Mechanical microscopy of cancer cells: TGF-ß induced epithelial to mesenchymal transition corresponds to low intracellular viscosity in cancer cells.

Viscosity is an essential parameter that regulates bio-molecular reaction rates of diffusion-driven cellular processes. Hence, abnormal viscosity levels are often associated with various diseases and malfunctions like cancer. For this reason, monitoring intracellular viscosity becomes vital. While several approaches have been developed for in vitro and in vivo measurement of viscosity, analysis of intracellular viscosity in live cells has not yet been well realized. Our research introduces a novel, natural frequency-based, non-invasive method to determine the intracellular viscosity in cells. This method can not only efficiently analyze the differences in intracellular viscosity post modulation with molecules like PEG or glucose but is sensitive enough to distinguish the difference in intra-cellular viscosity among various cancer cell lines such as Huh-7, MCF-7, and MDAMB-231. Interestingly, TGF-ß a cytokine reported to induce epithelial to mesenchymal transition (EMT), a feature associated with cancer invasiveness resulted in reduced viscosity of cancer cells, as captured through our method. To corroborate our findings with existing methods of analysis, we analyzed intra-cellular viscosity with a previously described viscosity-sensitive molecular rotor-based fluorophore-TPSII. In parity with our position sensing device (PSD)-based approach, an increase in fluorescence intensity was observed with viscosity enhancers, while, TGF-ß exposure resulted in its reduction in the cells studied. This is the first study of its kind that attempts to characterize differences in intracellular viscosity using a novel, non-invasive PSD-based method.

Exploring the extensive crosstalk between the antagonistic cytokines- TGF-ß and TNF-α in regulating cancer pathogenesis.

A plethora of cytokines are produced in the tumor microenvironment (TME) those play a vital role in cancer prognosis. Though it is completely contextual, cytokines produced from an inflammatory micro-environment can either modulate cancer progression at early stages of tumor development or in later stages cytokine derived cues can in turn control tumor cell invasion and metastasis. Therefore, understanding the crosstalk between the key cytokines regulating cancer prognosis is critical for the development of an effective therapy. In this regard, the role of transforming growth factor-beta (TGF-ß) in cancer is controversially discussed in general inhibition of TGF-ß promotes de novo tumorigenesis whereas paradoxically, TGF-ß can promote malignancy in already established tumors. Another important cytokine, TNF-α have intense crosstalk with TGF-ß from the fact that in a non-cancer context, TGF-ß promotes fibrosis whereas TNF-α has anti-fibrotic activity. We have recently reported that TGF-ß-induced differentiation of epithelial cells to mesenchymal type is suppressed by TNF-α through regulation of cellular homeostatic machinery- autophagy. Moreover, there are also rare reports of synergy between these two cytokines as well. The crosstalk between TGF-ß and TNF-α is not only limited to regulating cancer cell differentiation and proliferation but also includes involvement in cell death. In this review, we hence summarize the molecular mechanisms by which these two important cytokines, TGF-ß and TNF-α control cancer prognosis.

Transforming growth factor- ß mediated regulation of epigenome is required for epithelial to mesenchymal transition associated features in liver cancer cells.

Hepatocellular carcinoma (HCC) frequently unfolds under an inflammatory condition, which is a hub for a plethora of cytokines. A better understanding of the cytokine functions and their contributions to disease development is key to design of future therapeutic strategies and reduction of global HCC burden. In this context, one of the major cytokines present in the HCC tumour milieu is the transforming growth factor-ß (TGF-ß). One of its classical functions involve facilitation of epithelial to mesenchymal transition (EMT), in tumour cells, promoting an invasive phenotype. In spite of its clinical relevance, the cellular events associated with TGF-ß-induced EMT and its molecular regulation is poorly elucidated. Therefore, as part of this study, we treated HCC cells with TGF-ß and characterized the cellular processes associated with EMT. Interestingly, EMT triggered by TGF-ß was found to be associated with cytostasis and altered cellular metabolism. TGF-ß resulted in down-regulation of cell cycle-associated transcripts, like Cyclin A2 (CCNA2), and metabolic genes, like Glutamic-oxaloacetic transaminase 1 (GOT1) through epigenetic silencing. An overall increase in total histone repressive mark (H3K27me3) associated with a specific enrichment of H3K27me3 at the upstream promoter region of CCNA2 and GOT1 was observed after TGF-ß exposure, leading to their down-regulation. Importantly, TGF-ß-downstream signalling mediator- SMAD and chromatin repressive complex member-enhancer of zeste homolog 2 (EZH2) were found to co-immunoprecipitate and were required for the above effects. Overall, our findings reflect that HCC cells undergoing EMT, attain cytostasis and modulate metabolic demands to efficiently facilitate the EMT differentiation switch, and these events are regulated at the epigenomic level through TGF-ß-mediated signalling. Our results provide better understanding of cellular invasive features which can lead to development of novel therapeutic strategies.

