Intracellular P-glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells

Abstract

The 2 clones, LoVo 5 and LoVo 7, derived from untreated LoVo WT human colon adenocarcinoma cells and exhibiting different sensitivity to doxorubicin (DOX), were compared in order to identify possible determinants of intrinsic drug resistance. A multidrug resistant variant cell line, selected from LoVo WT cells by continuous exposure to DOX (LoVo DX), was also included in the study. Analysis of the expression and organization of cytoskeletal elements by flow cytometry and fluorescence microscopy evidenced a positive correlation between vimentin expression and DOX resistance in LoVo 7 and LoVo DX cells, whereas differences in actin, tubulin or cytokeratin did not seem to relate to drug response. The expression and localization of different drug transporters commonly implicated in drug resistance, i.e., the MDR1 gene product P‐glycoprotein (P‐gp), the multidrug resistance‐related protein MRP and the lung resistance‐related protein LRP were also investigated by means of flow cytometry and fluorescence microscopy, following labeling with specific monoclonal antibodies. Surface expression of P‐gp was only detected in LoVo DX cells, which also exhibited increased MRP and LRP protein levels. However, significant amounts of P‐gp were found at intracellular sites in the intrinsically resistant LoVo 7 clone. Modulation of P‐gp function by cyclosporin A was found to alter DOX accumulation and efflux in LoVo 7 cells, indicating that intracellular P‐gp plays a functional role in drug trafficking and suggesting possible implications in determining the intrinsic resistance displayed by this clone. Int. J. Cancer 87:615–628, 2000. © 2000 Wiley‐Liss, Inc.

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

“…23 LoVo DX cells were about 100 times more resistant to DOX treatment than LoVo WT cells. Concerning the surface P-gp expression examined by flow cytometry, LoVo WT cells were negative while MDR variants were strongly positive for P-gp labeling.…”

“…Cell suspensions were then incubated for 30 min at 4°C with MAb MRK16 (Kamiya, Seattle, WA). 23 The intracellular content of thiol groups was determined using CM-H 2 DCFDA (Molecular Probes, Eugene, OR). 28 CM-H 2 DCFDA stocks (10 mM) were prepared in ethanol.…”

“…Moreover, MDR cells also exhibited increased MRP and LRP protein levels, while no significant differences were observed in both intracellular GSH content and glutathione-S-transferase activity compared to sensitive cells. 23,24 In our study, LoVo WT and LoVo DX cells were treated with subcytotoxic concentrations of BSAO and spermine. Our results showed that (i) LoVo DX cells are more sensitive than LoVo WT cells to the cytotoxic effect of the enzymatic oxidation products of spermine, (ii) cytotoxicity was due mainly to the presence of H 2 O 2 and completely inhibited in the presence of both catalase and NAD-dependent ALDH and (iii) mitochondria appear to play a pivotal role in determining the differential response between sensitive and drug-resistant cells.…”

The network of P-glycoprotein and microRNAs interactions

“…66,69 Several studies have suggested that intracellular P-gp also contributes to drug resistance. 58,70 Indeed, studies using leukemic cell lines and AML patient samples have shown that the levels of total P-gp (protein or mRNA) correlated better with drug efflux than those of surface P-gp protein. 69 Furthermore, a strong association between intracellular P-gp and intrinsic resistance was observed in a human colon carcinoma clone, which did not express P-gp on the plasma membrane.…”

“…69 Furthermore, a strong association between intracellular P-gp and intrinsic resistance was observed in a human colon carcinoma clone, which did not express P-gp on the plasma membrane. 70 Nevertheless, other studies claim that intracellular P-gp does not seem to have a (major) role in drug resistance. 61,64 Therefore, it is important to better understand how P-gp is regulated, which seems to be a complex and highly controlled process.…”