| Hepatocellular carcinoma?HCC?is the fourth leading cause of cancer-related death in the world,and its incidence continues rising.A large number of studies have reported that fructose-1,6-bisphosphate aldolase A?ALDOA?is overexpressed in many cancers,especially in HCC,and the high expression of ALDOA is closely related to the overall survival rate of patients with HCC.However,there is no effective inhibitor of ALDOA at present.In our previous study,the active ingredients of traditional Ch inese medicine were widely screened,and it was found that polysaccharide HDPS-4II isolated from Holotrichia diomphalia Bates having highly affinity with ALDOA.In this study,we analyzed the structure of polysaccharide HDPS-4II and prepared its sulfated derivatives,further explored the activities of HDPS-4II and its sulfated derivatives against liver cancer with ALDOA as the target,which layed a foundation for the development of a new anti-tumor drug.Method1. The crude polysaccharide HDPS was prepared by alkali extraction and alcohol precipitation method,and was isolated and purified by anion-exchange and size exclusion chromatography.The primary structure was investigated by a combination of chemical and instrumental analysis methods.Congo red analysis,circular dichroism spectra and scanning electron microscopy were used to explore the advanced stru cture and surface morphology.2.HDPS-4II and its sulfated derivatives were prepared by the chlorosulfonic acid-pyridine method.These derivatives were identified by ultraviolet and visible spectrum?UV?and infrared?IR?.3.The affinities of HDPS-4II and its sulfated derivatives binding to ALDOA and ALDOB were determined by surface plasmon resonance?SPR?technique.Effects of HDPS-4II and its sulfated derivative?SHDPS-4II?on the proliferation of four kinds of human hepatoma cells were determined by MTT assay.Colorimetric method was used to determine the effects of SHDPS-4II,HDPS-4II and sulfated Dextran1000?SDextran1000?on the activities of three key enzymes of intracellular glycolysis,ALDO,phosphofructokinase?PFK?and hexokinase?HK?.4.The subcutaneous transplantation model of human hepatocellular carcinoma SMMC-7721 in nude mice was established to evaluate the anti-hepatoma activity of HDPS-4II,SHDPS-4II and SDetran1000?control?in vivo.Results1. The structure of HDPS-4II isolated from Holotrichia diomphalia Bates:HDPS-4II was a straight polysaccharide composed of?1-6?-?-D-Glcp and?1-4?-?-D-Glcp with a molar ratio of 10.0:1.0.HDPS-4II had a triple helix structure and its surface was reticulate and porous.2. Preparation of sulfated derivatives of HDPS-4II:the sulfated derivatives of dextrans?control?and HDPS-4II were prepared and named as SHDPS-4II,SDextran1000,SDextran3000 and SDextran4000.The se derivatives were identified by UV and IR,the characteristic absorption peaks appearing at about 268,264 and280 nm were corresponding to S-O and-SO3-groups respectively,and the characteristic peaks appearing at about 1240 and 800?850 cm-1were corresponding to stretching vibration ofgroup and C-O-S bond,respectively.The degrees of substitution of the derivatives were 1.3 0,0.97,0.75 and 0.90,respecti vely.3. The inhibitory effect of HDPS-4II and SHDPS-4II on ALDOA and their anti-hepatoma activity in vitro:The dissociation constant?KD?values of SHDPS-4 II and HDPS-4 II on ALDOA were 3.78 and 9.76?M,respectively.The results of anti-hepatoma activity in vitro showed that HDPS-4II,SHDPS-4II and SDetran1000 had significant anti-hepatoma effects,and inhibited the proliferation of hepatocellular carcinoma cells in concentration-dependent manners.Among them,SHDPS-4II and SDextran1000 had the most sign ificant inhibitory effect s on the proliferation of hepatoma cells HCCLM3 and Hep G2,respectively.The values of half maximal inhibitory concentration??were 1.63?M and 0.72?M,respectively.Furthermore,HDPS-4II,SHDPS-4II and SDextran1000 inhibited the activity of intracellular ALDO enzyme in concentration-dependent manners,and the inhibitory effect of SHDPS-4II was the highest.When the concentration of SHDPS-4II was 10?M,the inhibition rate was 80.18±1.38%.HDPS-4II,SHDPS-4 II and SDextran1000 could promote intracellular PFK and HK enzymes activities At the concentration of 10?M,the increase rates of HDPS-4II in PFK and HK activities were 295.44±5.91%and 117.11±8.22%,respectively.4. The nude mice subcutaneously inoculated with SMMC-7721 cells were used to evaluate the anti-hepatocellular carcinoma activity of HDPS-4II and SHDPS-4II in vivo.Results showed that 50 mg/kg HDPS-4II and 20 mg/kg SHDPS-4II significantly reduced the tumor growth with inhibition-percentage of39.22%and 51.78%,respectively.In summary,HDPS-4II is a homogeneous and linear glucan that exhibits significant anti-hepatocellular carcinoma effect in vivo and vitro,and SHDPS-4II,the sulfated derivatives of HDPS-4II,also exhibits higher anti-hepatocellular carcinoma activity. |
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