p38 MAPK inhibitor SB203580 enhances anticancer activity of PARP inhibitor olaparib in a synergistic way on non-small cell lung carcinoma A549 cells.

The Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib gives promising results against various types of cancers in clinical trials. The combination of drugs always increases therapeutic efficacy because of targeting multiple pathways of cancer progression. Our objective was to explore the potential synergistic anticancer activities of olaparib combined with p38 MAPK inhibitor (MAPKi) SB203580 on non-small cell lung carcinoma (NSCLC) A549 cells. The effects of the individual compound and their combination on cell survival, DNA damage as detected by γH2AX foci, expression of key proteins in Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair, caspase 3 activation, nuclear fragmentation and telomerase regulation were studied in A549 cells. The results showed that olaparib and SB203580 individually reduced cell viability in a dose-dependent manner but combined treatment synergistically reduced cell viability. Olaparib combined with SB203580 significantly reduced error-free HR repair via reducing MRE11-RAD50 and promoted error-prone NHEJ repair by increasing Ku70-Ku80 leading to increased DNA damage-induced apoptosis. Notably, the alteration of proteins in HR/NHEJ pathways, DNA damage and induction of apoptosis was significant by combined treatment but not by 1 µM olaparib treatment alone. In addition, combined treatment reduced telomerase activity more than single treatment via reducing telomerase subunits. These data implicated that the anticancer potential of olaparib was significantly increased by combining SB203580 through increasing DNA damage-induced apoptosis and inhibiting telomerase activity.

Ethanolic extract of leaf of Dillenia pentagyna reduces in-vitro cell migration and induces intrinsic pathway of apoptosis via downregulation of NF-κß in human NSCLC A549 cells.

Despite the advancement of the pharmaceutical industry, medicinal plants are still a reliable source of traditional medicines to cure a number of diseases. Various parts of Dillenia pentagyna are used in traditional medicine in India for treatment of various disorders including cancers, but detailed mechanisms are still unknown. Dried leaves of D. pentagyna were extracted with ethanol and termed as an ethanolic extract of leaves of D. pentagyna (EELDP). Our aim was to elucidate the role of EELDP in in-vitro cell migration and apoptosis in highly metastatic human lung adenocarcinoma A549 cells. We measured cell viability and in-vitro cell migration in three different human cancer cells A549, HeLa and U2OS treated with EELDP (0-0.6 mg/mL). However, A549 cells showed higher sensitivity to EELDP treatment. Hence we studied several key markers of metastasis and apoptosis pathway in A549 cells treated with EELDP. EELDP treatment significantly reduced in-vitro cell migration, wound healing, expression and activity of MMP-2, MMP-9 via reduction of nuclear factor kappa Beta (NF-κß). EELDP also reduced vimentin, N-cadherin and increased claudin-1. The intrinsic pathway of apoptosis was triggered by EELDP via the NF-κß pathway through the increase of the Bax to Bcl2 ratio, leading to the fall of mitochondrial membrane potential and subsequently induced release of cytochrome c, activation of caspase-3 followed by nuclear fragmentation in A549 cells. Furthermore, we observed change of a few markers of metastasis and apoptosis in other two cell types HeLa and U2OS treated with EELDP. These data implicate that the effect of EELDP is not cell-specific. Since only 0.1 mg/mL EELDP significantly reduces in-vitro cell migration and increases apoptosis, the active compound(s) present in EELDP is very much potent to control highly metastatic cancer.

Reduction of metastatic potential by inhibiting EGFR/Akt/p38/ERK signaling pathway and epithelial-mesenchymal transition after carbon ion exposure is potentiated by PARP-1 inhibition in non-small-cell lung cancer.

