Analysis Of Nuclear Morphology Research Articles

Transition metal oxide nanoparticles are effective in inhibiting lung cancer cell survival in the hypoxic tumor microenvironment

BackgroundThe transition metal oxide nanoparticles are in focus for their anti-cancer potential. In this study we have synthesized and characterized CuO, NiO and Fe2O3 nanoparticles and, investigated their cytotoxic potential in the heterogeneous tumour microenvironment. MethodsNanoparticles were synthesized by aqueous precipitation method and characterized with UV-Visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and X-ray diffraction (XRD). Cell viability of lung cancer cells (A549) grown in normoxia (18%O2) and hypoxia (1%O2) was determined for all nanoparticles. The mechanism of cell death was assessed by nuclear morphological analysis, flow cytometry analysis and western blotting. Generation of intracellular ROS in treated cells and its contribution to cell viability was determined. ResultsThe synthesized metal oxide nanoparticles were successfully characterized with SEM, spectroscopy and X-ray diffraction patterns. Cell viability of lung cancer cells was compromised in both normoxia and hypoxia. ROS generation was shown to contribute to cellular toxicity in CuO, but not NiO and Fe2O3. ConclusionWe have shown the therapeutic potential of CuO, NiO and Fe2O3 nanoparticles in non small cell lung cancer cells cultured in hypoxia, a relevant feature of solid tumors along with normoxia. The newly synthesized nanoparticles showed efficacy in both conditions. General significanceHypoxia drives metabolic alterations and epigenetic modifications in the tumor microenvironment. By using conditions that mimic tumour microenvironment, this study expands the possibility of using metal oxide nanoparticles as a therapeutic agent for lung cancer treatment.

Extracellular histones reduce survival and angiogenic responses of late outgrowth progenitor and mature endothelial cells

ESSENTIALS: Extracellular histones are highly augmented in sites of neovessel formation, such as regeneration tissues. We studied histone effect on survival and angiogenic activity of mature and progenitor endothelial cells. Extracellular histones trigger apoptosis and pyroptosis and reduce angiogenesis in vivo and in vitro. Histone blockade can be useful as a therapeutic strategy to improve angiogenesis and tissue regeneration. Extracellular histones are highly augmented in sites of neovessel formation, like regeneration tissues. Their cytotoxic effect has been studied in endothelial cells, although the mechanism involved and their action on endothelial colony-forming cells (ECFCs) remain unknown. To study the effect of histones on ECFC survival and angiogenic functions and compare it with mature endothelial cells. Nuclear morphology analysis showed that each human recombinant histone triggered both apoptotic-like and necrotic-like cell deaths in both mature and progenitor endothelial cells. While H1 and H2A exerted a weak toxicity, H2B, H3 and H4 were the most powerful. The percentage of apoptosis correlated with the percentage of ECFCs exhibiting caspase-3 activation and was zeroed by the pan-caspase inhibitor Z-VAD-FMK. Necrotic-like cell death was also suppressed by this compound and the caspase-1 inhibitor Ac-YVAD-CMK, indicating that histones triggered ECFC pyroptosis. All histones, at non-cytotoxic concentrations, reduced migration and H2B, H3 and H4 induced cell cycle arrest and impaired tubulogenesis via p38 activation. Neutrophil-derived histones exerted similar effects. In vivo blood vessel formation in the quail chorioallantoic membrane was also reduced by H2B, H3 and H4. Their cytotoxic and antiangiogenic effects were suppressed by unfractioned and low-molecular-weight heparins and the combination of TLR2 and TLR4 blocking antibodies. Histones trigger both apoptosis and pyroptosis of ECFCs and inhibit their angiogenic functions. Their cytotoxic and antiangiogenic effects are similar in mature endothelial cells and disappear after heparin addition or TLR2/TLR4 blockade, suggesting both as therapeutic strategies to improve tissue regeneration.

Quantification of apoptosis and necroptosis at the single cell level by a combination of Imaging Flow Cytometry with classical Annexin V/propidium iodide staining

Precisely identifying the type of programmed cell death is one of the key questions in contemporary biomedical research. We developed a straightforward approach allowing quantitative discrimination between two types of cell death on the single cell level: apoptosis and necroptosis. This method uses the combination of imaging flow cytometry with classical Annexin V/propidium iodide staining, which allows for the ascertainment of typical features of dying cells: exposure of the phospholipid phosphatidylserine and the loss of membrane integrity. Image-based analysis of nuclear morphology enables us to distinguish between secondary necrotic/late apoptotic and necroptotic cells directly in one assay. This is a major advantage compared to other contemporary approaches of necroptosis detection, which require a parallel application of several methods. This approach can be used for the quantitative assessment of cell death in cell and systems biology studies of signal transduction networks.

