The Mechanism Of Licochalcone A Inhibiting Osteosarcoma Through Oxidative Stress And Endoplasmic Reticulum Stress

Osteosarcoma is a differentiation disease caused by the genetic and epigenetic differentiation of mesenchymal stem cells into osteoblasts.It is the most common primary bone malignant tumor in children and adolescents aged 10-20 years.Osteosarcoma is a highly malignant tumor,which grows rapidly.15-20%of the patients find distant metastases at the first visit.The prognosis is poor,which brings great pain to the patients and their families.At present,the side effects of anticancer drugs are large,and it is easy to form multidrug resistance,and multidrug resistance is an important reason for the failure of osteosarcoma treatment.Therefore,to find a new effective and safe drug to improve the therapeutic effect of osteosarcoma is the key problem to be solved.Licochalcone A（LCA）is a compound existing in Chinese traditional herbal medicine Glycyrrhiza uralensis Fisch.Previous studies have shown that LCA has a variety of biological activities,such as anti-virus,anti-infection,anti-tumor,anti-angiogenesis and so on.In particular,the anti-tumor characteristics of LCA have been widely concerned.Many studies have pointed out that LCA can inhibit the proliferation of tumor cells,cause cell cycle arrest,promote apoptosis,anti-invasion and anti-migration and other anti-tumor effects to inhibit the occurrence and development of tumor.Endoplasmic reticulum（ER）is an important organelle involved in the regulation of calcium ion and protein synthesis,processing and transportation in cells.The abnormal protein folding in ER caused by external and internal factors will cause the abnormal function of ER,which is called ER stress.ROS activates endoplasmic reticulum stress through endoplasmic reticulum receptor proteins such as endoplasmic reticulum kinase（PERK）,transcription factor 6（ATF6）,eukaryotic translation initiation factor（eif-2α）and their downstream signaling pathways.Related studies have shown that endoplasmic reticulum stress plays an important role in the occurrence and development of tumor.Autophagy is a major degradation system in eukaryotic cells,which is involved in the degradation of long-lived proteins,organelles and other cellular contents.In addition,autophagy,as an important mechanism of endoplasmic reticulum associated degradation（ERAD）pathway,is involved in the degradation of misfolded proteins and proteinaggregates during ER stress.Autophagy is generally considered to be a mechanism of cell survival.However,over activated autophagy also induces cell death,which is called autophagic cell death.Antitumor drugs can induce tumor cell apoptosis through ER stress and autophagy.However,the specific mechanism of LCA inhibiting the proliferation of OS cells remains unclear.We intend to explore the effect of LCA on the proliferation of OS cells and the role of ER stress and autophagy in its regulation,so as to provide theoretical basis for clinical treatment of OS and the search for the target of combination therapy.This project is divided into four parts:the first part,taking OS cell line as the research object,to explore the inhibitory effect of LCA on the proliferation and cycle arrest of OS cells.The second part is to explore the role of oxidative stress in the apoptosis of OS cells induced by LCA.In the third part,we explored the role of ROS mediated ER stress in LCA-induced apoptosis and autophagy of OS cells.In the fourth part,we established a nude mouse model of subcutaneous heterotopic implantation of OS cells,and further explored the inhibitory effect of LCA on OS by histological methods.PART Ⅰ Licochalcone A inhibits Osteosarcoma cell proliferation and induces cell cycle arrest in vitroObjective:To study the effect of LCA on the proliferation of OS cells in vitro.Methods:We cultured OS cells.When the cell density reached 60%-80%,we used diferent concentrations of LCA for 24 hours and specific concentrations（40 μM）of LCA for different time to intervene.The viability of osteosarcoma cells was detected by CCK-8,and the effect of LCA on the proliferation of OS cells was detected by cell cloning experiment.Meanwhile,flow cytometry and Western blot were used to detect the effect of LCA on the cell cycle of osteosarcoma.Results:1.CCK-8 test results showed that with the increase of LCA concentration and the prolongation of LCA action time,the activity of osteosarcoma cells decreased continuously,showing a significant time and concentration dependence.2.Cell cloning experiment also showed that LCA could inhibit the proliferation of OS cells in a concentration dependent manner.3.Flow cytometry showed that the proportion of G2/M phase increased with the increase of LCA concentration.Western blot analysis showed that with the increase of drug concentration,cell cycle related proteins cdc25c decreased,p-cdkl,p-cdc25c,p21 increased,which further indicated that the cell cycle of OS cells was obviously blocked after treated with different concentrations of LCA.Conclusion:1.LCA can effectively inhibit the activity of OS cells,and its inhibitory effect depends on time and concentration.2.LCA can effectively inhibit the proliferation of OS cells in a concentration and time-dependent manner,which indicates that LCA may inhibit the viability of OS cells by inhibiting the proliferation of OS cells.3.LCA can induce cell cycle arrest in a concentration dependent manner.PART Ⅱ The role of oxidative stress in the apoptosis of osteosarcoma cells induced by Licochalcone AObjective:To study the role of oxidative stress in LCA induced apoptosis of OS cells.Methods:OS cells were cultured.When the cell density reached 60%-80%,different concentrations of LCA were used for drug intervention.The effects of LCA on mitochondrial membrane potential and apoptosis of OS cells were detected by flow cytometry,and the reactive oxygen species content of OS cells was detected by fluorescence microscope.Western blot was used to detect the effect of LCA on the expression of apoptotic protein in OS cells.The role of oxidative stress in LCA induced apoptosis of OS cells was detected by flow cytometry and Western blot.Results:1.The results of mitochondrial membrane potential showed that with the increase of LCA