Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Effect of mutations on the p53 IRES RNA structure Implications for de-regulation of the synthesis of p53 isoforms

Earlier we have demonstrated the presence of internal ribosome entry site (IRES) within tumor suppressor p53 mRNA. Here we have mapped the putative secondary structure of p53-IRES RNA using information from chemical probing and nuclease mapping experiments. Additionally, the secondary structure of the IRES element of the wild-type RNA was compared with cancer-derived silent mutant p53 RNAs. These mutations might result in the conformational alterations of p53-IRES RNAs. The results also indicate decreased IRES activities of the mutants as compared to wild-type RNA. Further, it was observed that some of the cytoplasmic trans-acting factors, critical for enhancing IRES function, were unable to bind mutant RNAs as efficiently as to wild-type. Our results suggest that hnRNP C1/C2 binds to p53-IRES and siRNA mediated partial silencing of hnRNP C1/C2 showed appreciable decrease in IRES function and consequent decrease in the level of the corresponding p53 isoform. Interestingly mutant p53 IRES showed lesser binding with hnRNP C1/C2 protein. Finally, upon doxorubicin treatment, the mutant RNAs were unable to show enhanced p53 synthesis to similar extent compared to wild type. Taken together, these observations suggest that mutations occurring in the p53 IRES might have profound implications for de-regulation of its expression and activity.

The Structure of the Thioredoxin−Triosephosphate Isomerase Complex Provides Insights into the Reversible Glutathione-Mediate Regulation of Triosephosphate Isomerase

Protein−protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin−triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or nmers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3) 8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.

Differential Activities of the Two Closely Related Withanolides, Withaferin A and Withanone: Bioinformatics and Experimental Evidences

Background and Purpose: Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library. Methodology/Principal Findings: In the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails.

Design and Synthesis of a Functional Derivative of the Triazinium Cation and Its Rhodium Complex that Shows Photoinduced DNA Cleavage Activity and Photocytotoxicity

The design and synthesis of an intensely blue rhodium(III) complex 3]+ of a new N,N-donor ligand, 8-(quinolin-8-ylamino)pyrido2,1-c]1,2,4]benzotriazin-11-ium, 2]+, which contains a planar pendant triazinium arm, is described. Structural characterization for 3]+ was carried out by using various spectroscopic techniques and single-crystal X-ray crystallography. The organometallic rhodium(III) compound shows a ligand-based reversible reduction at 0.65 V. The electrochemically reduced compound displays a single-line EPR spectrum that signifies the formation of ligand-based free radicals. Compound 3]+ shows a binding propensity to calf thymus DNA to give a Kapp value of 6.05X105 M1. The parent triazinium salt, pyrido2,1-c]1,2,4]benzotriazin-11-ium 1]+ and the ligand salt 2]+ exhibit photoinduced cleavage of DNA in UV-A light, whereas the reference Rh complex 3]+ photocleaves DNA with red light (647.1 nm). The compounds show photonuclease activities under both aerobic and anaerobic conditions. Mechanistic investigations under aerobic conditions with several inhibitors indicate the formation of hydroxyl radicals by means of a photoredox pathway. Under anaerobic conditions, it is believed that a photoinduced oxidation of DNA mechanism is operative. Compound 3]+ exhibits photocytotoxicity in HeLa cervical cancer cells to give IC50 values of (12+/-0.9) mu M in UV-A light at 365 nm and (31.4+/-1.1) mu M in the dark.

Molecular Design of Synthetic Benzimidazoles for the Switchover of the Duplex to G-quadruplex DNA Recognition

Benzimidazole derivatives are well known for their antibacterial, antiviral, anticonvulsant, antihistaminic, anthelmintic and antidepressant activities. Benzimidazole's unique base-selective DNA recognition property has been studied widely. However, most of the early benzimidazole systems have been targeted towards the binding of duplex DNA. Here we have shown the evolution and progress of the design and synthesis of new benzimidazole systems towards selective recognition of the double-stranded DNA first. Then in order to achieve selective recognition of the G-quadruplex DNA and utilize their potential as future anti-cancer drug candidates, we have demonstrated their selective cytotoxicity towards the cancer cells and potent telomerase inhibition ability.

Cyclic nucleotide signaling in intestinal epithelia: getting to the gut of the matter

The intestine is the primary site of nutrient absorption, fluid-ion secretion, and home to trillions of symbiotic microbiota. The high turnover of the intestinal epithelia also renders it susceptible to neoplastic growth. These diverse processes are carefully regulated by an intricate signaling network. Among the myriad molecules involved in intestinal epithelial cell homeostasis are the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). These cyclic nucleotides are synthesized by nucleotidyl cyclases whose activities are regulated by extrinsic and intrinsic cues. Downstream effectors of cAMP and cGMP include protein kinases, cyclic nucleotide gated ion channels, and transcription factors, which modulate key processes such as ion-balance, immune response, and cell proliferation. The web of interaction involving the major signaling pathways of cAMP and cGMP in the intestinal epithelial cell, and possible cross-talk among the pathways, are highlighted in this review. Deregulation of these pathways occurs during infection by pathogens, intestinal inflammation, and cancer. Thus, an appreciation of the importance of cyclic nucleotide signaling in the intestine furthers our understanding of bowel disease, thereby aiding in the development of therapeutic approaches.