BACKGROUND: Carbon ion (12C) radiotherapy is becoming very promising to kill highly metastatic cancer cells keeping adjacent normal cells least affected. Our previous study shows that combined PARP-1 inhibition with 12C ion reduces MMP-2,-9 synergistically in HeLa cells but detailed mechanism are not clear. To understand this mechanism and the rationale of using PARP-1 inhibitor with 12C ion radiotherapy for better outcome in controlling metastasis, we investigated metastatic potential in two non-small cell lung cancer (NSCLC) A549 and H1299 (p53-deficient) cells exposed with 12C ion in presence and absence of PARP-1 inhibition using siRNA or olaparib. METHODS: We monitored cell proliferation, in-vitro cell migration, wound healing, expression and activity of MMP-2, - 9 in A549 and p53-deficient H1299 cell lines exposed with 12C ion with and without PARP-1 inhibitor olaparib/DPQ. Expression and phosphorylation of NF-kB, EGFR, Akt, p38, ERK was also observed in A549 and H1299 cells exposed with 12C ion with and without PARP-1 inhibition using siRNA or olaparib. We also checked expression of few marker genes involved in epithelial-mesenchymal transition (EMT) pathways like N-cadherin, vimentin, anillin, claudin-1, - 2 in both NSCLC. To determine the generalized effect of 12C ion and olaparib in inhibition of cell's metastatic potential, wound healing and activity of MMP-2, - 9 was also studied in HeLa and MCF7 cell lines after 12C ion exposure and in combination with PARP-1 inhibitor olaparib. RESULTS: Our experiments show that 12C ion and PARP-1 inhibition separately reduces cell proliferation, cell migration, wound healing, phosphorylation of EGFR, Akt, p38, ERK resulting inactivation of NF-kB. Combined treatment abolishes NF-kB expression and hence synergistically reduces MMP-2, - 9 expressions. Each single treatment reduces N-cadherin, vimentin, anillin but increases claudin-1, - 2 leading to suppression of EMT process. However, combined treatment synergistically alters these proteins to suppress EMT pathways significantly. CONCLUSION: The activation pathways of transcription of MMP-2,-9 via NF-kB and key marker proteins in EMT pathways are targeted by both 12C ion and olaparib/siRNA. Hence, 12C ion radiotherapy could potentially be combined with olaparib as chemotherapeutic agent for better control of cancer metastasis.

α-Amino Acid Derived Benzimidazole-Linked Rhodamines: A Case of Substitution Effect at the Amino Acid Site toward Spiro Ring Opening for Selective Sensing of Al3+ Ions.

α-Amino acid derived benzimidazole-linked rhodamines have been synthesized, and their metal ion sensing properties have been evaluated. Experimentally, l-valine- and l-phenylglycine-derived benzimidazole-based rhodamines 1 and 2 selectively recognize Al3+ ion in aqueous CH3CN (CH3CN/H2O 4/1 v/v, 10 mM tris HCl buffer, pH 7.0) over the other cations by exhibiting color and "turn-on" emission changes. In contrast, glycine-derived benzimidazole 3 remains silent in the recognition event and emphasizes the role of α-substitution of amino acid undertaken in the design. The fact has been addressed on the basis of the single-crystal X-ray structures and theoretical calculations. Moreover, pink 1·Al3+ and 2·Al3+ ensembles selectively sensed F- ions over other halides through a discharge of color. Importantly, compounds 1 and 2 are cell permeable and have been used as imaging reagents for the detection of Al3+ uptake in human lung carcinoma cell line A549.

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

Synthesis, structure, spectroscopic characterization, and protein binding affinity of new water-soluble hetero- and homometallic tetranuclear [Cu(II)2Zn(II)2] and [Cu(II)4] clusters.

