Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

BODIPY-Tagged Platinum(II) Curcumin Complexes for Endoplasmic Reticulum-Targeted Red Light PDT.

[Pt(RB)(Cur)]NO3 (RBC), [Pt(IRB)(Cur)]NO3 (IRBC), and [Pt(L)(Cur)]NO3 (PBC), where HCur is curcumin, L is 1-benzyl-2-(2-pyridyl)benzimidazole, and RB and IRB are red-light-active non-iodo and diiodo-BODIPY tagged to L, respectively, were synthesized and characterized, and their anticancer activities were studied (BODIPY, boron-dipyrromethene). RBC and IRBC displayed BODIPY-centered absorption bands within 615-635 nm along with the respective curcumin bands at 445 and 492 nm in 10% dimethyl sulfoxide (DMSO)-Dulbecco's phosphate-buffered saline (DPBS). Emission bands were observed at 723 and 845 nm for RBC and IRBC, respectively, in 10% DMSO-DPBS. RBC (ΦΔ, 0.27) and IRBC (ΦΔ, 0.40) generated singlet oxygen in red light (λ = 642 nm) as evidenced from 1,3-diphenylisobenzofuran (DPBF) titrations. The formation of 1O2 from BODIPY and HO• from the curcumin was evidenced from the mechanistic pUC19 DNA photocleavage studies. The BODIPY complexes showed photocytotoxicity in A549, HeLa, and MDA-MB-231 cells while being less toxic in the dark [IC50: 1.3-6.9 µM, red light; 7.2-12.8 µM, 400-700 nm visible light]. The emissive RBC displayed localization in the endoplasmic reticulum (ER). Apoptotic cell death was evidenced from the Annexin-V/fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay and green fluorescence in red light in the Fluo-4 AM assay due to ER stress, and mitochondrial dysfunction was evidenced from the 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay in A549 cells.

Cobalt(III) Complexes for Light-Activated Delivery of Acetylacetonate-BODIPY, Cellular Imaging, and Photodynamic Therapy.

Cobalt(III) complexes [Co(TPA)(L1)](ClO4)2 (1), [Co(4-COOH-TPA)(L1)](ClO4)2 (2), [Co(TPA)(L2)]Cl2 (3), and [Co(4-COOH-TPA)(L2)]Cl2 (4) having acetylacetonate-linked boron-dipyrromethene ligands (L1, acac-BODIPY; L2, acac-diiodo-BODIPY) were prepared and characterized, and their utility as bioimaging and phototherapeutic agents was evaluated (TPA, tris-(2-pyridylmethyl)amine; 4-COOH-TPA, 2-((bis-(2-pyridylmethyl)amino)methyl)isonicotinic acid). HL1, HL2, and complex 1 were structurally characterized by X-ray crystallography. Complexes 1 and 2 on photoactivation or in a reducing environment (excess GSH, ascorbic acid, and 3-mercaptopropionic acid) released the acac-BODIPY ligand. They exhibited strong absorbance near 501 nm (ε ∼ (5.2-5.8) × 104 M-1 cm-1) and emission bands near 513 nm (ΦF ∼ 0.13, λex = 490 nm) in dimethyl sulfoxide (DMSO). Complexes 3 and 4 with absorption maxima at ∼536 and ∼538 nm (ε ∼ (1.2-1.8) × 104 M-1 cm-1), respectively, afforded high singlet oxygen quantum yield (ΦΔ âˆ¼ 0.79) in DMSO. Complexes 1-4 showed Co(III)-Co(II) redox responses near -0.2 V versus saturated calomel electrode (SCE) in dimethylformamide (DMF)-0.1 M tetrabutylammonium perchlorate (TBAP). The photocleavage of pUC19 DNA by complex 4 revealed the formation of both singlet oxygen and superoxide anion radicals as the reactive oxygen species (ROS). Confocal fluorescence microscopy showed the selective accumulation of complex 1 in the endoplasmic reticulum (ER) in A-549 cells. Complex 4 exhibited a high phototherapeutic index value (PI > 7000) in HeLa cancer cells (IC50 ∼ 0.007 µM in visible light of 400-700 nm, total dose ∼5 J cm-2). The ancillary ligands in the complexes demonstrated a structure-activity relationship and modulated the Co(III)-Co(II) redox potential, the complex solubility, acac-BODIPY ligand release kinetics, and phototherapeutic efficacy.

