3-(Tetrazol-5-yl)-2-imino-coumarins Derivatives: Synthesis, Characterization, and Evaluation on Tumor Cell Lines

The first report of new 3-(tetrazol-5-yl)-2-iminocoumarin derivatives is described. The title compounds were prepared in two steps and were obtained in good yields (55-93%). They have been fully characterized by 1H, 13C NMR, FTIR, UV-Visible and HRMS. They were tested for their antiproliferative activities against six representative human tumor cell lines (Huh 7-D12, Caco2, MDA-MB231, HCT 116, PC3 and NCI-H727) and HaCat keratinocytes. Among them, compound 5e was active on HCT 116 (IC50 15 μM).

EFFECT OF ASCORBIC ACID ON DNA SYNTHESIS,INTRACELLULAR ACCUMULATION OF ADM AND ADM RESISTANCE OF TUMOR CELL LINES

Objective: To determine the effect of ascorbic acid (AA) on DNA synthesis, intracellular accumulation of ADM and ADM resistance of tumor cell lines. Methods: K562, K562/ADM and KB cell lines were used to study the effect of ascorbic acid on DNA synthesis, intracellular accumulation of ADM and ADM resistance by fluid scintillometry, MTT method, spectrofluorophotometry and immunocytochemistry. Results: Results showed that AA was capable of inhibiting DNA synthesis of K562 and K562/ADM in a dosedependence fashion, but not KB cell line, and significantly reducing ADM sensitivity in K562 and KB cell lines, as well as potentiating obviously ADM resistance in K562/ADM cell line. Conclusion: These effects of AA may be closely correlated with significant elevation of intracellular accumulation of ADM in KB cell line, and significant reduction of that in K562 and K562/ADM cell lines but possibly not correlated with the expression of Pglycoprotein.

TIME-AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

Effects of Low Concentrations of Di-(2-ethylhexyl) and Mono-(2-ethylhexyl) Phthalate on Steroidogenesis Pathways and Apoptosis in the Murine Leydig Tumor Cell Line MLTC-1

The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line （MLTC-1） in vitro. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure to DEHP. All three genes （P450scc, P450c17, and 38HSD） under study showed an increased expression following exposure to DEHP or MEHP, although some insignificant inhibitory effects appeared in the 10μmol/L treatment group as compared with the controls. It was also found that DEHP or MEHP stimulated INSL3 mRNA and protein especially in the 0.001 μmol/L treatment group. Testosterone secretions were stimulated after the exposure to DEHP or MEHP. Alterations of steroidogenic enzymes and INSL3 in MLTC-1 cells might be involved in the biphasic effects of DEHP/MEHP on androgen production.

RADIATION-INDUCED APOPTOSIS OF TWO NASOPHARANGEALCARCINOMA CELL LINES

Objective: To study apoptosis induced by radiation in two nasopharyngeal carcinoma (NPC) cell lines, CNE and CNE-2. Methods: Hoechst 33342 staining, immuno-histochemical staining, RT-PCR, DNA dot blotting and Southern blotting were used to identify apoptosis. Results: A single dose of X-irradiation resulted in apoptosis, the apoptotic index (AI) was time- and dose-dependent. Different apoptotic responses existed in the two cell lines. Immunohistochemical staining showed that bcl-2 protein was strongly positive in CNE but negative in CNE-2. However, RT-PCR revealed p53 mRNA in CNE-2 but not in CNE. P53 and bcl-2 genes were both present in the two cell lines as shown by DNA blotting, but the 2.8 kb fragment of the p53 gene was much lower than the 5.6 kb fragment on CNE which was clearly shown in Southern hybridization, suggestive of partial deletion of p53 gene in CNE. Conclusion: Apoptotic response to radiation is different in two NPC cell lines. CNE is more radioresistant than CNE-2. Overexpression of bcl-2 protein and partial deletion of p53 gene may explain their difference in radiosensitivity.