Two new water-soluble hetero- and homometallic tetranuclear clusters, Na4[Cu2Zn2(ccdp)2(µ-OH)2]·CH3OH·6H2O (1) and K3[Cu4(ccdp)2(µ-OH)(µ-OH2)]·14H2O (2), have been synthesized in methanol-water at room temperature by exploiting the flexibility, chelating ability, and bridging potential of a carboxylate-rich dinucleating ligand, N,N'-bis(2-carboxybenzomethyl)-N,N'-bis(carboxymethyl)-1,3 diaminopropan-2-ol (H5ccdp). Complex 1 is obtained through the self-assembly of two monoanionic [CuZn(ccdp)](-)fragments, which are, in turn, exclusively bridged by two µ-OH(-)groups. Similarly, complex 2 is formed through the self-assembly of two monoanionic [Cu2(ccdp)](-) species exclusively bridged by one µ-OH(-) and one µ-OH2 groups. Complexes 1 and 2 are fully characterized in the solid state as well as in solution using various analytical techniques including a single-crystal X-ray diffraction study. The X-ray crystal structure of 1 reveals that two Cu(II) centers are in a distorted square-pyramidal geometry, whereas two Zn(II) centers are in a distorted trigonal-bipyramidal geometry. The solid-state structure of 2 contains two dinuclear [Cu2(ccdp)](-) units having one Cu(II) center in a distorted square-pyramidal geometry and another Cu(II) center in a distorted trigonal-bipyramidal geometry within each dinuclear unit. In the powder state, the high-field EPR spectrum of complex 1 indicates that two Cu(II) ions are not spin-coupled, whereas that of complex 2 exhibits at least one noninteracting Cu(II) center coordinated to a nitrogen atom of the ligand. Both complexes are investigated for their binding affinity with the protein bovine serum albumin (BSA) in an aqueous medium at pH ~7.2 using fluorescence spectroscopy. Synchronous fluorescence spectra clearly reveal that complexes 1 and 2 bind to the active sites in the protein, indicating that the effect is more pronounced toward tyrosine than tryptophan. Density functional theory calculations have been carried to find the Fukui functions at the metal sites in complexes 1 and 2 to predict the possible metal centers involved in the binding process with BSA protein.

High throughput screening of a new fluorescent G-quadruplex ligand having telomerase inhibitory activity in human A549 cells.

Identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy. The screened compound from ZINC database using docking studies was experimentally verified for its binding with three different telomeric G-quadruplex DNA sequences and anti-telomerase activity in A549 cells. Identified compound is an intrinsic fluorescent molecule, permeable to live cells and has a higher affinity to 22AG out of three different telomeric G-quadruplex DNA. It showed cytotoxicity and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has a good anti-cancer effect.

Leaf Extract and Active Fractions of Dillenia pentagyna Roxb. Reduce In Vitro Human Cancer Cell Migration Via NF-κB Pathway.

BACKGROUND: Different parts of Dillenia pentagyna have long been used in traditional medicines to cure several diseases including cancer. However, the mechanism(s) of anti-cancer effects are still unknown. We aimed to elucidate the anti-metastatic potential of ethanolic extracts of leaves of D. pentagyna (EELDP) and active fractions of it in highly metastatic human cancer cells. METHODS: We screened different HPLC fractions of EELDP based on their anti-metastatic effect. We used TLC and ESI-MS for determining the presence of various phytochemicals in EELDP and fractions. We monitored in vitro anti-metastasis effect of EELDP (0-0.6 mg/ml) and active fractions (0-0.050 mg/ml) on various human cancer cells like A549, HeLa, and U2OS. RESULTS: EELDP significantly reduced cell viability and cell migration in A549, HeLa, and U2OS cells. However, higher sensitivity was observed in A549 cells. We screened 2 active HPLC fractions F6 and F8 having anti-MMPs activity. EELDP and active fractions reduced metastasis via the NF-κB pathway, decreased the expression of Vimentin, N-cadherin, and increased the expression of Claudin-1. CONCLUSION: Significant reduction of metastasis by EELDP at a dose of 0.1 mg/ml or by active fractions at 0.050 mg/ml implicates that the active compound(s) present in crude or fractions are extremely potent to control highly metastatic cancer.

PARP1 modulates telomere sister chromatid exchange and telomere length homeostasis by regulating telomere localization of SLX4 in U2OS cells.

