Low-Level Doxorubicin Resistance in Benzo[a]pyrene-Treated KB-3-1 Cells Is Associated with Increased LRP Expression and Altered Subcellular Drug Distribution

doi:10.1006/taap.2000.8903

Toxicology and Applied Pharmacology

Volume 164, Issue 2, 15 April 2000, Pages 134-142

https://doi.org/10.1006/taap.2000.8903 Get rights and content

Abstract

The P-glycoprotein (P-gp)-negative epidermoid pharyngeal carcinoma cells KB-3-1 were grown in 0.25 mM benzo[a]pyrene (BaP) for 3 months and increased resistance to doxorubicin, but not to vinblastine, colchicine, or cisplatin, was found. Doxorubicin resistance was not altered by cyclosporin, the P-gp inhibitor. Intracellular accumulation of BaP or calcein, a substrate for P-gp and multidrug resistance protein (MRP), was not altered by inhibitors of the P-gp and MRP. The expression of cytochrome P450 (CYP) 1A1, lung-resistance-related protein (LRP), P-gp, and MRP was investigated. Overexpression of CYP1A and LRP, on the mRNA and protein levels, was found. BaP-treated KB-3-1 cells remained P-gp negative while the level of MRP was not altered. Subcellular accumulation of BaP was found to be localized in the cytoplasm and minimal in the nuclei in BaP treated cells. In contrast, even penetration of BaP to the nuclei and cytoplasm was found in untreated cells. Subcellular distribution of doxorubicin was altered following BaP treatment with localized accumulation of the cancer drug in cytoplasmic organelles but not in the nuclei. Our data suggested that LRP might play a protective role against toxic compounds. The correlation of increased expression of LRP, but not P-gp nor MRP, with decreased doxorubicin accumulation in the nuclear target suggests a pivotal role of this perinuclear transporter in the MDR phenotype of P-gp-negative cancer cells. These results also propose an alternative mechanism of cancer drug resistance emergence, namely, induction of LRP activity following treatment with BaP, an environmental toxicant and a carcinogen.

