Dataset Information

Effects of hypoxia on human cancer cell line chemosensitivity.

ABSTRACT:

Background

Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia.

Methods

A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA).

Results

Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs.

Conclusions

A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.

SUBMITTER: Strese S

PROVIDER: S-EPMC3707755 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Publications

Effects of hypoxia on human cancer cell line chemosensitivity.

Strese Sara S Fryknäs Mårten M Larsson Rolf R Gullbo Joachim J

BMC cancer 20130705

<h4>Background</h4>Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditi ...[more]

PMID: 23829203

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Project description:A total of 20 KYSE human esophageal squamous cell carcinoma cell lines (KYSE-30, -140, -150, -170, -180, -200, -220, -350, -410, -450, -510, -520, -590, -770, -850, -890, -1170, -1190, -1250, and -2270) were kindly provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). The KYSE cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. The exponentially growing cultured cells (2 x 10^6) were collected after two-washings with PBS. Total RNA was prepared from the frozen cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA). The quality of the RNA was checked using Agilent Technologies 2100 Bioanalyzer (Agilent, Palo Alto, CA). CodeLink Expression Bioarray System (Amersham Bioscience, Tokyo, Japan) was used according to the manufacturerâs protocol. Briefly, first-strand cDNA was generated from 1 Âµg of total RNA for each cell line using reverse transcriptase and a T7 primer, and then second-strand cDNA was produced using DNA polymerase mix and RNase H. cRNA was generated via an in vitro transcription reacrion using T7 RNA polymerase and biotin-11-UTP (Perkin Elmer, Boston, MA). After quantified by spectrometry and qualified using Agilent Technologies 2100 Bioanalyzer (Agilent), 10 Âµg of cRNA was fragmented and hybridized to a Uniset Human 20K I Bioarray containing 19,981 human probes with 108 positive and 300 negative bacterial control probes. After hybridization, the arrays were rinsed and labeled with Streptavidin-Cy5, scanned using Agilent DNA Microarray Scanner (Agilent), then analyzed with CodeLink Expression Analysis Software ver.2.3. Expression levels were normalized to the median expression value of the whole array spots except for the bacterial controls. To explore the genes responsible for chemosensitivity in cancer cells, expression profiles of various cancer cell lines were examined. Statistically comparing them with the chemosensitivity measured by in vitro MTT assay in each cell line, several candidate genes were selected and predicition models for anticancer efficacy were established using their expression levels.

Project description:A total of 20 KYSE human esophageal squamous cell carcinoma cell lines (KYSE-30, -140, -150, -170, -180, -200, -220, -350, -410, -450, -510, -520, -590, -770, -850, -890, -1170, -1190, -1250, and -2270) and a non-cancerous esophageal epithelial cell (HEEC-1)were kindly provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). The KYSE cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. HEEC-1 cells were cultured in Keratinocyte SFM medium with growth supplement containing 2.5 mg EGF and 25 mg bovine pituitary extract in 500 mL liquid basal medium (GIBCO BRL, Rockville, MD) and expanded by passage twice in a week. The exponentially growing cultured cells (2 x 10^6) were collected after two-washings with PBS. Total RNA was prepared from the frozen cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA), and mRNA was purified using MACS mRNA Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the supplierâs protocols. RIKEN human 21K array containing 20,784 clones with positive and negative controls was used to analyze gene expression of 20 KYSE esophageal cancer cell lines using HEEC-1 as reference. The target DNAs used to construct human 21 K array were the glycerol stock of cDNA clones purchased from ResGen (Invitrogen Corporation, Carlsbad, CA)). One-microgram poly(A) RNAs from KYSE cells and the reference cells were labeled with Cy3-dCTP and Cy5-dCTP, respectively, by random-primed reverse transcription. Arrays were laser-scanned using ScanArray 5000TM confocal laser scanner (GSI Lumonics, Billerica, MA), and the images were analyzed using ScanAlyzeTM (Stanford University). All experiments were performed in duplicate (_1 & _2). To explore the genes responsible for chemosensitivity in cancer cells, expression profiles of various cancer cell lines were examined. Statistically comparing them with the chemosensitivity measured by in vitro MTT assay in each cell line, several candidate genes were selected and predicition models for anticancer efficacy were established using their expression levels.

Dataset Information

Effects of hypoxia on human cancer cell line chemosensitivity.

Background

Methods

Results

Conclusions

Publications

Effects of hypoxia on human cancer cell line chemosensitivity.

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