As a trace element, selenium is essential for nutrition and exhibits a wide range of biological functions. It is also shown to have anticarcinogenic or preventive chemicals from inducing tumors. Selenium compounds have been reported to possess a wide range of biological and pharmacological activities including anti-inflammatory, antiviral, anti tumor and used to a lot of disease such as Keshan disease. These diverse pharmacological activities of selenium compounds have demonstrated that they have anti-tumor activity. It is commonly accepted that the mechanism is attributed partially to its strong antioxidative properties which may relate to apoptosis and selenium compounds served as one of the most potent preventive chemicals from inducing tumors could against the side effect of chemotherapeutic agents and enhance the activity of chemotherapeutic agents and diminish normal cells’ toxicity. Nevertheless, its clinical effectiveness is restricted due to the dose-limiting toxicity of normal and healthy cells action, studies have focused on searching for new selenium compound which possess higher anti-tumor activity and lower toxicity. Therefore, the purpose of this study was to investigate the anti-tumor activities of selenium compound in vitro as well as in vivo, and to explore the mechanism. The main experimental results were as follows:
1. Anti-proliferation activity of NaSeVO and SeMoV in vitro NaSeVO 0.625~20 mg/L and SeMoV 0.313~10 mg/L could significantly inhibite proliferation of K562 cells in vitro in a time and dose-dependent manner. The IC50 values were 14.41 (4.45-46.60) and 3.45 (2.29-5.22) mg/L after treated with NaSeVO 0.625~20 mg/L for 48 h and 72 h, respectively. The IC50 values were 14.41 (4.45~ 46.60) and 3.45 (2.29~5.22) mg/L after treated with SeMoV 0.313~10 mg/L for 48 h and 72 h, respectively. 2. Anti- tumor activity in vivo The anti-tumor effect of NaSeVO and SeMoV in vivo was evaluated by the inhibition rate of tumor mass. Results showed that i.p. NaSeVO 5 and 10 mg/kg had a significant anti-tumor effect on the growth of S180 with inhibition rate 26.8 % and 58.4 %, and 31.3 % and 47.4% on H22, respectively. Inhibition rate of SeMoV was 31.3 %、47.4% and 38.19%、39.17%, respectively. 3. Effect of NaSeVO and SeMoV on cell cycle distribution of K562 Result showed that the proportion of G0/G1 phase was increased at low concentration, whereas the proportion of G2/M was decreased. the proportion of G0/G1 phase was decreased and the proportion of G2/M was increased at high concentration after treatment with NaSeVO 0.625~20 mg/L for 24, 48 h. It also caused cells gathered in S phase. There are significantly sub-G occurred at high concentration, sub-G% were 3.3 and 3.5, respectively. The proportion of G0/G1 phase was increased and S was decreased at low concentration after treatment with SeMoV 0.313~10 mg/L for 24 h. the proportion of; whereas the proportion of S increased and G0/G1 was decreased at high concentration.
After treatment for 48h, the proportion of S increased and G0/G1 was decreased at high concentration. There are significantly sub-G occurred at 5mg/L（24h） , 1.25, 2.5 mg/L（48h）4. Morphological features of apoptosis Typical apoptosis character was present in the K562 cells treated with NaSeVO and SeMoV for 24h. Nuclear condensation, chromosome fragmentation and apoptosis bodies were observed by inverted microscope. The electron microscopic observation also revealed typical apoptotic features, including shrinkage of cellular and nuclear membranes, condensed heterochromatin around the nuclear periphery, and cytoplasmic vacuolation in the K562 cells treated with NaSeVO and SeMoV for 24 h. 5. Eexpression of Bax and Bcl-2 Buffy precipitate appearanced in endochylema or cell membrane was regarded as positive expression. The study of immunocytochemistry shows that the expression of bcl-2 is significantly inhibited by NaSeVO 10 mg/L and SeMoV 5 mg/L and bax increased. 6. Effect of NaSeVO and SeMoV on intracellular Ca2+, Mg2+ and ROS concentration, pH value and MMP The fluorescence intensity of intracellular Ca2+ and Mg2+ was greatly increased after treatment with NaSeVO and SeMoV as compared with control group. Similar to the change of intracellular Ca2+ and Mg2+, fluorescence intensity of intracellular ROS also increased. However, treatment with NaSeVO and SeMoV markedly lowered the fluorescence intensity of intracellular pH value and mitochondrial membrane potential. 7. Effect of NaSeVO and SeMoV on content of cytochrome C, IκBαand NF-κB Results of western-blotting assay showed that the intracellular content of cytochrome C and IκBαmarkedly increased and NF-κB decreased when treatment with NaSeVO and SeMoV for 24h.
Taken together, the above results suggest:
1. NaSeVO and SeMoV 5， 10 mg/kg has anti-tumor activity in vivo. 2. NaSeVO and SeMoV could inhibit the proliferation of K562 in vitro and arrest cell cycle. 3. The occurrence of sub-G and change of K562 morphology indicated that the mechanism of NaSeVO and SeMoV is attributed partially to apoptosis induced through the elevation of intracellular Ca2+, and Mg2+ concentration, and the reduction of pH value and mitochondrial membrane potential 4. NaSeVO and SeMoV could inhance the expression of bax and inhibit the expression of bcl-2. They also increased intracellular content of cytochrome C and IκBαand decreased NF-κB . In summary, the above results suggest: NaSeVO and SeMoV have anti-tumor activity in vivo and in vitro, the mechanism of which may relat to apoptosis.