On Antitumor Activity And Mechanism Of EPSs Produced By Five Species Of Algae From Desert Soil Crusts

Aim: Algae from desert environment are known to excrete amount of extracellular polymeric substances (EPSs) into the surrounding matrix when they grow. These EPSs are life support system, play important roles in protecting them from drought, oxidation, salt and radiation. And they are also key substance of gluing soil grains together during the formation process of algal crusts. The object of this study is to further investigate their function in antitumor and possible mechanism. Methods: Microcoleus vaginatus, Phormidium tenue, Scytonema javanicum, Desmoloccus olivaceus and Nostoc sp. are dominant species of algal crusts of Shapotou area, Ningxia Hui Autonomous Region of China. They have different cohesion in stabilization fine sand grains. Their EPSs were extracted, separated and purified. The skin cancer cell A431 was bought from Cell Collection Centre of Wuhan University. After culture and exposure to these EPSs, survival rate of A432 cell proliferation was measured by MTT assay. The cell cycle and membrane potential of mitochondrion (△Ψm) was determined by flow cytometry. The changes of cell morpha and ultrastructure were observed under inverted light microscope and transmission electron microscope. DNA laddering of apoptosis was performed by agarose gel electrophoresis. The expression of caspase-3, bcl-2 and bax were analyzed by immunohistochemistry, and antioxidation system including content of free radicals (O2), enzyme activity of SOD, CAT and POD were measured as well. Results: The EPSs from the five species all could kill A431 cancer cell, and at the dose-time-dependent pattern. Incubation of A431 cell with EPSs caused a G2/M stagnation in cell cycle progression. Membrane potential of mitochondria began fall after exposure to EPSs 24 h, whole mitochondria collapsed after 72h, broken after 96h. And all EPSs facilitated the expression of caspase-3 and bax protein, restrained the expression of bcl-2. So the result of immunohistochemistry further confirmed the apotpsis by EPSs induced. Agarose gel electrophoresis further showed DNA degrade into small fragments as laddering after 96 h. Light microscope showed the apoptosis process was cell diminish in size or becoming round or crescent shape first, then cell-cell juncture disappeared. TEM showed villus of cell surface disappeared first, cell membrane blebbing , and then nucleous aggregated, chromatin condensetion, andedged. Finally karyotheca broke, the aggregated chromatin spread into cytoplasm. During the whole process there were not apoptosis body formation. This apoptosis process was similar to most tumor cells induced by other anti-tumor agents. The content of free radicals increased after exposure to EPSs, and antioxidant enzyme activities also showed obviously changes. Immunohistochemistry assay showed that EPSs treatment up-regulated the expression of caspase-3 and bax protein, down-regulated that of bcl-2 protein. Conclusion: The EPSs had obvious antitumor activity, the stagnation of cell cycle and decrease of membrane potential of mitochondria might be the main reason of tumor cell apoptosis. The apoptosis process was closely related to content of free radicals, because the free radicals participated in the up-regulation of caspase-3 and bax and down-regulation of bcl-2. So the mechanism of apoptosis of cancer cell might be the reason was because of lost balance of antioxidant system.