The Relationship Between Structures Of Heparin-Like Glycosamionglycans And Tumor

Heparin-like compounds are poly-anion glycosaminoglycans, which include unfractionated heparin (UFH), low molecular weight heparin (LMWH), heparan sulfate (HS) and their derivatives of UFH/HS. They have been widely used in clinical to treat thrombosis and coagulation for many years. It has been found these compounds have other bioactivities in recent years, among which their anti-tumor activity is important. Interestingly, these compounds have complex structures and the differences of their structures (the size of the molecule, the content and position of sulfate group) are responsible for their different functions.In order to study the relationship between structures of heparin-like compounds and their functions, we extracted and purified HS from UFH byproducts, then analyzed some physico-chemical properties of HS, UFH, LMWH and PSH. IR, NMR, ESI-MS and HPLC were also emoployed to determine the structures of these four GAGs. And the effects on the proliferation of tumor cells and endothelial cells were also studied with MTT test. Moreover, heparin-like GAGs' effect on platelet aggregation in vitro was also studied. Finanally, ELISA method was used to observe the effect of heparin-like GAGs on p-selectin release and how these compounds influenced gelatinase activity was also studied.The results and conclusions of the studies are the following:1 The methods of HS extracting and purification from UFH by-product have been established.2 Some physico-chemical properties of UFH, LMWH, PSH and HS were determined. The relative molecular weight of the four comounds were 9789, 4200, 9733 and 12904 respectively; their SO427COO" value were 2.612, 2.663, 5.009 and 1.182 respectively; the content of uronic acid were 40.5%, 32.4%, 54.9% and 37.2% respectively; and their anti-coagulate activities were 155, 99, 32 and 48usp.u/mg respectively.3 IR and NMR were employed to analyze the structure characteristics of UFH, LMWH, PSH and HS. The results showed the major disaccharides in UFH were IdoA-2SO3-* GlcNSO3-6SO3 and UA-* GlcNAc-6SO3; those in LMWH were A 4'5UA-2SO3 - GlcNSO3-6SO3 and UA -* GlcNAc-6SO3; those in PSH were IdoA-2,3-di-SO3—GlcNSO3-3,6-di-SO3 and UA-2,3-di-SO3—GlcNAc-6SO3 and the counterpart in HS were GlcA-* GlcNAc and GlcA-* GlcNSO3. Besides, determined by HPLC, the disaccharide composition of HS was a-AUA-*GlcN, a-AUA -GlcNAc, a-AUA-GlcN-6SO3 and a-AUA-2SO3—GlcNSO3. One of the oligosaccharide structure in LMWHS was A4'5UA-* GlcNSO3/ GlcNH2-6-SO3-*GlcA— GlcNAc which was determined by ESI-MS.4 The results of the MTT test demonstrated the effects of those four heparin-like GAGs on tumor cell growth (Bel-7402) were different. The LMWH can inhibit the proliferation of Bel-7402 in a dose-dependent way and the inhibitory activity increased with the concentration. However HS accelerated the proliferation of tumor cells and the acceleration activity rose to a certain degree corresponding to the increase of concentration, and then decreased. UFH and PSH both inhibit tumor cell proliferation at higher concentration but accelerate the proliferation at lower concentration. So it is proposed that proper molecular size of heparin-like GAGs is critical for inhibitiing tumorcell proliferation while the content of sulfate group is not the important factor in this aspect. The same method was also employed to test the effects of the four heparin-like GAGs on proliferation of endothelial cells (ECV-204) but found only LMWH and UFH can inhibit the proliferation moderately at higher concentration while other two compounds showed slight acceleration effects.5 The results of tumor cells adhering to endothelial cells showed all of the four compounds could inhibit the adhesion action in a dose-dependent manner. The activities of UFH, LMWH and HS declined with the decrease of concentration but rose dramatically at the concentration of 1.6X 10"3mg/ml, then droped. PSH was much more effective than other three compounds and had dose-dependent.6 Platelet aggregation test results showed the inhibitory effect of PSH was the highest while HS had no significant inhibitory effect. It suggested that the content of sulfate group played an important role on the aggregation. However, these four GAGs had no significant effects on the release of P-selectin.7 The results on inhibitory gelatinase activity experiments showed that heparin-like compounds could inhibit the activity of gelatinase significantly in a dose-dependent manner. The activity-concentration turning point of UFH, LMWH, PSH and HS were 0.026mg/ml, 0.026mg/ml, 0.104mg/ml and 0.42mg/ml respectively. Below the concentration, the inhibitory activities were increased with the concentration increase, while above the concentration, the inhibitory activities were decreased with the concentration increase. Totally, the inhibitory effect of these four compounds is as follows: PSH>LMWH>UFH>HS. It suggested that the content of sulfate groups and the molecular size of heparin-like GAGs were the major two factorsresponsible for the inhibitory effect on gelatinase.