New 4-imino-4H-Chromeno[2,3-d]Pyrimidin-3(5H)-Amine: Synthesis, Cytotoxic Effects on Tumoral Cell Lines and in Silico ADMET Properties

The synthesis of new 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine in four steps including one step under microwave dielectric heating is reported. The structural identity of the synthesized compounds was established according to their spectroscopic analysis, such as FT-IR, NMR and mass spectroscopy. These new compounds were tested for their antiproliferative activities on seven representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3 and MCF7) and also on fibroblasts. Among them, only the compounds 6c showed micromolar cytotoxic activity on tumor cell lines (1.8 50 50 > 25 μM). Finally, in silico ADMET studies ware performed to investigate the possibility of using of the identified compound 6c as potential anti-tumor compound.

Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopusscaber, on lung adenocarcinoma (A549) cells

OBJECTIVE： Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma （A549） cells. METHODS： The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 50% inhibitory concentration （IC50） value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase- contrast microscopy. The induction of apoptosis was evaluated using acddine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling （TUNEL） assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly （ADP-ribose） polymerase was also analyzed. RESULTS： Deoxyelephantopin exhibited cytotoxicity to A549 cells （IC50 = 12.287 μg/mL）, however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways. CONCLUSION： These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

DEPLETION OF O^6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND POTENTIATION OF 1-(4-AMINO-2-METHYL-5- PYRIMIDINYL) METHYL-3 (2-CHLOROETHYL)-3-NITRO- SOUREA ANTITUMOR EFFECT BY STREPTOZOTOCIN

O6-methylguanine-DNA Methyltransferase (MGMT) can specifically repair the DNA demage Induced by chioroethylnitrosoureas (CENU) such at 1-(4-amino-2-methyt-pyrlmidinyl) methyl-3-(2-chloroethyl-)-3-nitrosourea (ACNU), constituting the molecular basis of tumor cell resistance to CENU. The present study demonstrated that sensitization of resistant tumor cells to ACNU could be achieved by streptozotocin (STZ) treatment which could deplete MGMT activity in vitro and in vivo. It suggested that depletion of the molecular basis of tumor cell resistance to chemotherapeutic agents might be a practicable way to improve the effectiveness of tumor chemotherapy.

Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

DNA double-strand break(DSB)is generally regarded as the most lethal of all DNA lesions after radiation.Ku80,DNA-PK catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)proteins are major DSB repair proteins.In this study,survival fraction at 2Gy(SF2)values of eight human tumor cell lines(including four human cervical carcinoma cell lines HeLa,SiHa,C33A,Caski,three human breast carcinoma cell lines MCF-7,MDA-MB-231,MDA-MB-453,and one human lung carcinoma cell line A549)were acquired by clone formation assay,and western blot was applied to detect the expressions of Ku80,DNA-PKcs and ATM protein.The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis.We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference.The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2(r=0.723,P=0.043),but Ku80 and ATM expression had no correlation with SF2(P>0.05).Thesefindings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.

Tumor blood vessels formation in osteosarcoma:vasculogenesis mimicry

BACKGROUND: Osteosarcoma is characterized by high neovascularization and a high propensity for metastasis through bloodstream. This study was to examine whether there is evidence for vasculogenic mimicry in osteosarcoma and to illustrate mechanism of tumor blood vessels formation in osteosarcoma. METHODS: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed with phase contrast microscope and transmission electron microscope (TEM). Morphometric studies using hematoxylin and eosin (HE) stain and CD31 immunohistochemical stain to show tumor-lined channels in human osteosarcoma were also performed. RESULTS: Observation with light microscope and TEM showed that highly aggressive osteosarcoma cell lines (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen, in the absence of endothelial cells or fibroblasts. Morphometric observation using HE stain and CD31 immunohistochemical stain showed that tumor cell-lined channels were also detected in vivo in osteosarcoma; by comparison, all vascular areas in the pedicle of osteochondroma or outside osteochondroma were endothelial-lined. CONCLUSION: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry.