Marked Inhibition of Cellular Proliferation in the Normal Human Esophageal Epithelial Cells and Human Esophageal Squamous Cancer Cells in Culture by Carotenoids: Role for Prevention and Early Treatment of Esophageal Cancer

Background: Globally Esophageal cancer is a common cancer arising from human esophageal mucosal tissue.Epidemiological studies suggest inverse correlation between carotenoid intake and incident risk of this devastatingmalignancy. Methods: In an effort to examine the modulatory role of carotenoids in human esophageal carcinogenesisat a cellular level, we examined the effects of α-carotene and β-carotenes, on cell proliferation and DNA synthesis inhuman esophageal epithelial (HEE) cells and human esophageal squamous cancer (HESC) cells in in-vitro cultures.HEE and HESC cells were incubated with different concentrations of α- and β-carotenes both individually and incombination. Results: Both Carotenes significantly inhibited (p<0.05) cellular proliferation and decreased DNAsynthesis in HEE and HESC cells. The effect of α- and β-carotene together on DNA synthesis in HEE and HESC cellswas significantly greater than either carotenoid alone, suggesting a synergistic effect. Greater magnitude of cellularinhibition of DNA synthesis was observed on HEE cells than HESC cells. Conclusion: Our results suggest that acombination of α-and β-carotene may provide a novel strategy for prevention and treatment of esophageal and upperaero digestive tract cancer in humans.     

三氧化二砷抑制恶性黑色素瘤B_(16)细胞生长及对端粒酶活性的影响

背景与目的:研究三氧化二砷(As2O3)对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的影响,探讨砷及其化合物治疗恶性黑色素瘤的作用机制,为临床治疗恶性黑色素瘤提供新的理论和实验依据。材料与方法:四甲基噻唑氮蓝(Methylthiazolyltetrazolium,MTT)比色法检测As2O3对B16细胞的生长抑制作用:端粒重序列扩增酶联免吸附实验(Telomericrepeatamplificationprotocolenzyme_linkedimmunosorbantassay,TRAR_ELISA)和聚内烯酰胺凝胶电泳银染(Telomericrepeatamplificationprotocol,polyacrylamidegelelectrophoresissilver_staining,TRAP_PAGE)法检测B16细胞的端粒酶活性。结果:As2O3可显著抑制B16细胞的生长并可下调端粒酶活性,其抑制作用有显著的时间和剂量依赖关系。结论:As2O3对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的抑制作用随药物浓度的增加和作用时间的延长而增强。     

尼莫地平对体外培养脑胶质瘤细胞增殖及DNA合成的影响

本文采用集落形成实验,细胞生长曲线和[~3H」-TdR掺入实验的方法观察了尼莫地平的体外抗肿瘤作用。结果表明,尼莫地平对恶性星形细胞瘤体外细胞系TJ_(861)细胞的集落形成率、细胞生长率及DNA合成均有较明显的抑制作用。     

Melanoma risk in relation to height, weight, and exercise (United States)

Height and weight and derivations thereof are positively associated with a number of cancers. While several authors have reported an increased risk of melanoma among people at the higher extremes of these measures, the association has not been fully explored. New cases of primary cutaneous melanoma in 1997 in western Washington State (n = 386) were compared to controls selected by random-digit dialing (n = 727). Each study participant completed a telephone survey, and data were collected on height, weight, sun-related melanoma risk factors, demographic characteristics, as well as habits such as diet and exercise. Risk of melanoma was analyzed by logistic regression with adjustment for age, hair color, lifetime sun exposure, and fruit and vegetable intake. An excess risk of melanoma was identified in men in the upper quartiles of height (OR = 2.4, 95% confidence interval (CI) = 1.3–4.5), weight (OR = 2.8, CI = 1.5–5.2), and body surface area (OR = 2.8, CI = 1.5–5.1) vs. the lowest quartiles. In women, no association was present for any anthropometric measure. In addition, we found that men and women exercising five to seven days per week were at a decreased risk of melanoma (OR = 0.7, CI = 0.5–1.0). The anthropometric findings are largely consistent with previous studies, while this is the first report of an association of exercise with melanoma risk. The mechanisms for the effect of exercise and for the difference between men and women in the effect of anthropometric factors are unknown. Future research in basic and epidemiologic science should focus on biochemical or behavioral explanations for these observations.     

