Anti-tumor Effect Of Evodiamine Derivatives And Their Nano Preparations On H1688 Small Cell Lung Cancer Cells

[Objective] To study the anti-tumor activity and mechanism of two different derivatives of evodiamine(Evodiamine nitro compound,END and Evodiamine amino derivative,EDA)and their nano-formulation evodiamine nitro derivative phospholipids(Evodiamine nitro derivative complex,ENPD and Evodiamine amino derivative complex,EAPD)on small cell lung cancer H1688 cells.[Methods] H1688 small cell lung cancer cell lines were cultured in vitro for experiments.Cell viability was measured by MTT at different time points after different concentrations of EVO,END,EAD,ENPD and EAPD.The apoptosis of H1688 cells and the mitotic cycle were investigated by flow cytometry.Fluorescence probe staining was used to investigate the changes of reactive oxygen species,calcium ion concentration and mitochondrial membrane potential in H1688 cells.The uptake behavior and main uptake sites of fluorescent-labeled nano-phospholipid complexes in cells were observed by means of organelle co-localization.Western blot was used to detect apoptosis-related proteins such as caspase 12,caspase 9,caspase 3,and cyt C,and endoplasmic reticulum stress-related proteins such as Chop and bim,and the expression of autophagy marker protein LC 3.Then the tumorigenesis in nude mice and H&E staining were used to further investigate the anti-tumor effects of evodiamine derivatives and their nano-phospholipid complexes on H1688 cell line of small-cell lung cancer in vivo.[Results] MTT results showed that END,EAD and their nanophospholipid complexes ENPD and EAPD inhibited H1688 small cell lung cancer cells in a time-and concentration-dependent manner,with the strongest effect of END and ENPD.Flow cytometry showed that END,EAD,ENPD and EAPD could induce apoptosis of H1688 cells,and block their mitosis in G2/M phase.Further detection of apoptosis-related indicators found that after treatment with the above drugs,the mitochondrial membrane potential decreased,the level of reactive oxygen species and intracellular calcium ion concentration increased in H1688 cells after treatment with the above drugs,showing obvious apoptosis.Western blot analysis showed that the expression of caspase 3/ 9/ 12 was up-regulated in H1688 cells after END and EAD treatment.However,caspase 3/ 9/ 12 was significantly up-regulated after ENPD and EAPD.And the release of Cyt C increased.At the same time,the autophagy marker protein LC 3 I/LC3 II value was significantly reduced after EVO,END,EAD,ENPD,EAPD treatment,especially after ENPD and EAPD.The above results indicated that END,EAD,ENPD and EAPD can induce apoptosis of H1688 cells through mitochondrial pathway,but END and EAD effects are much weaker than ENPD and EAPD.And autophagy can also be induced to occur.After investigating the behavior of nano-phospholipid complex in cells,it can be seen that ENPD and EAPD were mainly absorbed by the endoplasmic reticulum and aggregated in the cytoplasm.More aggregation of ENPD and EAPD in the vicinity of the endoplasmic reticulum induces more severe endoplasmic reticulum stress Tumor formation experiments in nude mice showed that all the compounds could inhibit weight loss and tumor growth in nude mice.[Conclusion] Both END and EAD,two different derivatives of evodiamine,had anti-tumor activity of H1688 in small-cell lung cancer cells,and the anti-tumor activity was more significant than that of EVO,the maternal drug,the END effect is stronger.The nanophospholipid complex ENPD and EAPD enhance the anti-tumor effect of END and EAD.The main anti-tumor mechanism of each compound is the apoptosis of H1688 cells induced by endoplasmic reticulum stress-mitochondrial pathway,which is also related to the enhancement of drug-induced autophagy.