| Carbohydrates are one of the three major nutrients(protein,sugar and lipid)in the human body and are widely found in nature.According to the number of monose units,carbohydrates can be divided into monosaccharides,oligosaccharides and polysaccharides.Oligosaccharides are smaller in molecular weight and simpler in structure than polysaccharides,and are theoretically easier to be absorbed by the human body.After polysaccharides are cracked into oligosaccharides,the solubility is enhanced,while some of the original biological activities are improved,and the quality control will be more stable.In addition,oligosaccharides are generally less toxic and safer than that of many polysaccharides and natural active substances.Grateloupia filicina are from the algae of Grateloupia filicina(Wulf.)C.Ag.and Grateloupia livida(Harv.)Yamada.Grateloupia filicina contains a variety of chemical components,of which carbohydrates,especially polysaccharides,are the main chemical components.The oligosaccharide G19 studied in this paper is derived from the degradation of the polysaccharide component G19 s in Grateloupia filicina C.Ag.In the preliminary screening of anti-tumor activity,it has been proved that G19 has an inhibitory effect on the growth of a variety of tumor cells,while in-depth research on glioma was carried out.The mechanism of G19 inhibiting the growth of glioma both in vivo and in vitro was clarified.However,theoretically due to the high hydrophilicity of oligosaccharides,it is difficult for them to penetrate the blood-brain barrier,which may not achieve good curative effects in practical applications.Therefore,we try to understand whether G19 has an inhibitory effect on other tumors that are not blocked by the blood-brain barrier.Hence,we chose gastroenterological tumors(pancreatic cancer)as the object of continued research.Pancreatic cancer is a highly metastatic,invasive malignant solid tumor with high mortality and poor prognosis.It’s very important therapeutic strategies to develop new drug targets and discover new potential lead compounds with low toxicity for the potential treatment of pancreatic cancer.In order to explore the specific effect of this oligosaccharide on pancreatic cancer,we first tested the effect of G19 on a variety of pancreatic cancer cells in vitro.MTT results showed that the inhibitory effect of oligosaccharide G19 on the four types of pancreatic cancer cells PANC-1,SW1990,Aspc-1,and Bx PC-3 was very significant in a does-dependent manner.The inhibition rate of PANC-1 and Bxpc-3 were 91.3% and 98.2%.The clone formation experiment showed that G19 could inhibit the number and size of clones formed by PANC-1 cells.Through the experiment of nude mice subcutaneous xenografted tumor,we found that the inhibition rates of the G19 at low concentration(10 mg/ml)and high concentration(20 mg/ml)treatment groups relative to the control group were 25.3 % and 38.7 %.What’s more,G19 did not show significant effect on the body weight,indicating that G19 has few side effects.The results of cell scratches and Transwell experiment showed that G19 could inhibit the migration and invasion of PANC-1 cells.The mechanism study indicated that G19 could down-regulate the p-β-Catenin,Snail,Slug,Vimentin,N-cadherin and up-regulate the E-cadherin to block the EMT process.Since it is reported that sulfated carbohydrate compounds have anticoagulant activity and can cause bleeding in the body,in order to detect whether G19 has a similar effect,capillary blood vessel method and tail trimming method was used to measure blood clotting time.However,the results showed that G19 had no effect on blood clotting activity in the body.So G19 has good safety.In order to explore the mechanism of G19 inhibiting the growth of pancreatic cancer cells,it was firstly found that G19 could induce the apoptosis of PANC-1 cells by Hoechst 33258 staining and Annexin V/PI double staining.RT-q PCR and Western blot results showed that after G19 treatment,the expression of apoptosis-related molecule Bcl-2 was down-regulated,and the expressions of Cleaved caspase-3,Caspase 9 and Bax were up-regulated.By detecting the upstream signaling pathway,it was found that G19 may induce apoptosis by activating SAPK/JNK,p38 MAPK,and p53.In addition,it was found that G19 induced autophagy in PANC-1 cells by MDC staining.Western blot results showed that the expressions of autophagy-related molecules ATG5,Beclin 1,and LC3 B were up-regulated after G19 treatment.AMPKα protein expression and phosphorylation were inhibited,indicating that G19 may promote autophagy through the negative regulation of m TOR.RNAseq results showed that G19 also had a great influence on PI3K-AKT,ECM-receptor interaction,FAK(Focal adhesion)signaling pathway.In summary,the oligosaccharide G19 can exert anti-pancreatic cancer effects by inhibiting cell proliferation,migration,inducing cell apoptosis and autophagy.The above studies show that oligosaccharide G19 has the potential to become an lead compounds of anti-pancreatic cancer drug.As we know,G19 belongs to oligosaccharide and previous studies says that the direct anti-tumor activity of G19’s original polysaccharide G19 s is not ideal.Are there other seaweed polysaccharides that have direct and better inhibitory activity on these tumors? Sargassum fusiforme(Harv.)Setch.is a brown algae plant belonging to the Sargassum genus.It has the functions of digesting food and removing blood stasis,promoting immune function,and lowering blood lipid and blood glucose.It has been reported that the isolated and purified Sargassum fusiforme(Harv.)Setch.polysaccharide had the effect of inhibiting liver cancer after sulfation modification.Hence,I screened the anti-tumor activity of the polysaccharide fractions isolated from Sargassum fusiforme.Since most sulfated polysaccharides have anti-angiogenesis effect.Angiogenesis is closely related to the occurrence and development of tumors.We first utilized the tube formation experiment to test whether polysaccharide from Sargassum fusiforme might have impact on angiogenesis.Indeed,polysaccharides SPW0 and SPW05 from Sargassum fusiforme could inhibit HMEC-1 cells from forming a tube on Matrigel in a concentration-dependent manner.Subsequent MTT assay shows that SPW0,SPW06,and SPW08 all have a good inhibitory effect on colorectal cancer cells HT29.Among them,SPW0 might inhibit HT29 cells growth,while had a relatively small inhibitory effect on normal liver cells LO2,suggesting that this component may be less toxic.The nude mouse xenograft model proved that the two components SPW0 and SPW05 do had a certain inhibitory effect on the growth of colorectal cancer in vivo.The inhibition rates were 41% and 38% respectively Subsequently,the homogeneous polysaccharide SPW0S2 was isolated from SPW0 polysaccharide.When the administration concentration was 1 mg/ml,the inhibition rate of HT29 cells was75.4%,and the toxicity to normal liver cells was less.Western blot experiments showed that SPW0S2 treatment inhibited the phosphorylation of AKT and PI3 K,increased the expression of p21,and inhibited the phosphorylation of β-Catenin,indicating that SPW0S2 may inhibit the growth of HT29 cells by affecting these molecules.In addition,through the tube formation experiments,it was found that SPW0S2 can also inhibit the formation of tube,but the mechanism needs to be further explored.The above studies have expanded the anti-tumor effect of G19,and further found that G19 has the potential to inhibit pancreatic tumor cell migration,invasion,and promote autophagy and apoptosis,laying a theoretical and material basis for the research and development of new anti-pancreatic cancer carbohydrate drugs.In addition,Sargassum fusiforme polysaccharide SPW0 and its homogeneous polysaccharide SPW0S2,both derived from algae,were shown to have good pharmacological activities to directly inhibit tumor growth and have anti-angiogenesis ability.It is suggested that algae-derived glycan compounds can be used as potential anti-tumor functional active substances,which provide useful scientific ideas for studying the structure-activity relationship of anti-tumor saccharides and developing innovative drugs. |
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