Schedule dependent tumour growth delay,DNA cross-linking and pharmacokinetic parameters in target tissues with cis-diamminedichloroplatinum(II) and etanidazole with or without hyperthermia or radiation

It has been reported previously that striking increases in tumour growth delay and cytotoxicity are seen when cis-diamminedichloroplatinurn(II) (CDDP) is combined with mild local hyperthermia (43°C, 30 min) and/or etanidazole (ETA). This paper reports a study of CDDP pharmacology and the in vivo tumour DNA cross-linking produced by these combinations. In C3H mice bearing the FSaIIC murine fibrosarcoma, Pt plasma pharmacokinetics were not significantly altered by any of the combination of treatments. Although ETA caused no significant change in CDDP tissue pharmacokinetics, treatment of the tumour-bearing limb with hyperthermia immediately following an i.p. injection of CDDP (10 mg/kg) resulted in an increased peak Pt concentration (3.5 versus 2.8 μg Pt/g tumour wet weight) and doubled the t_1/2 elimination of Pt (15 to 30 h) from the tumour. Similar heat-induced changes were observed in the Pt pharmacokinetics in skin. There was about a three-fold increase in the Pt area under the curve (AUC) for the tumour, a 1.5-fold increase in the AUC for skin and little change in the AUC for muscle with hyperthermia. When the tumour DNA cross-linking factor (CLF) was determined, it was found that local hyperthermia treatment (43°C, 30 min) increased the CLF of CDDP from 1.7 to 2.7 and hyperthermia (43°C, 1 h) further increased the CLF to 6.1. Misonidazole (MISO) (1 g/kg) increased the CDDP CLF to 2.0, 6.3 and 15.1 in conjunction with 37, 43 (30 min) and 43°C (1 h), respectively. ETA (1 g/kg) was more effective than MISO at increasing the CDDP CLF, producing CLFs of 2.8, 9.1 and 21.5 at 37, 43 (30 min) and 43°C (1 h), respectively. These changes in CLF were reflected in an increased tumour growth delay in the FSaIIC murine fibrosarcoma with CDDP (5 mg/kg) alone from 4.4 to 5.9 days with 43°C (30 min) and then to 11.9 days with ETA (1 g/kg) and 20.9 days with both ETA and local hyperthermia (43°C, 30 min). When CDDP, ETA and hyperthermia were added to a radiation schedule of 300 cGy daily for five days, it was found that giving ETA (1 g/kg), CDDP (5 mg/kg) and hyperthermia (43°C, 30 min) together on day 1 produced the largest tumour growth delay (43 days) and that other schedules which divided the dose of ETA over the other days of the radiation treatment (including one schedule with a second heat treatment on day 4) were significantly inferior. Thus, local hyperthermia increased tumour versus normal tissue exposure to Pt, probably by altering the vascular physiology of the tumour, and both hyperthermia and ETA markedly increased CDDP-induced DNA damage, leading to commensurate increases in tumour growth delay. These results suggest that the addition of both ETA and hyperthermia to CDDP should be effective clinically, and they indicate that treatment once a week with hyperthermia, CDDP and ETA at the maximum tolerated dose will be the most successful.     

人食管鳞癌EC9706细胞线粒体DNA中ND1基因突变的检测及意义

目的通过对人食管鳞癌EC9706细胞线粒体DNA（mtDNA）中ND1基因进行检测,分析其基因突变的意义。方法培养人食管鳞癌EC9706细胞,提取其mtDNA中的ND1基因,并测序。结果测序发现,人食管鳞癌EC9706细胞mtDNA中ND1基因的3971处出现点突变,由C突变为T。结论人食管鳞癌EC9706细胞中mtDNA的ND1基因的突变与食管癌发生、发展关系密切。     

Expression of Different Extracellular Matrix Components in Human Brain Tumor and Melanoma Cells in Respect to Variant Culture Conditions

