| The anticancer research of polysaccharide from Pinus massoniana pollen (PPM60) and its sulfated derivative (SPPM60) in our laboratory 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. PPM60 had the stronger inhibitory effects on the proliferation of K562 cell in vitro, while SPPM60 reduced its inhibitory effect. The mechanism of inhibition of K562 cells may be increased intracellular calcium and reactive oxygen species, blocking cells in G0/G1 phase and induce apoptosis of K562 cells. This research aims to determine the effects of PPM60 and SPPM60 on the proliferation and cell cycle of human HepG2 cells in vitro, and then to discuss the role of its in vitro anti-tumor mechanism. So as to provides a theoretical basis for cancer adjuvant therapy.Firstly, polysaccharide was extracted and precipitated by 60% alcohol from Pinus massoniana pollen, naming PPM60. Three components PPM60-A, PPM60-B, PPM60-C were obtained separately by molecular sieve Sephacryl S-400HR. PPM60 and the purified components were chemically modified by chlorosulfonic acid- pyridine method. The modified compounds were named SPPM60, SPPM60-A, SPPM60-B, SPPM60-C respectively, with1.611, 1.4, 1.45, 0.968 substituted degrees were 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. 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 and 400μg/mL, SPPM60 inhibited the growth of HepG2 remarkably by 38.21% and 39.16%, while PPM60 just had little cytotoxic activity. We compared two different degrees of SPPM60(SPPM60-1, SPPM60-2, DS are 1.226 and 1.611). After 72-hour treatment with 200μg/mL, SPPM60-1 and SPPM60-2 inhibited the growth of HepG2 remarkably by 40.25% and 53.34% respectively. Compared the inhibition rates between the high DS and the low DS, they had significant differences.Thirdly, the activitives of PPM60 and SPPM60-2 on HepG2 cells were studied by observing their effects on cell cycle. The analysis of cell cycle distribution was performed by flow cytometry. The result showed that SPPM60-2 could block HepG2 cells in G2/M phase, while the cell cycle distribution of PPM60 group had no remarkable change. According to the results of real-time PCR, SPPM60-2 could decrese the expression mRNA level of CDK1 and CyclinB, increase the expression of p53 and p21, as compared with control, which had obvious differences.While after treated with PPM60, the expression of mRNA level of the target genes, no significant differences compared with the control group.Fourthly, the activities of PPM60 and SPPM60-2 on HepG2 cells were studied by observing their influences on VEGF in cell culture supernatant. After treated with 200μg/mL SPPM60-2 and SPPM60B, the expression of VEGF were extremely significant reduced, compared with the control group.The results suggested that: (1)PPM60 had no inhibitory effect on HepG2 cells growth, however SPPM60 could inhibit cell growth, and with higher DS of sulfated polysaccharide had higher inhibition rate on HepG2 cells. (2)SPPM60-2 could efficiently inhibit the proliferation of hepatocarcinoma HepG2 cell, and its mechanism may related to down-regulation the mRNA level of CDK1, CyclinB and up-regulation of p53, p21, arrest cell cycle in the G2/M phase.(3)SPPM60-2 has certain antiangiogenic effect, while PPM60 group has no significant effect. |
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