Genome-wide DNA methylation changes in oral submucous fibrosis.

OBJECTIVE: Oral submucous fibrosis (OSF) is a debilitating potentially malignant condition of the buccal cavity characterized by extensive extracellular matrix deposition resulting in stiffness and trismus. As OSF is a progressive disease, we hypothesized that there would be extensive epigenetic changes in OSF tissues. MATERIALS AND METHODS: Using the Infinium HumanMethylation450 BeadChip Array, we analyzed gross DNA methylation changes in seven OSF tissues compared to five controls. Comparison with transcriptomic data and pathway analyses was conducted to find commonly regulated genes. RESULTS: A total of 3,294 differentially methylated regions mapping to 857 genes were identified. Comparison with transcriptome data revealed 38 downregulated-hypermethylated genes and 55 hypomethylated-upregulated genes. Using methylation-specific and qRT-PCR, aberrant hypomethylation and increased expression of FGF13, RPS6KA3, and ACSL4 genes were confirmed. Pathways involved in insulin signaling, ubiquitin-mediated proteolysis, nicotine addiction, and RAS/MAPK pathways were dysregulated, among others. Intriguingly, numerous genes located on the X chromosome were dysregulated in OSF tissues as the transcript for XIST gene was downregulated due to hypermethylation of the XIST promoter. CONCLUSIONS: This study highlights global epigenetic dysregulation of tissues of the oral cavity in OSF patients and hints at possible X chromosomal dysregulation, previously not implicated in the pathogenesis of OSF.

Differential gene expression in peritumoral brain zone of glioblastoma: role of SERPINA3 in promoting invasion, stemness and radioresistance of glioma cells and association with poor patient prognosis and recurrence.

PURPOSE: Glioblastoma (GBM) is a highly invasive tumor. Despite advances in treatment modalities, tumor recurrence is common, seen mainly in the peritumoral brain zone (PBZ). We aimed to molecularly characterize PBZ, to understand the pathobiology of tumor recurrence. METHODS/PATIENTS: We selected eight differentially regulated genes from our previous transcriptome profiling study on tumor core and PBZ. Expression of selected genes were validated in GBM (tumor core and PBZ, n = 37) and control (n = 22) samples by real time quantitative polymerase chain reaction (qPCR). Serine protease inhibitor clade A, member 3 (SERPINA3) was selected for further functional characterization in vitro by gene knockdown approach in glioma cells. Its protein expression by immunohistochemistry (IHC) was correlated with other clinically relevant GBM markers, patient prognosis and tumor recurrence. RESULTS: The mRNA expression of selected genes from the microarray data validated in tumor core and PBZ and was similar to publicly available databases. SERPINA3 knock down in vitro showed decreased tumor cell proliferation, invasion, migration, transition to mesenchymal phenotype, stemness and radioresistance. SERPINA3 protein expression was higher in PBZ compared to tumor core and also was higher in older patients, IDH wild type and recurrent tumors. Finally, its expression showed positive correlation with poor patient prognosis. CONCLUSIONS: SERPINA3 expression contributes to aggressive GBM phenotype by regulating pro-tumorigenic actions in vitro and is associated with adverse clinical outcome.

Maloplatin-B, a Cisplatin-Based BODIPY-Tagged Mito-Specific "Chemo-PDT" Agent Active in Red Light.

Maloplatin-B, a cisplatin-based complex, namely [Pt(A-BOD)(NH3)2](NO3) (Pt-A-BOD) with a pendant boron-dipyrromethene (BODIPY) moiety, where HA-BOD is a methyl malonyl chloride derived monostyryl BODIPY ligand, was designed and developed as near-IR light (600-720 nm) organelle-targeting photodynamic therapy agent. The complex [Pt(acac)(NH3)2](NO3) (Pt-Ac) was used as a control. Pt-A-BOD displayed an absorption band at 616 nm (ε = 2.9 × 104 M-1 cm-1) in 10% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (DMSO/DMEM, pH 7.2). This complex displayed a broad emission band within 650-850 nm with a λem value of 720 nm in 10% DMSO-DMEM (pH 7.2) upon excitation (λex) at 615 nm with a large Stokes shift. The fluorescence quantum yield (ΦF) value for Pt-A-BOD is 0.032 and for the ligand HA-BOD is 0.24. The BODIPY complex and ligand showed the formation of singlet oxygen as the ROS (reactive oxygen species) on irradiation with near-IR red light of 660 nm, as evidenced from a 1,3-diphenylisobenzofuran (DPBF) assay. The complex displayed remarkable apoptotic NIR light-induced PDT activity with half-maximum inhibitory concentration values (IC50) of 1.6-2.4 µM in A549 lung and HeLa cervical cancer cells, while it was less active in the dark. The cellular ROS generation by the complex in red light was ascertained by a DCFDA (2',7'-dichlorofluorescein diacetate) assay. Cellular imaging showed its localization primarily in the mitochondria of A549 cancer cells. The JC1 and Annexin-V FITC/PI assays carried out for A549 cancer cells treated with the BODIPY complex showed the alteration of mitochondrial membrane potential and apoptotic cell death on near-IR red light (600-720 nm) irradiation, respectively.

