Small molecule interactions with biomacromolecules: selective sensing of human serum albumin by a hexanuclear manganese complex - photophysical and biological studies.

A covalently bonded hexanuclear neutral complex, [Mn6(µ3-O)2(3-MeO-salox)6(OAc)2(H2O)4] (1), has been synthesized and characterized by single crystal X-ray diffraction analysis along with IR and HRMS studies. Complex 1 has been found to selectively interact with human serum albumin (HSA), a model transport protein. The interaction of 1 with HSA was investigated by monitoring the change in the absorbance value of HSA at λ = 280 nm with increasing concentration of 1. Likewise, fluorescence titrations were carried out under two conditions: (i) titration of a 5 µM solution of complex 1 with the gradual addition of HSA, showing a ∼9-fold fluorescence intensity enhancement at 424 nm, upon excitation at 300 nm; and (ii) upon excitation at 295 nm, titration of 5 µM HSA solution with the incremental addition of complex 1, showing a quenching of fluorescence intensity at 334 nm, with simultaneous development of a new emission band at 424 nm. A linear form of the Stern-Volmer equation gives KSV = 9.77 × 104 M-1 and the Benesi-Hildebrand plot yields the binding constant as KBH = 1.98 × 105 M-1 at 298 K. The thermodynamic parameters, ΔS°, ΔH°, and ΔG°, were estimated by using the van't Hoff relationship which infer the major contribution of hydrophobic interactions between HSA and 1. It was observed that quenching of HSA emission arises mainly through a dynamic quenching mechanism as indicated by the dependence of average lifetime 〈τ〉 on the concentration of 1. The changes in the CD (circular dichroism) spectral pattern of HSA in the presence of 1 clearly establish the variation of HSA secondary structure on interaction with 1. The most probable interaction region in HSA for 1 was determined from molecular docking studies which establish the preferential trapping of 1 in the subdomain IIA of site I in HSA and substantiated by the results of site-specific marker studies. Complex 1 was further evaluated for its antiproliferative effects in lung cancer A549 cells, which strictly inhibits the growth of the cells in both 2D and 3D mammospheres, indicating its potential application as an anticancer drug.

Effect of Lysyl Oxidase G473 A Polymorphism on Lysyl Oxidase and Total Soluble Collagen Expression in Oral Submucous Fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a debilitating collagen-metabolic disorder leading to submucosal fibrosis and trismus. Lysyl oxidase (LOX), a critical collagen biosynthetic enzyme, is up-regulated in OSF. Polymorphisms in the Lysyl oxidase gene have been associated with increased risk of OSF and might affect normal collagen synthesis, accumulation, or degradation, crucial in determining fibrosis severity. METHODS: One hundred OSF cases and 100 controls were genotyped for LOX G473A(Arg158Gln) polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The expression of LOX was estimated both by quantitative mRNA analysis and western blot. Total soluble collagen was evaluated from mucosal tissue obtained from OSF cases. Immunohistochemical (IHC) localization of type 1 collagen was performed in mucosal tissue obtained from patients carrying various genotypes. RESULTS: Heterozygous G473A genotype was significantly higher in OSF cases [2.063(95% CI =1.059-4.016)], among 26-40 years age-group [4.375(95% CI=1.323-14.267),p=0.029] and in male patients [2.38 (95% CI= 1.107-5.121), p= 0.042]. LOX expression was significantly higher in cases of the heterozygous or homozygous carrier (p <0.001). We found the total soluble collagen level significantly (p <0.001) higher among patients carrying GA or AA genotype. IHC revealed focal deposition of type1 collagen in the submucosal tissue; comparatively higher deposition was evident in mucosal tissue of OSF patients carrying AA genotype. CONCLUSIONS: These findings suggest LOX G473A polymorphism confers an increased risk of OSF and may affect collagen accumulation in OSF cases.

HSD17B7 gene in self-renewal and oncogenicity of keratinocytes from Black versus White populations.

Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte-derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black) versus White Caucasian (White) individuals have on average higher oncogenic and self-renewal potential, which are inversely related to mitochondrial electron transfer chain activity and ATP and ROS production. HSD17B7 is the top-ranked differentially expressed gene in HKCs and Head/Neck SCCs from individuals of Black African versus Caucasian ancestries, with several ancestry-specific eQTLs linked to its expression. Mirroring the differences between Black and White HKCs, modulation of the gene, coding for an enzyme involved in sex steroid and cholesterol biosynthesis, determines HKC and SCC cell proliferation and oncogenicity as well as mitochondrial OXPHOS activity. Overall, the findings point to a targetable determinant of cancer susceptibility among different human populations, amenable to prevention and management of the disease.

