Inhibitory Effect Of The Sulfated Derivative Of Polysaccharide From Pinus Massoniana Pollen On HepG2 Cell And Its Mechanism

The anticancer research of polysaccharide from Pinus massoniana pollen(PPM60) and its sulfated derivative(SPPM60) has indicated that, PPM60 and SPPM60 both inhibited the growth of S180 tumor in vivo by promoting immunity, and the activity of SPPM60 was much stronger. This research aims to determine the effects of PPM60 and SPPM60 on the proliferation and differentiation of human HepG2 cells in vitro, and then to give some theoretical and experimental roof for the research of SPPM60 anticancer function.Firstly, 2g PPM60 was chemically modified by chlorosulfonic acid- pyridine method. 3.72g SPPM60, a modified compound with 1.41 substituted degrees was obtained. The infrared ray(IR) spectrum of SPPM60 showed the characteristic absorptions of sulfated ester bond(C-O-S and S=O).Secondly, the activities of PPM60 and SPPM60 on HepG2 cells were studied by observing their effects on cell proliferation and morphology. MTT colourimetry was used to evaluate the influences of PPM60 and SPPM60 on HepG2 cell viability. SPPM60 inhibited the proliferation of HepG2 in dose- and time-dependent manner. After 72-hour treatment with 200μg/ml, SPPM60 inhibited the growth of HepG2 remarkably by 42.05%, while PPM60 just had little cytotoxic activity. Inverted microscope, Hematoxylin and Eosin staining, Ho.33342/PI double fluorescence labeled staining were used to observe cell morphology. SPPM60 induced HepG2 underwent some morphology changes as follows: the rate of cell proliferation reduced remarkably; cells became more sparser; contact inhibition appeared in some areas; the space between cells enlarged; adhesion ability weakened; the shape of cells became spindle or arborization; the number of nucleolus reduced. There were no morphology differences between PPM60 group and control group.Thirdly, the activities of PPM60 and SPPM60 on HepG2 cells were studied by observing their effects on cell cycle and cytosolic free calcium concentration([Ca²⁺]_i). The analysis of cell cycle distribution was performed by flow cytometry. The result showed that SPPM60 could stop HepG2 cells in G0/G1 phase. With an accumulation of cells in G0/G1 phase while a decreases of cells in S and G2/M phase, PPM60 had little effect on cell cycle. After cells were incubated with Fura-2/AM fluorescent probe, the concentration of cytosolic free calcium was measured by fluorescence spectrophotometer. In 40 minutes, there was no obvious change of [Ca²⁺]_i. But after 24-hour, 48-hour, 72-hour treatment, SPPM60 reduced [Ca²⁺]_i remarkably, while PPM60 still had no effect.Fourthly, the activities of PPM60 and SPPM60 on HepG2 cells were studied by observing their effects on cell biochemistry indexes. The expression of Alpha-fetoprotein(AFP) was determined by immunocytochemistry. The secretory amount of albumin(Alb) was detected by bromocresol green method. The specific activities ofγ-glutamyl transferase(γ-GT) was measured by diazo reaction colourimetry. Alkaline phosphatase(ALP) activities were detected by colourimetry. After treated with 200μg/ml SPPM60, the expression AFP was obviously reduced, the secretory amount of Alb was increased, the specific activity ofγ-GT was declined remarkably and the activity of ALP and heat-resistant ALP were reduced either. However, PPM60 group had no remarkable change.The results suggested that:(1)PPM60 had no inhibitory effect on HepG2 cells growth, however SPPM60 could inhibit cell growth. Sulfation endowed PPM60 anticancer activity.(2)SPPM60 could efficiently induce human hepatocarcinoma HepG2 cell differentiation to normal, and its mechanism may related to decrease [Ca²⁺]_i, prevent cell cycle, inhibit cell proliferation and growth. In addition, the superoxide anion radical (O₂^-.) and hydroxyl radical (.OH) scavenging effects of PPM60 and SPPM60 were examined in vitro by fluorescence spectrophotometric method. The results affirmed that PPM60 and SPPM60 had certain effect on scavenging free radicals. Furthermore, sulfated polysaccharide was more effective than polysaccharide at scavenging O₂^-., but was less effective than polysaccharide at scavenging .OH.