Anti-proliferative effect of fungal taxol extracted from Cladosporium oxysporum against human pathogenic bacteria and human colon cancer cell line HCT 15

Cladosporium oxysporum a new taxol producing endophytic fungus was identified and production of taxol were characterized using UV–visible spectroscopy (UV–vis), high-performance liquid chromatography (HPLC), infrared (IR) nuclear magnetic resonance spectroscopy (NMR (13C and 1H)) and liquid chromatography–mass spectrometry (LC–MS). The taxol biosynthetic gene (dbat) was evaluated for new taxol producing fungus. Antibacterial activity against six different human pathogenic bacteria was done by agar well diffusion method. The anticancer efficacy of isolated fungal taxol were also evaluated in human colon cancer cell HCT 15 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytotoxicity and nuclear morphology analysis. The isolated fungal taxol showed positive towards biosynthetic gene (dbat) and effective against both Gram positive as well as Gram negative. The fungal taxol suppress growth of cancer cell line HCT 15 with an IC50 value of 3.5μM concentration by 24h treatment. Thus, the result reveals that C. oxysporum could be a potential alternative source for production of taxol and have antibacterial as well as anticancer properties with possible clinical applications.

DNA methylation and differential gene regulation in photoreceptor cell death.

Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP.

Programmed cell death induced by (-)-8,9-dehydroneopeltolide in human promyelocytic leukemia HL-60 cells under energy stress conditions.

(+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.

Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities

The present study was aimed at biosynthesis of silver nanoparticles (AgNPs) using ethanolic extract of rose (Rosa indica) petals and testing their potential antibacterial activity using selective human pathogenic microbes, anticancer activity using human colon adenocarcinoma cancer cell line HCT 15 as well as anti-inflammatory activity using rat peritoneal macrophages in vitro. The biologically synthesized AgNPs were also characterized by UV–visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The characterized AgNPs showed an effective antibacterial activity against Gram negative (Escherichia coli, Klebsiella pneumoniae) than Gram positive (Streptococcus mutans, Enterococcus faecalis) bacteria. MTT assay, analysis of nuclear morphology, mRNA expression of Bcl-2, Bax and protein expression of caspase 3 as well as 9, indicated potential anticancer activity. In addition, green synthesized AgNPs also attenuated cytotoxicity, nuclear morphology and free radical generation (O2− and NO) by rat peritoneal macrophages in vitro. The results of our study show the potential green synthesis of silver nanoparticles in mitigating their toxicity while retaining their antibacterial activities.

Computational image analysis of colony and nuclear morphology to evaluate human induced pluripotent stem cells.

Non-invasive evaluation of cell reprogramming by advanced image analysis is required to maintain the quality of cells intended for regenerative medicine. Here, we constructed living and unlabelled colony image libraries of various human induced pluripotent stem cell (iPSC) lines for supervised machine learning pattern recognition to accurately distinguish bona fide iPSCs from improperly reprogrammed cells. Furthermore, we found that image features for efficient discrimination reside in cellular components. In fact, extensive analysis of nuclear morphologies revealed dynamic and characteristic signatures, including the linear form of the promyelocytic leukaemia (PML)-defined structure in iPSCs, which was reversed to a regular sphere upon differentiation. Our data revealed that iPSCs have a markedly different overall nuclear architecture that may contribute to highly accurate discrimination based on the cell reprogramming status.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Inhibition of transient receptor potential melastatin 7 (TRPM7) channel induces RA FLSs apoptosis through endoplasmic reticulum (ER) stress.

Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) apoptosis induced by thapsigargin in vitro. In this study, using a combination of Western blotting, RT-PCR, and nuclear morphology analysis, we investigated the influence and potential function of TRPM7 channels on the apoptosis induced by thapsigargin in RA FLSs. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in RA FLSs apoptosis. The expression of TRPM7 was significantly potentiated in RA FLSs. Co-incubation of RA FLSs with Gd(3+), 2-APB, or TRPM7-siRNA increased cell apoptosis. Furthermore, we found that suppression of TRPM7 channels also increased the expression CHOP and calpain and decreased the expression caspase-3. We conclude that suppression of TRPM7 channels may increase RA FLSs apoptosis in vitro, and this is associated with endoplasmic reticulum (ER) stress. Therefore, inhibition of TRPM7 could activate ER stress and induce RA FLSs apoptosis.