BODIPY-Ruthenium(II) Bis-Terpyridine Complexes for Cellular Imaging and Type-I/-II Photodynamic Therapy.

A series of multichromophoric ruthenium(II) complexes with the formulation [Ru(tpy-BODIPY)(tpy-R)]Cl2 (1-4), having a heteroleptic Ru(II)-bis-tpy (tpy = 4'-phenyl-2,2':6',2″-terpyridine) moiety covalently linked to a boron-dipyrromethene (BODIPY) pendant, have been prepared and characterized and their application as a phototherapeutic and photodetection agent in cancer therapy has been explored. Ligand L1 with a terpyridine-BODIPY moiety and complex 1 as its PF6 salt (1a) have been structurally characterized by a single-crystal X-ray diffraction study. Complex 1a has a distorted-octahedral RuN6 core with a Ru(II)-bis-terpyridine unit that is covalently linked to one photoactive BODIPY unit. The complexes exhibit strong absorbance near 502 nm (ε ≈ (3.7-7.8) × 104 M-1 cm-1) and high singlet oxygen sensitization ability, giving singlet oxygen quantum yield (ΦΔ) values ranging from 0.57 to 0.75 in DMSO. An emission-based study using complex 4 and Singlet Oxygen Sensor Green (SOSG) displays the formation of singlet oxygen inside the cells and also in the buffer medium upon light irradiation. DNA (pUC19) photocleavage experiments using ROS scavengers/stabilizers reveal photoinduced generation of singlet oxygen by a type-II process and of the superoxide anion radical by a type-I process. Complex 4 having a pendant biotin moiety as a cancer cell targeting group shows high photocytotoxicity with a remarkable phototherapeutic index (PI) value of >1400 in HeLa cancer cells with a low light dose activation (400-700 nm, 2.2 J cm-2). The complexes display reduced activity in noncancerous HPL1D cells. The emission property of the complexes is used for cellular imaging, thus making them suitable as next-generation theranostic PDT agents.

Both EZH2 and JMJD6 regulate cell cycle genes in breast cancer.

BACKGROUND: Strong evidences support the critical role of Jumonji domain containing 6 (JMJD6) in progression of breast cancer. Here we explore potential partners that coregulate gene expression, to understand additional pathways that are activated by higher amounts of JMJD6. METHODS: We used Gene Set Enrichment Analysis (GSEA) data to identify factors that display gene expression similar to cells treated with JMJD6 siRNA. Using chromatin immunoprecipitations (ChIP) against genomic regions that bind JMJD6 identified by in house and public database Encyclopaedia of DNA Elements (ENCODE), we confirmed JMJD6 occupancy by ChIP PCR. We tested the association of co-regulated genes with patient prognosis using The Cancer Genome Atlas (TCGA) datasets. RESULTS: JMJD6 profiles overlapped with those of Enhancer of Zeste homolog 2 (EZH2) and together they appear to co-regulate a unique cassette of genes in both ER+ and ER- cells. 496 genes including aurora kinases, which are currently being tested as novel therapeutic targets in breast cancer were co-regulated in MDA MB 231 cells. JMJD6 and EZH2 neither inter-regulated nor physically interacted with one another. Since both proteins are chromatin modulators, we performed ChIP linked PCR analysis and show that JMJD6 bound in the neighbourhood of co-regulated genes, though EZH2 data did not show any peaks within 100 kb of these sites. Alignment of binding site sequences suggested that atleast two types of binding partners could offer their DNA binding properties to enrich JMJD6 at regulatory sites. In clinical samples, JMJD6 and EZH2 expression significantly correlated in both normal and tumor samples, however the strongest correlation was observed in triple-negative breast cancer (TNBC) subtype. Co-expression of JMJD6 and EZH2 imposed poorer prognosis in breast cancer. CONCLUSIONS: JMJD6 and EZH2 regulate the same crucial cell cycle regulatory and therapeutic targets but their mechanisms appear to be independent of each other. Blocking of a single molecule may not axe cell proliferation completely and blocking both JMJD6 and EZH2 simultaneously may be more effective in breast cancer patients.

