The in Vitro and in Vivo Antitumor Effect Associating With the Hippo-yap Signaling Pathway of Gallic Acid and Its Combination With Icotinib Hydrochloride on the Non-small Cell Lung Cancer Cellular and Xenograft Tumor-bearing Nude Mouse Models
Authors: Wang Dapeng , Guo Yinghua , Liu Yuan , Zhang Bin , Bai Po , Su Xiaolei , Jiang Xuege , Han Sarengerile , Fang Tingzheng , Zhang Xuelin , Wang Junfeng , Liu Changting 
Published: June 2021

Abstract
Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used as first-line therapy for patients with EGFR mutant non-small cell lung cancer (NSCLC). However, EGFR-TKIs treatment of NSCLC will inevitably produce acquired drug resistance. It greatly limits the therapeutic effect of TKI. Recent studies have found that some natural drugs combined with TKI in the treatment of NSCLC may not only improve the efficacy but also reduce the occurrence of acquired drug resistance. This study intends to investigate the inhibitory effect of Gallic acid, (a natural plant extract) combined with EGFR-TKI on the growth of NSCLC cell lines and xenograft tumor-bearing nude mouse models and its possible mechanism.Methods: Immunohistochemical method was used to detect the expression of Caspase-3, Caspase-9, Bcl-2, Cox-2, YAP and p-YAP in tissues of human non-small cell lung cancer (NSCLC). NSCLC cell lines A549 and PC9 were co-cultured with Gallic acid and / or Icotinib hydrochloride to test the antitumor effect. Cell proliferation was measured by MTT assay. In addition, apoptosis was measured by flow cytometry (FCM). In addition, intracellular calcium concentration in A549/PC9 cells was detected by calcium fluorescence probe. Besides, cell migration was detected by Transwell assay. Moreover, expression of related key genes was detected by qPCR, and expression of Caspase-3, Caspase-9, Bcl-2, Cox-2, YAP and p-YAP was determined by Western blotting. The anticancer effect of Gallic acid combined with Icotinib hydrochloride in vivo was studied by xenograft tumor-bearing nude mouse models.Results: 6 cases of human non-small cell carcinoma were analyzed by immunohistochemical staining. As a result, the expression of Caspase-3, Caspase-9, Bcl-2, Cox-2, YAP protein in lung cancer tissues was lower than that in normal tissues. However, the expression of p-YAP protein in cancer tissues was higher than that in normal tissues; MTT analysis showed that Gallic acid had a good inhibitory effect on NSCLC. A549 and PC9 cells in a concentration-dependent manner; Gallic acid combined with Icotinib hydrochloride significantly decreased the cell survival rate compared with single drug intervention. Treatment with Gallic acid and/or Icotinib hydrochloride induced apoptosis, accompanied by increased expression of Caspase-3, Caspase-9, Bcl-2, Cox-2 and YAP, and inhibited cell migration by down-regulating p-YAP. Gallic acid combined with Icotinib hydrochloride significantly increased the concentration of intracellular Ca2+ compared with the control group. The involvement of Ca2+ in the process of apoptosis may be related to p-YAP, which may be the key to the apoptosis of lung cancer cells induced by Gallic acid combined with Icotinib hydrochloride. Gallic acid combined with Icotinib hydrochloride promoted apoptosis of A549 and PC9 cells by down-regulating the expression of phosphorylated YAP. Dephosphorylation of signal transduction leads to the formation and activation of YAP and enhances the anti-proliferation response. The combined administration of Gallic acid and Icotinib hydrochloride inhibited the growth of tumor and decreased the expression of p-YAP in the xenograft tumor-bearing nude mouse models.Conclusion: Gallic acid may enhance the proliferation inhibition and apoptosis induction effect of Icotinib hydrochloride on lung cancer cell lines. Besides, Gallic acid combined with Icotinib hydrochloride may be a potential treatment in the treatment of NSCLC. In addition, Gallic acid is promising as a potential antineoplastic drug in the treatment of lung cancer.