A novelin vitroplatform for the study of SN38-induced mucosal damage and the development of Toll-like receptor 4-targeted therapeutic options

Abstract

Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapyinduced gut toxicity. Currently, there are no validated in vitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5-15) were seeded into either 0.6 cm 2 , 0.4 mm pore mixed-cellulose transwell hanging inserts or 1.12 cm 2 , 0.4 mm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions in vitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells (P ¼ .0377). SN38 underwent more rapid degradation in the presence of cells (À76.04 AE 1.86%) compared to blank membrane (À48.39 AE 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.

Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration

“…Immunofluorescence staining was conducted as previously described by Wardill et al . 74 . The AQP1 primary antibody used for immunofluorescence was H-55 rabbit polyclonal (Santa Cruz Biotechnology, Dallas, TX, USA).…”

Dacomitinib‐induced diarrhea: Targeting chloride secretion with crofelemer

“…To test whether dacomitinib increased intestinal epithelial cell Cl – secretion, short‐circuit current ( I sc ) was measured in T84 cells in symmetrical physiological solutions in Ussing chambers. Once adequate resistance was reached (determined by TEER of > 1000 Ω cm 2 ), T84 monolayers were then placed onto 1.12 cm 2 aperture sliders (Physiologic Instruments; P2302) and mounted into Ussing chambers and continuously bathed in an oxygenated, glucose‐fortified Ringer's solution at 37°C. Cells were voltage clamped to zero potential difference by the application of short‐circuit current ( I sc ) and baseline established.…”

Orally Deliverable Nanotherapeutics for the Synergistic Treatment of Colitis-Associated Colorectal Cancer

“…CPT-11 (irinotecan) is a standard-of-care chemotherapeutic for the treatment of colorectal cancer in the clinic. However, this drug causes severe side effects, such as severe diarrhea and colitis, limiting dose intensification 8, 26. Treatment with SN38, which is the active metabolite of CPT-11, has been reported to produce inflammatory cytokines 27, 28.…”

“…Clinically, systemic administration of chemotherapy CPT-11 causes serious toxicity in patients with colorectal cancer. Enteritis, hematochezia, and diarrhea are the most common side effects, which impede long-term medications 8, 26. We anticipate that these orally deliverable nanoparticles have a high safety margin and could be used for the long-term treatment of CAC.…”