OBJECTIVE: Poly(ADP-ribose) polymerase1 (PARP1) interacts and poly(ADP-ribosyl)ates telomere repeat binding factor 2 (TRF2), which acts as a platform to recruit a large number of proteins at the telomere. Since the discovery of TRF2-SLX4 interaction, SLX4 is becoming the key player in telomere length (TL) maintenance and repair by telomere sister chromatid exchange (T-SCE). Defective TL maintenance pathway results in a spectrum of diseases called telomeropathies like dyskeratosis congenita, aplastic anemia, fanconi anemia, cancer. We aimed to study the role of SLX4 and PARP1 on each other's telomere localization, T-SCE, and TL maintenance in human telomerase-negative osteosarcoma U2OS cells to understand some of the molecular mechanisms of telomere homeostasis. MATERIALS AND METHODS: We checked the role of SLX4 and PARP1 on each other's telomere localization by telomere immunofluorescence. We have cloned full-length wild-type and catalytically inactive mutant PARP1 to understand the role of poly(ADP-ribosyl)ation reaction by PARP1 in telomere length homeostasis. TL of U2OS cells was measured by Q-FISH. T-SCE was measured by Telomere-FISH. KEY FINDINGS: We observed that SLX4 has no role in the telomere localization of PARP1. However, reduced localization of SLX4 at undamaged and damaged telomere upon PARP1 depletion was reversed by overexpression of exogenous wild-type PARP1 but not by overexpression of catalytically inactive mutant PARP1. PARP1 depletion synergized SLX4 depletion-mediated reduction of T-SCE. Furthermore, SLX4 depletion elongated TL, and combined insufficiency of SLX4 with PARP1 further elongated TL. CONCLUSION: So, PARP1 controls SLX4 recruitment at telomere by poly(ADP-ribosyl)ation reaction, thereby regulating SLX4-mediated T-SCE and TL homeostasis.

Novel 2D/2D g-C3N4/Bi4NbO8Cl nano-composite for enhanced photocatalytic degradation of oxytetracycline under visible LED light irradiation.

A novel visible-light-responsive 2D/2D g-C3N4/Bi4NbO8Cl nano-composite photocatalyst was hydrothermally synthesized using g-C3N4 and Bi4NbO8Cl. Various characterization techniques were employed to characterize the as-synthesized g-C3N4/Bi4NbO8Cl nano-composites. The photocatalytic efficiency of the nano-composite materials was assessed by the degradation of an emerging pharmaceutical pollutant, oxytetracycline (OTC), under visible LED light irradiation. It was observed that at an optimum mass ratio of 20% (g-C3N4 to Bi4NbO8Cl), the 20g-C3N4/Bi4NbO8Cl nano-composite produced the highest photocatalytic degradation efficiency toward OTC. The photocatalytic degradation of OTC (20 mgL-1) by 20g-C3N4/Bi4NbO8Cl (1 gL-1), under 60 min of visible LED light irradiation was 87%, which was about 1.2 and 1.8 times higher as compared to that of pure Bi4NbO8Cl and g-C3N4, respectively. This improved performance was associated with the formation of type-II heterojunction, which resulted in better visible-light absorption and reduced recombination of photogenerated electron-hole pairs. Moreover, it was observed that after four cycles of degradation experiments, the nano-composite was stable. The results of this work not only demonstrate the construction of 2D/2D g-C3N4/Bi4NbO8Cl nano-composite for successful low-cost and energy-efficient photocatalytic degradation of recalcitrant pollutant but also motivate the production of similar photocatalysts targeting environmental remediation.

Gamma ray-induced in vitro cell migration via EGFR/ERK/Akt/p38 activation is prevented by olaparib pretreatment.

Purpose: Radiotherapy using gamma ray is still the main therapeutic modality for the treatment of various cancers. However, local recurrence and increase of metastasis after radiotherapy is still a major therapeutic challenge. Aim of this work was to check cell migration along with activity and expression of some marker proteins involved in epithelial-mesenchymal transition (EMT) pathway in three different human cancer cells after exposure with gamma radiation in combination with PARP inhibitor olaparib.Materials and methods: Here, we presented cell viability, in vitro cell migration, activity of MMPs by gelatin zymography, expression of few EMT marker proteins and the signaling cascade involved in transcriptional regulation of MMPs after gamma irradiation with and without olaparib pretreatment in highly metastatic three human cancer cell lines-A549, HeLa and U2OS.Results: We observed that gamma irradiation alone increased in vitro cell migration, MMP-2,-9 activity, expression of N-cadherin, vimentin and the signaling molecules EGFR, ERK1/2, Akt, p38 that enhanced NF-kB expression in all three cell types. Olaparib treatment alone reduced in vitro cell migration along with reduction of expression of all the above-mentioned marker proteins of the EMT pathway. However, 4 h olaparib pretreatment prevented gamma ray induced activation of all these marker proteins in all three cell types.Conclusions: This data implicates that olaparib treatment in combination with gamma therapy could be promising in protecting patients from gamma-induced metastasis.

