Research On Antitumor Mechanisms Of Extracelluar Polymeric Substances From Desert Soil Algae

Some research results have indicated that the extracelluar polymeric substances (EPSs)play very important roles not only in adapting to the stress of radiation,high temperature and aridity and also in the form of the soil biological crust in arid and semarid area.There is,however,little report on the biomedical activity of the EPSs. Our experiments revealed the antitumor activity of EPSs by assaying the cell viability and the morphological changes and the DNA damage in the human epidermoid carcinoma human A431 cells.At the same time,we investigated the antitumor mechanism in A431 cells after the treatment of EPSs.The oxidant system of A431 cells was disordered after the treatment of the EPSs after various concentrations and times,and the different expression of the apoptotic genes was detected by western blotting.The main content and results are as followings.(1)The cell viability was detected by MTT assay and the median inhibitory concentration was 2.75mg/mL,5.32mg/mL,4.68mg/mL,9.44mg/mL and 4.25mg/mL after the treatment of EPSs from M.vaginatus,Nostoc sp.,D. olvaceus P.tenue,and S.javanicum for 96 hours respectively.Although there are very important inhibitory effect was found after treated by the five species of EPSs,it is exhibited significant difference among them because of their various composes.(2)The morphological changes were observed through HE staining,AO/EB staining and Giemsa staining.As the results in other research about apoptosis, the typical apoptotic characteristics such as reduction of cellular volume, condensation of chromatin,fragmentation of the nucleus,plasma membrane blebbing,and maintenance of an intact plasma membrane until the very late stage of the process was detected.Our results suggest that EPSs could induce apoptosis in A431 cells and have antitumor activity.(3)The DNA damage was detected by comet assay.After the treatment of 6mg/mL EPSs,DNA in A431 cells was serious damaged and cometic tail appear in the treated groups.The percentage of cometic cells and length of cometic tail were significant difference compared with control group (p＜0.05),especially EPS-M and EPS-S,the ratio amount to 77.8%and 76.2%respectively.(4)The SOD and GPX activity,the content of GSH which act as small molecular antioxidant and MDA are all changed after treated with various concentrations for different hours.This result indicated that the antioxidant system in A431 cells was disturbed by EPSs and this disturbance alleviated the ability of antioxidant and contributed to the early apoptosis phase.(5)The expression of Caspase-9 and Caspase-3 was detected by Western blotting. EPS-N,P and S can up-regulated the expression of Caspase-9 with the dose-dependent manners while EPS-M and EPS-D didn't similar to them. The latter cleaved the Pro-caspase-9 at the dose of 2mg/ml and the effect was decreased slighted when the dose of EPSs was increased.The expression of Caspase-3 show dose dependent manners after the treatment of EPS-M, EPS-N,EPS-D and EPS-P while the expression of Caspase-3 wasn't changed drastically treated with EPS-S for 72 hours.This result suggested that EPSs increased the expression of Caspase-9 and Caspase-3 and induced apoptosis in A431 cells by promoting the caspase-dependent path way.This effect may be the major cause of antitumor activity of EPSs.(6)Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Caspase-3,Bcl-2 and Bax.EPSs were able to induce the apoptosis in A431 cells.They can reduce the expression of apoptosis-regulated gene Bcl-2,and increase the expression of apoptosis-regulated gene Bax and Caspase-3.Among the EPSs from five desert soil algae,the EPS from M.vaginatus show the most significant antitumor activity.The positive ratio of the expression of Bcl-2 was decreased under the treatment of EPS-S from 72.6±6.45%to 6.5±1.67%, whereas the positive ratio of Bax was increased under the treatment of EPS-S from 11.27±1.48%to 86.5±13.05%and Caspase-3 was increased under the treatment of EPS-M from 8.2±0.91%to 81.2±5.16%.These results show significant difference comparing with control groups(p＜0.05).Our findings indicated that the mechanism of antitumor of EPSs may be through inducing the apoptosis in A431 cells.In the apoptosis,the mitochondrial path way was promoted.