Mechanism Study Underlying The Role Of Glypican-6 In Pancreatic Cancer Cells And The Polysaccharide Of RP02-1 Against Pancreatic Cancer

Pancreatic cancer is a common malignant tumor with 5-year survival rate less than 5%.Pancreatic cancer has the characteristics of easy recurrence and metastasis,poor prognosis,high mortality and high drug resistance.Our previous research showed that glycosylphosphatidylinositol Glypican-6(GPC6)was highly expressed in pancreatic cancer tissues,however the underlying mechanism is still vague.The mechanism of occurrence and development of pancreatic cancer is still unclear,while the lack of efficient and low-side drug intervention is still a challenge for the treatment of pancreatic cancer.Gemcitabine,as the first-line treatment for pancreatic cancer,is often accompanied by serious side effects.Therefore,screening for low-toxic and highly effective anti-pancreatic cancer drug candidates and understanding the pathogenesis of pancreatic cancer are the core tasks for the treatment of pancreatic cancer.This subject mainly studies the function and mechanism of glycosylphosphatidylinositol proteoglycan GPC6 in pancreatic cancer and the effect of natural drug polysaccharides on pancreatic cancer.GPC6 is a heparan sulfate proteoglycan,and its sugar chain(HS chain)is a co-receptor for cell growth factors.Previous studies suggested that the HS chain played important roles in tumor growth,invasion and metastasis.Hence theoretically,exogenous sugar chain intervention may inhibit tumor cell growth and invasion.Pectin is a sort of polysaccharide with the most complex structure on the cell wall of plants and plays multiple functions.The main chain of pectin is consisted of galacturonic acid with neutral sugar side chains.The complex structure makes them have many functions.Pectin can be used not only as a component of functional food,but also as a wide range of pharmaceutical research compounds.In previous studies,various biological activities of pectin have been discovered,such as anti-tumor,immunity enhancement,anti-aging,antioxidation and anti-diabetes,etc.In addition,compared with chemotherapy drugs,pectin has lower cytotoxicity but against pancreatic cancer,showing a better advantage.Therefore,further investigation on the activity and mechanism of pectin against pancreatic cancer may provide new ideas for anti-pancreatic cancer research,especially for the anti-pancreatic cancer new drug development.The roots of Polygala tenuifolia are traditional Chinese medicine for calming nerves and improving memory.The chemical structure study suggests that they mainly contain triterpenoid saponins,sugars,ketones,as well as minor alkaloids,coumarin,lignin,etc.Basically,they have a variety of pharmacological effects with the potential to be developed.Modern medical research shows that Polygala tenuifolia has antidementia,anti-depression,anti-tumor,anti-oxidation and memory improvement functions.The polysaccharide from is one of the active components extracted from the roots of Polygala tenuifolia.However,the polysaccharide with anti-tumor effect in this plant is rarely reported so far.We isolated and purified the pectin polysaccharide RP02-1 from Polygala tenuifolia,and examine the effects of RP02-1 against the five tumors including pancreatic cancer,non-small cell lung cancer,colon cancer,breast cancer and liver cancer.The inhibition rates of As PC-1 and Bx PC-3 were 41.83% and 57.54%,respectively.In addition,RP02-1 had no significant effect on the growth of normal liver cells and pancreatic ductal epithelial cells.The clone formation experiment showed that RP02-1 could inhibit the number and size of clones formed by Bx PC-3 and As PC-1 cells.The results of cell scratches and Transwell experiments showed that RP02-1 inhibited the migration of Bx PC-3 and As PC-1 pancreatic cancer cells.The results of Annexin V / PI double staining method and Hoechst 33258 staining method suggested that RP02-1 might induce As PC-1 and Bx PC-3 cells apoptosis.The mechanism study indicated that the expression of apoptosis-related functional molecule Bcl-2 was downregulated after RP02-1 treatment;while Bax and Cleaved Caspase-3 were up-regulated after the same treatment.Interestingly,polysaccharide RP02-1 could also inhibit the autophagy of Bx PC-3 cells.RT-q PCR and Western blot analysis revealed that the expressions of autophagy-related molecules Beclin-1,Atg5 and LC3 B were significantly downregulated after treatment with RP02-1.To further confirm the anti-pancreatic cancer effect of RP02-1,in vivo experimental studies were employed.Through the experiment of pancreatic cancer cell Bx PC-3 subcutaneous xenografted tumor,we found that the inhibition rates of the RP02-1 at low concentration(5 mg / kg)and high concentration(50 mg / kg)treatment groups relative to the control group were 49.58% and 55.45%,respectively.However,RP02-1 did not show any significant effect on the body weight of the mice,indicating that RP02-1 had few significant side effects,indicating very good safety.In summary,we isolated and purified a pectin RP02-1 from Polygala tenuifolia and uncovered that RP02-1 might have anti-pancreatic cancer activity.This study suggested that RP02-1 could exert anti-pancreatic cancer effects by inhibiting cell proliferation,impairing cell migration,inducing apoptosis,and impeding autophagy.These studies suggest that RP02-1 has the potential to become a leading compound to be developed as anti-pancreatic cancer drugs.The above studies indicate that RP02-1 can exert anti-pancreatic cancer activity.However,the current research on the pathogenesis of pancreatic cancer lacks effective drug targets and biomarkers.Therefore,we selected proteoglycans on the surface of cell membranes to study their mechanism of action in the development of pancreatic cancer.The previous studies in this group have shown that GPC6 is highly expressed in pancreatic cancer and plays important roles in cell proliferation,migration,invasion and EMT.This project focuses on the study of GPC6 signaling pathway implicated in molecular mechanism underlying the action of promoting pancreatic cancer development.To examine,the efficiency of knockdown and overexpressed GPC6 in PANC-1 and SW1990 cell lines and corresponding control stable transfected cell lines were detected by Western blot.Through immunofluorescence experiments,we showed that the expression level of FOXO3 a was up-regulated by knocking down GPC6 in SW1990 cells,meanwhile,the transcription factor FOXO3 a was involved in the knockdown of GPC6-mediated anti-pancreatic cancer activity in PANC-1cells.Further mechanism research suggested that after knocking down GPC6,the Kras/ MEK / ERK / FOXO3 a signaling pathway were blocked,thereby promoting FOXO3 a expression and further exerting its anti-pancreatic cancer activity.In conclusion,the study shows that the pectin polysaccharide RP02-1 may be a lead compound against pancreatic cancer,and GPC6 may be a new potential target for anti-pancreatic cancer drug development.The study provides new evidences to understand that glycans play key roles in the development of pancreatic cancer,and also lays a good foundation for the polysaccharides based innovative drugs development.