Cationic porphyrin derivatives for application in photodynamic therapy of cancer

Current studies in photodynamic therapy (PDT) against cancer are focused on the development of new photosensitizers (PSs), with higher phototoxic action. The aim of this study was to compare the therapeutic efficiency of tri-cationic meso-substituted porphyrin derivatives (Tri-Py+–Me–PF, Tri-Py+–Me–Ph, Tri-Py+–Me–CO2Me and Tri-Py+–Me–CO2H) with the well-known tetra-cationic T4PM. The phototoxic action of these derivatives was assessed in human colon adenocarcinoma cells by cell viability, intracellular localization and nuclear morphology analysis. In the experimental conditions used we determined that after light activation –PF, –Ph and –CO2Me cause a more significant decline of cell viability compared to –CO2H and T4PM. These results suggest that the nature of the peripheral substituent influences the extent of cell photodamage. Moreover, we have demonstrated that PS concentration, physicochemical properties and further light activation determine the PDT response. All porphyrins were clearly localized as a punctuated pattern in the cytoplasm of the cells, and the PDT scheme resulted in apoptotic cell death after 3 h post-PDT. The tri-cationic porphyrin derivatives Tri-Py+–Me–PF, Tri-Py+–Me–Ph and Tri-Py+–Me–CO2Me showed a promising ability, making them good photosensitizer candidates for oncological PDT.

Apoptosis mediated anti-proliferative effect of compound isolated from Cassia auriculata leaves against human colon cancer cell line

A compound was isolated from Cassia auriculata leaves and characterized by high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC–MS), UV–vis spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR). The in vitro anticancer effect of the compound isolated from C. auriculata was evaluated in human colon cancer cells HCT 15 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cytotoxicity, nuclear morphology analysis and measurement of lactate dehydrogenase. The isolated compound 4-(2,5 dichlorobenzyl)-2,3,4,5,6,7 hexahydro7(4 methoxyphenyl)benzo[h][1,4,7] triazecin8(1H)-one showed 50% inhibition of HCT 15 cells when tested at 20μg/ml after 24h incubation. Cytotoxicity, nuclear morphology and lactate dehydrogenase assays clearly show potent anticancer activity of the isolated compound against colon cancer. Thus, the in vitro findings suggest that the compound isolated from C. auriculata leaves have potent anti-cancer properties with possible clinical applications.

Quantitative Analysis of Tendon Structural Integrity in Chickens using Fourier Transform-Second Harmonic Generation Imaging

Degenerative changes in tendons are common in human and land-dwelling vertebrates of all species, and are associated with both age and use. Tendon rupture and inflammation are common causes of lameness in broilers and breeders at multiple ages and has become a very important issue in the poultry industry attempts to analyze tendon health have relied primarily on visual assessment. The ability to conduct meaningful experiments on causes and interventions would be much improved by a direct assay of tendon structural integrity. The objective of this study is to test whether we can use Fourier transform-second harmonic generation (FT-SHG) imaging technique to quantify the differences in collagen fiber organization and nuclei morphology between normal and injured chicken tendons. Tendon injury/degeneration was induced by rearing chickens in wire-floor pens and feeding them an oxidized fat diet. Injured tendons had greater collagen fiber degradation/degeneration; more randomly organized collagen fibers, higher cellularity and more circular-shaped nuclei than normal tendons, indicating loss of collagen tertiary structure and lateral compression in injured tendons. Therefore, analysis of collagen fiber organization and nuclear morphology using FT-SHG imaging is a potential diagnostic tool to evaluate tendon structural health and to test the effects of nutrients and intervention strategies on the structural development of tendons in chickens.

Nuclear morphometric and morphological analysis of exfoliated buccal and tongue dorsum cells in type-1 diabetic patients

