The Role And Mechanisms Of Temozolomide-Perillyl Alcohol Conjugate In Mitophagy For Non-Small Cell Lung Cancer

Background:TMZ-POH,a novel temozolomide analog,is developed based on the conjugation of temozolomide(TMZ)and perillyl alcohol(POH).The anti-tumor activity of TMP-POH in non-small cell lung cancer mainly depends on its ability to induce the massive accumulation of reactive oxygen species(ROS)in tumor cells.Accumulated ROS can cause mitochondrial damage and imbalance between mitochondrial fusion and fission and then activate mitophagy to remove the dysfunctional mitochondria to maintain cell survival.Therefore,we propose a hypothesis that TMZ-POH induces a large accumulation of ROS in non-small cell lung cancer to play an anti-tumor role,which may be achieved by impairing mitophagy.Objective:We aim to clarify the role and mechanism of TMZ-POH in regulating mitophagy in NSCLC cell lines,and to observe the effects of TMZ-POH on apoptosis and radiation sensitivity in NSCLC cell lines.Methods : Western blot assay was used to detect proteins related to autophagy,mitochondrial fusion and fisson,lysosomal function and membrane transport.The autophagosomes,mitochondria and lysosomes were observed by transmission electron microscopy and immunostaining.Cell apoptosis and immunofluorescence probe were detected by flow cytometry.Results:In A549,SPCA1,H460 and H520 cell lines,TMZ-POH can significantly increase the expression of LC3B-? expression in a concentration-dependent manner.After using Baf.A1,Western Blot and immunofluorescence reveal that LC3B-? expression has no obvious change in TMZ-POH treatment group.Compared with the control group,BECN1 and MTOR pathway related proteins MTOR,pho-MTOR and P70S6 K were not significantly changed in the TMZ-POH treatment group.In A549 and SPCA1 cell lines,mitochondria were labeled with COX ? and TOM20,and was observed by immunofluorescence.The TMZ-POH was able to induce more damaged mitochondria.In the mitochondrial protein,TMZ-POH can induce LC3B-? significantly expression,but in the cytoplasmic protein TMZ-POH can only induce LC3B-? slightly expression.Immunofluorescence showed that TOM20 and Parkin had better co-localization with LC3B-? in the TMZ-POH treatment group.In A549 and H460 cell lines,the SQSTM1 expression were significantly up-regulated in the TMZ-POH treatment group.The effect of flow cytometry in detecting TMZ-POH was similar to that of baf.A1,which could significantly enhance the fluorescence intensity of MTG.In A549 cell lines,the co-localization relationship between lysosomes and mitochondria in the TMZ-POH treatment group was observed by immunofluorescence.The A549 and SPCA1 cells infected with mRFP-GFP-LC3 adenovirus were observed by immunofluorescence.The TMZ-POH treatment group showed the yellow fluorescence of autophagosomes instead of the red fluorescence of autophagosomes.In A549 and SPCA1 cells,the fluorescence intensity of LTR in the TMZ-POH treatment group was significantly decreased,and the expression of EEA1 and mature CTSD in the TMZ-POH treatment group was significantly down-regulated.In A549 cells,the co-localization relationship between LAMP1 and LC3 B in TMZ-POH treatment group was significantly decreased.Western Blot confirmed that the activity of RAB7 A in the TMZ-POH treatment group was decreased,and expressions of LAMP1,LAMP2,RAB7 A,RILP were significantly decreased,while the expression of RAB7 A in the TMZ-POH treatment group was up-regulated by Mevalonate pathway agonist MAV and GGOH.In A549 and SPCA1 cells,3MA can significantly upregulated the ability of TMZ-POH in inducing apoptosis.Compared with the 0Gy control group,the proportion of apoptosis in the TMZ-POH treatment group combined with 10 Gy radiation was significantly increased in a concentration-dependent manner.Conclusion:The TMZ-POH induces the aggregation of autophagosomes,which is involved in the occurrence of autophagy but not affected by the MTOR pathway.The TMZ-POH can lead to the imbalance between mitochondrial fusion and fission in NSCLC cell lines,damage the mitochondria,and then induce the occurrence of mitophagy.Meanwhile,TMZ-POH impairs Mitophagy flux by inducing lysosomal dysfunction and the expression of RAB7 A.More importantly,our data demonstrated TMZ-POH sensitized cancer cell to irradiation induced apoptosis.We clarified the mechanism of anti-tumor effects of TMZ-POH in NSCLC cell lines and demonstrated the possibility of this new compound as a potential radiotherapy sensitizer.