Concurrent Treatment with Anti-DLL4 Enhances Antitumor and Proapoptotic Efficacy of a γ-Secretase Inhibitor in Gastric Cancer

Abstract

The Notch signaling pathway has been identified as a therapeutic target for cancers. γ-Secretase inhibitors (GSIs) have been progressively recognized as potential anticancer drugs. The present study aimed to investigate the effects of anti-delta like legend 4 (anti-DLL4) treatment on the anticancer efficacy of GSIs in gastric cancer. SGC-7901-GFP human gastric cancer cells were tested for DLL4 expression by rosette formation test and immunofluorescence, and then were treated with anti-DLL4 antibody N-[N-(3,5-difluorophenacetyl)-L-ananyl]-S-phenylglycine t-butyl ester (DAPT, a type of GSI), or a combination of anti-DLL4 antibody and DAPT. The effects of in vitro treatments on cell apoptosis, cell cycle, and cell invasion were analyzed. For in vivo study, an orthotopic mouse model of gastric cancer was established with green fluorescence expressing SGC-7901. Ultrasound targeted microbubble destruction was used to treat tumor-bearing mice with anti-DLL4 antibody conjugated microbubbles, DAPT, and a combination of the two. Real-time fluorescence imaging was performed to assess tumor cell inhibition in each group. Following in vivo treatments, apoptosis of tumor cells and the expression of apoptosis-related genes BAX, Bcl-2, and P53 were detected by TUNEL and immunohistochemical staining. In vivo combined treatment of anti-DLL4 and DAPT led to a higher rate of cell apoptosis and greater inhibition of cell invasion than that observed with DAPT treatment alone. DAPT and anti-DLL4 combination therapy resulted in decreased cell distribution at G1 phase and increased cell distribution at S phase, compared to the untreated control group (P < .01). In vivo combined therapy with anti-DLL4 and DAPT significantly increased tumor growth inhibition and tumor cell apoptosis when compared to DAPT therapy alone (P < .05). In addition, combined treatment significantly increased expression of BAX and P53 and reduced Bcl-2 expression (P < .05). Conversely, treatment with DAPT alone only increased expression of BAX and P53 (P < .05), suggesting that the reduction of Bcl-2 expression may play an important role in the synergetic antitumor and proapoptosis effects of the combined treatment. Concurrent treatment with anti-DLL4 enhances the antitumor and proapoptotic efficacy of the γ-secretase inhibitor in gastric cancer both in vitro and in vivo.

<p>The Role of DLLs in Cancer: A Novel Therapeutic Target</p>

“…89 DLL4/Notch signaling maintains the self-renewal and invasion ability of GC cells, which can be inhibited by DLL4 knockdown. 88,95,96 In contrast, DLL4 is expressed in the vascular endothelium of ccRCC. Endothelial DLL4 in ccRCC can on one hand promote tumor angiogenesis through VEGF activation, 92,[97][98][99] or activate Notch signaling in tumor cells, thus inducing hematogenous metastasis.…”

DR-5 and DLL-4 mAb Functionalized SLNs of Gamma-Secretase Inhibitors- An Approach for TNBC Treatment

“…On the other hand, naïve GSI only enhanced the expression of BAX and P53, suggesting that the reduced Bcl-2 expression had a significant function in synergistic antitumor and proapoptotic effects. 33 Drug delivery using SLNs is an accepted approach for targeted drug delivery to improve efficacy and reduce off-target side effects. SLNs as a nanocarrier, enhance the bioavailability, and provide control release.…”

“…On the other hand, naïve GSI only enhanced the expression of BAX and P53, suggesting that the reduced Bcl-2 expression had a significant function in synergistic antitumor and proapoptotic effects. 33 …”

“…[30][31][32] It was recently reported that a combination of DLL4 mAb with GSIs has synergetic proapoptotic effects. 33 Death receptor 5 (DR-5) is a member of tumor necrosis factor (TNF) receptor superfamily, overexpressed explicitly on the surface of TNBC cells. Therefore, it can be used as a target for effective drug delivery in TNBCs.…”

“… 30 - 32 It was recently reported that a combination of DLL4 mAb with GSIs has synergetic proapoptotic effects. 33 …”