Targeting PKM2 Promotes The Therapeutic Sensitivity Of Propranolol In Infantile Hemangioma And Its Underlying Mechanisms

Background and purpose:Infantile hemangioma(IH)is the most common benign tumour of infancy.Propranolol has now become the first-line therapy for IH.Recent studies have shown that reduced therapeutic sensitivity to propranolol in some patients has been an important factor in the failure of IH treatment,but the exact underlying mechanisms are yet to be fully elucidated.A number of researches have shown that an important key glycolytic enzyme,pyruvate kinase M2(PKM2),plays an important role in regulating tumor progression and therapeutic sensitivity.The purpose of this study was to elucidate the effect of PKM2 on the development of IH and to further investigate the mechanism of the role of PKM2 in the therapeutic sensitivity of IH.Methods:1.Hemangioma endothelial cells(HemECs)were firstly isolated and cultured,and HemECs were transfected with PKM2 overexpression or knockdown lentivirus,and then the effects of knockdown or overexpression of PKM2 on the biological behaviour of HemEC cells were examined by CCK assay,EdU staining,cell scratching assay and tuber formation assay.Murine hemangioma model was constructed to further verify the regulatory role of PKM2 on hemangioma progression through in vivo experiments.2.To detect the expression of PKM2 in IH tissue samples with different sensitivity to propranolol using immunohistochemistry.HemECs were transfected with PKM2 overexpression lentivirus,and then the effects of propranolol on the proliferation and apoptosis of HemECs overexpressing PKM2 were detected by CCK assay,Western Blot and flow cytometry;the inhibitory effects of the combination of shikonin and propranolol on PKM2 overexpressing HemECs were examined by CCK assay,Western Blot and flow cytometry.Murine hemangioma model was constructed,and the inhibitory effects of propranolol and shikonin on hemangiomas were further verified by in vivo experiments.Results:1.In vitro experiments confirmed that the proliferation,migration and angiogenic ability of HemECs were inhibited after knocking down PKM2 in HemECs;in contrast,the proliferation,migration and angiogenic ability of PKM2 overexpressing HemECs were significantly increased.In in vivo experiments,the PKM2 knockdown group showed significantly lighter tumor colour,lower microvessel density(MVD)values and lower VEGF expression in tissue samples than the negative control group,while the PKM2 overexpression group showed significantly darker tumor colour,higher MVD values and higher VEGF expression in tissue samples than the negative control group.2.In IH tissue samples with different sensitivity to propranolol,PKM2 expression was found to be significantly higher in the samples with reduced sensitivity.HemECs overexpressing PKM2 showed reduced sensitivity to propranolol and increased IC50 concentrations of the drug,and apoptosis was significantly reduced compared to the control group.When combined with shikonin,the sensitivity of HemECs overexpressing PKM2 to propranolol was significantly increased,and apoptosis was significantly higher than that of the propranolol alone group.In vivo experiments confirmed that the tumour colour faded more significantly,the MVD value decreased and the expression of apoptosis-associated proteins Bax and Cleaved caspase3 increased in tissue samples after the combination of propranolol with shikonin compared to propranolol alone.There was no significant decrease in body weight in all groups of nude mice,and no significant structural damage to the heart,lung,liver,kidney and spleen tissues in all groups.Conclusion:PKM2 promoted the proliferation,migration and angiogenesis of HemECs,and PKM2 significantly promoted IH progression in vivo.When combined with shikonin,it significantly enhanced the inhibitory effect of propranolol on HemECs,which was further confirmed in in vivo experiments,improving the therapeutic sensitivity of propranolol on IH without significant systemic toxicity,with potential clinical application.