Inhibition Of H460 Cell Proliferation By 8-hydroxyquinoline And Its Analogs

Cu is an essential trace element in human and animal tissues. The copper ion is also an integral part of many metalloenzymes. It has been found the serum copper concentration correlates with tumor incidence and burden, malignant progression and recurrence in a variety of human cancer. Copper ions seem to play on important role in the stimulation of angiogenesis. Therefore, copper became one of the targets in antiangiogenic cancer treatment. In vitro and in vivo studies have confirmed the efficacy of copper-reducing and copper-chelating agents in antiangiogenic treatment. Based on the experimental data coming from in vitro and in vivo studies, it seems reasonable to use copper chelators or copper-reducing agents in antiangiogenic treatment alone or in combination with other therapeutic approaches like surgery or classic chemotherapy, such as tetrathiomolybdate (TM), Captoril,Zinc and Trientine . However, there are certain limitations of using anti-copper compounds in antitumour treatment. One of them is the long time of drug administration required to produce therapeutic effects. In addition, more work has to be done to explore the exact mechanisms of action of copper-reducing agents and even more important, to define the effects of a long-term copper deficiency.In recently a kind of organic-copper compounds, (8-hydroxyquinoline) Ox and (5-chloro-7-iodo-8-hydroxyquinoline) CQ were found to inhibit the chymotrypsin-like activity of the proteasome in vitro and in human tumor cell culture. Proteasome inhibition and apoptosis were also studied in copper-containing cancer breast cells treated with 8-hydroxyquinoline. CQ is capable of binding copper, forming new complexes that have proteasome-inhibitory to cancer breast cells. Dou concludes the complex between CQ and copper in tumor cellular is a novel proteasome inhibitor. CQ also has the effects on the viability of eight human cancer cell lines. Varia group analyzed the crystal structure of CQ-Cu (II) complex by the way of X ray diffractometry and proved the binding of CQ - Cu (II) is depended on the 8- hydroxyl and N of pyridine. Based on the research referred we presumed the 8- hydroxyl of Ox and CQ is bind to Cu (II) and has antitumor bioactivity to cancer cells. While 8- hydroxyl is substituted it is unable to bind Cu (II) and have no bioactivity. The content of this paper consists four parts. In the first part, the anti-proliferative effects of Ox and CQ on H460 cell line was studied in copper-enrichment or not. Especially the mechanism of anti-proliferative effects of Ox and CQ on H460 was discussed in this paper. In the second part, the 8- hydroxyl of Ox and CQ was substituted by alkane and the library of 8-OHQ analogs was synthesized by combinatory chemistry approach. The library of Ox analogs were screened for their antitumor activity on H460 cell line. In the third part, the mechanism of antitumor activity for Ox, CQ and Ox analogs were discussed. In the last part the feasibility of a self-calibrated LC/MS/UV method to determine the absolute amount of compounds in their storage and screening lifecycle was evaluated.The major research work and results of the paper are as follows:Part 1 the anti-proliferative effects on H460 cell line were compared under three treatments and UV-Vis titration was used to study the binding constant between Ox, CQ and Cu (II). The copper level of H460 per cell was determined under three treatments. The cytotoxic activity of Ox, CQ was investigated and the Ox was more potent than CQ on H460 cell line. Moreover the cytotoxic activity was more active for the ligands in copper-enrichment than without copper. The chelators were able to increase the level of copper of per cell when the cell line was cultured in copper-enrichment medium and the binding constants took great effect on the copper level of H460 cell line, which also influenced the cell viability. The binding constant of Ox-Cu²⁺ is larger than the binding constant of CQ-Cu²⁺ so Ox is easier to combine with Cu (II) than CQ, which raised the Cu level of cell line.Part 2, the Ox analogs were found inactive on H460 cell line when 8-hydroxyl was substituted by alkine. While six of 8-hydroxyquinoline derivatives and eleven of 8-hydroxyquinoline analogs were active on H460 cell line when mixed with Cu (II). By contrast to their IC50 values of six 8-hydroxyquinoline derivatives and eleven of 8-hydroxyquinoline analogs compounds 1#, B, C and 115 # were particularly potent on H460 cell line but were less than Ox and CQ. They are unable to combine with Cu (II) by UV-vis titration.Part 3 In contrast the anti-proliferative effects of Ox, CQ and Ox analogs in four different treatments are more potent to inhibit the cell viability when fully mixed with Cu more time and were determined higher level of Cu in per cell. It was found that the intracellular Cu level was connected with binding constants in four different treatments. In contrast the binding constant of Ox-Cu²⁺ is larger so higher level of Cu²⁺ were transferred into cells than CQ. However 115# was capable to transfer Cu²⁺ into the cells even though it didn't combine with Cu (II). In addition Ox, CQ and OX analogs at low concentrations in the presence of copper or not generates reactive oxygen species (ROS). The level of intracellular ROS was not the main mechanism of inhibition of H460 cells proliferation.Part 4 HPLC/MS/UV detector was applied to determine the self-calibrated quantification for the library of 50 pure compounds. There are several immediate applications of this method: 1) quantification of storage compounds at the time of screening to determine the real concentration for primary screening and IC50 measurements; 2) re-analyze compound collection by quantification of compound loss due to decomposition, freeze/thaw, and other effects during storage.Conclusions:1) Ox and CQ are potent to inhibit H460 cells proliferation. The anti-tumor proliferative effect is related with binding constant2) Ox analogs are inactive on H460 cell line but it is highly potent on H460 cell line in the presence of Cu (II).3) Ox, CQ and Ox analogs generates reactive oxygen species in the presence of Cu (II) nor not. The generation of ROS was not the main mechanism of inhibition of H460 cells proliferation.4) The self-calibrated LC/MS/UV method to determine the absolute amount of compounds in their storage and screening lifecycle is feasible but it has some limitation.