Reasearch About Ofloxacin Rhodanine Derivatives Induces Apoptosis Of Human Hepatocarcinoma SMMC-7721 Cells

Since Quinolones has born in 1962, fluoroquinolone antibacterial has been widely used by clinical application. Quinoline carboxylic acid structure is the skeleton of quinolones. It acts through the mechanisms of inhibiting bacterial DNA gyrase activity, and eukaryotes topoisomerase Ⅱ and DNA gyrase have highly homology. At the same time, fluoroquinolone carboxylic acid have a certain effect of promoting apoptosis on the tumor cells. Related studies have shown that the C-3 carboxyl of fluoroquinolone is an effective site of fluoroquinolones to be modified, so we piece it together with different active pharmacophores, This research group has already acquired several antitumor compounds with Strong activity, Such as cinnamic aldehyde ofloxacin acyl hydrazone, the methoxy cinnamaldehyde levofloxacin acyl hydrazone, benzaldehyde levofloxacin schiff base, etc. They can obviously inhibit the proliferation of tumor cells as well as induce the apoptosis of liver cancer cells by mitochondria or（and）receptor pathway. And each of their 24 hours’ IC₅₀ value concentration is under 10 μmol/L. Application of pBR322 plasmid DNA electrophoresis experiment has proved that cinnamic aldehyde ofloxacin acyl hydrazone derivative compounds can lead to DNA damaging, as the DNA topoisomerase II agents. Because of the large molecular weight, low solubility, bad cells absorbing and the other reasons of the synthetic compounds,which needs Further improvement,without going on further application development.Furthermore, as the structure five yuan heterocyclic ketone, Rhodanine is similar to flavonoids. If increasing some kinds of side chains to the substituent of rhodanine, we can find its solubility increases.Thus Rhodanine is often used to the synthesis of new drugs. There were not any reports of piecing Rhodanine and fluoroquinolone together up to now at home or abroad. Then we designed and synthesized a series of fluoroquinolone-rhodanine new compounds with advantages of good solubility and smaller molecular weight, prospecting to find clinical candidate compounds with significant antitumor activity.Objective Through the screening of dozens of new synthesized fluoroquinolone-rhodanine derivatives, to find and select the compounds which has the most significant proliferation inhibition on the human liver cancer cells SMMC-7721. Preliminary study it’s effect of apoptosis induction, and to provideexperimental foundation and theoretical basis for developing and utilizing such compounds.Methods MTT Method to detect fluoroquinolone derivatives’ and other drugs’ effects on cell proliferation; DAPI fluorescent staining method to detect the nucleus morphological changes of SMMC-7721 cell after disposed by the selected compounds. TUNEL method to detect the apoptosis rate;PI staining and Flow cytometry to detect changes of the cell cycle. Western Blot method to measure the expression quantity changes of apoptosis signaling pathways related protein as p53 and Caspase-3.Results In the prophase, after a large number of screening, we find out twelve kinds of fluoroquinolone derivatives that have obvious antitumor activities, among which R1, R3, R4 and R5 have more significantly inhibiting effects on cell proliferation, among which 6-（3-Benzyl-4-oxo-2-thioxo-thiazol idin-5-ylidenemethyl）-9-fluoro-3-methyl-10-（4-methyl-piperazin-1-yl）-2,3-dihydro-7-oxo-7-hydro-pyrido[1,2,3-de][1,4]benzoxazine, that is ofloxacin Rhodanine, R3, of which the inhibition of cell proliferation effects is the most pronounced. At the concentration range of 2-20 mmol/L, it can significantly inhibit the proliferation of SMMC-7721 cells, CaCO-2 cells and EC-9706 cells. 24 hours’ IC₅₀ value concentration of SMMC-7721 cells reached 3.893 mmol/L（r²= 0.8304）, with 48 hours’ of 3.849 mmol/L（r²= 0.7706）and 72hours’ of 2.892 mmol/L（r²= 0.7558）. And 24 hours’ IC₅₀ value concentration of EC-9706 cells and Ca CO-2cells respectively is 4.181 mmol/L（r²= 0.8833）, 3.408 mmol/L（r²= 0.9601）. As a reference model, The 24hours’ IC₅₀ value concentration of Sunitinib acts on SMMC-7721 cells is 8.075 mmol/L（r²= 0.9845）. The 24hours’ IC₅₀ value concentration of ofloxacin acts on SMMC-7721 cells is 240.137 mmol/L（r²= 0.8005）.The 24 hours’ IC₅₀ value concentration of R3 acts on L-02 cells is 33.959 mmol/L（r²= 0.9309）. DAPI staining to observe the nuclear shape changes, results show that, after R3 affecting on SMMC-7721 cells,SMMC-7721 cells showed the nucleus pycnosis, fractures, condensed chromatins, marginalized chromatins and changes of apoptosis bodies, ect, which is the morphological characteristics of apoptosis. TUNEL shows that, the apoptosis rate of each R3 treatment group of the consentration separately of 2.688mmmol/L, 3.893 mmmol/L and 5.639 mmmol/L is（34.44±4.53）%,（48.40±7.52）% and（62.27±6.86）%. The apoptosis rate increases significantly compared with the control group. Cell cycle detection results show that, The research has groups of the control group and the treatment group with the concentration of R3 respectively of 2.688 m mmol/L, 3.893 m mmol/L and 5.639 m mmol/L. Their G₀/G₁ phase cell ratios is respectively of（36.08±1.82）%,（54.24±9.08）%,（62.84±4.74）% and（69.78±4.60）%; and Their S phase cellratio is respectively of（28.50±3.33）%,（17.31±6.66）%,（30.65±4.71）% and（24.79±4.03）%; and Their G₂/M phase cell ratio is respectively of（35.41±3.62）%,（28.45±3.30）%,（6.53±0.54）% and（5.34±1.01）%; and Their Sub-G₁ phase cell ratio is respectively of（2.44±0.44）%,（11.31±4.08）%,（18.90±2.37）% and（31.15±4.86）%. The changes of G₀/G₁ phase, G₂/M phase and Sub-G₁ phase compared with the control group with significant difference, as changes of S phase compared with the control group with no significant difference. It shows that after the SMMC-7721 cells proposed by R3, the cell cycle is blocked at G₁-S phase testing point. Western Blot results show that, After the disposing of R3, p53 protein expression increased, so as the protein expression of Caspase-3 and it’s active pyrolysis fragments.Conclusions （1） As the modified fluoroquinolone derivative, R3 can significantly inhibit the proliferation of the tumor cells mainly by blocking the G₁-S phage and has fair effects about better selectively inhibiting the tumor cells.（2） R3 obviously induces the apoptosis of the human liver cancer cells SMMC-7721.