Regulation of ß-catenin by IGFBP2 and its cytoplasmic actions in glioma.

PURPOSE: IGFBP2 is one of the highly expressed genes in glioblastoma (GBM). It has both IGF dependent and independent activities. IGF independent actions are mediated by the activation of integrin signalling through its RGD motif present at C-terminal domain. One of the actions of IGFBP2 is to regulate ß-catenin by the inactivation of GSK3ß, which preferentially accumulates in the cytoplasm. The mechanism of nuclear ß-catenin regulation by IGFBP2 and role of cytoplasmic ß-catenin is not clear. We aimed to understand the mechanism in GBM cell lines. METHODS: The gene expression studies were performed by RT-PCR, western blot analysis; the knockdown of genes was performed by shRNA transfection; RNAIP and luciferase reporter assays were utilized to study the cytoplasmic regulation of genes by ß-catenin; neurosphere assays were performed to study the stemness of cells. RESULTS: IGFBP2 overexpression or treatment in GBM cells regulates ß-catenin, TRIM33 (E3 ubiquitin ligase) and Oct4 genes. TRIM33 was induced by IGFBP2. ß-catenin was found to accumulate predominantly in the cytoplasm and nuclear ß-catenin was depleted by IGFBP2 induced TRIM33. IGFBP2 regulated cytoplasmic ß-catenin binds to 3' UTR of Oct4 RNA. IGFBP2 was also able to induce stemness of glioma cells. CONCLUSIONS: IGFBP2 induces TRIM33 which regulates the nuclear ß-catenin protein. In addition, IGFBP2 stabilizes the cytoplasmic ß-catenin which is involved in the regulation of Oct4 transcript and consequently induction of stemness of glioma cells.

Ruthenium(II) Conjugates of Boron-Dipyrromethene and Biotin for Targeted Photodynamic Therapy in Red Light.

The ruthenium(II) complexes [RuCl(L1)(L3)]Cl (1), [RuCl(L1)(L4)]Cl (2), [RuCl(L2)(L4)]Cl (3), [RuCl(L1)(L5)]Cl (4), and [RuCl(L2)(L5)]Cl (5) of NNN-donor dipicolylamine (dpa) bases (L4, L5) having BODIPY (boron-dipyrromethene) moieties, NN-donor phenanthroline derivatives (L1, L2), and benzyldipicolylamine (bzdpa, L3) were prepared and characterized by spectroscopic techniques and their cellular localization/uptake and photocytotoxicity studied. Complex 1, as its PF6 salt (1a), has been structurally characterized with help of a single-crystal X-ray diffraction technique. It has a RuN5Cl core with the Cl bonded trans to the amine nitrogen atom of bzdpa. The complexes showed intense absorption spectral bands near 500 nm (ε ≈ 58000 M-1 cm-1) in 2 and 3 and 654 nm (ε ≈ 80000 M-1 cm-1) in 4 and 5 in 1/1 DMSO/DPBS (v/v). Complex 5 having biotin and PEGylated-disteryl BODIPY gave a singlet oxygen quantum yield (ΦΔ) of ∼0.65 in DMSO. Complex 5 exhibited remarkable PDT (photodynamic therapy) activity (IC50 ≈ 0.02 µM) with a photocytotoxicity index (PI) value of >5000 in red light of 600-720 nm in A549 cancer cells. The biotin-conjugated complexes showed better photocytotoxicity in comparison to nonbiotinylated analogues in A549 cells. The complexes displayed less toxicity in HPL1D normal cells in comparison to A549 cancer cells. The emissive BODIPY complexes 3 and 5 (ΦF ≈ 0.07 in DMSO) showed significant mitochondrial localization.

