Chabita Saha

A review on structure–affinity relationship of dietary flavonoids with serum albumins

Flavonoids are a class of plant secondary metabolites and among thousands of flavonoids few are considered as dietary flavonoids. Serum albumin (SA), the most abundant protein in plasma, functions as the most important carrier of vital drugs, including dietary flavonoids. The binding affinity of dietary flavonoids to SA is demonstrated to be governed by structure–affinity relationship (SAR) and its bioavailability. The present review summarizes the interactions of flavonoids categorized as flavanol, flavonol, flavone, isoflavone, flavanones, and anthocyanidins with SAs (bovine serum albumin and human serum albumin) in light of SAR. The key findings are: (1) the position and degree of hydroxylation highly influence the affinity of flavonoids to SAs, (2) glycosylation decreases and substitution of methoxy group increases the affinity of flavonoids for SAs, (3) catechin gallates have higher binding affinity to SAs than catechins and gallocatechins, (4) inorganic metal ions modulate the binding affinity of flavonoids to SAs, and (5) hydrophobic interaction plays a major role in the interactions of all flavonoids with SAs.

Influence of Galloyl Moiety in Interaction of Epicatechin with Bovine Serum Albumin: A Spectroscopic and Thermodynamic Characterization

The health benefits stemming from green tea are well known, but the exact mechanism of its biological activity is not elucidated. Epicatechin (EC) and epicatechin gallate (ECG) are two dietary catechins ubiquitously present in green tea. Serum albumins functionally carry these catechins through the circulatory system and eliminate reactive oxygen species (ROS) induced injury. In the present study ECG is observed to have higher antioxidant activity; which is attributed to the presence of galloyl moiety. The binding affinity of these catechins to bovine serum albumin (BSA) will govern the efficacy of their biological activity. EC and ECG bind with BSA with binding constants 1.0×106 M−1 and 6.6×107 M−1, respectively. Changes in secondary structure of BSA on interaction with EC and ECG have been identified by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic characterization reveals the binding process to be exothermic, spontaneous and entropy driven. Mixed binding forces (hydrophobic, electrostatic and hydrogen bonding) exist between ECG and BSA. Binding site for EC is primarily site-II in sub-domain IIIA of BSA and for ECG; it is site-I in sub-domain IIA. ECG with its high antioxidant activity accompanied by high affinity for BSA could be a model in drug designing.

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.

Intercalation and Induction of Strand Breaks by Adriamycin and Daunomycin: A Study with Human Genomic DNA

The anticancer drugs Adriamycin (ADR) and Daunomycin (DNM) of the anthracycline family are effective in treating a variety of cancers. Although their interactions with other cellular targets may play a role in the selective cytotoxicity of these drugs, it is generally believed that intercalation with DNA is essential for their activity. However, a relationship has not yet been established between intercalation and cellular processes leading to cytotoxicity. The present study was designed to investigate the relationship, if any, between intercalation and DNA strand breaks. ADR and DNM were observed to be strong intercalators of human genomic DNA by absorption and fluorimetric methods that were further substantiated by rise in thermal melting temperature. DNM is the better intercalator of the two, which is also evident from circular dichroic spectral changes. DNA strand breaks, considered to be an index of genotoxicity, was assayed by single cell gel electrophoresis (SCGE; comet assay). ADR and DNM induced equivalent genotoxicity in normal human lymphocytes at a clinically used dose, which was observed to be independent of intercalation efficiency though positively correlated to yield of reactive oxygen species.

Biophysical studies of mutated K562 DNA (erythroleukemic cells) binding to adriamycin and daunomycin reveal that mutations induce structural changes influencing binding behavior

K562 cells are erythroleukemic cells derived from a chronic myeloid leukemia patient in blast crisis. Comparison of the genome from K562 cells and normal human genome has been very useful strategy, in uncovering eight genes, implicated in acute myeloid leukemia (AML). These genes carry mutations in K562 genome and the role of these mutations in the progression and treatment of AML is still not known. Consequences of these mutations on drug DNA binding are also not known exactly. In the present study, mutation induced structural changes in K562 genome, compared to normal genome, are identified by Fourier transform infra red (FTIR) and circular dichroism (CD) spectroscopy. These structural changes in native K562 DNA favor stronger binding with binding constants 2.0 × 108 and 1.9 × 109 M−1 with antileukemic drugs adriamycin and daunomycin (DNM), respectively, compared to normal DNA. On binding, these drugs disrupt the native B form structure of normal DNA to a greater extent, compared to A-like structure of K562 DNA. Fluorescence and absorption studies reveal higher intercalation as well as mixed groove binding of these drugs with K562 DNA compared to normal DNA. Among the drugs, DNM has higher affinity for K562 DNA.

