Interactions of Cisplatin and Daunorubicin at the chromatin level

Unexpectedly, the widely used anticancer agents Cisplatin (Cis-Pt) and Daunorubicin (Dauno) exhibited cell type- and concentration-dependent synergy or antagonism in vitro. We attempted to interpret these effects in terms of the changes elicited by the drugs in the chromatin, the target held primarily responsible for the cytotoxicity of both agents. We measured the effect of Cis-Pt on the levels of Dauno in different cell compartments, the effect of Cis-Pt on Dauno-induced nucleosome eviction, and assessed the influence of Dauno on DNA platination in flow- and laser scanning cytometry as well as in laser ablation-inductively coupled plasma-mass spectrometry assays. We show that the two drugs antagonize each other through a decrease of interstrand crosslinks upon co-treatment with Dauno, and also via the diminished Dauno uptake in the presence of Cis-Pt, and both effects are observed already at low Dauno concentrations. At high Dauno concentrations synergy becomes dominant because histone eviction by Dauno intercalation into the DNA is enhanced in the presence of co-treatment with Cis-Pt. These interactions may have an impact on the efficacy of combination treatment protocols, considering the long retention time of DNA adducts formed by both agents

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/104 bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [14C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [14C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/107 bp (∼1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues

DNA-Destabilizing Agents as an Alternative Approach for Targeting DNA: Mechanisms of Action and Cellular Consequences

DNA targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia, both in terms of drug brands and prescription volumes. Small DNA-interacting molecules share the ability of certain proteins to change the DNA helix's overall organization and geometrical orientation via tilt, roll, twist, slip, and flip effects. In this ocean of DNA-interacting compounds, most stabilize both DNA strands and very few display helix-destabilizing properties. These types of DNA-destabilizing effect are observed with certain mono- or bis-intercalators and DNA alkylating agents (some of which have been or are being developed as cancer drugs). The formation of locally destabilized DNA portions could interfere with protein/DNA recognition and potentially affect several crucial cellular processes, such as DNA repair, replication, and transcription. The present paper describes the molecular basis of DNA destabilization, the cellular impact on protein recognition, and DNA repair processes and the latter's relationships with antitumour efficacy

A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues

A HPLC method was validated to quantify doxorubicin associated to DNA from tissue.Successfully applied to an in vivo mouse-based pharmacokinetic study.Important tool for future studies evaluating intracellular pharmacokinetics.Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid–liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2 μm, 2.0 × 100 mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10 ng/mL and is shown to be linear up to 3000 ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72 h after administration of PEGylated liposomal doxorubicin (Doxil®; PLD) at 6 mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin

Interstrand Crosslink Repair: New Horizons of DNA Damage Repair

Since the dawn of civilization, living organisms are unceasingly exposed to myriads of DNA damaging agents that can temper the ailments and negatively influence the well-being. DNA interstrand crosslinks (ICLs) are spawned by various endogenous and chemotherapeutic agents, thus posing a somber menace to genome solidity and cell endurance. However, the robust techniques of damage repair including Fanconi anemia pathway, translesion synthesis, nucleotide excision and homologous recombination repair faithfully protect the DNA by removing or tolerating damage to ensure the overall survival. Aberrations in such repair mechanisms adverse the pathophysiological states of several hereditary disorders i.e. Fanconi Anemia, xeroderma pigmentosum, cerebro-oculo-facio-skeletal syndrome and cockayne syndrome etc. Although, the recognition of ICL lesions during interphase have opened the new horizons of research in the field of genetics but still the detailed analysis of conditions in which repair should occur is largely elusive

A Sensitive High Performance Liquid Chromatography (HPLC) Assay for the Quantification of Doxorubicin Bound to DNA

Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of intracellular active form of doxorubicin. Thus, we have developed a high performance liquid chromatography (HPLC) methodology in order to quantify the concentrations of doxorubicin that are bound to DNA in tumors and tissues as an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate bound drug; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2 um, 2.0 x 100 mm) analytical column; and a gradient mobile phase of 0.1% formic acid in water and acetonitrile. The assay has a lower limit of quantification (LLOQ) of 10 ng/mL and is shown to be linear up to 3,000 ng/mL. We demonstrated the suitability of this assay for doxorubicin bound to DNA in vivo by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72 hours after administering PEGylated liposomal doxorubicin (Doxil®; PLD) IV at 6 mg/kg. This HPLC assay allows for a sensitive and simple intracellular quantification of doxorubicin as compared to other methods and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle carriers.Doctor of Philosoph

Evaluating the efficacy of DNA repair biomarkers to assess human cell response to chemotherapy using imaging flow cytometry

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Chemotherapy and radiotherapy are widely accepted as common forms of treatment for cancers. The majority of cancer patients receive chemotherapy alone or in combination with radiotherapy. Most chemotherapeutic drugs cause DNA damage to the rapidly dividing cancer cells but normal cells are also damaged in the process. Therefore DNA repair levels in tumour and normal cells may determine the success of the treatment. The aim of this work was to evaluate the use of DNA repair biomarkers for assessing responses to chemotherapeutic drugs. The novel technique of imaging flow cytometry was employed to analyse the induction and resolution of γ-H2AX and RAD51 DNA repair biomarkers in DNA repair normal cell lines MRC5-SV1 and NB1-HTERT, an ATM-deficient cell line AT5BIVA (derived from an Ataxia Telangiectasia patient) and an XPF-deficient cell line GM08437B. Two cell lines were also developed, MRC5-SV1R and NB1-HTERTR which had been made resistant to HN2. A range of chemotherapeutic drugs, Adriamycin, Cisplatin and Nitrogen Mustard which have different modes of action were examined in this work. We have demonstrated distinct differences in γ-H2AX and RAD51 foci induction and resolution between the two DNA repair normal cell lines following exposure to different chemotherapeutic drugs. Additionally, it was demonstrated that both the resistant and sensitive cell lines have elevated γ-H2AX and RAD51 expression profiles in comparison to the parental cell lines over a 48 hour period post treatment with the cross-linking agent HN2. It is concluded that while both the γ-H2AX and Rad51 biomarkers may be useful for determining chemotherapeutic response, a larger cohort of cell lines and tumour samples is required for further analysis