Studies On Antibacterial Activities, And Antitumor Activities Of Pyrazolone Complexes, And Its Apoptotic Mechanism On Tumor Cells

Pyrazolone is a five membered lactum ring structure, containing two Nitrogens and one ketonic group. During the discovery of pyrazolone and its derivatives, they were only known as a kind of NSAIDs. But in recent time they played versatile role in several complications like cerebral ischaemia and cardiovascular diseases. The chemistry of pyrazolone has gained increasing attention due to its diverse pharmacological properties such as cytotoxic, anti-inflammatory, antimicrobial, antioxidant, antifungal, antiviral,oral hypoglycaemic activity, etc. Pyrazolones are believed to be involved in various biochemical and physiological reactions and thus the scientific research programs are continuously pouring in with respect to improvised synthetic techniques to prepare numerous pyrazolone derivatives. From a last decade a lot of work is going on the pyrazolone nucleus. Scientists have developed large number of new compounds related to this moiety and screened them for their different pharmacological activities to get a molecule of desired pharmacological activity. Their peer extensive and versatile profile have made the pyrazolones favorites for newer drug development. Keeping in view the increasing importance of these derivatives, a need to review the progress regarding pyrazolone was felt.4-acyl pyrazolone is a kind of excellent extractantcan for variety of metal ions as its wide biological activities, and usually play an important role in the development of science as new pesticides or anticancer drugs. This paper is studied on acyl pyrazolone and hydrazine or aldehyde hydrazone Schiff base metal complexes for the antitumor and antimicrobial function selection. Its interaction with DNA was studied and the relationship between the biological activity, and the in vivo antitumor tumor-burdened mice model was studied. The antitumor signaling pathway was studied and discussed. The research in this paper includes the following three parts:1. Studies on Antitumor Activities,Antibacterial Activities of the Pyrazolone Complexes and interactions with DNA Eleven kinds of pyrazolone complexes were screened for antitumor activity and antibiotic activity. Their antitumor activity in vitro were evaluated by examining cytotoxicity against human tumor cell lines. The results indicated that human esophageal cancer Eca-109cell lines and human cervical carcinoma HeLa cell lines were inhibited by five kinds of pyrazolone complexes. The complex Cd(Ⅱ)of N-(1-phenyl-3-methyl-4-propenylidene-5-pyrazolone)-salicylidene hydrazide (Cd-PMPP-SAL)showed better antiproliferative activity to human cancer cells. The antibiotic activity of the pyrazolone complexes were detected by cylinder-plate method. Five kinds of pyrazolone complexes have antibacterial activities for Staphylococcus aureus, Dist ribution, Klebsiella penumoniae and Shigella flexneri. The complex Cd-PMPP-SAL showed better antibacterial activities against Staphylococcus aureus with a MIC value of15μg·mL-1. In addition, the interaction relationship between the pyrazolone complexes and DNA were studied by UV-vis spectra and Fluorescence spectra. The results showed that interaction between the pyrazolone complex and DNA caused a hypochromic effect of the DNA ultraviolet absorption.2. Cd-PMPP-SAL induces apoptosis in human esophageal cancer cells Eca-109via the mitochondrial pathwayIn this study the Cd-PMPP-SAL complex was screened for its good antitumor activity. The human esophageal cancer cells Eca-109was screened for its sensitivity to the complexes. Here we investigated its tumor apoptosis effects and underlying mechanisms on human esophageal cancer cells Eca-109in vitro and the express level of related protein, the results showed that Cd-PMPP-SAL complex inhibited cell viability and induced cell apoptosis in Eca-109cell lines for its IC50value of3.5μg/mL in a dose-and time-dependent manner. The result of Hoechst33258fluorescence assay showed the apoptosis cells were deep dyed, The result of DNA ladder showed that DNA was gradually cut into small pieces, The result of LDH showed that the cells Apoptosis was more than cells necrotic. The detect of flow cytometry showed cell pathway is apoptosis. Cd-PMPP-SAL induced apoptosis by collapsing the mitochondrial membrane potential, inducing cytochrome c release from mitochondria, reducing the ratio of Bcl-2/Bax, increasing activation of caspase-3,-7,-9and enhancing levels of ROS. The apoptotic effects of Cd-PMPP-SAL on Eca-109cells were partially blocked by a caspase-3and caspase-9inhibitor. These findings indicate that Cd-PMPP-SAL can inhibit cell proliferation and induce apoptosis of Eca-109cells and may be a novel effective candidate as chemotherapeutic agent against human esophageal cancer.3. The antitumor effect and apoptotic mechanism of Cd-PMPP-SAL againist B16cells in vivo and in vitro.The anti-tumor apoptosis mechanism of Cd-PMPP-SAL to mice melanoma cells B16in vitro and in vivo, layed the groundwork for the drugs in the clinical study. The results shows that the cell proliferation of B16cells treated by Cd-PMPP-SAL was obvious. The inhibition IC50value concentration is4.15μg/mL; B16cells occurred early apoptosis induction after treated by Cd-PMPP-SAL; The apoptosis rates of B16cells was significantly greater than the necrosis rates; DNA Ladder agarose electrophoresis method was used to detect cell DNA fragment after treated with Cd-PMPP-SAL, the result showed that the nucleus DNA had been gradually cut into small pieces of180-200bp; Hoechst33258fluorescent assay was used to observe cells morphology changes, the experimental results showed the change of apoptotic cells and the visible of apoptotic body occurred; The caspase activity method was used to detect the cell caspase protein expression. The results showed that the B16cells apoptosis was induced by Cd-PMPP-SAL, the caspase family protein caspase-3,-7,-9was activated; The study of antitumor apoptosis mechanism of Cd-PMPP-SAL in vivo used C57mice B16cell transplantation tumor model, mice tumor suppression was made, the weight and the volume of tumor in mice was tested, the inhibition rate of tumor was calculated and mice spleen weight was measured, the results showed that Cd-PMPP-SAL inhibited the tumor volume growth in vivo, but the mice weight and spleen weight did not changed significantly. The result suggests that Cd-PMPP-SAL has no obvious side effects in vivo; The paraffin section and HE stain was made to measure the damage of mice tumor, liver, lungs; Using immunohistochemical assay to detect the expression level of the VEGF and FGF2protein. The results showed Cd-PMPP-SAL loosed structure between the tumor cells the cells angiogenesis atrophy in mice, but the liver and lung tissue had no obvious damage; The immunohistochemical of mice tumor biopsies detection results showed that Cd-PMPP-SAL decreased VEGF and FGF2protein expression in mice tumor. The result of TUNEL showed that there were less apoptotic cells in tumor tissues.