Transcriptome profiling reveals PDZ binding kinase as a novel biomarker in peritumoral brain zone of glioblastoma.

PURPOSE: Peritumoural brain zone (PT) of glioblastoma (GBM) is the area where tumour recurrence is often observed. We aimed to identify differentially regulated genes between tumour core (TC) and PT to understand the underlying molecular characteristics of infiltrating tumour cells in PT. METHODS: 17 each histologically characterised TC and PT tissues of GBM along with eight control tissues were subjected to cDNA Microarray. PT tissues contained 25-30% infiltrating tumour cells. Data was analysed using R Bioconductor software. Shortlisted genes were validated using qRT-PCR. Expression of one selected candidate gene, PDZ Binding Kinase (PBK) was correlated with patient survival, tumour recurrence and functionally characterized in vitro using gene knock-down approach. RESULTS: Unsupervised hierarchical clustering showed that TC and PT have distinct gene expression profiles compared to controls. Further, comparing TC with PT, we observed a significant overlap in gene expression profile in both, despite PT having fewer infiltrating tumour cells. qRT-PCR for 13 selected genes validated the microarray data. Expression of PBK was higher in PT as compared to TC and recurrent when compared to newly diagnosed GBM tumours. PBK knock-down showed a significant reduction in cell proliferation, migration and invasion with increase in sensitivity to radiation and Temozolomide treatment. CONCLUSIONS: We show that several genes of TC are expressed even in PT contributing to the vulnerability of PT for tumour recurrence. PBK is identified as a novel gene up-regulated in PT of GBM with a strong role in conferring aggressiveness, including radio-chemoresistance, thus contributing to recurrence in GBM tumours.

Molecular pathways regulated by areca nut in the etiopathogenesis of oral submucous fibrosis.

Many oral mucosal lesions are due to substance abuse, such as tobacco and areca nut, amongst others. There is considerable evidence that oral lesions/disorders such as some leukoplakias, most erythroplakias, and submucous fibrosis have malignant potential, with a conversion rate of 5%-10% over a 10-year period. There have been several reports on possible biomarkers that predict malignant conversion of the oral lesions associated with these disorders. Management of these is mostly surgical removal of the lesion followed by observation, and in some cases treatment by antioxidants and anti-inflammatory agents. Oral submucous fibrosis is due to excessive deposition of extracellular matrix in the connective tissue plus, particularly, collagens. The deposition of collagen leads to stiffness of the affected regions and results in difficulty in mouth opening. Areca nut chewing is proposed as the most probable etiological factor in the manifestation of oral submucous fibrosis. Several studies suggest involvement of proinflammatory cytokines, dysregulated by areca nut, in the development of the disease. Amongst these, transforming growth factor-ß is in the forefront, which is also shown to be involved in fibrosis of other organs. This review addresses the molecular mechanisms involved in oral submucous fibrosis development and provides a model for the regulation of transforming growth factor-ß by areca nut. It provides an exemplar of the role of modern molecular techniques in the study of oral disease.

Diplatinum(II) Catecholate of Photoactive Boron-Dipyrromethene for Lysosome-Targeted Photodynamic Therapy in Red Light.

The binuclear platinum(II) boron-dipyrromethene (BODIPY) complex [{Pt(dach)}2(µ-Dcrb)] (DP), where dach is 1,2-diaminocyclohexane and H4Dcrb is a morpholine-conjugated BODIPY-linked dicatechol bridging ligand, was prepared for lysosome organelle targeting and near-IR (NIR) light (600-720 nm) induced photocytotoxic activity. The platinum complex [Pt(dach)(cat)] (CP), where H2cat is catechol, was synthesized and used as a control complex without bearing the BODIPY unit. The complex DP displayed a band at 660 nm (ε = 2.1 × 104 M-1 cm-1) in the red region of the UV-visible spectrum recorded in 10% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (DMSO/DMEM, pH 7.2). The complex DP and the BODIPY ligand displayed emission in 10% DMSO-DMEM (pH 7.2) giving an λem value of 668 nm (λex = 650 nm) with a ΦF value of 0.02 for DP and 0.16 for H4Dcrb (ΦF, fluorescence quantum yield). Titration experiments using 1,3-diphenylisobenzofuran (DPBF) indicated that the complex DP and H4Dcrb on irradiation with near-IR light of 600-720 nm generated singlet oxygen (1O2) as the ROS (reactive oxygen species). The complex DP showed significant lysosomal localization and remarkable apoptotic photodynamic therapy (PDT) effects, giving half-maximal inhibitory concentration values (IC50) within 0.6-3.4 µM in HeLa cervical cancer, A549 lung cancer, and MDA-MB231 multidrug resistant cancer cells, while being essentially nontoxic in the dark and in the HPL1D immortalized lung epithelial normal cells. The acridine orange assay using A549 cells showed lysosomal membrane permeabilization by the complex DP under near-IR light (600-720 nm). This complex on near-IR light (600-720 nm) activation in A549 cells induced apoptotic cell death, as observed from an Annexin-V FITC assay.

