Years, lots of mAbs to HER2 extracellular domain have been made (Shawver et al., 1994;

Years, lots of mAbs to HER2 extracellular domain have been made (Shawver et al., 1994; Pedersen et al., 2015; Shen et al., 2011; Ceran et al., 2012; Fu et al., 2014; De Lorenzo et al., 2005). Two of them, trastuzumab and pertuzumab, had been approved by FDA for therapy of individuals with HER2 overexpressed breast cancer (AmiriKordestani et al., 2014). MonoclonalDepartment of Immunology, School of Public Wellness, Tehran University of Medical Sciences, 2Monoclonal Antibody Analysis Center, Avicenna Investigation Institute, ACECR, Tehran, Iran. For Esfenvalerate Technical Information Correspondence: [email protected] and [email protected] Asian Pacific Journal of Cancer Prevention, VolTahereh Soltantoyeh et alantibodies targeting HER2 affect cancer cell development by way of two big mechanisms: 1) direct mechanism through abrogating cell signaling, cell cycle arrest, preventing receptor dimerization and inducing receptor internalization and degradation; 2) indirect mechanism involving activation from the immune method including antibody L-Cysteine Cancer dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (Szymanska et al., 2016; Mortenson and Fu, 2013). Typically, mAbs to a variety of epitopes of HER2 might have distinctive effect on downstream signaling pathways (Yip et al., 2003) and inhibit cell growth far more potently than individual mAbs (Meng et al., 2016; Nahta et al., 2004). Amongst numerous signaling pathways that are activated by ErbB receptors, Ras, Raf, MEK, ERK12, PI3K and PLC1 pathways play essential roles in cell proliferation (Yarden et al., 2001; Zhou et al., 2013; Nahta et al., 2004; AppertCollin et al., 2015). In our prior research, we developed and characterized quite a few antiHER2 mAbs including two inhibitory (1T0 and 2A8) mAbs which displayed superior antitumor activity in combination with trastuzumab (Tahmasebi et al., 2013; Kazemi et al., 2011). In this study, we investigated the effect of these inhibitory mAbs at the same time as one particular stimulatory mAb (1H9) alone and in mixture with trastuzumab on two key intracellular downstream signaling pathways of HER2. In addition, we evaluated the impact of mAbs on HER2 degradation which is another mechanism for cell growth inhibition (Mortenson and Fu, 2013).Materials and MethodsCell culture, antibody therapy and cell lysate preparation To determine the impact of mAbs therapy on AKT and ERK phosphorylation, 306 BT474 cells (National Cell Bank of Iran, Pasteur Institute, Tehran, Iran) were seeded in T25 culture flask and fed with RPMI1640 culture medium (Gibco, California, USA) containing 20 FBS fetal bovine serum (Gibco) and 10 ml insulin (Sigma Aldrich, St Louis, MO, USA), for 48 h at 37 inside a 5 CO2 humidified atmosphere. Then, cells were treated with 50 ml of 1T0, 2A8, 1H9 (created in our earlier works) (Tahmasebi et al., 2013; Kazemi et al., 2011), trastuzumab (GenentechRoche) and pertuzumab mAbs alone and 25 ml of each mAb in mixture with 25 trastuzumab for 24 h. Trastuzumab, pertuzumab (GenentechRoche) and their mixture were utilised as controls. Just after incubation, cells were washed with icecold PBS, trypsinized and lysates have been prepared applying mammalian protein extraction reagent (MPER, Thermo Fisher Scientific, California, USA). HaltTM protease and phosphatase inhibitor (Thermo Fisher Scientific) was added straight away prior to preparation of cell lysates based on the manufacturer’s instruction. Protein concentration of cell lysates was determined utilizing BCA protein assay kit (Thermo Fisher Scientific). Analysis of AKT.