| Introduction Among all kinds of tumors, 1-2% are bone tumor; the incidence is not high. However, since China has a large number of population, a huge number of patients are suffering from the disease of bone tumor. Meanwhile, other tumors and carcinoma with a high incidence like lung cancer, breast cancer, and prostatic cancer are more likely to have metastases to bone. Thus in total, the number of people having bone cancer is impressively large. At present, surgeries are no longer the only option to treat bone cancer. Doctors are developing the combining treatments to cure it, among which the development of new types of chemotherapeutic drugs brings the treatment to a new era. In 1960 s, Schiff base were found to suppress tumors and microbials, and have the function to catalyze. In recent years, researchers developed the compounds of Schiff base-based and platinum or cuprum, which have shown strong effect against tumor. In this study, a new type coordination compound of Schiff base Zinc(SBZCC) was synthesized, and its inhabiting effect on osteosarcoma or other common metastatic tumors to bone was tested, as well as associated mechanism.Objective To develop a new type of coordination compound of Schiff base zinc, and estimate its apoptosis effect on MG-63 human osteosarcoma cells and MCF-7 human breast cancer cells. To investigate the apoptosis mechanism and associated signal pathway.Methods(1) The syntehsis of SBZCC.(2) The cytotoxicity of the novel SBZCC was tested in MG-63 and MCF-7 cells. We treated MG-63 and MCF-7 cells with 50 μ mol/L cisplatin or different concentrations of SBZCC for variuos durations and assessed cell viability with CCK-8 assay. In order to determine whether SBZCC induces cell apoptosis, we treated MG-63 and MCF-7 cells with 10, 50, or 70 μ mol/L SBZCC for 4, 24 and 48 h, and determined cell apoptosis by flow cytometry. Tumor cell morphology was observed by inverted microscope, and photos were taken.(3) The chimeric reaction between the new SBZCC and DNA was determined through indensity analysis of DNA after the anarose gel electrophoresis. Flow cytometer was also used for detecting the change of mitochondrial membrane potential. Western Blot was introduced to detect the expression change of apoptosis related proteins Bax, Bcl-2, caspase-9, Cyt-c, Fas, Fas-L, caspase-8 and caspase-3. The effect of the new SBZCC inducing the apoptosis to MG-63 and MCF-7 cells was quantified through time and doses. The anti-tumor activity of SBZCC was also tested in mice, by examining the murine weight and the size of the tumor. Quantitative realtime-PCR was then introduced to examine the expression of apoptosis-associated genes in the tumor in the mice.Results(1) Using salicylaldehyde, salicylic hydrazide, zinc acetate as the raw meterials, the SBZCC was prepared through by water bath heating reflux and precipitation.(2) The CCK-8 assay indicated that, increased concentration and prolonged treating time was correlated with the enhanced suppressive effects of SBZCC against the proliferation of MG-63 and MCF-7 cells. In MG-63 cells, the most significant effect(51.37% suppression) of SBZCC was oserved at 70 μmol/L for 48 h. Similar suppression(60.87%) was observed in MCF-7 under the same condition. No >50% suppression was detected under other conditions for both cells. Flow cytometry based on Annexin V-FITC showed that the new SBZCC, with doses of 10 μmol/L, 50 μmol/L, 70 μmol/L and treating time of 4, 24, 48 h, significantly(P<0.05) induced apoptosis in MG-63. Apparently, time dependency was also applied for SBZCC inducing apoptosis in MG-63. The highest apoptosis rate(29.79±1.36%, P<0.05) in MG-63 was observed at 70 μmol/L for 48 h. Again, similar effect(30.29±1.77%, P<0.05) was observed in MCF-7 cells under the same condistion. It was determined that the SBZCC in both cells blocked the cell cycle from G1 to S or G2; meanwhile, it also reduced the amount of cells at S phage. Both in turn inhibited the proliferation and the growth of tumor cells. Morphology of apoptosis among SBZCC-treated cells was also confirmed through normal microscopy.(3) The Western Blot results suggested that, given same treatment time, the ratio of Bax/Bcl-2 in MG-63 cells was correlated with the concentration of SBZCC. The decrease of Cyt-C in motichodria and its increase in cytoplasm was also detected in a SBZCC concentration-dependent manner. Clear bands of cleaved caspase-3 and 9 were observed. Given the same dose of SBZCC, the ratio of Bax/Bcl-2 in MG-63 cells was correlated with prolonged treatment. The decrease of Cyt-C in motichodria and its increase in cytoplasm was also detected in a time-dependent manner. Clear bands of cleaved caspase-3 and 9 were also observed. Companied with the increase of SBZCC concentration, the gray ratio of Fas and Fas-L was increased from 0.48±0.06 to 1.83±0.90, and from 0.29±0.02 to 0.85±0.07, respectively. This was time dependent. Clear bands of cleaved caspase-3 and 8 were observed. Similar results were also observed in MCF-7 cells. The decrease of Cyt-C in motichodria and its increase in cytoplasm was also detected in a SBZCC concentration-dependent manner. Clear bands of cleaved caspase-3 and 9 were observed. Given the same dose of SBZCC, the ratio of Bax/Bcl-2 in MG-63 cells was correlated with prolonged treatment. The decrease of Cyt-C in motichodria and its increase in cytoplasm was also detected in a time-dependent manner. Clear bands of cleaved caspase-3 and 9 were also observed. Companied with the increase of SBZCC concentration, the gray ratio of Fas and Fas-L was increased, which was time dependent. Clear bands of cleaved caspase-3 and 8 were observed.(4) The novel SBZCC treatment could significantly inhibit the growth of the MG-63 human osteosarcoma cells and MCF-7 human breast cancer cells in tumor-bearing nude mice without significant hematological toxicity as well as hepatotoxicity and nephrotoxicity. m RNA levels of Bax, Bcl-2, Caspase-8, Caspase-9, and Caspase-3 were correlated with the concentration of SBZCC.Conclusions(1) The new zinc complex of schiff base can induce the apoptosis in MG-63 bone sarcoma cells and MCF-7 human breast cancer cell;(2) The SBZCC induces apoptosis through mitochondrial pathway and death receptor pathway. |
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