JMJD6 induces HOTAIR, an oncogenic lincRNA, by physically interacting with its proximal promoter.

Using microarray analysis, we found that HOX transcript antisense intergenic RNA (HOTAIR) is up-regulated by Jumonji domain containing-6 (JMJD6), a bifunctional lysyl hydroxylase and arginine demethylase. In breast cancer, both JMJD6 and HOTAIR RNAs increase tumor growth and associate with poor prognosis but no molecular relationship between them is known. We show that overexpression of JMJD6 increased HOTAIR expression and JMJD6 siRNAs suppressed it in ER+ MCF-7, triple negative MDA-MB-231 and non-breast cancer HEK 293 cells. Therefore, JMJD6 regulates HOTAIR independent of ER status. Using various deletion constructs spanning (-1874 to +50) of the HOTAIR promoter, we identified pHP216 (-216 to +50 bp) as the smallest construct that retained maximal JMJD6 responsiveness. In ChIP assays, JMJD6 bound this region suggesting that JMJD6 may be directly recruited to the HOTAIR promoter. Mutant JMJD6H187A that is devoid of enzymatic activity could bind this site but failed to induce transcription. ChIP and electromobility shift assays identified a JMJD6 interaction region from (-123 to -103 bp) within the HOTAIR promoter. In tumor samples but not normal breast tissue, the expression of JMJD6 linearly correlated with HOTAIR suggesting that JMJD6-mediated up-regulation may occur specifically in tumors. Further, concurrent high expression of both genes correlated with poor survival when individual expression of either gene showed no significant association in TCGA datasets. We propose that high JMJD6 expression may achieve higher levels of HOTAIR in breast tumors. Further, since high levels of HOTAIR promote metastasis and death, blocking JMJD6 may be useful in preventing such events.

Reduction Responsive Nanovesicles Derived from Novel α-Tocopheryl-Lipoic Acid Conjugates for Efficacious Drug Delivery to Sensitive and Drug Resistant Cancer Cells.

Two novel α-tocopheryl-lipoic acid conjugates (TL1 and TL2) were synthesized for the anticancer drug, doxorubicin (DOX), delivery. Both conjugates were able to form stable nanovesicles. The critical aggregation concentration (CAC) was determined using 4-(N,N-dimethylamino)cinnamaldehyde (DMACA) as a fluorescence probe. Formation of highly packed nanovesicles was characterized by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy and microviscosity measurements. The morphologies of nanovesicles were visualized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The response of nanovesicles to reducing environment of cells was probed by the addition of dithiothreitol (DTT), which was followed by the increase in the hydrodynamic diameter under dynamic light scattering (DLS) measurements. The encapsulation efficiency of a commonly used anticancer drug, doxorubicin (DOX), in nanovesicles was found to be ∼60% and ∼55% for TL1 and TL2, respectively (TL1-DOX and TL2-DOX). Also, the cumulative drug (DOX) release from DOX-encapsulated nanovesicles in response to biological reducing agent glutathione (GSH) was ∼50% and ∼40% for TL1-DOX and TL2-DOX, respectively, over a period of 10 h. Both TL1-DOX and TL2-DOX delivered the anticancer drug, doxorubicin (DOX), across the DOX-sensitive and DOX-resistant HeLa (HeLa-DOXR) cells in an efficient manner and significantly more efficaciously than the drug alone treatments, especially in HeLa-DOXR cells. The nanovesicle mediated DOX treatment also showed significantly higher cell death when compared to DOX alone treatment in HeLa-DOXR cells. Blood compatibility of the nanovesicles was supported from clotting time, hemolysis, and red blood cell (RBC) aggregation experiments for their potential in vivo applications. Concisely, we present biocompatible and responsive nanovesicles for efficacious drug delivery to drug-sensitive and drug-resistant cancer cells.

