OBJECTIVE: To detect the changes of heat shock protein(HSP) expression in human hepatocellular carcinoma HepG2 cells after treated by quercetin through a proteomics strategy termed SILAC (stable isotope labeling by amino acids in cell culture)-MS (mass spectrometry). METHODS: HepG2 cells cultured in d3-labeled DMEM medium were passaged for more than ten generations to reach an enough high labeling ratio. MTT assay was used to assess the inhibitory effect of quercetin on proliferation of HepG2 cells. In SILAC, total protein was extracted from control HepG2 cells and those treated by 50 µmol/L quercetin for 48 h, and then mixed to a 1:1 ratio. After in-gel digestion and idenfication by LC-MS/MS analysis, quantification informations of changed proteins were acquired by searching on Mascot 2.0 program (MatrixScience Ltd., London) against SWISS-PROT protein database. To ensure a high confidence level for identification, those peptides with Mascot scores below the threshold value were excluded from analysis and not included in the list of quantified proteins (P < 0.01). Protein abundance was calculated as ratios of the peak intensity of the fragment ions from the labeled versus the unlabeled peptides. RT-PCR was uesd to verify the reliability of HSPs changes by quercetin treatment from the SILAC-MS results. RESULTS: After passaged for ten generations, the d3-labeling ratio was above 95%. MTT showed that quercetin inhibited the proliferation of HepG2 cells obviously, with a IC(50) close to 50 µmol/L, and in a dose-dependent and time-dependent manner. The MS showed that the expression of almost all heat shock family proteins was down-regulated a lot. The expression of HSP90 exposed to quercetin for 48 h was decreased to 49.3% of the normal HepG2 cells, and the expression of HSP70 was decreased to 43.6% of the normal Hep G2 cells. Quantitation information showed that the expression of HSP90α, HSP76, HSP60 and HSP27 was declined to 59.3%, 44.2%, 51.3% and 62.6%, respectively. Those results demonstrated that the quantification for changed protiens by SILAC-MS was correct. CONCLUSIONS: Quercetin can exert a significant inhibitory effect on whole expression of heat shock proteins in HepG2 cells. We suppose this maybe one of the pathways through which quercetin plays an important anti-tumor role. SILAC-MS is a reliale technique and can be used to quantify the changes of whole protein spectrum in HepG2 cells before and after treatment with some exogeneous factors.
OBJECTIVE: To detect the changes of heat shock protein(HSP) expression in humanhepatocellular carcinoma HepG2 cells after treated by quercetin through a proteomics strategy termed SILAC (stable isotope labeling by amino acids in cell culture)-MS (mass spectrometry). METHODS: HepG2 cells cultured in d3-labeled DMEM medium were passaged for more than ten generations to reach an enough high labeling ratio. MTT assay was used to assess the inhibitory effect of quercetin on proliferation of HepG2 cells. In SILAC, total protein was extracted from control HepG2 cells and those treated by 50 µmol/L quercetin for 48 h, and then mixed to a 1:1 ratio. After in-gel digestion and idenfication by LC-MS/MS analysis, quantification informations of changed proteins were acquired by searching on Mascot 2.0 program (MatrixScience Ltd., London) against SWISS-PROT protein database. To ensure a high confidence level for identification, those peptides with Mascot scores below the threshold value were excluded from analysis and not included in the list of quantified proteins (P < 0.01). Protein abundance was calculated as ratios of the peak intensity of the fragment ions from the labeled versus the unlabeled peptides. RT-PCR was uesd to verify the reliability of HSPs changes by quercetin treatment from the SILAC-MS results. RESULTS: After passaged for ten generations, the d3-labeling ratio was above 95%. MTT showed that quercetin inhibited the proliferation of HepG2 cells obviously, with a IC(50) close to 50 µmol/L, and in a dose-dependent and time-dependent manner. The MS showed that the expression of almost all heat shock family proteins was down-regulated a lot. The expression of HSP90 exposed to quercetin for 48 h was decreased to 49.3% of the normal HepG2 cells, and the expression of HSP70 was decreased to 43.6% of the normal Hep G2 cells. Quantitation information showed that the expression of HSP90α, HSP76, HSP60 and HSP27 was declined to 59.3%, 44.2%, 51.3% and 62.6%, respectively. Those results demonstrated that the quantification for changed protiens by SILAC-MS was correct. CONCLUSIONS:Quercetin can exert a significant inhibitory effect on whole expression of heat shock proteins in HepG2 cells. We suppose this maybe one of the pathways through which quercetin plays an important anti-tumor role. SILAC-MS is a reliale technique and can be used to quantify the changes of whole protein spectrum in HepG2 cells before and after treatment with some exogeneous factors.
Authors: Seyed Mohammad Ali Mirazimi; Fatemeh Dashti; Mohammad Tobeiha; Ali Shahini; Raha Jafari; Mehrad Khoddami; Amir Hossein Sheida; Parastoo EsnaAshari; Amir Hossein Aflatoonian; Fateme Elikaii; Melika Sadat Zakeri; Michael R Hamblin; Mohammad Aghajani; Minoodokht Bavarsadkarimi; Hamed Mirzaei Journal: Front Pharmacol Date: 2022-04-06 Impact factor: 5.988