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Folate Receptor upregulation of tumor cells and its effect on folate conjugate uptake

Author: ZhangTong
Tutor: ZhangQiQing
School: Peking Union Medical College , China
Course: Biomedical Engineering
Keywords: folate receptor dexamethasone targeted therapy HeLa cells
CLC: R73-3
Type: Master's thesis
Year: 2010
Downloads: 363
Quote: 1
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Abstract

Background:Virtually all of the experimental approaches in folate receptor-targeted therapeutics and imaging depend on the ability of the target tissues to express an adequate level of the receptor. Indeed in all of the animal models of successful FR-targeted therapeutics, the target tumors express a uniform and optimal level of FR. However, quantitative studies of FR expression in human tumors have revealed considerable variability and non-uniformity in FR expression. A number of studies have shown that a variety of innocuous transcription modulators may be used to selectively up-regulate FRs in tumors that are positive for the receptors without causing de novo synthesis of the receptors in FR negative tissues. In particular, various nuclear receptor ligands, either alone or in combination with inhibitors of histonedeacetylase (HDAC), has proven to be useful modulators of FR expression and could be used in combination with virtually any FR-targeted therapeutic agent to improve outcome.Purpose:In the study, the endogenous FR-a in tumor cells was up-regulated by dexamethasone (Dex). The effect of Dex on the FR-mediated endocytosis was determined by Flow Cytometry.1. The effect of dexamethasone on FR-a expression of HeLa and SKOV-3 cellsThe FR-a expression of different tumor cell lines (HeLa, MCF-7 and SKOV-3) was evaluated by RT-PCR, Western Blotting and Flow Cytometry. The data showed that HeLa and SKOV-3 were FR-a positive, while no FR-a was detected of MCF-7 cells.The dexamethasone (dose range tested was 5 nM to 100 nM) showed no significant cytotoxity in HeLa and SKOV-3 cells by WST-1 assay. HeLa and SKOV-3 cells were treated with either vehicle alone or dexamethasone (5 nM to 100 nM) for 96 hours, at the end of which, mRNA for FR-a was measured by real-time reverse transcription-PCR, the total protein from cell lysates were measured by Western Blotting, the cells were also incubated with anti-FR antibody for the FCM assay. The results showed in a progressive increase in the expression of both endogenous FR-a mRNA membrane FR-a protein detected by FCM. However, in SKOV-3 cells, after the treatment of Dex, data showed that endogenous FR-a mRNA increased, while no significant change in the expression of membrane FR-a protein.2. The effect of folate receptor content on cell surface to targeting efficiency HeLa cells were incubated with coumarin 6 loaded folic acid conjugated micell (FM) or coumarin 6 loaded micell in folate-free RPMI 1640 media (with or without 1 mM folic acid) for a period time at 37℃. Cellular uptake was then examined by fluorescence microscopy or flow cytometry. The cellular uptake of FM was also determined with cells exposed for 96 hours to 100 nM Dex. Overall fluorescence was much greater in cells treated with FM than in those treated with M. Cellular uptake of FM was blocked by 1 mM free folic acid, indicating that uptake was mainly due to folate receptor mediated endocytosis. At the same time the uptake of FM was further increased when HeLa cells were primarily cultured with 100 nM Dex for 96 hours.

Folate Receptor upregulation of tumor cells and its effect on folate conjugate uptake

Abstract

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