concentration,the damage of mitochondria in OS cells increased significantly.2.Fluorescent probe DCFH-DA showed that the content of ROS increased in a concentration dependent manner after LCA treatment.3.LCA was used to treat OS cells.Flow cytometry showed that the apoptosis rate increased with the increase of LCA concentration.NAC can inhibit the apoptosis rate of OS cells induced by LCA.4.Western blot analysis showed that with the increase of LCA concentration,the expression of apoptosis related proteins cleaved-caspase-3,cleaved-caspase-8,cleaved-caspase-9 and cleaved PARP increased,and mitochondrial protein Bax/Bcl-2 increased NAC could inhibit the decrease of apoptotic protein.Conclusion:1.LCA can damage the mitochondria of OS cells and induce the production of ROS in OS cells.2.LCA can induce OS cell apoptosis in a concentration and time-dependent manner.3.LCA can induce OS cell apoptosis by inducing ROS production.PART Ⅲ The role of ROS mediated endoplasmic reticulum stress in Licochalcone A induced apoptosis and autophagy of osteosarcoma cellsObjective:To study the role of ROS mediated ER stress in LCA induced apoptosis and autophagy of OS cells.Methods:OS cells were cultured.When the cell density reached 60%-80%,different concentrations of LCA were used for drug intervention.The change of Ca²⁺ content in osteosarcoma cells was detected by flow cytometry.Ca²⁺inhibitor 2-APB was used for further experiment,and the change of apoptosis rate was detected by flow cytometry.Western blot was used to detect the effect of LCA on ER stress-related protein expression in osteosarcoma cells.NAC,an inhibitor of reactive oxygen species,was used for further experiment.Western blot was used to detect the effect of oxidative stress on ER stress in osteosarcoma cells induced by LCA.In order to further study the effect of ER stress on osteosarcoma cell apoptosis induced by LCA,flow cytometry was used to detect the effect of LCA on osteosarcoma cell apoptosis induced by ER stress inducer TM and ER stress inhibitor TUDCA.The expression of autophagy protein was detected by Western blot and the autophagosome was observed by transmission electron microscope.In order to observe the induction of autophagy pathway by LCA more intuitively,we constructed OS cells stably expressing mRFP-GFP-LC3 for further experiment.In order to clarify the role of ER stress in apoptosis and autophagy of OS cells induced by LCA,and to further explore the effect of autophagy on ER stress and apoptosis induced by LCA,OS cells were pretreated with ER stress inhibitor（TUDCA）and its effects on ER stress,apoptosis and autophagy of OS cells induced by LCA was observed.RNA interference was used to study the effect of siChop on LCA induced apoptosis and autophagy.Apoptosis and autophagy protein expression were detected by Western blot.At the same time,we detected the apoptosis of OS cells induced by LCA after 3-MA pretreatment by flow cytometryResults:1.With the increase of LCA treatment time,Ca²⁺ in OS cells increased significantly,and Ca²⁺inhibitor could reduce the apoptosis rate of OS cells induced by LCA.2.ER stress-related proteins were detected by Western blot.The results showed that the expression of GRP78,perk,eIF2 α,ATF4 and chop increased with the prolongation of LCA treatment time.Reactive oxygen species inhibitor NAC could reduce the expression of ER stress-related proteins,and ER stress inducer TM could significantly increase the apoptosis rate of OS cells.However,ER stress inhibitor TUDCA decreased the apoptosis rate of OS cell 143B induced by LCA.3.Western blot analysis of autophagy protein showed that LCA could significantly up regulate the expression of LC3-Ⅱ protein in OS cells in a time-dependent manner.Transmission electron microscopy further confirmed that LCA could induce autophagy.4.Western blot results showed that inhibition of ER stress could reduce apoptosis and autophagy protein expression induced by LCA.Conclusion:1.LCA can increase the content of Ca²⁺in OS cells,and Ca²⁺can promote the apoptosis of OS cells.2.LCA induced ER stress and promoted apoptosis of OS cells,ROS promoted ER stress.3.ER stress is involved in the apoptosis and autophagy of OS cells induced by LCA,and the autophagy induced by LCA can inhibit the apoptosis of OS cells.PART Ⅳ Effect of Licochalcone A on osteosarcoma in nude miceObjective:To investigate the anti OS effect of LCA in nude mice.Methods:10 BALB/C male nude mice（4-6 weeks old,16-20g）were selected and the 143B cell suspension（1×107 cells/ml）diluted to 100 μLwith cold PBS was transplanted into the armpit of mice.When the axillary tumors were visible,the mice were randomly divided into two groups:control group（DMSO group）and LCA treatment group（30mg/kg/D）,with 5 mice in each group.The drug was given every other day for 15 days.During the administration,the body weight and tumor volume of mice were measured every 2 days.After 15 days of administration,blood was drawn to detect the liver and kidney function of mice,and all mice were killed.The tumor tissue of xenograft was carefully removed,weighed and fixed.At the same time,the important organs（heart,liver,spleen,lung and kidney）of mice were taken out for fixation.The histological changes of important organs were detected by H&E staining,and the proliferation of osteosarcoma was detected by Ki67 and TUNEL immunofluorescence.Results:1.The volume of the tumors in the control group were higher than LCA groups（P<0.05）.2.The outcomes manifested that more TUNEL positive cells were found in the xenografts treated with LCA in comparison with the control group.Meanwhile,the expression of Ki67 was dramatically reduced in the xenograft model treated with LCA in comparison with the control group.3.The results of AST,ALT,BUN and CREA between the control and LCA groups were maintaining the normal functions.The histological analysis indicated that LCA did not show any significant changes of the major organs at the end of this study.Conclusion:1.LCA can significantly reduce the tumor volume of OS bearing mice.2.Immunofluorescence results showed that LCA could significantly inhibit the proliferation and induce apoptosis of OS cells in vivo.3.The results of liver and kidney function and histology of important organs showed that LCA was relatively safe.