Lycorine Induces Apoptosis And G1 Arrest Through ROS/P38 Signaling Pathway In Osteosarcoma Cells

Osteosarcoma is an aggressive malignant neoplasm that primarily affects teenagers and young adults,and is one of the leading causes of cancer mortality in the pediatric population.Although multi-drug chemotherapy treatment after surgery has increased overall 6-year survival rate by about 50%as compared with surgery alone,conventional chemotherapy has many disadvantages.This includes the high incidence of recurrence,strong chemo-resistance,chemotherapy collateral toxicity to normal tissues which can lead to heart damage,neutropenia,thrombocytopenia,and anaemia,and other severe side effects.In fact,current treatment approaches have reached a survival plateau and attempts to improve osteosarcoma prognosis have proven unsuccessful.Thus,there is clear evidence that development of new agents with high efficacy and fewer side effects to provide better prognostic outcome is urgently needed.Lycorine(LY)is a natural alkaloid extracted from Amaryllidaceae plant species that exhibits many biological benefits including anti-tumor,anti-inflammatory,anti-viral and anti-malarial effects.LY potent anti-tumorigenic effects,both in vitro and in vivo,with selective cytotoxicity demonstrated in multiple myeloma,cervical cancer,prostate cancer,bladder cancer,breast cancer,leukemia and hepatocellular carcinoma.Importantly,the anti-tumorigenic effects of LY were observed at pharmacological doses with minimal toxicity to normal cells.Mechanistically,LY invokes many biological mechanisms including but not limited to inhibition of protein synthesis,induction of cell cycle arrest and suppression of cell proliferation leading to activation of pro-apoptotic cascades.Apoptosis(programmed cell death)is essential in regulating cellular growth and tissue development,and a key player of the innate tumor-suppression mechanism.Defects or inactivation of the apoptotic pathway is a major driving force for malignant transformation allowing neoplastic cells to survive beyond their normal lifespans,permitting tumor metastasis and development of resistance to anti-cancer drugs.With its importance in oncogenesis,targeting various aspects of apoptosis remains popular target of many cancer treatment strategies.Indeed,all current clinically used anti-cancer drugs exert their biological effects via the restoration of apoptotic signaling pathways towards normality as to eliminate malignant cells.Despite demonstrating potent and promising anti-tumorigenic effects against myriad of cancers,the effect of LY on osteosarcoma have yet to be established.Thus in this study,we investigated the effects of LY on osteosarcoma in vitro and in vivo,and the mechanisms via which it exerts these effects.This study is composed of three part as follows:(1)Osteosarcoma cell lines are used to investigate the inhibition effects of LY on osteosarcoma cells proliferation and induction of apoptosis and cell cycle arrest of osteosarcoma cells.(2)The research of mechanism of LY's inhibition on osteosarcoma cells.(3)Further verification of LY's effects on osteosarcoma in vivo through using xenograft osteosarcoma mouse model.Part I:Lycorine inhibits proliferation and induces apoptosis and G1 cell cycle arrest in human osteosarcoma cells in vitroObjective:To investigate lycorine's effects on osteosarcoma cell proliferation,apoptosis and cell cycle.Methods:Osteosarcoma cells were treated with different concentration of LY for 24h and 48h before the examination of LY's cytotoxicity.Colony-forming assay was used to further investigate LY's repression on osteosarcoma cell proliferation.Osteosarcoma cells were treated with different concentration of LY(0,2.5,5 and 10?M)for 24h or treated with 10?M for different time duration(0,16h,32h,and 48h)before the examination of apoptosis and cycle arrest through flow cytometry to test if LY exhibited its effects by dose-or/and time-dependent manner.The expression of apoptosis-and cell cycle-associated proteins were tested through western blot.Results:In this study,we found that LY showed inhibition on osteosarcoma and got the IC50 of LY on four different osteosarcoma cell lines.LY showed stronger cytotoxicity on SJSA-1 and U20S than HOS and MG63.LY exhibited dose-dependent and time-dependent cytotoxic effects on human osteosarcoma cell-lines SJSA-1 and U20S,induced G1 phase cell cycle arrest and cellular death via apoptosis.LY also induced the expression of Bax which is associated with apoptosis and P21 which is a robust inhibitor of cell cycle programming and the cleavage of caspase-3 and PARP.Conclusion:Collectively,our data suggests that LY exhibit dose-dependent and time-dependent cytotoxic effects on human osteosarcoma cell-lines SJSA-1 and U20S,induced G1 phase cell cycle arrest and cellular death via apoptosis.Part ?:Lycorine inhibits proliferation and induces apoptosis and G1 cell cycle arrest in human osteosarcoma cells in vitroObjective:To investigate the mechanism of lycorine's inhibition on proliferation and induction of apoptosis and G1 cell cycle arrest in human osteosarcoma cells.Methods:We investigated the phosphorylation change of JNK,P38 and ERK in osteosarcoma cells after LY treatment by using western blot.Immunofluorescence was used to examine the location of P53in osteosarcoma cells before and after LY treatment.As we found LY mainly promoted the phosphorylation of P38,the P38 inhibitor SB203580 was used to test LY's effects.We also examined the production of reactive oxygen species(ROS)in osteosarcoma cells after LY treatment and the use of ROS scavenger NAC to verify it.Results:Lycorine treatment of osteosarcoma cells promoted the phosphorylation of P38,but had no significant effect on the phosphorylation of JNK and ERK.The phosphorylation of P38 by lycorine was partially antagonistic to the P38 specific inhibitor SB203580,and SB203580 partially antagonistic to the lycorine-induced apoptosis of osteosarcoma cells and G1 phase block.Lycorine can promote the phosphorylation of protein P53 and promote its entry into the nucleus.Lycorine can promote the production of reactive oxygen species in osteosarcoma cells,and the use of reactive oxygen scavenger NAC can antagonistic lycorine-induced apoptosis and G1 phase arrest of osteosarcoma cells,as well as antagonistic phosphorylation of P38 and P53.Conclusion:Lycorine induces the robust production of ROS,which further lead to the phosphorylation of P38 followed by P53 phosphorylation.Then phosphorylated P53 translocates to nucleus and promotes the expression of Bax and P21,the cleavage of caspase-3 and PARP.As a result,apoptosis and cell cycle arrest occur.Part ?:Lycorine inhibits proliferation and induces apoptosis and G1 cell cycle arrest in human osteosarcoma cells in vivoObjective:To investigate lycorine's effects on human osteosarcoma cells in vivo.Methods:A volume of 100?L U20S cell suspension(density of 1x107 cells/mL)in cold PBS was injected subcutaneously into the dorsal area of BALB/c-nu mice.When the tumors in the dorsal area were macroscopically observed,mice were randomly separated into four groups:control group(injected with PBS)and three LY-treated groups containing 5 mg/kg/d,10 mg/kg/d or 15 mg/kg/d respectively(n=6 mice per group).The mice then received intraperitoneal injections of LY or PBS every day for 25 days.Mice body weight and tumor volume were measured every five days during the process.After 25 days,all mice were sacrificed,and the xenograft tumors were finely excised,weighed,frozen in liquid nitrogen and processed for subsequent protein extraction or fixed for immunohistochemical analysis.Results:Compared with PBS-treated control,the tumor volumes and weights of LY-treated groups were significantly reduced.There were no significant differences in body weights of mice throughout experimental period nor did we observe any cytotoxic effects of LY on normal tissues(heart,kidney,liver and lung)in non-tumor bearing mice.Conclusion:Lycorine can also inhibit osteosarcoma cells in vivo.