TGF-ß induces changes in breast cancer cell deformability.

Mechanical properties of cells are shown to regulate cell behaviors leading to phenotypic changes that may aid in the development and progression of disease. In this study, we used atomic force microscopy (AFM) indentation with a spherical probe to characterize the elastic and viscoelastic properties of invasive (MDA-MB-231) and noninvasive (MCF-7) breast cancer cells treated with transforming growth factor-ß (TGF-ß). We also used confocal fluorescence imaging to investigate the sub-membrane cytoskeletal structure of the cells. Results showed significant alterations in moduli of both cell types after 24 h TGF-ß treatment which had a context dependent response; moduli for MDA-MB-231 decreased whereas MCF-7 demonstrated stiffening response. Stress relaxation tests showed increased fluid-like nature of MDA-MB-231 following TGF-ß treatment and lower fluidity for MCF-7 cells. We also observed significant alterations in the expression and orientation of actin stress fibers with TGF-ß treatment which correlated with the changes in cell mechanics. Less invasive MCF-7 cells had a delayed overall increase in cell deformability after 48 h exposure to TGF-ß; a similar trend was observed for MDA-MB cells. These changes may be important to facilitate migration, for instance, during metastasis of cancer cells through submicron sized spaces.

Traction cytometry: regularization in the Fourier approach and comparisons with finite element method.

Traction forces exerted by adherent cells are quantified using displacements of embedded markers on polyacrylamide substrates due to cell contractility. Fourier Transform Traction Cytometry (FTTC) is widely used to calculate tractions but has inherent limitations due to errors in the displacement fields; these are mitigated through a regularization parameter (γ) in the Reg-FTTC method. An alternate finite element (FE) approach computes tractions on a domain using known boundary conditions. Robust verification and recovery studies are lacking but essential in assessing the accuracy and noise sensitivity of the traction solutions from the different methods. We implemented the L2 regularization method and defined a maximum curvature point in the traction with γ plot as the optimal regularization parameter (γ*) in the Reg-FTTC approach. Traction reconstructions using γ* yield accurate values of low and maximum tractions (Tmax) in the presence of up to 5% noise. Reg-FTTC is hence a clear improvement over the FTTC method but is inadequate to reconstruct low stresses such as those at nascent focal adhesions. FE, implemented using a node-by-node comparison, showed an intermediate reconstruction compared to Reg-FTTC. We performed experiments using mouse embryonic fibroblast (MEF) and compared results between these approaches. Tractions from FTTC and FE showed differences of ∼92% and 22% as compared to Reg-FTTC. Selection of an optimum value of γ for each cell reduced variability in the computed tractions as compared to using a single value of γ for all the MEF cells in this study.

Glucose-Appended Platinum(II)-BODIPY Conjugates for Targeted Photodynamic Therapy in Red Light.

Platinum(II) complexes [Pt(L1)(R-BODIPY)]Cl (1) and [Pt(L2)(R-BODIPY)]Cl (2), where R-BODIPY is 8-(4-ethynylphenyl)-distyryl-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3, L1 is 4'-phenyl-2,2':6',2″-terpyridine, and L2 is (2,2':6',2″-terpyridin-4'-oxy)ethyl-ß-d-glucopyranoside, were synthesized and characterized, and their photocytotoxicity was studied. The phenylacetylide complex [Pt(L1)(C≡CPh)]Cl (3) was prepared and used as a control. Complexes 1 and 2 showed near-IR absorption bands at 713 nm (ε = 3.47 × 104 M-1 cm-1) and 715 nm (3.2 × 104 M-1 cm-1) in 10% dimethyl sulfoxide (DMSO)-Dulbecco's Modified Eagle's Medium (DMEM) (pH 7.2). The BODIPY complexes are emissive in 10% DMSO-DMEM at pH 7.2 with λem (λex, Φf) = 822 nm (710 nm, 0.022) for complex 1 and λem (λex, Φf) = 825 nm (710 nm, 0.026) for complex 2. They generated singlet oxygen (1O2) in red light as evidenced from 1,3-diphenylisobenzofuran (DPBF) titration experiments. The singlet oxygen quantum yield (ΦΔ) values for 1 and 2 were ∼0.6 signifying their photosensitizing ability. They were remarkably photodynamic therapy (PDT) active in red light showing significant red light-induced cytotoxicity in cervical HeLa, lung cancer A549, and breast cancer MCF-7 cells (IC50: 2.3-24.7 µM in light) with negligible dark toxicity (IC50 > 100 µM). A significant enhancement in cellular uptake was observed for 2 having glucose-appended terpyridine ligand compared to 1. The confocal microscopy showed significant mitochondrial localization of the complexes as evidenced from the JC-1 assay. The complexes released the photoactive R-BODIPY ligand upon red light-irradiation as evidenced from the mass and 1H NMR spectral studies. Complex 2 is remarkable in satisfying the essential requirements of targeted PDT in red light.

