To gather further insight into the interaction between P-glycoprotein (Pgp) and its substrates, 167 compounds were analyzed in multidrug resistant human colon carcinoma cells. These compounds were selected from the National Cancer Institute Drug Screen repository using computer-generated correlations with known Pgp substrates and antagonists. The compounds were prospectively defined as Pgp substrates if cytotoxicity was increased > or =4-fold by the addition of cyclosporin A (CsA) and as Pgp antagonists if inhibition of efflux increased rhodamine accumulation by 4-fold. Among the 84 agents that met either criterion, 35 met only the criterion for substrates, 42 met only the criterion for antagonists, and only seven met both criteria. Thus, compounds interacting with Pgp form two distinct groups: one comprising cytotoxic compounds that are transported and have poor or no antagonistic activity and a second comprising compounds with antagonistic activity and no evidence of significant transport. Vinblastine accumulation and kinetic studies performed on a subset of 18 compounds similarly differentiated substrates and antagonists, but inhibition of 3H-azidopine labeling and induction of ATPase activity did not. These data support an emerging concept of Pgp in which multiple regions instead of specific sites are involved in drug transport.
Development of colorectal cancer (CRC) involves a series of genetic alterations with altered expression of proteins and cell signaling pathways. Here, we identified that galectin‐4 (gal‐4), a marker of differentiation, was down‐regulated in CRC. The goal of this work was to determine the function of gal‐4 in CRC. Toward this goal, the human colon biopsies and tissue microarrays containing a gradient of pathology were analyzed for gal‐4 expression by immunohistochemistry. Cell proliferation, migration, motility, forced expression, knockdown, cell cycle and apoptosis assays were used to characterize gal‐4 function. Immunohistochemistry identified that gal‐4 expression was significantly down‐regulated in adenomas and was essentially absent in invasive carcinomas. Forced expression of gal‐4 in gal‐4 −ve cells induced cell cycle arrest and retarded cell migration and motility. Further, gal‐4 sensitized the cells to camptothecin‐induced apoptosis. Gal‐4 knockdown resulted in increased cell proliferation, migration and motility. Gal‐4 was found to be associated with Wnt signaling proteins. Finally, gal‐4 expression led to down‐regulation of Wnt signaling target genes. This study demonstrates that loss of gal‐4 is a common and specific event in CRC. This study also shows that gal‐4 exhibits tumor suppressive effects in CRC cells in vitro. Through its ability to interact with and down‐regulate the functions of Wnt signaling pathway, gal‐4 reveals a new dimension in the control of the Wnt signaling pathway. Thus, gal‐4 may prove to be an important molecule in understanding the biology of CRC.
The epithelial Na+ channel (ENaC) that mediates regulated Na+ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (INa) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline INa by approximately 50% and INa could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of α and γ ENaC were sequentially substituted with glycines. This scan yielded two valine residues in γ ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an ∼20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P1 position residue for PE and substitution of alanine 190 in the γ subunit eliminated INa activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the γ subunit (γTh) allowed for INa activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T176-S198) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the γTh but not the wild-type γ subunit. These results demonstrate that γ subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the γ subunit results in ENaC activation.
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