Background:Diabetes mellitus type 1 that results from immunologically mediated damage to the β-cells in the pancreas. Diabetes mellitus is characterized by recurrent or persistent hyperglycemia. Hyperglycemia can be associated with salivary gland dysfunction and alterations in the oral epithelial cells.Aim:The aim of this study was to evaluate the qualitative and quantitative changes in buccal and tongue dorsum epithelial cells using an exfoliative cytology method in type 1 diabetic patients.Materials and Methods:We performed light microscopic analysis of the buccal and tongue dorsum smears in thirty type 1 diabetic patients and thirty healthy individuals. The oral smears were stained using Papanicolaou method for cytological examination and nuclear morphometric analysis. In each case, the mean nuclear area, perimeter, length, breadth, and roundness factor were evaluated in each smear using the image analysis software (Q Win, Leica™).Results:The nuclear area, length, breadth, and perimeters were significantly higher in the diabetic group from tongue dorsum smear than that of the control group (P < 0.05). In the cytological examination, karyorrhexis-karyolysis-karyopyknosis, binucleation, nuclear membrane irregularity, cytoplasmic polymorphism, perinuclear halo were observed in oral smears with type 1 diabetic patients. Binucleation (P = 0.002) and nuclear membrane irregularity (P = 0.024) were significantly more common in buccal smears of diabetic group. Furthermore, the sensitivity of buccal mucosa was significantly higher in the diabetic group (P = 0.006).Conclusion:The light microscopic and nuclear morphometric study indicates that type 1 diabetes can produce morphological and nuclear morphometric changes in the oral mucosa that are noticeable with exfoliative cytology.

Induction of apoptosis by c9, t11-CLA in human endometrial cancer RL 95-2 cells via ERα-mediated pathway

Numerous studies have shown that conjugated linoleic acid (CLA) can inhibit cancer cells growth and induce apoptosis in vitro and in vivo. The aim of the present study was to investigate the effects of CLA, including cis9, trans11-conjugated linoleic acid (c9, t11-CLA) and trans10, cis12-conjugated linoleic acid (t10, c12-CLA), on apoptosis of human endometrial cancer RL 95-2 cells and its related mechanisms.The MTT analysis was used to evaluate the effect of CLA isomers on the viability of endometrial cancer RL 95-2 cells. We then estimated the apoptosis by Morphological observation and Annexin V-FITC/PI staining and flow cytometry. We also used Western blot analysis to assess the expression of caspase-3, Bax, Bcl-2 proteins and the activation of Akt/p-Akt and ERα/p-ERα. Propylpyrazole-triol (PPT), a selective ERα agonist was used to confirm the induction of apoptosis by c9, t11 CLA may relate to ERα-mediated pathway.In CLA-treated RL 95-2 cells, we found that c9, t11-CLA inhibited viability and trigged apoptosis, as judged from nuclear morphology and flow cytometric analysis. The expression of caspase-3 and the ratio of Bax/Bcl-2 were significant increased, but no obvious change was observed about Akt and p-Akt in c9, t11-CLA-treated cells. However, the expression of total ERα level in RL 95-2 cells-treated with c9, t11-CLA was unchanged, while in the concentration of 80mM, c9, t11-CLA down-regulated the protein expression level of p-ERα. Then PPT has the antagonistic action on growth inhibitory effect in RL 95-2 cells incubated with c9, t11-CLA.This study demonstrated that c9, t11- CLA could induce apoptosis in RL 95-2 cells, and may involve in ERα-mediated pathway. These results indicated that c9, t11- CLA could induce apoptosis of endometrial cancer cells and may be potential agents for the treatment of endometrial cancer.

Correlation analysis of nuclear morphology, cytokeratin and Ki‐67 expression of urothelial carcinoma cells

We aimed to delineate the morphogenesis of aberrant nuclear features of urothelial carcinoma (UC) cells in association with cytokeratin (CK) expression patterns and cell proliferation activity. Correlation analysis of the nuclear area by morphometry and the expression patterns of CK5, CK20 and Ki-67 by triple immunofluorescence analysis was applied to 1699 cells from five low-grade and seven high-grade cases of UC. The majority of UC cells showed aberrant cellular differentiation represented by abnormal CK expression patterns of CK5+ / CK20+ (40.5%) or CK5- / CK20+ (56.0%). CK5+ / CK20- cells, a phenotype of cancer stem/progenitor cells, represented a very small population (1.9%) and showed a low proliferation activity. Ki-67+ cells showed a significantly different CK expression pattern compared with that of Ki-67(-) cells. The nuclear areas of CK5- / CK20+ cells (71.3 ± 25.9 μm2) were significantly larger than those of CK5+ / CK20+ cells (66.6 ± 25.5 μm2). Negativity for CK5 was related to the grade of UC and an increased number of CK5- / CK20+ / Ki-67+ cells was related to a higher malignant potential. We conclude the nuclear morphology is related to cell differentiation represented by CK expression and cell proliferative activity.