Sustained androgen receptor signaling is a determinant of melanoma cell growth potential and tumorigenesis.

Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II-associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex.

NOTCH1 gene amplification promotes expansion of Cancer Associated Fibroblast populations in human skin.

Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses cancer/stromal cells expansion. Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.

CSL controls telomere maintenance and genome stability in human dermal fibroblasts.

Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.

A differentially selective probe for trivalent chemosensor upon single excitation with cell imaging application: potential applications in combinatorial logic circuit and memory devices.

A new rhodamine 6G-benzylamine-based sensor (L1), having only hydrocarbon skeletons in the extended part, was synthesized and characterized by single-crystal X-ray crystallographic study. It exhibited excellent selective and sensitive recognition of trivalent metal ions M3+ (M = Fe, Al and Cr) over mono- and di-valent and other trivalent metal ions. A large enhancement of the fluorescence intensity for Fe3+ (41-fold), Al3+ (31-fold) and Cr3+ (26-fold) was observed upon the addition of 3.0 equivalent of these metal ions into the probe in H2O/CH3CN (4 : 1, v/v, pH 7.2) with naked eye detection. The corresponding Kf values were evaluated to be 9.4 × 103 M-1 (Fe3+), 1.34 × 104 M-1 (Al3+) and 8.7 × 103 M-1 (Cr3+). Quantum yields of the L1, [L1-Fe3+], [L1-Al3+] and [L1-Cr3+] complexes in H2O/CH3CN (4 : 1, v/v, pH 7.2) were found to be 0.012, 0.489, 0.376 and 0.310, respectively, using rhodamine-6G as standard. LODs for Fe3+, Al3+ and Cr3+ were determined by 3σ methods and found to be 1.28, 1.34 and 2.28 µM, respectively. Cyanide ion scavenged Fe3+ from the [Fe3+-L1] complex and quenched its fluorescence via its ring-closed spirolactam form. Advanced level molecular logic devices using different inputs (2 and 4 inputs) as advanced level logic gates and memory devices were constructed. The large enhancement in fluorescence emission of L1 upon complexation with M3+ metal ions makes the probe suitable for the bio-imaging of M3+ (M = Fe, Al and Cr) in living cells.

Androgen receptor functions as transcriptional repressor of cancer-associated fibroblast activation.

The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other's expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.

Genome-wide DNA methylation profile identified a unique set of differentially methylated immune genes in oral squamous cell carcinoma patients in India.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the common malignancies in Southeast Asia. Epigenetic changes, mainly the altered DNA methylation, have been implicated in many cancers. Considering the varied environmental and genotoxic exposures among the Indian population, we conducted a genome-wide DNA methylation study on paired tumor and adjacent normal tissues of ten well-differentiated OSCC patients and validated in an additional 53 well-differentiated OSCC and adjacent normal samples. RESULTS: Genome-wide DNA methylation analysis identified several novel differentially methylated regions associated with OSCC. Hypermethylation is primarily enriched in the CpG-rich regions, while hypomethylation is mainly in the open sea. Distinct epigenetic drifts for hypo- and hypermethylation across CpG islands suggested independent mechanisms of hypo- and hypermethylation in OSCC development. Aberrant DNA methylation in the promoter regions are concomitant with gene expression. Hypomethylation of immune genes reflect the lymphocyte infiltration into the tumor microenvironment. Comparison of methylome data with 312 TCGA HNSCC samples identified a unique set of hypomethylated promoters among the OSCC patients in India. Pathway analysis of unique hypomethylated promoters indicated that the OSCC patients in India induce an anti-tumor T cell response, with mobilization of T lymphocytes in the neoplastic environment. Survival analysis of these epigenetically regulated immune genes suggested their prominent role in OSCC progression. CONCLUSIONS: Our study identified a unique set of hypomethylated regions, enriched in the promoters of immune response genes, and indicated the presence of a strong immune component in the tumor microenvironment. These methylation changes may serve as potential molecular markers to define risk and to monitor the prognosis of OSCC patients in India.

Neo-tanshinlactone D-ring modified novel analogues induce apoptosis in human breast cancer cell via DNA damage.

