Hypoxia-induced Dysregulation Of Local Renin-angiotensin System And Its Effect On Invasion Of Lung Cancer Cells

Background: Lung cancer is a leading cause of cancer deaths globally,invasion and metastasis is associated with treatment failure and poorprognosis. Local renin-angiotensin system (RAS), in addition to systemicRAS, may influence cancer risk, tumor cellular proliferation, apoptosis,angiogenesis, and inflammation. RAS components exist and are oftendysregulated in lung cancer tissues, however little is known about themechanism of this dysregulation. Hypoxia is a hallmark of solid tumors,intratumoral hypoxia affects every major aspect of cancer biology. RAScomponents are expressed in different human cancer tissues. Previous studyshave shown that hypoxia can regulate RAS expression in somatic cells andinfluence proliferation and migration. The aim of this study is to investigate:(1) influence of hypoxia on local RAS regulation in lung cancer cells,(2)influence of hypoxia on migration of lung cancer cells,(3) effect of Captopriland Losartan on hypoxia-induced migration of lung cancer cells.Methods:(1) In this study, Mouse Lewis lung carcinoma cells (LLCs) and A549human lung cancer cells were cultured under hypoxic conditions(2%O2-93%N2-5%CO2) in a hypoxia incubator or in the presence of ahypoxia mimetic agent, cobalt chloride (CoCl2). HIF-1α protein levels weredetected in LLCs and A549cells cultured in normoxia and hypoxia exposure.(2) We investigated (i)the expression of the RAS components includingAngiotensin II (AngII), angiotensin converting enzymes (ACE and ACE2)and angiotensin II receptors (AT1R and AT2R) in LLCs and A549cellsexposured to hypoxia in vitro and (ii) the effect of Captopril, an ACEinhibitor, and Losartan, an Angiotensin type I receptor blocker to RASexpression under hypoxia. AngII concentrations in the culture medium andcellular extract were detected by RIA kits. Real time PCR and Western blotanalysis were performed to detect the presence of ACE, ACE2, AT1R, andAT2R expression.(3)A549cells were pretreatmented with CoCl2. Migrationof A549cells was tested by transwell, levels of VEGF-a and MMP2weredetected by Western blot analysis, effects of ACE inhibitor and AT1R blockeron migration and VEGF-a, MMP2expression under hypoxia were tested.Results: HIF-1α protein accumulated in LLCs and A549cells exposured tohypoxia. Ang II concentration in cellular extract related to total cellularprotein is higher under hypoxia than normoxia, however in cell culturemedium there were no statistical differences simultaneously. Increased mRNA expression of ACE, ACE2, AT1R, and AT2R in LLCs and A549cellswere detected during hypoxia, which did not match with the protein variation.ACE protein expression was upregulated during hypoxia, however ACE2protein continously declined. CoCl2dose-dependently increased ACE anddecreased ACE2protein expression. AT1R and AT2R protein expression wasregulated in a bidirectional way during hypoxia. In the early phase (6h), theAT1R protein decreased while AT2R protein increased markedly. As hypoxiaexposure went on, AT1R protein expression began to rise and AT2R proteinexpression began to decline, both showing time-dependent regulation byhypoxia. Captopril and losartan both upregulated ACE2and AT2R proteinexpression in LLCs under hypoxia. Hypoxia exposure enhanced migrationand protein levels of VEGF-a and MMP2in A549cells, which were inhibitedby captopril or losartan.Conclusions: These results indicate that hypoxia exposure up-regulatedendogenous Ang II level, ACE, AT1R expression, and down-regulated ACE2and AT2R expression in lung cancer cells. ACEI and ARB inhibithypoxia-induced migration in A549cells, which might be ascribed todown-regulation VEGF-a and MMP2. Local RAS might be a potentialherapeutic target to hypoxic lung cancer cells.