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H2O2 exposure, both triggers apoptosis in retinal neuronal cell line RGC-5 (retinal ganglion cell-5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC-5 under these conditions. Sub-confluent cells were treated either with H2O2 or maintained in SFM (serum-free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC-5 by 72 h serum starvation and 500 M H2O2 exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H2O2 treatment. Serum starvation did not alter JNK1 (c-Jun N-terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho-JNK) was evident. Conversely, H2O2 exposure blocked the expression and activation of JNK1 to phospho-JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC-5 cells may follow different signalling pathways when challenged with serum starvation and H2O2.

Biological evaluation of a new family of aminosteroids that display a selective toxicity for various malignant cell lines

This study investigated the antineoplasic potential of a new family of aminosteroids. The antiproliferative activity of seven 5α-androstane-3α,17β-diol derivatives selected from a screening study was measured on nine cancerous cell lines (HL-60, K-562, LNCaP, PC-3, Shionogi, MCF-7, MDA-MB-231, BT-20, and OVCAR-3) and two normal cell lines (peripheral blood lymphocytes and WI-38). The aminosteroids efficiently inhibited the cell growth of seven cancer cell lines [inhibitory concentration (IC(50)) values=0.2-6.4 µmol/l] and showed weak toxicity on normal cell lines. Two representative aminosteroids were tested and found to induce apoptosis and a G0/G1 cell cycle block in HL-60-treated cells, but not terminal myeloid differentiation. By a nuclear morphology analysis with fluorescence microscopy, typical apoptotic morphological changes were exhibited by treated cells. One aminosteroid tested in vivo (xenograft model) reduced the breast cancer (MCF-7 cells) tumor growth induced in nude mice. Furthermore, the information gathered suggests that this family of aminosteroids induced growth inhibition cells by arresting the cell cycle and triggering apoptosis.

Insulin catalyzes the curcumin-induced wound healing: An in vitro model for gingival repair

Objectives:Human gingival fibroblasts (hGFs) play a major role in the maintenance and repair of gingival connective tissue. The mitogen insulin with IGFs etc. synergizes in facilitating wound repair. Although curcumin (CUR) and insulin regulate apoptosis, their impact as a combination on hGF in wound repair remains unknown. Our study consists of: 1) analysis of insulin-mediated mitogenesis on CUR-treated hGF cells, and 2) development of an in vitro model of wound healing.Materials and Methods:Apoptotic rate in CUR-treated hGF cells with and without insulin was observed by AnnexinV/PI staining, nuclear morphological analysis, FACS and DNA fragmentation studies. Using hGF confluent cultures, wounds were mechanically created in vitro and incubated with the ligands for 48 h in 0.2% fetal bovine serum DMEM.Results:CUR alone showed dose-dependent (1–50 μM) effects on hGF. Insulin (1 μg/ml) supplementation substantially enhanced cell survival through up-regulation of mitogenesis/anti-apoptotic elements.Conclusions:The in vitro model for gingival wound healing establishes that insulin significantly enhanced wound filling faster than CUR-treated hGF cells over 48 h. This reinforces the pivotal role of insulin in supporting CUR-mediated wound repair. The findings have significant bearing in metabolic dysfunctions, e.g. diabetes, atherosclerosis, etc., especially under Indian situations.

Automated Sputum Cytometry for Detection of Intraepithelial Neoplasias in the Lung

Background: Despite the benefits of early lung cancer detection, no effective strategy for early screening and treatment exists, partly due to a lack of effective surrogate biomarkers. Our novel sputum biomarker, the Combined Score (CS), uses automated image cytometric analysis of ploidy and nuclear morphology to detect subtle intraepithelial changes that often precede lung tumours.Methods: 2249 sputum samples from 1795 high-risk patients enrolled in ongoing chemoprevention trials were subjected to automated quantitative image cytometry after Feulgen-thionin staining. Samples from normal histopathology patients were compared against samples from carcinomain situ(CIS) and cancer patients to train the CS.Results: CS correlates with several lung cancer risk factors, including histopathological grade, age, smoking status, and p53 and Ki67 immunostaining. At 50% specificity, CS detected 78% of all highest-risk subjects—those with CIS or worse plus those with moderate or severe dysplasia and abnormal nuclear morphology.Conclusion: CS is a powerful yet minimally invasive tool for rapid and inexpensive risk assessment for the presence of precancerous lung lesions, enabling enrichment of chemoprevention trials with highest-risk dysplasias. CS correlates with other biomarkers, so CS may find use as a surrogate biomarker for patient assessment and as an endpoint in chemoprevention clinical trials.