亡的发生,用琼旨糖凝胶电泳进一步检测发生凋亡的特征性ＤＮＡ降解。流式细胞术定量分析用不同剂量氨氯吡咪处理的卵巢癌细胞诱导的凋亡发生率。同时检测被处理细胞细胞内ｐＨ。结果 氨氯吡咪处理的卵巢癌细胞在形态学上显示出凋亡细胞的特征,琼脂糖凝胶电泳显示特征性ＤＮＡ阶梯图。流式细胞术检测表明,０．０１￣５μｍｏｌ     

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression

Human carcinomas were shown to express mRNA and protein for IL-2R alpha, beta and gamma chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rbeta- and gamma-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rbeta and gamma proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5-10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT-PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.     

Antitumor and immunomodulatory effect of coumarin and 7-hydroxycoumarin against Sarcoma 180 in mice

The antitumor effect of peroral treatment with coumarin and its main metabolite in humans 7-hydroxycoumarin (7-OHC) against Sarcoma 180 in mice was studied. Both agents inhibited tumor growth and increased survival time of tumor-bearing animals. The antitumor effect was better when coumarins were administered prior to tumor inoculation suggesting that the immunomodulatory potential of coumarins might exceed their well-known direct cytostatic activity. The additive effect of coumarins in combination with a suboptimal LPS dose in tumor growth inhibition was demonstrated. Coumarin treatment enhanced the macrophage migration activity in the presence and absence of LPS and increased nitric oxide release. In vitro, coumarins induced IL-12 in murine macrophages and additively increased the LPS-induced IL-12 release. These data indicate that the immunomodulatory activity of coumarins contributes to their direct cytostatic effect and demonstrate their potential to combine as immunostimulators with other antitumor agents.     

NS-398, a selective cyclooxygenase 2 inhibitor,inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines

Cyclooxygenase 2 (COX-2) has been suggested to be associated with liver carcinogenesis. Several reports have shown that NSAIDs inhibit the growth of hepatocellular carcinoma cell lines. There is little evidence of how COX-2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth-inhibitory mechanism of a selective COX-2 inhibitor, NS-398, in 4 hepatocellular carcinoma cell lines by studying cell growth, COX-2 and proliferating cell nuclear antigen (PCNA) expression, cell cycle distribution and the evidence of apoptosis. NS-398 inhibited the growth of all 4 cell lines in a time- and dose-dependent manner and the inhibitory effects were independent of the level of COX-2 protein expression. PCNA expression was downregulated by NS-398 in a dose-independent manner. NS-398 caused cell cycle arrest in the S phase with a reduction in cell numbers and cell accumulation in the G0/G1 phase, for all 4 cell lines. No evidence of apoptosis was observed in our present study. Our findings suggest that a selective COX-2 inhibitor might serve as an effective tool for the chemoprevention and treatment of hepatocellular carcinomas. A reduction in cell number in the S phase may be an important event in cell cycle arrest caused by NS-398 in hepatocellular carcinoma cell lines.     

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。     

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine?;) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1?h, 42°C). In IGROV-1 cells, a low concentration (15?µg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27^Kip1 and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

三氧化二砷诱导的结肠癌细胞凋亡及其与细胞周期的关系

目的：探讨三氧化二砷（Ａｓ２Ｏ３）对结肠癌细胞的凋亡诱导作用及其与细胞周期的关系。方法：采用透射电镜、丫啶橙荧光染色,ＴｄＴ及流式细胞仪检测药物作用前后细胞在形态学及细胞周期２等方面的变化。结果：形态学观察发现Ａｓ２Ｏ３诱导的ＬｏＶｏ细胞死亡呈现凋亡特征,研究表明随经物作用时间延长,凋亡细胞比例逐渐增加。Ａｓ２Ｏ３在低浓度时主要干扰细胞在Ｓ期的通过,高浓度时则选择性诱导Ｓ期细胞凋亡。结论：Ａｓ２ｏ     

In vivo and in vitro invasiveness of human lung carcinoma cell lines.

Eight cell lines derived from human non-small cell lung carcinomas were used to compare their in vivo invasiveness, in vitro chemoinvasive abilities and type IV collagenase activity. For the evaluation of the in vivo invasive potential, the tumor cells were seeded into deepithelialized rat tracheas and transplanted subcutaneously into nude mice. The invasive behavior of the cells was observed at 4, 8 and 12 weeks and assessed histologically by determination of the levels of penetration of tumor cells into the different layers of the tracheal wall. Except for two cell lines that did not grow at all in vivo, there was a very good correspondence between the levels of in vivo tracheal wall penetration and the in vitro chemoinvasion assay using fibronectin as chemoattractant and Matrigel as barrier. This also correlated very well with the capacity of the cells to secrete type IV collagenase. The in vivo evaluation of invasion using tracheal transplants, although requiring several weeks of experimentation, proved to be very reliable, yielding homogeneous results with little internal variation, and is proposed as a dependable in vivo invasion assay that closely mimics the in vivo human conditions in which most carcinomas develop and eventually invade neighboring tissues.     

Characterization of DNA damage and cytotoxicity induced in two human colon carcinoma cell lines by cyclodisone

Cyclodisone is an active alkylating antitumor agent that is being considered for Phase 1 clinical trials in humans and is currently undergoing toxicological evaluation. Cyclodisone was found to be more toxic to human colon carcinoma cells of the Mer- phenotype (BE) than cells of the Mer+ phenotype (HT-29). DNA interstrand cross-links were observed in the sensitive cell line but only at concentrations which were extremely toxic. No DNA interstrand cross-links were observed in the resistant cell line. Total DNA cross-links, which reflect both DNA interstrand and DNA-protein cross-linking, were observed in either cell type but were greater in quantity and persisted longer in the sensitive BE cell line, when compared to those produced in the resistant HT-29 cell line. DNA strand breaks were also observed in both cell types and were found to be protein associated. The mechanism of action of cyclodisone would appear to be related to the presence of total DNA cross-links and might involve an, as yet, unidentified DNA-protein interaction.