Molecular Mechanism Associated With Radiochemotherapy Resistance Of Lung Cancer And Tumor Targeted Nanotherapy

Lung cancer is a predominant cause of cancer death in both men and women.Despite recent progress,lung cancer therapeutic outcomes remain unsatisfactory.Lung cancer is heterogeneous and histologically divided into two types:small cell lung carcinoma(SCLC)and non-small cell lung carcinomas(NSCLC).NSCLC comprise 85%of lung cancer cases and constitute a heterogeneous population of squamous,adenocarcinoma,and large cell carcinomas.The main treatment methods of NSCLC include surgery,chemotherapy,radiotherapy and gene targeted therapy.Chemotherapy based on platinum-based drugs,particularly cisdiammine-dichloroplatinum(?)(cisplatin),alone or in combination with other methods,are most widely used in treating patients with nonresectable NSCLC.Cisplatin treatment induces DNA lesions,which can lead to cell cycle arrest,DNA repair,senescence,and apoptotic death,which is collectively called DNA damage response.Cisplatin treatment initially seems successful,but often chemoresistance develops and therapy fails.Radiotherapy(RT)is another treatment for lung cancer.RT is thought to locally control tumor cells by direct physical DNA damage,or indirect insults from reactive oxygen species.However,similar to cisplatin-resistance development,cancer cells may acquire resistance to radiation-induced apoptosis by dynamic interplay and regulation of multiple pro-survival factors,and therefore such radioresistant development leads to therapy failure.While many mechanisms in developing cisplatin-resistance and radioresistance have been suggested,it is not known whether these developments share the same molecular mechanisms and genetic modification profiles.In this study,we compared expression profiles of several DNA repair and anti-apoptosis associated molecules in cisplatin-resistant(A549CisR and H292CisR)and radioresistant(A549R26 and H292R22)cells vs.parental cells and tried to find the up-regulation profiles of these molecules in the cell sets which were distinctively different.Neuroendocrine differentiation(NED)is defined as morphological changes and positive staining with NED markers such as neuroamines,neuropeptides,neuron specific enolase,chromogranin A,synaptophysin,Pro-opiomelanocortin,and CD56.NED has been observed in solid tumors,including lung cancer,colorectal cancer,breast cancer,prostate cancer(PCa),and pancreatic cancer.Whether therapy resistance and NED is correlated depends on the cancer cell type.While it was suggested that NED is involved in chemoresistance in PCa,the SCLC that show high levels of NED characteristics are known to be extremely chemosensitive and radiosensitive.Whether NED of NSCLC is associated with therapy resistance and is a step towards more malignant behavior has not been resolved.In the second part,we tested whether NED is associated with radioresistant cells(A549R26-1 and H157R24-1).Chemotherapy is a widely adopted anticancer modality in the clinical setting,owing to the wide application and accessibility of chemo-drugs.However,inefficient drug delivery severely impedes the efficacy of chemotherapy,which leads to serious adverse effect and limited clinical outcomes.Nanomedicine is an attractive approach to overcome obstacles related with chemodrugs because it can improve the drug solubility,prolong the circulation time in the blood,enhance tumor accumulation,and enable controlled release.Nevertheless,cancer selectivity as well as sensitivity still remains critical challenges that would undermine the therapeutic efficacy of chemodrugs and cause undesired systemic toxicity.Herein,in the third part,we developed a dual hypoxiaresponsive drug delivery system to enable photodynamic therapy(PDT)-induced drug release and drug activation intermediated via PDT-induced hypoxia.Part ? Correlation between DNA repair and anti-apoptosis associated molecules and radiochemotherapy resistance in NSCLCObjectives:To investigate correlation between DNA repair and anti-apoptosis associated molecules and radiochemotherapy resistance in NSCLCMethods:we developed cisplatin-resistant(A549CisR and H292CisR)and radioresistant(A549R26 and H292R22)sub-line NSCLC cells by long term treatment of parental cells with cisplatin or radiation,and screened the expression of DNA repair and anti-apoptosis related molecules by qPCR analyses to reveal the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells,compared with parental cells.Western blot analysis of parental vs.resistant cells and the IHC staining of tumor tissues of A549P,A549CisR,and A549R26 cell-derived xenografts in mice were used to confirm the results.Finally,specific inhibitors of the up-regulated moleculars or specific molecular knocked down cells were used to verify the recovery of cisplatin resistance or radiosensitivity.Results:Our data showed no cross-resistance between two sets of cell lines,indicating that molecular mechanisms of developing each resistance may be different.In qPCR analyses of screening DNA repair and cell survival-associated molecules,we identified NF?B and TNFa as the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells,respectively,compared with parental cells.Western blot analysis of parental vs.resistant cells and the IHC staining of tumor tissues of A549P,A549CisR,and A549R26 cell-derived xenografts in mice confirmed such results.Studies using specific inhibitors of NF?B and YNF? and experiments using NF?B and TNF?