Effect of Substitution at Amine Functionality of 2,6-Diaminopyridine-Coupled Rhodamine on Metal-Ion Interaction and Self-Assembly.

2,6-Diaminopyridine-coupled rhodamines 1 and 2 have been synthesized, and the effect of substitution on amine functionality toward metal-ion interactions and self-assembly is thoroughly investigated. Both the compounds effectively recognize different metal ions of biological significance fluorimetrically and colorimetrically with a high degree of selectivity and sensitivities. While compound 1 is sensitive to Fe3+ ions, compound 2 is responsive to both Fe3+ and Al3+ ions in aqueous CH3CN (4/1, v/v; 10 mM tris HCl buffer, pH 6.8). The sensing mechanism involves the metal-ion chelation-induced spirolactam ring opening of the rhodamine scaffold that results in both color and fluorescence changes, while the extent of interactions with the metal ions is truly governed by the chemical structure of the compounds. Both 1 and 2 are proficient in detecting Fe3+ and Al3+ ions in human lung cancer cells (A549). As new findings, unlike 1, compound 2 formed a faint pink gel in the toluene-hexane mixture solvent (1:1, v/v), and the gel state of 2 selectively recognizes Ag+ ions by exhibiting a phase change from gel to purple sol. Experimental findings establish the role of the formamide moiety in forming the self-assembly.

Induction of apoptosis by the inhibitors of poly(ADP-ribose)polymerase in HeLa cells.

To investigate the role of poly(ADP-ribose)polymerase (PARP) in the physiological condition of cell growth, we studied the ability of PARP inhibitors to induce apoptosis. Benzamide (BA) and 4-amino-1,8-naphthalimide (NAP), two well-known inhibitors of PARP, treatment increased nuclear fragmentation and caspase-3 activity in HeLa (Human cervical cancer cell line) cells. The increase of cellular NAD(+) level was observed in HeLa cells treated with BA in comparison with untreated control cells. For unrevealing the specific PARP family member responsible for such induction of apoptosis we knocked down and over-expressed PARP-1 gene in HeLa cells. PARP-1 knock down cells were sensitive to BA induced nuclear fragmentation and caspase-3 activation while exogenous expression of PARP-1 rendered cells resistant to BA induced apoptosis. This result indicated that inhibition of PARP-1 resulted in induction of apoptosis.

Sensitization of chondrosarcoma cells with PARP inhibitor and high-LET radiation.

Chondrosarcoma is a malignant tumor that arises from cartilaginous tissue and is radioresistant and chemoresistant to conventional treatments. The preferred treatment consists of surgical resection, which might cause severe disabilities for the patient; in addition, this procedure might be impossible for inoperable locations, such as the skull base. Carbon ion irradiation (hadron therapy) has been proposed as an alternative treatment, primarily due to its greater biological effectiveness and improved ballistic properties compared with conventional radiotherapy with X-rays. The goal of this study was to characterize the genetic mutations of a grade III chondrosarcoma cell line (CH2879) and examine the cellular responses to conventional radiotherapy (X-rays) and hadron therapy (proton and carbon ions) in the presence of the PARP inhibitor Olaparib. To better understand PARP inhibition, we first analyzed the formation of poly-ADP ribose chains by western blot; we observed an increase in its signal after irradiation, which disappeared on addition of the PARP inhibitor. PARPi enhanced ratio of approximately 1.3, 1.8, and 1.5 following irradiation of cells with X-rays, protons, and C-ions, respectively, as detected by clonogenic assay. The decrease in cell survival was confirmed by proliferation assay. The radiosensitivity of CH2879 cells was associated with mutations in homologous recombination repair genes, such as RAD50, SMARCA2 and NBN. This study demonstrates the capacity of the PARP inhibitor Olaparib to radiosensitize mutated chondrosarcoma cells to conventional photon irradiation, proton and carbon ion irradiation.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

Inhibition of telomerase activity by reduction of poly(ADP-ribosyl)ation of TERT and TEP1/TP1 expression in HeLa cells with knocked down poly(ADP-ribose) polymerase-1 (PARP-1) gene.

