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Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

ABSTRACT: Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjunction with [(1)H-(15)N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.

SUBMITTER: Stuart CH

PROVIDER: S-EPMC3983131 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

Stuart Christopher H CH Horita David A DA Thomas Michael J MJ Salsbury Freddie R FR Lively Mark O MO Gmeiner William H WH

Bioconjugate chemistry 20140206 2

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjun ...[more]

PMID: 24450459

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Project description:Background:Multidrug resistance (MDR) is a major obstacle in breast cancer treatment. The predominant mechanism underlying MDR is an increase in the activity of adenosine triphosphate (ATP)-dependent drug efflux transporters. Sulbactam, a ?-lactamase inhibitor, is generally combined with ?-lactam antibiotics for treating bacterial infections. However, sulbactam alone can be used to treat Acinetobacter baumannii infections because it inhibits the expression of ATP-binding cassette (ABC) transporter proteins. This is the first study to report the effects of sulbactam on mammalian cells. Methods:We used the breast cancer cell lines as a model system to determine whether sulbactam affects cancer cells. The cell viabilities in the present of doxorubicin with or without sulbactam were measured by MTT assay. Protein identities and the changes in protein expression levels in the cells after sulbactam and doxorubicin treatment were determined using LC-MS/MS. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was used to analyze the change in mRNA expression levels of ABC transporters after treatment of doxorubicin with or without sulbactam. The efflux of doxorubicin was measures by the doxorubicin efflux assay. Results:MTT assay revealed that sulbactam enhanced the cytotoxicity of doxorubicin in breast cancer cells. The results of proteomics showed that ABC transporter proteins and proteins associated with the process of transcription and initiation of translation were reduced. The mRNA expression levels of ABC transporters were also decreased when treated with doxorubicin and sulbactam. The doxorubicin efflux assay showed that sulbactam treatment inhibited doxorubicin efflux. Conclusions:The combination of sulbactam and doxorubicin enhances the cytotoxicity of doxorubicin in the breast cancer cells by inhibiting the expression of ABC transporter proteins and proteins associated with the process of transcription and initiation of translation, and blocking the efflux of doxorubicin. Co-treatment of doxorubicin and sulbactam can be used in breast cancer treatment to decrease the prescribed dose of doxorubicin to avoid the adverse effects of doxorubicin.

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Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

Publications

Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

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