Local tumor invasion into the surrounding brain tissue is a major characteristic of malignant gliomas. These processes critically depend on the interaction of tumor cells with various extracellular matrix (ECM) components. Because only little quantitative information about expression of ECM gene products in general and expression in response to alterations of the surrounding environment is available, the present study was designed. Four human glioblastoma cell lines (U373MG, U138MG, U251MG, GaMG) as well as four human melanoma cell lines (MV3, BLM, 530, IF6) were tested with semiquantitative RT-PCR for their ability to express mRNA of different human ECM components (fibronectin, decorin, tenascin, collagen I, collagen IV, versican). In addition, two human medulloblastoma (MHH-Med 1, MHH-Med 4) and two fibrosarcoma (HT1080, U2OS) cell lines were analyzed. Cells which were grown in DMEM medium containing 10% FCS expressed most of the analyzed protein components. When the same medium, but depleted of ECM proteins by filtrating through a membrane with cut-off at >100 kD was used, basal mRNA expression of the ECM proteins was changed in most of the examined cell lines. Using serum free conditions, most of the cell lines again showed a variation in the expression pattern of mRNA encoding for the different ECM proteins compared to the other medium conditions. Comparing different cell lines from one tumor entity or different tumor groups, ECM expression was heterogeneous with regard to the different tumor entities as well as within the entities themselves. Migration assays revealed heterogeneous responses between the different cell lines, ECM components and culture conditions, making it difficult to correlate ECM expression patterns and migratory behavior.Our results revealed that all examined cell lines are able to produce ECM proteins in vitro. This suggests that tumor cells can modulate their microenvironment in vitro which has to be taken into consideration for studies related to migration and invasion.     

Antitumor Activity and Cellular Accumulation of a New Platinum Complex, (—)-(R)-2-Aminomethylpyrrolidine(1,l-cyclobutanedicarboxylato)platinum(II) Monohydrate, in Cisplatin-sensitive and -resistant Murine P388 Leukemia Cells

We have examined the cytotoxicity and accumulation of (—)-(U)-2-aminomethylpyrrolidine(1,l-cyclobutanedicarboxylato)platinum(II) monohydrate (DWA2114R) in parent and cisplatin-resistant mouse P388 leukemia cells (P388 and P388/DDP), in comparison with those of cisplatin (CDDP) and carboplatin (CBDCA). The degrees of resistance to CDDP and CBDCA, expressed as the ratio of IC₅₀for P388/DDP celts to IC₅₀ for P388 cells, were 75–33 and 100-27, respectively, under the conditions of 2–24 h exposure to each drug at a density of 10⁶ cells/ml. The corresponding values (25–7) for DWA2114R were relatively low. Accumulations of CDDP and CBDCA were reduced in P388/DDP cells; however, no reduction in accumulation of DWA2114R was observed at various exposure periods and concentrations of the drugs. The accumulations of CDDP in P388 and P388/DDP cells at drug concentrations corresponding to the IC₅₀ values for drug exposure periods of 2–24 h were 0.41–0.97 and 13.1–33.7 ng Pt/10⁷ cells, respectively, suggesting that an intracellular mechanism of resistance against CDDP could be activated in P388/DDP cells. P388/DDP cells also showed relatively low resistance to DWA2114R via this mechanism in comparison with CDDP and CBDCA. From the relationship between structure and activity of several Pt-complexes, these different properties of DWA2114R compared with CDDP and CBDCA could be due not only to the differences in carrier ligand structure but also to the properties of the whole molecule associated with the carrier ligand and leaving group.     

Augmentation of the Susceptibility of Human Leukemia to Lymphokine-Activated Killer (LAK) Cells by Exposure of the Leukemic Target Cells to Cytotoxic Drugs in Vitro and in Vivo

Experimental studies have shown that interleukin-2-induced lymphokine-activated killer (LAK) cells are able to lyse fresh noncultured leukemia cells and that human leukemia cells have a distinct susceptibility to LAK-cell-mediated cytolysis. Cytolysis is considerably lower with fresh noncultured leukemia cells than with the leukemia cell lines K562 and Daudi. For therapeutic considerations it would be desirable to achieve as much cytolysis as possible. The current study revealed that incubating leukemia cells with cytotoxic drugs in vitro significantly augments their susceptibility to the lytic effect of LAK cells and, more importantly that exposing leukemia cells to anticancer agents in vivo during induction chemotherapy also increases their sensitivity to LAK-cell-mediated cytolysis. These results support a possible benefit from combining chemotherapy with immunotherapeutic approaches in leukemia treatment.     

