| Esophageal cancer is the eighth most common cancer in the world and the sixth leading cause of cancer death.Esophageal cancer is one of the most unknown and dangerous malignant tumors in the world,The main reason is its extremely aggressive and low survival rate.Because early esophageal cancer has no obvious clinical symptoms,most esophageal cancers are diagnosed only when they develop to advanced stages.In recent years,the incidence of esophageal adenocarcinoma in the world has increased year by year.At present,the treatment of patients with esophageal cancer is mainly through two main ways of chemotherapy and radiotherapy,and chemotherapy is the first choice.Although chemotherapy can inhibit the proliferation of tumor cells in a specific range,because traditional anti-cancer drugs are difficult to distinguish between tumor cells and normal cells,anti-tumor drugs may inhibit normal cell proliferation while inhibiting tumor cells,thereby affecting the body great damage.Because the internal structure of cancer cells and cancerous tissues is extremely cumbersome,it is difficult for ordinary anti-tumor drugs to produce obvious effects.In addition,due to the shortcomings of poor solubility of ordinary antitumor drugs in water,inability to circulate in the body for a long time,and low bioavailability,the tumor cannot be completely cured.Therefore,there is an urgent need to find a more effective method to treat esophageal cancer.In recent years,hyaluronic acid,as a biomaterial,has aroused great interest.Hyaluronic acid(HA)is a linear polysaccharide formed by the repeated connection of glucuronic acid and N-acetylglucosamine through glycosidic bonds.Hyaluronic acid is also the most common biomacromolecule in the connective tissue of all vertebrates.Due to the good physical and chemical properties of Hyaluronic acid,such as biocompatibility,degradability,non-toxicity and non-immunogenicity,Therefore,hyaluronic acid is extremely widely used in biomedicine.Hydrogen sulfide is an important mediator involved in various physiological functions,plays an important role in cancer pathophysiology,and can inhibit the proliferation of a variety of cancer cells.Due to the high incidence of esophageal cancer and its poor prognosis,we urgently need to develop new drugs that are more novel,safe,effective,and fewer prognostic adverse reactions to prevent and treat esophageal cancer and reduce the patient's mortality rate.Objective: HA-ADT is synthesized by hyaluronic acid and small molecule hydrogen sulfide donor ADT-OH,which can improve the solubility,stability and targeting of small molecule drug ADT-OH,thereby improving the bioavailability of ADT-OH to a certain extent.The mechanism of inhibition of HA-ADT on esophageal cancer cells(KYSE30 and KYSE70)was explored.Methods: with trans-anethole and sulfur as raw materials,ADT was obtained by reaction under certain conditions;then,ADT was demethylated to get ADT-OH;finally,ADT-OH and HA were chemically reacted to get HA-ADT.In this paper,the structure,particle size,potential and appearance of the compound were analyzed by 1H-NMR,FT-IR,particle size,Zeta potential and TEM,and the in vitro release of hydrogen sulfide was detected.In vitro experiments: MTT colorimetry,CCK-8 and Ed U kits were used to explore the effects of Na HS,GYY4137 and HA-ADT on the proliferation of KYSE30 and KYSE70 cells;The effects of Na HS,GYY4137 and HA-ADT on the migration and invasion of KYSE30 and KYSE70 cells were observed by Wound healing,Transwell and Invasion experiments;Colony formation assay was used to observe the effect of three kinds of hydrogen sulfide donors on the clone ability of esophageal cancer cells;TUNEL assay and flow cytometry were used to detect the effect of hydrogen sulfide donor on apoptosis of human esophageal cancer cells;Flow cytometry and Western blot were used to detect the effect of hydrogen sulfide donor on cell cycle;The effects of hydrogen sulfide donors on the expression of apoptosis protein,PI3K/AKT/m TOR pathway,Wnt/?-Catenin pathway and autophagy related protein of KYSE30 and KYSE70 were observed by Western blotting.In vivo experiments: human esophageal cancer cells KYSE30 and KYSE70 were inoculated into the armpit of nude mice to observe the effect of hydrogen sulfide donor on the tumorigenesis rate of human esophageal cancer cells;Histological morphology of tumor was observed by HE staining,the expression of CD31,Ki67,Cleaved caspase-3,Beclin-1 and p21 in four groups of tumor tissues were observed by IHC experiment,and their differences were compared.Results: 1.Observe the results of 1H-NMR and FT-IR and calculate,the grafting rate of HA-ADT reached 19%.2.Combined with the results of particle size,Zeta potential and TEM,wecan see that the particle size of HA-ADT is relatively small,the particle size distribution is uniform,the surface has negative electric charge,HA-ADT is round,the size is uniform,which shows that the compound has good stability.3.The in vitro release results of hydrogen sulfide suggest that HA-ADT can slowly and continuously release H2 S.4.According to MTT,CCK-8,Ed U results and statistics,in the range of 0?300?M,hyaluronic acid has no significant cytotoxicity,HA-ADT can better inhibit the activity and proliferation of KYSE30 and KYSE70 cells.5.The results of Wound healing assay,Transwell and Invasion suggested that the cell migration and invasion ability of HA-ADT group was significantly reduced.6.The effect of hydrogen sulfide donors on cell cycle was observed by flow cytometry and Western blotting,the results showed that HA-ADT significantly inhibited the cell cycle of esophageal cancer cells.7.Colony formation experiments showed that HA-ADT significantly reduced the cloning ability of esophageal cancer cells.8.TUNEL assay,flow cytometry and expression of apoptosis related proteins showed that HA-ADT significantly accelerated the apoptosis of KYSE30 and KYSE70 cells.9.Western blotting showed that the expression of Beclin-1 decreased in HA-ADT group,while the expression of Lc3A/B and P62 increased.10.Western blotting showed that HA-ADT significantly reduced the expression of p-PI3K/p-AKT/p-m TOR and Wnt3a/p-??Catenin/p?Gsk?3?.11.Xenograft tumors in nude mice showed that the volume of transplanted tumors in the HA-ADT group was significantly reduced.12.The results of hematoxylin-eosin staining showed that compared with the control group,the HA-ADT group had smaller nuclei,more interstitial,less nuclear division,and a larger range of necrosis.13.According to the immunohistochemical results of Ki67,CD31,Cleaved caspase-3,Beclin-1 and p21,the HA-ADT significantly reduced the microvessel density of the transplanted tumor tissue,while increasing the apoptosis index,regulating autophagy,and inhibiting the process of cell cycle.Conclusion: The HA-ADT is formed by chemically linking HA and ADT-OH.The chemical properties of HA-ADT are stable and the solubility in water is good.In vitro and in vivo experiments showed that HA-ADT could inhibit the migration,invasion and cycle of tumor cells and promote the apoptosis of esophageal cancer cells better than Na HS and GYY4137 when the concentration of hydrogen sulfide donors was 200 ?M.Therefore,HA-ADT can be used as a potential new drug to inhibit tumor growth. |
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