In Figure?Figure5(B),5(B), Western blot analysis showed a remarkable decreased expression of MGr1-Ag/37LRP and inhibited the phosphorylation of FAK, AKT, and ERK, as well as subsequently downregulating expression of the Bcl-2 protein compared to the respective control groups. CAM-DR in gastric cancer. MGr1-Ag/37LRP might be a potential effective reversal target to MDR in gastric cancer. is still unsolved, mainly because these drug-resistant models lack consideration of the role of the RAF709 tumor microenvironment. Increasing evidence suggests that the tumor microenvironment is the primary site leading to relapse after chemotherapy. Adhesion tumor cells to ECM components, such as fibronectin via 1-integrin, have been shown to confer resistance to a host of chemotherapeutic drugs.1,2 This anti-apoptotic phenomenon, called cell adhesion-mediated drug resistance (CAM-DR) is a form RAF709 of de novo drug resistance.3C5 Therefore, identification of mediators of cell adhesion may elucidate novel targets for GC therapy and inhibition of these targets could potentially overcome CAM-DR. Our laboratory previously reported MGr1-Ag as an upregulated protein in GC drug-resistant cell line SGC7901/VCR,6,7 and was identified as the 37-kDa laminin (LN) receptor precursor (37LRP).8 It has been shown to exhibit high laminin-binding activity, which is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis.9,10 We first reported that MGr1-Ag/37LRP may promote MDR of GC cells by decreasing intracellular drug accumulation and inhibiting drug-induced apoptosis.11 Further study suggested that MGr1-Ag could prompt CAM-DR through interaction with LN. However, the MGr1-Ag-initiated intracellular signal transduction pathway is still unknown. Focal adhesion kinase (FAK) carries out proteinCprotein interaction adaptor functions at sites of cell attachment to the ECM, contributing to focal-adhesion scaffolding, and also transmits adhesion-dependent signals into the cell interior.12 Several studies have indicated that FAK has a direct role in tumor growth and survival by activating survival pathways of PI3K/AKT and MAPK/ERK.13,14 Here, we show that apoptosis induced by chemotherapeutic drugs may by partly inhibited by survival pathways of PI3-kinase/AKT and MAPK/ERK activated by the interaction of FAK and MGr1-Ag/37LRP after the adhesion of MGr1-Ag/37LRP to LN, which is MGr1-Ag/37LRP’s ligand in the ECM of the gastric tumor microenvironment. We undertook these studies to characterize the role and the molecular mechanism of MGr1-Ag/37LRP in CAM-DR of GC cells to implicate a potential effective reversal target to MDR of GC. Materials and Methods Assessment of tumor growth Approximately 1??106 SGC7901/VCR cells were inoculated s.c. with 0.1?mL Matrigel (Sigma-Aldrich, St. Louis, MO, USA) in the flank region of 6C8-week-old male athymic nude mice (Experimental Animal Center, Fourth Military Medical University, Xi’an, China) using a 27-gauge needle under halothane anesthesia. When tumors reached 200?mm3, mice were randomly selected for treatment with vincristine alone (VCR), MGr1-Ag/37LRP antisense oligonucleotide (ASO) plus vincristine (VAS), scrambled ASO plus vincristine (VNS), MGr1-Ag/37LRP siRNA vector plus vincristine (VSM), scrambled RNA vector plus vincristine (VSP), mAb MGr1 plus vincristine (VAb), control antibody MGb2 plus vincristine (VIg), or bearing tumor without any treatment (TB). Each experimental group consisted of 10 mice. After randomization, 10?mg/kg MGr1-Ag/37LRP or scrambled ASO, 0.2?mg/kg MGr1-Ag/37LRP JAM2 siRNA or scrambled RNA vector, and 100?mg/kg antibody MGr1 or MGb2 was injected i.p. once every 3?days for 36?days for treatment groups.15,16 A total of 0.6?mg/kg micellar vincristine was given i.v. three times per week from days 7 to 14 and from days 21 to 28.17 Tumor volume measurements were taken once every 4?days for 40?days and calculated by RAF709 the formula length??width??depth??0.5236 before being sampled.18 Data points were expressed as average tumor volume levels SE. All animal procedures were carried out according to the guidelines of the Chinese Council on Animal Care and with appropriate institutional certification. Half of the transplanted tumors in every group were dissected and fixed in formalin for immunohistochemical studies. Half of the tumors were also immediately harvested in cold isopentane, frozen in liquid nitrogen, and kept at ?80C for subsequent Western blot analysis. Statistical analysis Each.