Comparative analysis of antioxidant and antiproliferative activities of crude and purified flavonoid enriched fractions of pods/seeds of two desert legumes Prosopis cineraria and Cyamopsis tetragonoloba.

Cyamopsis tetragonoloba and Prosopis cineraria are two legumes of the semi-arid region of Indian subcontinent which are unexplored with respect to their medicinal potential. Moreover, there is considerable lack in the comparative analysis of the biological properties of crude and enriched fractions obtained from the pods and seeds. Therefore, this study aims in investigating the effect of purification on the antioxidant and anticancerous activities of the extracts from the two legumes. This is the first study to purify an enriched methanolic fraction using Amberlite XAD7HP column chromatography followed by analysis using Thin Layer Chromatography. This matrix provided an economic and time efficient isolation of flavonoids and isoflavonoids from the seeds and pods of the above mentioned legumes. In addition, antioxidant activity carried out using DPPH assay showed that purification process did not contributed to enhanced antioxidant potential. However, inverse results were obtained during anticancerous activity assay on Huh-7 cell lines.

Interleukin-6 Induced Proliferation Is Attenuated by Transforming Growth Factor-ß-Induced Signaling in Human Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is often associated with an inflammatory setting. A plethora of cytokines are secreted in this milieu, actively contributing to the progression of the disease; however, the extent of cytokine interaction and how it contributes to HCC development remains an enigma. In this regard, our analysis of available patient-derived data suggests that cytokines like interleukin-6 (IL-6) and transforming growth factor-beta (TGF-ß) are enriched in HCC. We further analyzed the effect of these cytokines independently or in combination on HCC cells. Importantly, IL-6 was found to induce a STAT-3-dependent proliferation and mediate its pro-proliferative effects through activation and direct interaction with the p65 subunit of NFkB. Alternatively, TGF-ß was found to induce a SMAD-dependent induction of epithelial to mesenchymal transition (EMT) coupled to growth arrest in these cells. Interestingly, the simultaneous addition of IL-6 and TGF-ß failed to profoundly impact EMT markers but resulted in attenuation of IL-6-induced pro-proliferative effects. Analysis of the putative molecular mechanism revealed a decrease in IL-6 receptor (IL-6R) transcript levels, reduced expression of IL-6-induced STAT-3, and its nuclear localization upon addition of TGF-ß along with IL-6. Consequently, a reduced p65 activation was also observed in combination treatment. Importantly, SMAD levels were unperturbed and the cells showed more TGF-ß-like features under combination treatment. Finally, we observed that TGF-ß resulted in enrichment of repressive chromatin mark (H3K27me3) coupled to growth arrest, while IL-6 induced an open chromatin signature (H3K4me3) associated with an enhanced expression of EZH2. Overall, for the first time, we show that TGF-ß attenuates IL-6-induced effects by regulating the receptor level, downstream signaling, and the epigenome. Understanding the complex interactions between these cytokines can be imperative to a better understanding of the disease, and manipulation of cytokine balance can act as a prospective future therapeutic strategy.

Facile Incorporation of "Aggregation-Induced Emission"-Active Conjugated Polymer into Mesoporous Silica Hollow Nanospheres: Synthesis, Characterization, Photophysical Studies, and Application in Bioimaging.

