Cardamonin Inhibits MTORC1 Activated By Amino Acids Through Raptor

Aim 1 To confirm that Raptor plays an important role in amino acids signaling that regulates the activity of mTORC1.2 To explore whether the effect that cardamonin(CAR)inhibits the proliferation of SKOV3 cells is related to the inhibition of amino acid-mediated activation of mTORC1.Methods 1 The expression of Raptor in SKOV3 cells was interfered by sh RNA Human ovarian cancer SKOV3 cells were infected with appropriate amount of Raptor-sh RNA lentivirus vectors.The infected SKOV3 cells were screened by puromycin,then Raptor expression level was detected by Western Blot to identify the interferential effect.2 Cell culture Human ovarian cancer SKOV3 cells and Lv-Raptor-sh RNA infected SKOV3 cells were cultured in High-glucose DMEM medium contained 10 % fetal bovine serum,100 ?g·m L-1 streptomycin and 100 U·m L-1 penicillin.Both two cell lines were cultured at 37 ? with 5 % CO2 and then digested by 2.5 % trypsin and passaged when the adherent ones grew to 80 % ~ 90 %.3 Groups and drugs intervention 3.1 Normal conditions group,CAR group(Cardamonin: 10,30 ?M),RAP group(Rapamycin: 0.1 ?M)and AZD group(AZD8055: 0.1 ?M)were set in SKOV3 cell line to detect expression of mTORC1 related protein.3.2 Normal conditions group,CAR group(10,30 ?M),Amino acid starvation group(-AA,150 min),amino acid stimulation group(+AA,150 min),RAP(0.1 ?M)+ AA group(150 min),AZD(0.1 ?M)+ AA group(150 min)and CAR(10,30 ?M)+ AA group(150 min)were set in SKOV3 cells(detecting colocalization of m TOR and LAMP2,the activity of mTORC1 and cells' proliferation)and Lv-Raptor-sh RNA infected SKOV3 cells(detecting the cells' proliferation).3.3 Normal conditions group,Amino acid starvation group(-AA,150 min),amino acid stimulation group(+AA,150 min),CAR(30 ?M)+ AA group(150 min)were set in Lv-Raptor-sh RNA infected SKOV3 cells and normal SKOV3 cells to measure colocalization of m TOR and LAMP2,and activity of mTORC1 signaling pathway.4 Measurements and methodology 4.1 Inverted microscope was used to observe the cells' morphology and growth;4.2 Fluorescence microscope was used to detect fluorescence expression rate of lentivirus infected SKOV3 cells;4.3 Laser scanning confocal microscope was used to observe colocalization of m TOR and LAMP2;4.4 MTT method was used to measure the cells' proliferation;4.5 Western Blot was used to detect the expression of m TOR,p-m TOR,Raptor,4E-BP1,p-4E-BP1,S6K1 and p-S6K1.Results 1 CAR strikingly inhibited the proliferation of SKOV3 cells induced by amino acids(P < 0.01).2 The expression of p-m TOR,p-S6K1 and Raptor in SKOV3 cells were significantly inhibited by CAR(P < 0.05)3 The sequences of recombinant lentiviral vector were correct.Fluorescence expression rates of SKOV3 cells in both Lv-Raptor-sh RNA group and negative control group were more than 95 % after screening by puromycin,and Raptor expression level in Lv-Raptor-sh RNA group was significantly lower than normal control group and negative control groups(P < 0.01).4 CAR strikingly inhibited the proliferation of SKOV3 cells and Lv-Raptor-sh RNA infected SKOV3 cells in the presence of amino acids with a higher inhibition ratio in former one(P < 0.01).5 CAR significantly inhibited colocalization of m TOR and LAMP2 at lysosomal membranes and the expression of p-m TOR and p-S6K1 in amino acids stimulating condition(P < 0.05).6 The level of m TOR lysosomal location in Lv-Raptor-sh RNA infected SKOV3 cells was lower than that in SKOV3 cells,and it got much lower when Lv-Raptor-sh RNA infected SKOV3 cells were incubated with CAR(P < 0.05).7 The expression of p-m TOR and p-S6K1 in Lv-Raptor-sh RNA infected SKOV3 cells were lower than that in SKOV3 cells.CAR strikingly inhibited the phosphorylation of m TOR and S6K1 both in SKOV3 cells and Lv-Raptor-sh RNA infected SKOV3 cells which stimulated by amino acids,and had a higher inhibition ratio in SKOV3 cells.The expression of Raptor was much lower when Lv-Raptor-sh RNA infected SKOV3 cells were incubated with CAR(P < 0.05).Conclusions 1 CAR inhibited the SKOV3 cells' proliferation,which was closely related to the low expression of Raptor.2 CAR inhibited mTORC1 activited by amino acids through Raptor.