Anti-gastric Cancer Effect And The Mechanism Of Novel Mitochondrial-targeted Compound BODIPY-TPA

Aims: BTPA(BODIPY-TPA),a novel mitochondrial-targeted compound was synthesized and characterized in order to study the effect of BTPA on mitochondrial homostatsis and mitochondrial function,then further study the crosstalk between mitochondrial redox balance and anti-tumor effect to explore the possible mechanism of such action.It is expected to develop a low-toxic and high-effective anti-tumor compound that integrates diagnosis and treatment.Methods: Nanoparticles of novel mitochondrial-targeted agent BTPA was designed and synthesized,then characterized by UV absorption,fluorescence spectroscopy,nuclear magnetic resonance,mass spectrometry,and transmission electron microscopy.MitoTracker Red and Hoechst33342 fluorescent probe were used for co-localization to detect the mitochondrial targeting of BTPA.MCF-7,Patu8988,MFC,AGS and BGC-823 cells were treated with different concentrations of BTPA for 24 h.Cell viability was determined by MTT assay and the inhibition rate was calculated.The IC50 values were calculated and compared,and the selectivity of BTPA for normal human epithelial cells and tumor cells were compared.Tumor animal model was established for experiments in vivo.Different concentrations of BTPA and positive control drug pentafluorouracil(5-Fu)were intraperitoneally injected.After the mice was sacrificed,the tumor tissues and immune organs were used to evaluate the visceral index.The effects of BTPA on tumor tissues and PCNA were analyzed by HE staining and immunohistochemistry.The binding of BTPA to mitochondrial thioredoxin reductase TrxR2 was detected by molecular docking assay.The expression of TrxR2 in cells and tumor tissues was detected by western blot.The activity of total thioredoxin reductase TrxR was detected by the kit.The lipid peroxidation level of cells was detected by the MDA kit.The mitochondrial function-related experiments including JC-1 probe for mitochondrial membrane potential detection,and the kit for intracellular ATP levels detection and the Clark oxygen electrode for oxygen consumption of cellular respiration detection.Specific kits wer used to detect the levels of intracellular catalase,glutathione reductase and superoxide dismutase in order to study the effect of BTPA on mitochondrial redox balance.DHE and MitoSOX probes were used to detect the accumulation of reactive oxygen species in cytoplasm and mitochondria.Hoechst,Annexin V/PI were used to detect apoptosis,and NAC pre-protection was also used to analyze the mechanism of apoptosis.Western blot was used to detect the changes of apoptosis-related proteins and PI3K/Akt signaling pathway aims to study the specific mechanism of BTPAinduced apoptosis.Results: We obtained a uniform morphology of BTPA nanoparticles that selectively localize in the mitochondria of tumor cells.BTPA can effectively inhibit a variety of tumor cells while has less side effect on human normal cells.In vivo experiments showed that BTPA exhibited significant anti-proliferation effect on gastric cancer under the condition that it did not affect the normal immune organs(spleen,thymus,liver)of mice.Tumor tissue immunohistochemistry and HE staining results also confirmed the anti-proliferation effect of BTPA.Molecular docking experiments showed that BTPA has a certain binding affinity to mitochondrial thioredoxin reductase TrxR2,and western blot analysis showed that BTPA down-regulated TrxR2 expression in vitro and in vivo,but cytosolic thioredoxin reductase TrxR1 had no change.While the activity of the total thioredoxin reductase TrxR in the cells did not change much,the cells still produced strong oxidative stress because there is an obvious increase in lipid peroxidation levels.Mitochondrial function-related experiments showed that BTPA treatment produced significant mitochondrial dysfunction in gastric cancer cells,manifested by decreased mitochondrial membrane potential and decreased cellular ATP and suppressed cellular respiration.In addition,the cellular antioxidant system including catalase,glutathione reductase and superoxide dismutase levels were significantly down-regulated,and mainly showed down-regulation of mitochondrial antioxidants.Western blot analysis showed that the PI3K/Akt signaling pathway was also down-regulated,cell ROS and mitochondrial ROS were significantly accumulated,BTPA caused significant apoptosis,and mitochondria-mediated endogenous apoptosis.The pre-protection of NAC inhibited the production of this apoptosis.This indicated that oxidative stress is the direct cause of BTPA-induced apoptosis.Conclusion: BTPA has good mitochondrial targeting and has a good anti-gastric effect.This anti-tumor activity may be related to the destruction of mitochondrial redox balance,which is characterized by down-regulation of TrxR2 and down-regulation of mitochondrial antioxidant capacity.Strong oxidative stress further down-regulating PI3K/Akt signaling pathway,further aggravating intracellular accumulation of reactive oxygen species,which formed a vicious circle in the mitochondria then induced mitochondrial dysfunction and mitochondria-mediated endogenous apoptosis.