Typical aggregation-induced emission (AIE) luminogens tetraphenylethylene (TPE) and triphenylamine have been used to construct an AIE-active conjugated polymer, namely, poly(N,N-diphenyl-4-(4-(1,2,2-triphenylvinyl)styryl)aniline) (PTPA), which consist of D-π-A architecture by Wittig polymerization. We fabricated mesoporous silica hollow nanospheres (MSHNs) which were encapsulated with the AIE-active polymer for applications in cellular imaging. It exhibits a positive solvatochromism effect by increasing solvent polarity, supported by theoretical calculation using density functional theory. The structure of the monomers and polymer was confirmed by Fourier transform infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. Considering the advantage of high brightness in the fluorescence of PTPA, it was encapsulated into MSHNs by a noncovalent approach, and the surface was functionalized with an anti-EpCAM (antiepithelial cell adhesion molecule) aptamer through conjugation with γ-glycidoxypropyltrimethoxysilane for targeting cancer cells specifically. The aptamer-functionalized Apt-MSHNs exhibited excellent biocompatibility with the liver cancer-Huh-7 cells used for this study and was efficiently internalized by these cells. Because EpCAM are overexpressed in multiple carcinomas, including liver cancer, these aptamer-conjugated AIE MSHNs are therefore good candidates for targeted cellular imaging applications.

Autophagy inhibition potentiates SAHA­mediated apoptosis in glioblastoma cells by accumulation of damaged mitochondria.

Glioblastoma multiforme (GBM), often referred to as a grade IV astrocytoma, is the most invasive type of tumor arising from glial cells. The main treatment options for GBM include surgery, radiation and chemotherapy. However, these treatments tend to be only palliative rather than curative. Poor prognosis of GBM is due to its marked resistance to standard therapy. Currently, temozolomide (TMZ), an alkylating agent is used for treatment of GBM. However, GBM cells can repair TMZ­induced DNA damage and therefore diminish the therapeutic efficacy of TMZ. The potential to evade apoptosis by GBM cells accentuates the need to target the non­apoptotic pathway and/or inhibition of pro­survival strategies that contribute to its high resistance to conventional therapies. In recent studies, it has been demonstrated that HDAC inhibitors, such as vorinostat (suberoyl anilide hydroxamic acid; SAHA) can induce autophagy in cancer cells, thereby stimulating autophagosome formation. In addition, a lysosomotropic agent such as chloroquine (CQ) can result in hyper­accumulation of autophagic vacuoles by inhibiting autophagosome­lysosome fusion, which can drive the cell towards apoptosis. Hence, we postulated that combination treatment with SAHA and CQ may lead to increased formation of autophagosomes, resulting in its hyper­accumulation and ultimately inducing cell death in GBM cells. In the present study, we demonstrated that CQ co­treatment enhanced SAHA­mediated GBM cell apoptosis. Inhibition of the early stage of autophagy by 3­methyladenine pre­treatment reduced cell death confirming that apoptosis induced by CQ and SAHA was dependent on autophagosome accumulation. We also demonstrated that autophagy inhibition led to enhanced ROS, mitochondria accumulation and reduced mitochondrial membrane potential resulting in cell death. The present study provides cellular and molecular evidence concerning the combined effect of SAHA and CQ which can be developed as a therapeutic strategy for the treatment of glioblastoma in the future.

Genetic variability, associations, and path analysis of chemical and morphological traits in Indian ginseng [Withania somnifera (L.) Dunal] for selection of higher yielding genotypes.

BACKGROUND: The study was carried out to assess the genetic variability present in ashwagandha and to examine the nature of associations of various traits to the root yield of the plant. METHODS: Fifty-three diverse genetic stocks of ashwagandha (Withania somnifera) were evaluated for 14 quantitative characteristics. Analysis of variance, correlation, and path coefficient analysis were performed using the mean data of 2 years. RESULTS: Analysis of variance revealed that the genotypes differed significantly for all characteristics studied. High heritability in conjunction with high genetic advance was observed for fresh root weight, 12 deoxywithastramonolide in roots, and plant height, which indicated that selection could be effective for these traits. Dry root weight has a tight linkage with plant height and fresh root weight. Further, in path coefficient analysis, fresh root weight, total alkaloid (%) in leaves, and 12 deoxywithastramonolide (%) in roots had the highest positive direct effect on dry root weight. CONCLUSION: Therefore, these characteristics can be exploited to improve dry root weight in ashwagandha genotypes and there is also scope for the selection of promising and specific chemotypes (based on the alkaloid content) from the present germplasm.