Relationship between Cell-killing Efficiency and Number of Platinum Atoms Binding to DNA, RNA, and Protein Molecules in HeLa Cells Treated with cis-Diamine(glycolato)platinum(II)

HeLa S-3 cells were treated with ^195mPt-radiolabeled cis -diamine(glycolato)platinum(II) (254-S) under various conditions, and the relationship between the lethal effect and the numbers of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentrations for the cells treated with 254-S at 37°C for 0.5, 1, 2, and 3 h were 67.1, 47.0, 26.8 and 8.1 μ M ₁, respectively. Using identically treated cells, we determined the numbers of Pt atoms combined with DNA, RNA, and protein molecules after fractionation of the cells. In this way, the D₀ values (D₀, the dose that causes an average of one lethal event per member of the population), expressed as the drug concentration, could he related to the number of Pt atoms combined with each fraction. The efficiency of the Pt atom in killing the cells, expressed as the reciprocal of the D₀ values, was then calculated for each fraction. The results suggested that DNA was the primary target for cell killing by 254-S. The target volumes for DNA were 3.96, 4.97, and 11.77 × 10⁴ nucleotides for 1-, 2-, and 3-h treated cells, respectively. In terms of the target volume, the cell-killing effects of 254-S were comparable to those of cis -diamine-dichloroplatinum(II) (CDDP), for which the target volumes under identical conditions were determined to be 5.17, 5.71, and 10.3 × 10⁴ nucleotides, respectively, while in terms of the mean lethal dose (D₀), the cell-killing effects of 254-S were lower than those of CDDP by a factor of 5.1 (47.0/9.3), 4.0 (26.8/6.7), or 2.5 (8.1/3.2) for 1-, 2-, or 3-h treatment, respectively.     

Cell-killing Efficiency and Number of Platinum Atoms Binding to DNA, RNA and Protein Molecules of HeLa Cells Treated with Combinations of trans-Diammedichloroplatinum(II) and Hyperthermia

The effect of hyperthermia on the cell-killing efficiency of Pt atoms binding to DNA, RNA and protein molecules was examined. HeLa S-3 cells were treated with ^195mPt-radiolabeled trans -diaminedichloro-platinum(II) (TDDP) for 60 min at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentration (D₀) of TDDP for a 60-min treatment at 0, 25, 37, 40, 42 and 44°C was 1714,1016, 302, 179,125 and 42.5 μ M , respectively. (D₀ is defined as the dose that would give an average of one lethal event per member of the population; for further details, see Fig. 1). By using identically treated cells, the numbers of Pt-atoms combined with DNA, RNA and protein molecules were determined in the subcellular fractions. Thus, the D₀'s given as the drug concentrations were replaced with the number of Pt-atoms combined in each fraction. Then, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt-atoms combined and was calculated for each molecule. The efficiency for DNA was 0.206, 0.273, 0.779,1.28,1.77 and 5.14×10³ nucleotides, respectively, for the conditions described above. It seemed that hyperthermia potentially interacted not only with bifunctional, but also with monofunctional bonds. Thus, it was concluded that TDDP was markedly less cytotoxic than cis -diaminedichloroplatinum (CDDP) at 37°C, but was more cytotoxic than CDDP at 44°C.     

Inhibition of Nucleotide Excision Repair by Fludarabine in Normal Lymphocytes in vitro, Measured by the Alkaline Single Cell Gel Electrophoresis (Comet) Assay

Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-ara-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2–h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 μ M. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.     

Sequence-dependent Termination of Mammalian DNA Polymerase Reaction by a New Platinum Compound, (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) Monohydrate

We examined the mechanisms of the inhibition of DNA synthesis by a new platinum compound, (-)-( R )-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis- diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase α were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase β was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.     

