PNA-TPP Targeting To Inhibit Mitochondrial Fusion Of Lung Adenocarcinoma Stem Cells In Tumor-Bearing Model To Enhance Sensitivity Of Cisplatin

In recent decades,lung cancer has shown a gradually increase in the morbidity and mortality,which has become one of malignancies serious threat to people’s health.Especially,the incidence of lung adenocarcinoma increased rapidly,which has replaced the lung squamouscarcinoma as the most common pathological types nowadays.Although chemotherapy still plays a significant role in lung cancer treatment,the survival and prognosis of the patients are not ideal.Now it is generally believed that the existence of lung cancer stem cells which leads to chemotherapy resistance,recurrence and metastasis.Interestingly,mitochondrial morphology and distribution of continuous dynamic changing organelles,within fine regulation of the mitochondrial inner and outer membrane fusion and fission,which may play an key role in energy metabolism, mt DNA stability and integrity, structural integrity,invasion and metastasis,in addition,many studies illustrated that mitochondrial fusion may act an important role in resistance to the apoptosis that chemotherapeutic drug induced.In our previous study,the lung cancer stem cells presented more active mitochondrial fusion phenomenon than general lung cells, which might become a hot therapeutic target.Therefore,targeting to inhibit mitochondrial fusion of lung cancer stem cells,which might enhance therapeutic sensitivity of cisplatin and improve the survival rate.Therefore, on the basis of previous study, our study aims to combined peptide nucleic acid- triphenyl phosphate complexes with cisplatin in the treatment of immunodeficient mice tumor model, to further verify the peptide nucleic acid-triphenyl phosphate complex could target to inhibit mitochondrial fusion and promote chemosensitivity, which throws light on targeting therapy of lung cancer stem cells.Objective:1. To investigate the mitochondrial energy metabolism-related features and expression mitochondrial fusion related protein of lung adenocarcinoma stem cells after interfering with peptide nucleic acid- triphenylphosphine complexes.2.By constructing tumor-bearing model, to explore the influence of peptide nucleic acid-triphenyl phosphate complexes on targeting to inhibit mitochondriafusion as well as the efficacy of cisplatin in the treatment of tumor-bearing mice.Methods:A549 cell spheres were formed through serum-free culture.Peptide nucleic acid-triphenylphosphonium(PNA-TPP) complexes were constructed and synthesized by solid phase method,observing the transfection of PNA-TPP and morphological changes of mitochondria in lung cancer stem cells under Laser Confocal Microscop(LCM). To explore changing of the content of ATP, and the expression of mitochondrial energy metabolism protein(ATP6 and ND6) and mitochondrial fusion related protein MFN1,MFN2,OPA1 and ATAD3 A after the transfection and interference of PNA-TPP,The appropriate number of A549 cells and A549 sphere cells were injected into NOD-SCID subcutaneously of immunodeficient mice respectively, Combining PNA-TPP with cisplatin to treat tumor bearing mice, to assess the tumor volume changes and treatment effect, to detect tumor tissue ATP,ROS and cytochrome c, to investigate the expression of fusion protein in tumor tissues in Immuno Histochemistry(IHC).Result:1. After the design and synthesis of PNA-TPP complex,high pressure liquid chromatography analysis showed, a single peak presented at 14.44 min, no significant impurity peak,The mass spectrometry analysis showed: the complex molecular weight was 4398.7, which met the design requirements.2. A549 cell spheres were induced by A549 cells in serum-free medium(SFM),After transfecting with PNA-TPP,Cellular ATP content decreased(P<0.05),Laser scanning confocal microscope showed mitochondrial division level increased,consistent with mitochondria,Western Blot showed mitochondrial protein ATP6 and ND6 expression significantly inhibited(P <0.05), Western Blot showed mitochondrial fusion related protein MFN1, MFN2, OPA1 and ATAD3 A protein expression decreased(P <0.05).3. Nude mice were constructed by A549 cells and A549 spheres.Compared with the saline group and cisplatin treatment group, treating with PNA-TPP plus cisplatin in tumor-bearing mice, the tumor volume were significant smaller in the fourth and the fifth week(P<0.05),tissue ATP content decreased(P<0.05), PNA-TPP were consistent with the distribution of mitochondria in tumor tissue,Immunohistochemical showedmitochondrial energy metabolism protein ATP6 and ND6,mitochondrial fusion protein expression inhibited in tumor tissue(P<0.05),ROS levels increased significantly(P<0.05), cytoplasmic cytochrome c increased significantly in tumor tissue(P<0.05), the apoptosis cells increased significantly in TUNEL detection.Conclusion:1.compared with A549 cells, mitochondrial fusion related protein expressed higher in A549 spheres, After interfering with PNA-TPP, energy metabolism reduced,mitochondrial fusion protein inhibited significantly.2.PNA-TPP targeted to inhibit the mitochondrial fusion,which contributes to promote cisplatin chemotherapy sensitivity in lung cancer cells and lung adenocarcinoma stem cells.