Pyriplatin-Boron-Dipyrromethene Conjugates for Imaging and Mitochondria-Targeted Photodynamic Therapy.

Monofunctional pyriplatin analogues cis-[Pt(NH3)2(L)Cl](NO3) (1-3) having boron-dipyrromethene (BODIPY) pendants (L) with 1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4'-difluoroboradiazaindacene moieties were designed and synthesized, and their photocytotoxic properties were studied. The Pt-BODIPY conjugates displayed an absorption band within 505-550 nm and a green emissive band near 535 nm in 1% DMSO/DMEM (Dulbecco's modified Eagle's medium) buffer. Complex cis-[Pt(NH3)2(4-Me-py)Cl](NO3) (4) was used as a control for determining the structural aspects by X-ray crystallography. The mono- and diiodinated BODIPY complexes 2 and 3 showed generation of singlet oxygen on light activation as evidenced from the 1,3-diphenylisobenzofuran (DPBF) titration experiments. The cytotoxicity of the BODIPY complexes was tested against A549 (human lung cancer), MCF-7 (human breast cancer), and HaCaT (human skin keratinocyte) cells in dark and visible light (400-700 nm, 10 J cm-2). While complexes 2 and 3 showed excellent photocytotoxicity (IC50 ≈ 0.05 µM), they remained essentially nontoxic in the dark (IC50 > 100 µM). The emissive bands of 1 and 2 were used for cellular imaging by confocal microscopy study, which showed their mitochondrial localization. This was further supported by platinum estimation from isolated mitochondria and mitochondrial depolarization through a JC-1 assay. The photomediated apoptotic cell death was evidenced from flow cytometric assays, annexin-V/FITC-PI (fluorescein isothiocyanate-propidium iodide) and cell cycle arrest in sub-G1 and G2/M phases. The complexes bind to 9-ethylguanine as a model nucleobase to form monoadducts. A mechanistic study on DNA photocleavage activity using pUC19 DNA showed singlet oxygen as the reactive oxygen species (ROS). The combination of photodynamic therapy with DNA cross-linking property enhanced the anticancer potential of the monofunctional BODIPY-conjugates of pyriplatins.

DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression.

Long non-coding RNAs (lncRNAs) are emerging as important players in regulation of gene expression in higher eukaryotes. DDX5/p68 RNA helicase protein which is involved in splicing of precursor mRNAs also interacts with lncRNAs like, SRA and mrhl, to modulate gene expression. We performed RIP-seq analysis in HEK293T cells to identify the complete repertoire of DDX5/p68 interacting transcripts including 73 single exonic (SE) lncRNAs. The LOC284454 lncRNA is the second top hit of the list of SE lncRNAs which we have characterized in detail for its molecular features and cellular functions. The RNA is located in the same primary transcript harboring miR-23a∼27a∼24-2 cluster. LOC284454 is a stable, nuclear restricted and chromatin associated lncRNA. The sequence is conserved only in primates among 26 different species and is expressed in multiple human tissues. Expression of LOC284454 is significantly reduced in breast, prostate, uterus and kidney cancer and also in breast cancer cell lines (MCF7 and T47D). Global gene expression studies upon loss and gain of function of LOC284454 revealed perturbation of genes related to cancer-related pathways. Focal adhesion and cell migration pathway genes are downregulated under overexpression condition, and these genes are significantly upregulated in breast cancer cell lines as well as breast cancer tissue samples suggesting a functional role of LOC284454 lncRNA in breast cancer pathobiology.