Inhibitors of poly(ADP-ribose) polymerase (PARP) have been shown to reduce the telomerase activity in cultured cells. To find out the specific member of the PARP family, which participates in the regulation of telomerase activity, in the present investigation, we knocked down the PARP-1 gene by siRNA in HeLa cells and studied the telomerase activity. Reduction of expression of PARP-1 by siRNA increased cellular NAD(+) level and decreased general poly(ADP-ribosyl)ation of proteins. Telomerase activity decreased in cells with knocked down PARP-1 gene. Besides, we observed that telomerase reverse transcriptase (hTERT) was poly(ADP-ribosyl)ated in HeLa cells and such modification was decreased in cells with reduced PARP-1 expression. In addition, the expression of telomerase-associated protein 1 (TEP1/TP1) subunit of human telomerase holoenzyme reduced significantly in PARP-1 knock down HeLa cells. Thus, PARP-1 modulates the telomerase activity by altering poly(ADP-ribosyl)ation of TERT and/or the expression of TEP1/TP1.

Patients with Congenital Limb Anomaly Show Short Telomere, Shutdown of Telomerase and Deregulated Expression of Various Telomere-Associated Proteins in Peripheral Blood Mononuclear Cells-A Case Series.

Congenital limb anomalies are outcome of improper bone formation during embryonic development when cells divide, differentiate with high rate. So, telomerase activity is essential to maintain telomere length for such highly dividing cells. Here, we report four cases of congenital limb anomalies with detailed structures of limbs along with other clinical manifestations of age less than two years. We compared telomere length, expression of telomerase and telomere-associated genes of Peripheral Blood Mononuclear Cells (PBMC) in patient and four age-matched normal individual. Patient-1 was diagnosed with congenital limb hypogenesis ectrodactyly sequence, an autosomal dominant disorder, showing absence of digits and fibula in upper and lower limb respectively. Both mother and grandmother of Patient-1 showed similar hypogenesis of limbs. Patient-2 showed bilateral clenched hand with arthrogryposis, microcephaly and holoprosencephaly. Both Patient-3 and Patient-4 has no radius in upper limb. Additionally, Paient-3 showed right sided orbital Space Occupying Lesion (SOL) and Paranasal Sinuses (PNS) whereas Patient-4 showed fused kidney with fanconi anaemia. Furthermore, all the patients showed shorter telomere length, inactive telomerase and de-regulated expression of telomere-associated proteins in PBMC compared with age-matched control group. So, we can conclude that congenital limb anomalies may be linked with telomeropathy and a study with large number of samples is required to firmly establish such association.

Insight into the binding of a non-toxic, self-assembling aromatic tripeptide with ct-DNA: Spectroscopic and viscositic studies.

The report describes the synthesis, self-association and DNA binding studies of an aromatic tripeptide H-Phe-Phe-Phe-OH (FFF). The peptide backbone adopts ß-sheet conformation both in solid and solution. In aqueous solution, FFF self-assembles to form nanostructured aggregates. Interactions of this peptide with calf-thymus DNA (ct-DNA) have been studied using various biophysical techniques including ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The value of mean binding constant calculated from UV and fluorescence spectroscopic data is (2.914 ± 0.74) x 103 M-1 which is consistent with an external binding mode. Fluorescence intercalator displacement (FID) assay, iodide quenching study, viscosity measurement and thermal denaturation study of DNA further confirm the groove binding mode of peptide, FFF with ct-DNA. MTT cell survival assay reveals very low cytotoxicity of the peptide toward human lung carcinoma cell line A549.

Inhibition of telomerase activity and induction of apoptosis by curcumin in K-562 cells.