Mutation Induced Conformational Changes in Genomic DNA from Cancerous K562 Cells Influence Drug-DNA Binding Modes

Normal human genomic DNA (N-DNA) and mutated DNA (M-DNA) from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with anticancer drugs; adriamycin (ADR) and daunomycin (DNM). Isothermal calorimetric thermograms representing titration of ADR/DNM with N-DNA and M-DNA on analysis best fitted with sequential model of four and three events respectively. From Raman spectroscopy it has been identified that M-DNA is partially transformed to A form owing to mutations and N-DNA on binding of drugs too undergoes transition to A form of DNA. A correlation of thermodynamic contribution and structural data reveal the presence of different binding events in drug and DNA interactions. These events are assumed to be representative of minor groove complexation, reorientation of the drug in the complex, DNA deformation to accommodate the drugs and finally intercalation. Dynamic light scattering and zeta potential data also support differences in structure and mode of binding of N and M DNA. This study highlights that mutations can manifest structural changes in DNA, which may influence the binding efficacy of the drugs. New generation of drugs can be designed which recognize the difference in DNA structure in the cancerous cells instead of their biochemical manifestation.

Antagonistic effects of black tea against gamma radiation-induced oxidative damage to normal lymphocytes in comparison with cancerous K562 cells

Abstract The potential of naturally occurring antioxidants to reduce the cellular oxidative damage induced by ionizing radiation has been studied for more than a decade for their pharmacological application during cancer treatment. It is already known that radioprotective efficacy of phytochemicals might influence various end points of radiation damage. Flavonoids are well-known natural radioprotectors, and their biological effects depend upon their chemical structure. In the present study, radioprotective effect of black tea rich in flavonoids was evaluated against gamma radiation-induced oxidative damage on normal lymphocytes and compared with erythroleukemic K562 cells. Pre-treatment with black tea extract (BTE) significantly reduced radiation-induced loss of cell viability, generation of reactive oxygen species, mitochondrial dysfunction, activation of caspase-3 and apoptosis in normal lymphocytes compared to K562 cells. BTE also regulates the activity of endogenous antioxidant enzymes. The changes in the mRNA expression of bax, bcl2, p53 and Nrf2 were also followed to evaluate regulation of radiation-induced apoptosis by BTE. These findings suggest that black tea may have the potential of a natural radioprotective agent which can be used as adjunct with radiation during cancer treatment.

Abstract C59: Use of dietary antioxidant supplement during cancer therapy- pros and cons.

Black and green teas are rich in antioxidants and its flavonoids like thearubigins, epicatechins and catechins are known to have higher antioxidant efficacy than vitamin C. Despite significant research investigating the use of tea as dietary antioxidant supplement during conventional chemotherapy, no conclusion has been drawn regarding its mechanism of action and safety. In the present study chemopreventive effects of black tea extract (BTE) were evaluated on normal lymphocytes and compared with cancerous K562 cells. Chemotherapeutic drugs (Adriamycin and Daunomycin) act by generating oxidative stress and inducing apoptosis. The level of reactive oxygen species (ROS), apoptosis and gene expression in untreated and BTE treated cells were followed by different biochemical assays, flow cytometry and reverse transcription PCR. Results evidence that efficient scavenging of intracellular ROS by BTE resulting in reduced apoptotic cell population in normal lymphocytes. Gene expression of p53, bax, bcl2 and nrf2 show variation in untreated and BTE treated cells. Activity of endogenous antioxidant enzymes like SOD, GST and catalase is modified on tea treatment. Similar observations were recorded to a lower extent in K562 cells, suggesting BTE conferred protection to cancerous cells also. It can be summarized here that tea as a dietary supplement during chemotherapy shows interference in chemotherapeutic efficacy of the drugs in vitro. More in vitro, in vivo and clinical trials would shed light on the mechanism of action of the dietary antioxidant supplement during chemotherapy and its application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C59. Citation Format: Debjani Ghosh, Subrata Dey, Chabita Saha. Use of dietary antioxidant supplement during cancer therapy- pros and cons. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C59.

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).