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The strong staining of MRP1 at plasma membranes in several multidrug-resistant tumor cell lines explains its transporter function.26,27 The strong staining of MRP1 in the nuclei of MEC cells, but not on the nuclear membrane, indicates that MRP1 may not work as a nucleocytoplasmic transporter in MEC, as lung-resistance-related protein (LRP) did in an MDR phenotype of P-gp-negative cancer cells, which is located on the nuclear membrane, and leads to shifted subcellular distribution of chemotherapeutic agents.29 The reversed resistance of MC3/5FU-S cells to cisplatin and taxol, which are not substrates of MRP1,30 indicates that MRP1 may also affect MDR through non-transporter mechanism in MEC.
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And the intracellular fluorescent intensity from FITC-PEG-MWCNTs in these treated cells at these confocal layers was at similar level. Considering that the multidrug-resistant cancer cells, HepG2-DR and K562-DR, expressed much more drug efflux pumps (Kawai et al., 1994; Cheng et al., 2000) than the parental HepG2 and K562 cells, the similar amounts of PEGylated MWCNTs observed in both multidrug-resistant cells suggest that PEGylated MWCNTs were effectively taken up and accumulated in the multidrug-resistant cells. The overcoming of multidrug resistance made PEGylated MWCNTs a promising drug carrier for efficient drug delivery in multidrug resistance related cancer chemotherapy.
The acquisition of multidrug resistance poses a serious problem in chemotherapy, and new types of transporters have been actively sought to overcome it. In the present study, poly(ethylene glycol)-conjugated (PEGylated) multi-walled carbon nanotubes (MWCNTs) were prepared and explored as drug carrier to overcome multidrug resistance. The prepared PEGylated MWCNTs penetrated into mammalian cells without damage plasma membrane, and its accumulation did not affect cell proliferation and cell cycle distribution. More importantly, PEGylated MWCNTs accumulated in the multidrug-resistant cancer cells as efficient as in the sensitive cancer cells. Intracellular translocation of PEGylated MWCNTs was visualized in both multidrug-resistant HepG2-DR cells and sensitive HepG2 cells, as judged by both fluorescent and transmission electron microscopy. PEGylated MWCNTs targeted cancer cells efficiently and multidrug-resistant cells failed to remove the intracellular MWCNTs. However, if used in combination with drugs without conjugation, PEGylated MWCNTs prompted drug efflux in MDR cells by stimulating the ATPase activity of P-glycoprotein. This study suggests that PEGylated MWCNTs can be developed as an efficient drug carrier to conjugate drugs for overcoming multidrug resistance in cancer chemotherapy.
Cytotoxicity determination without photochemical artifacts
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A study was performed to improve cytotoxicity determinations by eliminating flavin-mediated photosensitization from tests with KB cells, NCI-H69 cells, P-glycoprotein expressing KBC5-8 cells, MRP1-expressing H69AR cells, and A240286S human lung adenocarcinoma cells. Growth inhibition by cis-platin, doxorubicin, etoposide, gemcitabine, taxol, vincristine, vinblastine, and vinorelbine was determined under flavin-protecting conditions using flavin-free culture media with fetal bovine serum as the only source of flavins. As compared to conventional tests, the IC₅₀ values determined under flavin-protecting conditions reflected increased apparent drug cytotoxicities, and were flawlessly reproducible. Flavin-mediated photosensitization should, therefore, be strictly eliminated from in vitro experiments involving cytotoxic and other drugs.
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In vitro studies have shown that technetium-99m methoxyisobutylisonitrile (Tc-99m MIBI) is a transport substrate for the P-glycoprotein (Pgp) pump. Therefore, Tc-99m MIBI uptake of tumors can be used to predict chemotherapy response in lung cancers. However, whether lung resistance-related protein (LRP) expression affects tumor accumulation and efflux of Tc-99m MIBI in lung cancers is not known. Our aim was to use Tc-99m MIBI uptake of tumors to predict Taxol based chemotherapy response of advanced non-small cell lung cancer (NSCLC) and to compare Pgp or LRP expression. Before chemotherapy with Taxol, 30 patients with stage IIIb or IV NSCLC were enrolled in this study. Chemotherapy response was evaluated in the third month after completion of treatment by clinical and radiological methods. No significant differences were found for other prognostic factors (age, sex, body weight loss, performance status, tumor cell type, and tumor stage) between the 15 patients with good response and the other 15 patients with poor response. Early chest single photon emission computed tomography (SPECT) was performed 10 min after intravenous injection of Tc-99m MIBI. Early tumor-to-normal lung (T/L) uptake ratios were calculated quantitatively on Tc-99m MIBI chest SPECT images. Immunohistochemical analyses were performed on multiple nonconsecutive sections of the biopsy specimens to determine Pgp and LRP expressions. The T/L uptake ratios on early Tc-99m MIBI chest SPECT images of 15 patients with good response were significantly higher than those of the other 15 patients with poor response. Significantly higher T/L uptake ratios were found in patients with negative than positive Pgp expression (p<0.05). However, no significant differences of T/L uptake ratios were found between patients with negative and positive LRP expressions (p>0.05). We concluded that Tc-99m MIBI chest SPECT could accurately predict Taxol base chemotherapy response of patients with advanced NSCLC. In addition, The Tc-99m MIBI tumor uptake was related to Pgp but not LPR expression in NSCLC.
PTEN associates with the vault particles in HeLa cells
2002, Journal of Biological Chemistry
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Evidence has been emerging recently that both MVP and the vault particle are frequently up-regulated in multidrug-resistant cancer cells (reviewed in Ref. 37). Although it has not been firmly proven that the drug-resistance phenotype is directly caused by the overexpression of vaults, some studies have demonstrated that reduction of MVP expression reversed the drug-resistance phenotype, and overexpression of MVP could induce drug resistance (48-50). Proteins responsible for drug resistance usually belong to the transmembrane transporters, such as P-glycoprotein (P-gp), MDR protein 1 (MRP1), and breast cancer-resistance protein (BCRP), which act by decreasing intracellular drug concentrations (reviewed in Ref. 37).
PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTEN-binding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. Co-immunoprecipitation confirmed the interaction between PTEN and MVP in transfected mammalian cells. More importantly, we found that a significant portion of endogenous PTEN associates with vault particles in human HeLa cells. Deletion mutation analysis demonstrated that MVP binds to the C2 domain of PTEN and that PTEN interacts with MVP through its EF hand-like motif. Furthermore, the in vitro binding experiments revealed that the interaction of PTEN with MVP is Ca²⁺-dependent.
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¹: To whom correspondence should be addressed at Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong. Fax: (852) 2788 7406. E-mail: [email protected].

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Regular ArticleLow-Level Doxorubicin Resistance in Benzo[a]pyrene-Treated KB-3-1 Cells Is Associated with Increased LRP Expression and Altered Subcellular Drug Distribution

Abstract

Biochem. Pharmacol.

Cell

J. Biol. Chem.

Adv. Enzyme Regul.

Biochem. Pharmacol.

Dev. Biol.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Structure

Blood

Eur. J. Cancer

Trends Cell Biol.

J. Biol. Chem.

Protein kinase C: A worthwhile target for anticancer drugs

Anticancer Drugs

Evidence that vault ribonuceloprotein particles localise to the nuclear pore complex

J. Cell Sci.

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cells

Br. J. Cancer

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1537 cells

Br. J. Cancer

Correlation between growth inhibition and intranuclear doxorubicin and 4′-iododoxorubicin quantitated in living K562 cells by microspectrofluorometry

Cancer Res.

Resistance to adriamycin: relationship of cytotoxicity to drug uptake and DNA single- and double-strand breakage in cloned cell lines of adriamycin-sensitive and resistant P388 leukemia

Cancer Res.

Regular Article
Low-Level Doxorubicin Resistance in Benzo[a]pyrene-Treated KB-3-1 Cells Is Associated with Increased LRP Expression and Altered Subcellular Drug Distribution