Telomerase, a reverse transcriptase that maintains telomere length, is highly activated in tumor cells and practically absent in somatic cells and hence considered a potential marker for tumorigenesis. A connection between telomerase activity and resistance to apoptosis has been established. Telomerase, therefore, has been proposed to represent a novel and potentially selective target for cancer therapy. Several synthetic compounds have been developed in recent years with a view to inhibit telomerase activity with telomere shortening below a critical length resulting in apoptosis. Such compounds are always highly toxic. Many plant-derived products act through the induction of apoptosis as a mechanism to suppress carcinogenesis. Curcumin, a phenolic compound isolated from the rhizome of the plant Curcuma longa Linn., has been reported to possess anti-tumor, apoptotic and anti-angiogenic properties. Apoptosis has emerged as the major mechanism by which anti-tumor agents eliminate pre-neoplastic cells or cells progressed to malignancy. The present study was undertaken to examine the mechanism of curcumin-induced apoptosis in human leukemia cell line K-562 with particular emphasis on the role of curcumin on telomerase activity. Induction of apoptosis by curcumin is initiated by the release of cytochrome c from mitochondria into the cytosol, and evidenced by the increase in DNA content in the sub-G1 region as obtained from FACS analysis. Apoptosis is mediated by the activation of caspases 3 and 8, up-regulation of the apoptotic gene bax with concomitant down-regulation of the anti-apoptotic gene bcl-2. Using TRAP assay it has been observed that curcumin inhibits telomerase activity in a dose and time-dependent manner, the inhibition being due to suppression of translocation of telomerase reverse transcriptase (TERT), a catalytic subunit, from cytosol to nucleus. Most significantly, the inhibition of telomerase activity by curcumin correlates with several parameters of apoptosis. The results suggest that telomerase status plays an important role in the induction of apoptosis in K-562 cells by curcumin.

PARP-1 depletion in combination with carbon ion exposure significantly reduces MMPs activity and overall increases TIMPs expression in cultured HeLa cells.

BACKGROUND: Hadron therapy is an innovative technique where cancer cells are precisely killed leaving surrounding healthy cells least affected by high linear energy transfer (LET) radiation like carbon ion beam. Anti-metastatic effect of carbon ion exposure attracts investigators into the field of hadron biology, although details remain poor. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are well-known radiosensitizer and several PARP-1 inhibitors are in clinical trial. Our previous studies showed that PARP-1 depletion makes the cells more radiosensitive towards carbon ion than gamma. The purpose of the present study was to investigate combining effects of PARP-1 inhibition with carbon ion exposure to control metastatic properties in HeLa cells. METHODS: Activities of matrix metalloproteinases-2, 9 (MMP-2, MMP-9) were measured using the gelatin zymography after 85 MeV carbon ion exposure or gamma irradiation (0- 4 Gy) to compare metastatic potential between PARP-1 knock down (HsiI) and control cells (H-vector - HeLa transfected with vector without shRNA construct). Expression of MMP-2, MMP-9, tissue inhibitor of MMPs such as TIMP-1, TIMP-2 and TIMP-3 were checked by immunofluorescence and western blot. Cell death by trypan blue, apoptosis and autophagy induction were studied after carbon ion exposure in each cell-type. The data was analyzed using one way ANOVA and 2-tailed paired-samples T-test. RESULTS: PARP-1 silencing significantly reduced MMP-2 and MMP-9 activities and carbon ion exposure further diminished their activities to less than 3 % of control H-vector. On the contrary, gamma radiation enhanced both MMP-2 and MMP-9 activities in H-vector but not in HsiI cells. The expression of MMP-2 and MMP-9 in H-vector and HsiI showed different pattern after carbon ion exposure. All three TIMPs were increased in HsiI, whereas only TIMP-1 was up-regulated in H-vector after irradiation. Notably, the expressions of all TIMPs were significantly higher in HsiI than H-vector at 4 Gy. Apoptosis was the predominant mode of cell death and no autophagic death was observed. CONCLUSIONS: Our study demonstrates for the first time that PARP-1 inhibition in combination with carbon ion synergistically decreases MMPs activity along with overall increase of TIMPs. These data open up the possibilities of improvement of carbon ion therapy with PARP-1 inhibition to control highly metastatic cancers.