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BMB Rep. 2014;47(5):274-9.

Establishment and characterization of bortezomib-resistant U266 cell line: constitutive activation of NF-κB-mediated cell signals and/or alterations of ubiquitylation-related genes reduce bortezomib-induced apoptosis.

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제1저자 박주원 · 교신저자 윤성수

PMID 24286313 PMC4163865 DOI 10.5483/bmbrep.2014.47.5.134 IF 4.1 혈액종양내과
소속 (3)
  1. Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea.
  2. Biomedical Research Institute, Seoul National University Hospital, Seoul 110-799, Korea.
  3. Cancer Research Institute, Seoul National University College of Medicine; Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-799, Korea.

Abstract

Bortezomib has been known as the most promising anti-cancer drug for multiple myeloma (MM). However, recent studies reported that not all MM patients respond to bortezomib. To overcome such a stumbling-block, studies are needed to clarify the mechanisms of bortezomib resistance. In this study, we established a bortezomib-resistant cell line (U266/velR), and explored its biological characteristics. The U266/velR showed reduced sensitivity to bortezomib, and also showed crossresistance to the chemically unrelated drug thalidomide. U266/velR cells had a higher proportion of CD138 negative subpopulation, known as stem-like feature, compared to parental U266 cells. U266/velR showed relatively less inhibitory effect of prosurvival NF-κB signaling by bortezomib. Further analysis of RNA microarray identified genes related to ubiquitination that were differentially regulated in U266/velR. Moreover, the expression level of CD52 in U266 cells was associated with bortezomib response. Our findings provide the basis for developing therapeutic strategies in bortezomib-resistant relapsed and refractory MM patients.

Figures

Fig. 1.
Fig. 1. U266/velR reduced sensitivity to bortezomib. (A) Flowchart for isolation of resistant clone to bortezomib. Detailed description about the flowchart can be found in Materials and Methods. (B) After U266 colonies were visible to the eye on the agar surface, colonies were observed, using an inverted microscope with a 10X objective lens. (C) U266 and U266/velR cells were treated for 72 h, with either 10 nM bortezomib, or 160 μM thalidomide. A cell proliferation assay was performed, as described in Materials and Methods. Data represented the mean ± SEM of 3 independent experiments. (D) U266 and U266/velR cells were treated with indicated concentrations of bortezomib for 24 h, and each 30 μg of whole-cell extracts was analyzed for PARP, procaspase-3 and GAPDH, by Western blot. (E) U266 and U266/velR cells were treated with indicated concentrations of bortezomib for 24 h, and the cells were then harvested, and lysed. These cells lysates were subjected to caspase-3 activity assay, using caspase-3 colorimeric activity assay kit. Data represented the mean ± SEM of 3 independent experiments (*P ＜ 0.05; **P ＜ 0.01).
Fig. 2.
Fig. 2. NF-κB-mediated acquired bortezomib resistance in U266/velR. (A) U266 cells were treated with 2 nM bortezomib on an intermittent schedule (1 day with bortezomib, followed by 2 days without bortezomib). After 7, 14, and 21 days of post-treatment of bortezomib, Cells were lysed and subjected to immunoblotting with p-ERK, ERK, p-p65, IκBα, and GAPDH, respectively. (B) U266 and U266/velR cells were treated with the indicated concentrations of bortezomib for 24 h, and whole-cell extracts were subjected to immunoblotting with p65, IκBα, and GAPDH.
Fig. 3.
Fig. 3. U266/velR increased CD138- population. (A) After U266 and U266/velR cells were labeled with anti-human CD138, FACS analysis was performed, to determine CD138 expression of the cell lines. Percentage of CD138- U266 was represented by the mean ± SEM of 3 independent experiments. (B) CD138+ and CD138- U266 cells were isolated, using MACS. Cell proliferation was measured after CD138+ and CD138- cells were treated with 10 nM bortezomib for 72 h, using CCK-8 cell proliferation kit. Data represented the mean ± SEM of 3 independent experiments.
Fig. 4.
Fig. 4. Genetic characterization in U266/velR. U266 and over-expression of CD52 in U266 (U266/CD52) were treated with 10 nM bortezomib for 72 h, using CCK-8 cell proliferation kit. Data represented the mean ± SEM of 3 independent experiments.

그림 출처: PubMed Central 공개 논문 (PMC4163865)

그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
BMB reports · eISSN 1976-670X
MeSH
Antineoplastic Agents / pharmacology · Apoptosis / drug effects / genetics · Boronic Acids / pharmacology · Bortezomib · Cell Line, Tumor · Drug Resistance, Neoplasm* / genetics / physiology · Gene Expression Regulation, Neoplastic / drug effects · Humans · Multiple Myeloma / drug therapy / genetics / metabolism · NF-kappa B / metabolism · Pyrazines / pharmacology · Signal Transduction / drug effects · Syndecan-1 / metabolism · Thalidomide / pharmacology · Ubiquitination
Substances
Antineoplastic Agents · Boronic Acids · NF-kappa B · Pyrazines · SDC1 protein, human · Syndecan-1 · Thalidomide · Bortezomib
참고문헌
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