Stable and Potent Analogues Derived from the Modification of the Dicarbonyl Moiety of Curcumin

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.

RECQL4 and p53 potentiate the activity of polymerase and maintain the integrity of the human mitochondrial genome

Germline mutations in RECQL4 and p53 lead to cancer predisposition syndromes, Rothmund-Thomson syndrome (RTS) and Li-Fraumeni syndrome (LFS), respectively. RECQL4 is essential for the transport of p53 to the mitochondria under unstressed conditions. Here, we show that both RECQL4 and p53 interact with mitochondrial polymerase (Pol gamma A/B2) and regulate its binding to the mitochondrial DNA (mtDNA) control region (D-loop). Both RECQL4 and p53 bind to the exonuclease and polymerase domains of Pol gamma A. Kinetic constants for interactions between Pol gamma A-RECQL4, Pol gamma A-p53 and Pol gamma B-p53 indicate that RECQL4 and p53 are accessory factors for Pol gamma A-Pol gamma B and Pol gamma A-DNA interactions. RECQL4 enhances the binding of Pol gamma A to DNA, thereby potentiating the exonuclease and polymerization activities of Pol gamma A/B2. To investigate whether lack of RECQL4 and p53 results in increased mitochondrial genome instability, resequencing of the entire mitochondrial genome was undertaken from multiple RTS and LFS patient fibroblasts. We found multiple somatic mutations and polymorphisms in both RTS and LFS patient cells. A significant number of mutations and polymorphisms were common between RTS and LFS patients. These changes are associated with either aging and/or cancer, thereby indicating that the phenotypes associated with these syndromes may be due to deregulation of mitochondrial genome stability caused by the lack of RECQL4 and p53. Summary: The biochemical mechanisms by which RECQL4 and p53 affect mtDNA replication have been elucidated. Resequencing of RTS and LFS patients' mitochondrial genome reveals common mutations indicating similar mechanisms of regulation by RECQL4 and p53.

Evaluation of the Genotoxic and Antigenotoxic Potential of the Alkaloid Punarnavine from Boerhaavia diffusa

Boerhaavia diffusa is a traditional herbal medicine extensively used in the Ayurveda and Unani forms of medicine in India and many parts of the world. Different parts of the plant are used as an appetizer, alexiteric, eye tonic, for flushing out the renal system, and to treat blood pressure. This study was conducted to evaluate the in vivo genotoxic and/or antigenotoxic potential of punarnavine, a separated alkaloid from the root of B. diffusa using toxicity studies (OECD guideline 474, 1997). The genotoxic and antigenotoxic potential of punarnavine was assayed using the comet assay on lymphocytes, liver, spleen, brain, and bone marrow as well as using the micronucleus test in bone marrow cells including the in vitro chromosomal aberration test. The results demonstrated that none of the tested doses of punarnavine showed genotoxic effects by the comet assay, or clastogenic effects in the micronucleus test. On the other hand, for all cells evaluated, the three tested doses of punarnavine promoted inhibition of DNA damage induced by cyclophosphamide. Based on these results, we concluded that punarnavine, an alkaloid from the Boerhaavia diffusa root, has no genotoxic or clastogenic effects in our experimental conditions. However, it caused a significant decrease in DNA damage induced by cyclophosphamide. It is suggested that the antigenotoxic properties of this alkaloid may be of great pharmacological importance and beneficial for cancer prevention.

Evaluation of hexane and ethyl acetate extracts of the sponge Jaspis diastra collected from Mauritius Waters on HeLa cells

Objectives Based on previous screening results, the cytotoxic effect of the hexane (JDH) and ethyl acetate extracts (JDE) of the marine sponge Jaspis diastra were evaluated on HeLa cells and the present study aimed at determining their possible mechanism of cell death. Methods Nuclear staining, membrane potential change, flow cytometry analysis of cell cycle distribution and annexin V staining were undertaken to investigate the effects of JDE and JDH. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance were used to characterize an isolated bioactive molecule. Key findings JDE displayed an IC50 25 times more significant than the JDH. Flow cytometry analysis revealed JDE induced apoptosis in HeLa cells accompanied by the collapse of mitochondrial membrane potential. Fractionation of JDE resulted in the isolation of the known cytotoxic cyclodepsipeptide, Jaspamide. Conclusions Taking our results together suggest that JDE can be valuable for the development of anticancer drugs, especially for cervical cancer. Further investigations are currently in progress with the aim to determine and isolate other bioactive compounds from this extract.

New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer

for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of 04 DNA even at a lower concentration of the stabilizing K+ ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

Cyclic Cationic Peptides Containing Sugar Amino Acids Selectively Distinguishes and Inhibits Maturation of Pre-miRNAs of the Same Family

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.