-knocked down cells showed that inhibition or knockdown of NF?B overcame cisplatin-resistance,while inhibition or knockdown of TNF? increased radiosensitivity of radioresistant lung cancer cells.Conclusions:NF?B and TNFa may be used as markers of the prognosis/diagnosis of individual resistance development during lung cancer treatmentPart ? Correlation between neuroendocrine differentiation and radioresistance in NSCLCObjectives:To investigate the correlation between neuroendocrine differentiation and radioresistance in NSCLCMethods:We developed radioresistant cell lines A549R26-1 and H157R24-1,and verified whether morphological changes of NED exist in these two cell lines.We also detected whether there is higher expression of NED markers in radioresistant A549R26-1-derived tumor tissue than in parent cell-derived tumor tissue by IHC staining.Next,we examined the levels of cAMP and IL-6 by Western blot analysis and ELISA.At the same time,inhibitor intervention and qPCR were used to detect the related molecular signal pathway.Results:We observed higher NE-like features in radioresistant A549(A549R26-1)and H157(H 157R24-1)cells than in parental cells.Also higher NED marker expressions were detected in A549R26-1 cell-derived tumors than in A549 cell-derived tumors.In mechanism studies,we found that NED induction in A549R26-1 and H157R24-1 cells was accompanied by increased intracellular cAMP and IL-6 levels.Treatment of radioresistant lung cancer cells with the inhibitor(SQ22536)of acetylate cyclase(AC)which is the enzyme responsible for the cAMP production,or the neutralizing antibody(Ab)of IL-6,resulted in decreased NE-like features in radioresistant lung cancer cells.In addition,we found MEK/Erk is the signaling pathway that triggers the cAMP-and IL-6-mediated NED induction in radioresistant lung cancer cells.Finally,we found that MEK/Erk signaling pathway inhibition decreased NED in radioresistant cells.Radioresistant lung cancer cells exhibiting high NE-like features also showed higher radioresistance and higher metastatic potential than parental cells.When we inhibited cAMP-,or IL-6-mediated pathways,or the downstream MEK/Erk signaling pathway,radiosensitivity of radioresistant lung cancer cells was significantly increased and their metastatic potential was significantly reduced.In vivo mouse studies,reducing NED by treating mice with the MEK/Erk inhibitor increased radiosensitivity.IHC staining of tumor tissues lowered expressions of the NED/epithelial-mesenchymal transition(EMT)/metastatic markers when mice were treated with the MEK/Erk inhibitor.Conclusions:We discovered that cAMP-and IL-6-mediated pathways are important in development of radiation-induced NED of NSCLC cells,so directly blocking these signaling pathways or alternatively targeting downstream MEK/Erk signaling pathways effectively inhibits the NED process and lowers the metastatic potential of these cellsPart III Dual hypoxia-responsive nanomedicine upon tumor targeted photodynamic therapy induced hypoxia aggravationObjectives:Developed a dual hypoxia-responsive drug delivery system which enabled photodynamic therapy(PDT)-induced drug release and drug activation intermediated via PDT-induced hypoxia.Verified the high efficiency and selectivity of antitumor therapy in vitro and in vivoMethods:We synthesized the amphiphilic polyethylenimine-alkyl nitroimidazole(PA)and hyaluronic acid-chlorin e6(HA-Ce6),which were self-assembled to tumor-targeting and hypoxia-dissociable nanoparticles(NPs),encapsulated bioreductive chemodrug,tirapazamine(TPZ).The size and morphology of nanoparticles were monitored by dynamic laser scanning(DLS)and transmission electron microscopy(TEM),respectively?MTT test was used to measure the antitumor effect of the drug delivery system in vitro.The antitumor effect in vivo of nanodrug delivery system was verified by tumor inhibition curve,hematoxylin and eosin(H&E)staining and TUNEL stainingResults:After systemic administration,the obtained PA/HA-Ce6@TPZ NPs enabled effective tumor accumulation due to HA-mediated cancer targeting.Upon receptor-mediated endocytosis,light irradiation(660 nm,10 mW/cm2)produced high levels of reactive oxygen species to mediate PDT and generated a severe local hypoxic environment to dissociate the NPs and selectively release TPZ,as a consequence of hypoxia-triggered hydrophobic-to-hydrophilic transformation of ANI.In the meantime,TPZ was activated under hypoxia,finally contributing to a synergistic anticancer treatment between PDT and hypoxia-strengthened bioreductive chemotherapy.Conclusions:We report the development of a dual hypoxia-responsive drug delivery system which demonstrates cooperative anticancer effect between PDT and PDT-activated bioreductive chemotherapy intermediated via PDT-induced hypoxia.With dual control over drug release and drug activity,this strategy enables highly selectively drug delivery in tumors to maximize the therapeutic efficacy and minimize the side toxicity.Finally,the work of this paper is summarized,and the future research topics are prospected.