| Rabdosia rubescens belongs to the genus Rabdosia of Labiatae,and its main anti-tumor active components are diterpenes,such as oridonin,Rabdosia rubescens An and so on.A large number of studies have shown that the above diterpenoids have good inhibitory effects on a variety of tumor cells,and these effects are related to cell cycle arrest,apoptosis,autophagy,regulation of oncoproteins,ROS aggregation,regulation of MAPKs and PI3K/Akt signal pathways and normal expression of Micro RNAs.However,the anti-tumor mechanism of this kind of compounds is very complex,and it is not completely clear at present.In the previous stage,our research group designed and synthesized 14-amino acid ester derivatives of Camellia oleifera.On this basis,this study carried out the anti-tumor activity test and metabonomic analysis of a series of derivatives,and further explored the anti-tumor molecular mechanism of these compounds.1.The results of anti-proliferative activity detection by MTT method for 72 h showed that the compound activity DOPT-043>DOPT-003>DOPT-010>Oridonin;the four compounds have good effects on Eca-109 and MGC-803,especially on Eca-109.The anti-proliferative activities of DOPT and DOPT-043 were tested for 24 h,48 h and 72 h.And we found that their anti-proliferative effects increased with the prolongation of the action time and the increase of the compound concentration.2.The screening criteria for differential metabolites in the non-target metabolome is FC>1.5 or FC<0.67,p<0.05 and VIP>1.Three groups of differential metabolites are obtained.The common feature is that the metabolites with relatively increased content are mainly fatty acids and polyamines,the metabolites with relatively decreased content are mainly amino acids and organic acids;the main affecting metabolic pathways are amino acid metabolism and sphingolipid metabolism.3.Cell colony formation experiments show that DOPT-043 can inhibit the formation of Eca-109 colonies,and the inhibitory effect is positively correlated with the concentration of DOPT-043.The cell scratch test showed that the migration ability of Eca-109 was negatively correlated with the concentration of DOPT-043.Flow cytometry experiments showed that the effect of DOPT-043 on Eca-109-induced apoptosis and intracellular ROS levels was proportional to the concentration,but its effect on cell cycle arrest was not obvious.WB results showed that the expressions of pro-apoptotic proteins Bax,Bid,Cyto-C,Cleaved-Caspase 3,Cleaved-Caspase 7,Cleaved-Caspase 9,and Cleaved-PARP were all up-regulated,and the expression of inhibitory apoptosis protein Bcl-2 was down-regulated;The expression of E-cadherin increased while the expression of interstitial marker proteins N-cadherin,MMP2 and Vimentin decreased;the expression of spermine oxidase SMOX was up-regulated.In summary,we found that: the 14-amino acid ester derivative of Aegilopsis caryophylla has good anti-tumor activity in vitro,among which DOPT-043 has the best activity,and this series of compounds is selective for Eca-109.The inhibitory effect of DOPT-043 on Eca-109 is time-and concentration-dependent,which can obviously promote the apoptosis of cancer cells and inhibit the migration of cancer cells.The possible anti-tumor mechanism of DOPT-043 is: by inhibiting tricarboxylic acid cycle energy supply,activating fatty acid oxidative metabolism to compensate energy supply and enhancing amino acid metabolism,resulting in increased conversion between spermine and spermidine,thereby generating a large amount of reactive oxygen species(ROS).ROS can further cause cellular DNA damage and promote the up-regulation of p53 expression;it can up-regulate the pro-apoptotic proteins Bax and Bid,down-regulate the inhibitory protein Bcl-2,promote the release of Cyto C,activate pro-enzyme Caspase 9,and then activate pro-enzyme Caspase 3 and Caspase 7,initiates the Caspase cascade to further cleave PRAP,triggering apoptosis and thereby exerting an anti-proliferative effect. |
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