Neo-tanshinlactone (NTL) a natural product is known for its specificity and selectivity towards the breast cancer cells. By NTL D-ring modification approach, 13 new analogues were synthesized (1A-1M). Among them 1J showed the best anticancer activity in MCF-7 (ER+, PR+/-, HER2-), SKBR3 (ER-, PR-, HER2+) and MDA-MB-231 (ER-, PR-, HER2-) cells lines with IC50 value 11.98nM, 23.71nM, and 62.91nM respectively. 1J showed minor grove binding interaction with DNA at AT-rich region and induced DNA double strand breaks (DDSBs). This had triggered several key molecular events involving, activation of ATM, Chk2 and p53, reduction in mitochondrial potential (Δψm) leading to caspase-3 and PARP cleavage mediated apoptosis. These results along with other biochemical studies strongly suggest that novel NTL analogue 1J caused DNA cleavage mediated apoptosis in the breast cancer cells and this may serve as potential lead for future breast cancer treatment.

Oxime Based Selective Fluorescent Sensor for Arsenate Ion in a Greener Way with Bio-Imaging Application.

A newly designed oxime based probe, 2,4-dihydroxyacetophenone-oxime (DHAO), was found to recognize H2AsO4- selectively with ∼3 fold "turn-on" fluorescence enhancement and LOD of 29 µM, K = (2.10 ± 0.54) × 104 M-1 in purely aqueous medium. The structures of the DHAO and its adduct with H2AsO4- were optimized by DFT calculations. Intracellular imaging of As(V) in HepG2 cells demonstrate the possibilities of in vitro/in vivo applications for selective monitoring of such species.

6-Gingerol inhibits Vibrio cholerae-induced proinflammatory cytokines in intestinal epithelial cells via modulation of NF-κB.

Context The effect of 6-gingerol (6G), the bioactive component of Zingiber officinale Roscoe (Zingiberaceae), in the reduction of Vibrio cholerae (Vibrionaceae)-induced inflammation has not yet been reported. Materials and methods Cell viability assay was performed to determine the working concentration of 6G. Elisa and RT-PCR were performed with Int 407 cells treated with 50 µM 6G and 100 multiplicity of infection (MOI) V. cholerae for 0, 2, 3, 3.5, 6 and 8 h to determine the concentration of IL-8, IL-6, IL-1α and IL-1ß in both protein and RNA levels. Furthermore, the effect of 50 µM 6G on upstream MAP-kinases and NF-κB signalling pathways was evaluated at 0, 10, 15, 30, 60 and 90 min. Results The effective dose (ED50) value of 6G was found to be 50 µM as determined by cell viability assay. Pre-treatment with 50 µM 6G reduced V. cholerae infection-triggered levels of IL-8, IL-6, IL-1α and IL-1ß by 3.2-fold in the protein level and two-fold in the RNA level at 3.5 h. The levels of MAP-kinases signalling molecules like p38 and ERK1/2 were also reduced by two- and three-fold, respectively, after 30 min of treatment. Additionally, there was an increase in phosphorylated IκBα and down-regulation of p65 resulting in down-regulation of NF-κB pathway. Conclusion Our results showed that 6G could modulate the anti-inflammatory responses triggered by V. cholerae-induced infection in intestinal epithelial cells by modulating NF-κB pathway.

Surfactant modulated aggregation induced enhancement of emission (AIEE)--a simple demonstration to maximize sensor activity.

A new type of easily synthesized rhodamine-based chemosensor L(3), with potential NO2 donor atoms, selectively and rapidly recognizes Hg(2+) ions in the presence of all biologically relevant metal ions and toxic heavy metals. A very low detection limit (78 nM) along with cytoplasmic cell imaging applications with no or negligible cytotoxicity indicate good potential for in vitro/in vivo cell imaging studies. SEM and TEM studies reveal strongly agglomerated aggregations in the presence of 5 mM SDS which turn into isolated core shell microstructures in the presence of 9 mM SDS. The presence of SDS causes an enhanced quantum yield (φ) and stability constant (Kf) compared to those in the absence of SDS. Again, the FI of the [L(3)-Hg](2+) complex in an aqueous SDS (9 mM) medium is unprecedentedly enhanced (∼143 fold) compared to that in the absence of SDS. All of these observations clearly manifest in the enhanced rigidity of the [L(3)-Hg](2+) species in the micro-heterogeneous environment significantly restricting its dynamic movements. This phenomenon may be ascribed as an aggregation induced emission enhancement (AIEE). The fluorescence anisotropy assumes a maximum at 5 mM SDS due to strong trapping (sandwiching) of the doubly positively charged [L(3)-Hg](2+) complex between two co-facial laminar microstructures of SDS under pre-miceller conditions where there is a strong electrostatic interaction that causes an improved inhibition to dynamic movement of the probe-mercury complex. On increasing the SDS concentration there is a phase transition in the SDS microstructures and micellization starts to prevail at SDS ≥ 7.0 mM. The doubly positively charged [L(3)-Hg](2+) complex is trapped inside the hydrophobic inner core of the micelle which is apparent from the failure to quench the fluorescence of the complex on adding 10 equivalents of H2EDTA(2-) solution but in the absence of SDS it is quenched effectively.