In vitro Antitumor Activity, Intracellular Accumulation, and DNA Adduct Formation of cis-[((1R, 2R)-1,2-Cyclohexanediamine-N, N')bis(myristato)] Platinum (II) Suspended in Lipiodol

SM-11355, cis -[((1 R ,2 R )-1,2-cyclohexanediamine- N , N' )bis(myristato)] platinum (II), is a lipophilic platinum complex under clinical development that targets primary hepatocellular carcinoma using Lipiodol as a carrier. SM-11355 was compared with cisplatin (CDDP) using an in vitro evaluation system capable of examining the release characteristics and the cytotoxicity of drugs suspended in Lipiodol. SM-11355 suspended in Lipiodol (SM-11355/Lipiodol) and CDDP suspended in Lipiodol (CDDP/Lipiodol) showed cytotoxic activity against rat ascites hepatoma AH-109A cells in a dosedependent manner. Their IC₅₀ values following 7-day exposure were 22.3 and 0.40 μg/ml, respectively. Following the subsequent 7-day exposure, from day 7 to day 14 after preparation of the suspension, SM-11355/Lipiodol showed an almost equivalent activity, but CDDP/Lipiodol did not show any activity at all. SM-11355/Lipiodol showed a sustained release into the culture medium over the course of a 14-day exposure. Following the exposure to CDDP/Lipiodol, the platinum concentration in the medium was at its maximum on the first day and remained constant thereafter. Intracellular platinum uptake and formation of platinum-DNA adducts were dependent on the release characteristics of each drug suspension. For SM-11355/Lipiodol, the drug release, intracellular drug uptake, and formation of platinum-DNA adducts over the course of the subsequent 7-day exposure were similar to those observed during the first 7 days. DPC, one of the compounds released from SM-11355/Lipiodol, was taken up by cells and showed formation of platinum-DNA adducts. Thus, this study suggests that SM-11355/Lipiodol may release active platinum compound(s) that bind to nuclear DNA and mediate the cytotoxic activity of SM-11355/Lipiodol.     

Effect of a Copper (II) Complex on The Induction of Apoptosis in Human Hepatocellular Carcinoma Cells

Objectives: In the present study, we aimed to identify the anti-proliferative potential of [Cu(L)(2imi)] complex[L = 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole] against HepG2 cells as anin vitro model of human hepatocellular carcinoma and normal mouse fibroblast L929 cells. Methods: The cytotoxicand apoptotic effects of [Cu(L)(2imi)] complex on HepG2 cells and normal fibroblasts (L929) were examined by MTTassay and flow cytometry, respectively. Results: Cytotoxicity induced by [Cu(L)(2imi)] complex was time dependent.Also, there was a positive correlation between cytotoxicity and an increase in Cu complex concentration. For HepG2cells, the cell viability percentage was 50% at 58 μg/mL after 24 h treatment, whereas in the same concentration andconditions, the viability percentage was surprisingly higher (about 100%) for L929 cells. Also, after 48 h treatment,the viability percentage of HepG2 cells at 55 μg/mL concentration was 50% in contrast with 89.3% for L929 cells inthe same conditions. Flow cytometry findings suggest that [Cu(L)(2imi)] complex is capable of decreasing cancer cellviability through apoptosis and did not efficiently activate the necrosis process. Conclusions: Finally, we found that[Cu(L)(2imi)] complex possess the potential for development as an anti-cancer drug for human hepatocellular carcinoma.     

Expression of DNA Polymerase α and Leu3a Molecules in Growing and Saturated Cultures of Human Leukemic Cells: Phenotype Analysis of Proliferative Cells by Flow Cytometry

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase α (pol α), was selected as a marker for proliferative cells, and Leu3a molecule as a cell-surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein-conjugated anti-pol α antibody, developed by us, and commercially available phycoerythrin-conjugated anti-Leu3a antibody. The optimal fixative for both proteins was phosphate-buffered 2% paraformaldehyde. The pol α-positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol α-linked fluorescence was observed. On the other hand, the pol α-positive population in the saturated culture was ca. 70%, with weaker pol α-linked fluorescence. Thus, the population of pol α-positive cells and the amount of pol α in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a-positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate-conjugated anti-pol α antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non-proliferative cells became easier by simultaneous labeling with antibodies against pol α and phenotype-speciflc proteins.