Effect Of The Polysaccharide From Pinus Massoniana Pollen And Its Sulfated Derivative On K562 Cell

The anticancer research of polysaccharides from Pinus massoniana pollen(PPM60) and its sulfated derivative(SPPM60)has indicated that, SPPM60 inhibited the growth of human HepG2 cells in vitro,prevent cell cycle, inhibit cell proliferation and growth;PPM60 had no inhibitory effect on HepG2 cells growth. This research aims to determine the effects of PPM60 and SPPM60 on the proliferation and differentiation of human K562 cells, and then to give some theoretical and experimental roof for the research of SPPM60 anticancer function.Firstly, 50 mg PPM60-A,PPM60-B,PPM60-C,PPM60-D were chemically modified by chlorosulfonic acid-pyridine method. 61.8 mg,79.0 mg,63.2 mg,71.9 mg SPPM60-A, SPPM60-B,SPPM60-C,SPPM60-D, modified compounds with 1.28,1.63,1.31,1.49 substituted degrees were obtained.Secondly,the activities of PPM60 and SPPM60 on K562 cells were studied by observing their effects on cell proliferation and cell cycle. MTT colourimetry demonstrated that PPM60 inhibited the proliferation of K562 was stronger than SPPM60. After 48-hour treatment with 400μg/ml, PPM60 inhibited the growth of K562 remarkably by 32%, while SPPM60 just had 16%. Wright stain and Ho.33342/PI double fluorescence labeled staining were used to observe cell morphology. PPM60 induced K562 underwent some morphology changes as follows: cell size and nuclear diameter were reduced, chromatin condensated, cytoplasm was more abundant, reduction in the proportion of nuclear plasma, cell membrane integrity but did not appear smooth, and certain apotosia cells. There were no apparent morphology differences between SPPM60 group and control group. The analysis of cell cycle distribution was performed by flow cytometry. The result showed that PPM60 could stop K562 cells in G0/G1 phase. With an accumulation of cells in G0/G1 phase and an inducement to cell apotosis. SPPM60 had little effect on cell cycle.Thirdly, the activities of PPM60 and SPPM60 on K562 cells were studied by observing their effects on cytosolic free calcium concentration([Ca2+]i) and reactive oxygen species(ROS). After cells were incubated with Fura-2/AM or DCFH-DA fluorescent probe, the concentration of cytosolic free calcium was measured by fluorescence spectrophotometer. After 24-hour, 48-hour, 72-hour treatment, PPM60 increased [Ca2+]i and ROS remarkably, while SPPM60 induced [Ca2+]i or had no apparent effect. The activities of PPM60 and SPPM60 on K562 cells were studied by observing their effects on cell superoxide dismutase(SOD). Within 72 hours, PPM60 induced SOD vitality while there were no conspicuous differences between SPPM60 group and control group. After 12-hour, 24-hour, 48-hour, 72-hour treatment, PPM60 had more effect than SPPM60 on increasing MDA .The results suggested that:(1) PPM60 had more inhibitory effect on K562 cells,sulfation reduced PPM60 anticancer activity.(2)PPM60 could efficiently induce human leukemia cell apotosis and its mechanism may related to increase [Ca2+]i and ROS, prevent cell cycle, inhibit cell proliferation and growth.