Di-oxime based selective fluorescent probe for arsenate and arsenite ions in a purely aqueous medium with living cell imaging applications and H-bonding induced microstructure formation.

A 2-hydroxy-5-methyl-benzene-1,3-dicarboxaldehyde di-oxime based turn-on blue emission fluorescent probe was found to recognize both AsO2(-) and H2AsO4(-) in a purely aqueous medium in intra and extra-cellular conditions. Self-organization of the ligand in the absence and presence of AsO2(-) and H2AsO4(-) was investigated by DLS, optical microscopy, optical fluorescence microscopy and FE-SEM methods.

Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients.

Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in management of the disease and improve survival rates. The comet assay has been successfully utilised to detect DNA damage in oral premalignant or malignancy. However, due to the invasive nature of collecting blood, it may be painful for many unwilling patients. This study compares the micronucleus (MN) assay in oral buccal mucosa cells with the comet assay in peripheral blood cells in a subset of oral habit-induced precancer and cancer patients. For this, MN assay of exfoliated epithelial cells was compared with comet assay of peripheral blood leucocytes among 260 participants, including those with oral lichen planus (OLP; n = 52), leukoplakia (LPK; n = 51), oral submucous fibrosis (OSF; n = 51), oral squamous cell carcinoma (OSCC; n = 54) and normal volunteers (n = 52). Among the precancer groups, LPK patients showed significantly higher levels of DNA damage as reflected by both comet tail length (P < 0.0001) and micronuclei (MNi) frequency (P = 0.0009). The DNA damage pattern in precancer and cancer patients was OLP < OSF < LPK < OSCC, and with respective oral habits, it was multiple habits > cigarette + khaini > cigarette smokers > areca + khaini > areca. There was no significant difference in the comet length and MNi frequency between males and females who had oral chewing habits. An overall significant correlation was observed between MNi frequency and comet tail length with r = 0.844 and P < 0.0001. Thus, the extent of DNA damage evaluation by the comet assay in peripheral blood cells is perfectly reflected by the MN assay on oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions.

A novel chromo- and fluorogenic dual sensor for Mg(2+) and Zn(2+) with cell imaging possibilities and DFT studies.

A diformyl-p-cresol (DFC)-8-aminoquinoline based dual signaling probe was found to exhibit colorimetric and fluorogenic properties on selective binding towards Mg(2+) and Zn(2+). Turn-on fluorescent enhancements (FE) as high as 40 fold and 53 fold in 9 : 1 MeCN/water (v/v) at pH 7.2 in HEPES buffer for Mg(2+) and Zn(2+), respectively, were observed. The binding constants determined from the fluorescence titration data are: K = (1.52 ± 0.21) × 10(5) M(-1) and (9.34 ± 4.0) × 10(3) M(-2) at n = 1 and 0.5, for Mg(2+) and Zn(2+), respectively. The L : M binding ratios were also determined by Job's method, which support the above findings. This is further substantiated by HRMS analysis. Due to solubility in mixed organo-aqueous solvents as well as cell permeability it could be used for the in vitro/in vivo cell imaging of Mg(2+) and Zn(2+) ions with no or negligible cytotoxicity. This probe could be made selective towards Mg(2+) over Zn(2+) in the presence of TPEN, both under intra- and extracellular conditions and is superior to other Mg(2+) probes which suffer from selectivity of Mg(2+) over Ca(2+) or Zn(2+). Furthermore the dissociation constant (Kd = 6.60 µM) of the Mg(2+)-() complex is far lower than the so far reported Mg(2+) probes which fall in the mM range.

Erratum to: Detection of a novel mutation in exon 20 of the BRCA1 gene.

[This corrects the article DOI: 10.2478/s11658-013-0110-3.].

Detection of a novel mutation in exon 20 of the BRCA1 gene.

Hereditary breast cancer constitutes 5-10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate.