Characterization of a Zeolite-Y-Encapsulated Zn(II)Salmphen Complex with Targeted Anticancer Property.

Resistance and severe side effects of classical chemotherapeutic drugs are major challenges to cancer therapy. New therapeutic agents and combination therapy are considered potential solutions that enhance the efficacy of the drug as well as reduce drug resistance. The success of a platinum-based anticancer drug, cisplatin, has paved the way to explore metal-centered anticancer therapeutic agents. Herein, the zeolite-Y-encapsulated Zn(II)Salmphen complex is synthesized using a flexible ligand approach. The Zn(II)Salmphen complex and its encapsulation within the supercage of zeolite-Y were characterized by elemental analysis, Fourier transform infrared (FTIR) spectroscopy, UV-vis, fluorescence, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), NMR, and high-resolution mass spectrometry (HRMS) techniques. Elemental analysis, PXRD, and SEM, all together confirm the integrity of the zeolite framework after the encapsulation of Zn(II)Salmphen complex in it, and elemental analysis provides the Si/Al ratio and Zn content present. FTIR and XPS studies indicate the successful encapsulation of the complex. NMR and HRMS studies confirm that the Zn(II)Salmphen complex is dimer; however, within the supercage of zeolite-Y, it is expected to exist as a monomer. The extent of structural modification of the encapsulated Zn(II)Salmphen complex is intimated by electronic spectroscopic studies. The free-state Zn(II)Salmphen is a fluorescent complex, and even the encapsulated Zn(II)Salmphen complex, when taken in dimethyl sulfoxide (DMSO), shows fluorescence. In comparison to cisplatin, encapsulated Zn(II)Salmphen complex displays comparable cytotoxicity (IC50 = 2.0 ± 0.5 µg/mL at 48 h) toward breast cancer cell line, whereas free Zn(II)Salmphen has better cytotoxicity (IC50 = 1.5 ± 0.5 µg/mL at 48 h). Importantly, elemental analysis has revealed that the IC50 value, if calculated only in terms of Zn(II)Salmphen within Zn(II)Salmphen-Y, is as low as 54.59 ng/mL, indicating a very high efficacy of the drug. Interestingly, a 48 h treatment with the encapsulated Zn(II)Salmphen complex shows no toxicity toward immortal noncancerous keratinocyte cells (HaCaT), whereas cisplatin has an IC50 value of 1.75 ± 0.5 µg/mL. Internalization studies indicate that zeolite-Y targets cancer cells better than it does noncancerous ones. Hence, cellular uptake of the zeolite-encapsulated Zn(II)Salmphen complex in cancer cells is more than that in HaCaT cells, resulting in the generation of more reactive oxygen species and cell death. Significant upregulation of DNA damage response protein indicates that DNA-damage-induced cellular apoptosis could be the mechanism of drug action. Overall, the zeolite-encapsulated Zn(II)Salmphen complex could be a better alternative to the traditional drug cisplatin with minimal effect on noncancerous HaCaT cells and can also be utilized as a fluorescent probe in exploring the mechanistic pathway of its activity against cancer cells.

Nature-inspired Enzyme engineering and sustainable catalysis: biochemical clues from the world of plants and extremophiles.

The use of enzymes to accelerate chemical reactions for the synthesis of industrially important products is rapidly gaining popularity. Biocatalysis is an environment-friendly approach as it not only uses non-toxic, biodegradable, and renewable raw materials but also helps to reduce waste generation. In this context, enzymes from organisms living in extreme conditions (extremozymes) have been studied extensively and used in industries (food and pharmaceutical), agriculture, and molecular biology, as they are adapted to catalyze reactions withstanding harsh environmental conditions. Enzyme engineering plays a key role in integrating the structure-function insights from reference enzymes and their utilization for developing improvised catalysts. It helps to transform the enzymes to enhance their activity, stability, substrates-specificity, and substrate-versatility by suitably modifying enzyme structure, thereby creating new variants of the enzyme with improved physical and chemical properties. Here, we have illustrated the relatively less-tapped potentials of plant enzymes in general and their sub-class of extremozymes for industrial applications. Plants are exposed to a wide range of abiotic and biotic stresses due to their sessile nature, for which they have developed various mechanisms, including the production of stress-response enzymes. While extremozymes from microorganisms have been extensively studied, there are clear indications that plants and algae also produce extremophilic enzymes as their survival strategy, which may find industrial applications. Typical plant enzymes, such as ascorbate peroxidase, papain, carbonic anhydrase, glycoside hydrolases and others have been examined in this review with respect to their stress-tolerant features and further improvement via enzyme engineering. Some rare instances of plant-derived enzymes that point to greater exploration for industrial use have also been presented here. The overall implication is to utilize biochemical clues from the plant-based enzymes for robust, efficient, and substrate/reaction conditions-versatile scaffolds or reference leads for enzyme engineering.

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.

Epigenetic adaptations in drug-tolerant tumor cells.

Traditional chemotherapy against cancer is often severely hampered by acquired resistance to the drug. Epigenetic alterations and other mechanisms like drug efflux, drug metabolism, and engagement of survival pathways are crucial in evading drug pressure. Herein, growing evidence suggests that a subpopulation of tumor cells can often tolerate drug onslaught by entering a "persister" state with minimal proliferation. The molecular features of these persister cells are gradually unraveling. Notably, the "persisters" act as a cache of cells that can eventually re-populate the tumor post-withdrawal drug pressure and contribute to acquiring stable drug-resistant features. This underlines the clinical significance of the tolerant cells. Accumulating evidence highlights the importance of modulation of the epigenome as a critical adaptive strategy for evading drug pressure. Chromatin remodeling, altered DNA methylation, and de-regulation of non-coding RNA expression and function contribute significantly to this persister state. No wonder targeting adaptive epigenetic modifications is increasingly recognized as an appropriate therapeutic strategy to sensitize them and restore drug sensitivity. Furthermore, manipulating the tumor microenvironment and "drug holiday" is also explored to maneuver the epigenome. However, heterogeneity in adaptive strategies and lack of targeted therapies have significantly hindered the translation of epigenetic therapy to the clinics. In this review, we comprehensively analyze the epigenetic alterations adapted by the drug-tolerant cells, the therapeutic strategies employed to date, and their limitations and future prospects.

Transcriptomic analysis reveals differential adaptation of colorectal cancer cells to low and acute doses of cisplatin.

Over the years, the landscape of cisplatin-based cancer treatment options has undergone continuous transitions. Currently, there is much debate over the optimum dose of cisplatin to be administered to cancer patients. In clinical practice, it can extend from repeated low sub-toxic doses to a few cycles of acute high drug doses. Herein, the molecular understanding of the overall cellular response to such differential doses of cisplatin becomes crucial before any decision making; and it has been a grey area of research. In this study, colorectal cancer (CRC) cells were treated with either- a low sub-toxic dose (LD; 30 µM) or a ten times higher acute dose (HD; 300 µM) of cisplatin, and thereafter, the cellular response was mapped through RNA sequencing followed by transcriptomic analysis. Interestingly, we observed that the tumor cells' response to varying doses of cisplatin is distinctly different, and they activate unique transcriptional programs. The analysis of differentially regulated or uniquely expressed transcripts and corresponding pathways revealed a preferential enrichment of genes associated with chromatin organization, oxidative stress, senescence-associated signaling, and developmentally-active signaling pathways in HD; whereas, modulation of autophagy, protein homeostasis, or differential expression of ABC transporters was primarily enriched in LD. This study is the first of its kind to highlight cellular transcriptomic adaptations to different doses of cisplatin in CRC cells. Consequently, since, protein homeostasis was found to be deeply affected after cisplatin treatment, we further analyzed one of the primary cellular protein homeostatic mechanisms- autophagy. It was activated upon LD, but not HD, and served as a pro-survival strategy through the regulation of oxidative stress. Inhibition of autophagy improved sensitivity to LD. Overall, our study provides a holistic understanding of the distinct molecular signatures induced in CRC cells in response to differential cisplatin doses. These findings might facilitate the design of tailored therapy or appropriate drug dose for enhanced efficacy against CRCs.

Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer-associated deaths worldwide. Though recent development in targeted therapy has improved NSCLC prognosis, yet there is an unmet need to identify novel causative factors and appropriate therapeutic regimen against NSCLCs. METHODS AND RESULTS: In this study, we identify key molecular factors de-regulated in NSCLCs. Analyze their expression by real-time PCR and immunoblot; map their localization by immuno-fluorescence microscopy. We further propose an FDA approved drug, chloroquine (CQ) that affects the function of the molecular factors and hence can be repurposed as a therapeutic strategy against NSCLCs. Available NSCLC mutation data reflects a high probabilistic chance of patients harboring a p53 mutation, especially a gain of function (GOF)-R273H mutation. The GOF-P53 mutation enables the P53 protein to potentially interact with non-canonical protein partners facilitating oncogenesis. In this context, analysis of existing transcriptomic data from R273H-P53 expressing cells shows a concomitant up-regulation of Yes-associated protein (YAP) transcriptional targets and its protein partner TEAD1 in NSCLCs, suggesting a possible link between R273H-P53 and YAP. We therefore explored the inter-dependence of R273H-P53 and YAP in NSCLC cells. They were found to co-operatively regulate NSCLC proliferation. Genetic or pharmacological inhibition of YAP and GOF-P53 resulted in sensitization of NSCLC cells. Further analysis of pathways controlled by GOF-P53 and YAP showed that they positively regulate the cellular homeostatic process- autophagy to mediate survival. We hence postulated that a modulation of autophagy might be a potent strategy to curb proliferation. In accordance to above, autophagy inhibition, especially with the FDA-approved drug- chloroquine (CQ) resulted in cytoplasmic accumulation and reduced transcriptional activity of GOF-P53 and YAP, leading to growth arrest of NSCLC cells. CONCLUSION: Our study highlights the importance of GOF-P53 and YAP in NSCLC proliferation and proposes autophagy inhibition as an efficient strategy to attenuate NSCLC tumorigenesis.

Silver nanoparticle-induced alteration of mitochondrial and ER homeostasis affects human breast cancer cell fate.

Breast cancer is one of the most frequent forms of cancer. Although different treatment modalities are available, none has proved to be a game-changer. In this context, nanomedicine is one of the hot research areas, with different nano-formulations being explored as a therapeutic strategy against breast cancer. Herein, silver nanoparticles (AgNPs) have shown prospects with their anti-tumor properties and are currently being explored aggressively; however, the underlying molecular mechanisms of AgNP action remain to be unearthed. As part of this study, human breast cancer cells- MCF7 were exposed to AgNPs (∼9 nm), and the effect of the same was explored on mitochondrial and endoplasmic reticulum (ER) dynamicity. We observed that the AgNPs co-localize with mitochondria and cause mitochondrial membrane depolarization, ROS generation, and destabilized mitochondrial homeostasis. Also, the NPs were found to enhance ER stress. We further found that increased ER stress is linked to the disruption of mitochondrial dynamics. Overall, our study shows that the AgNPs can effectively cause apoptosis of MCF-7 cells by regulating the mitochondrial-ER dynamicity. The results provide an insight into the mechanisms via which AgNPs act and can be used in developing a potential chemotherapeutic agent.

Analysis of Transcriptomic Data Generated from Drug-Treated Cancer Cell Line.

Resistance to chemotherapy is one major obstacle in current cancer treatment. Therefore, understanding the molecular basis of the acquisition of resistance is vital for the design and development of appropriate cancer therapy. Importantly, acquisition of resistance is not a single-step process, and the molecular signature of cells dynamically changes during this process. With the advent of next-generation omic technologies, today one can precisely map the molecular alterations not only in a population of tumor cells but also at the single-cell level as they attain chemo-resistance. In this chapter, we describe a detailed transcriptomic pipeline following next-generation sequencing for mapping alteration in expression during the process of attainment of resistance. We provide comprehensive information on the process to (1) track the differential expression of transcripts, (2) understand the gene ontology functions, (3) filter out candidate key genes, (4) identify the pathways regulated by them, and (5) generate a map of their probable interactions. We assume that our analytical method will be useful for research in this direction.

Dynamic alterations of H3K4me3 and H3K27me3 at ADAM17 and Jagged-1 gene promoters cause an inflammatory switch of endothelial cells.

Histone protein modifications control the inflammatory state of many immune cells. However, how dynamic alteration in histone methylation causes endothelial inflammation and apoptosis is not clearly understood. To examine this, we explored two contrasting histone methylations; an activating histone H3 lysine 4 trimethylation (H3K4me3) and a repressive histone H3 lysine 27 trimethylation (H3K27me3) in endothelial cells (EC) undergoing inflammation. Through computer-aided reconstruction and 3D printing of the human coronary artery, we developed a unique model where EC were exposed to a pattern of oscillatory/disturbed flow as similar to in vivo conditions. Upon induction of endothelial inflammation, we detected a significant rise in H3K4me3 caused by an increase in the expression of SET1/COMPASS family of H3K4 methyltransferases, including MLL1, MLL2, and SET1B. In contrast, EC undergoing inflammation exhibited truncated H3K27me3 level engendered by EZH2 cytosolic translocation through threonine 367 phosphorylation and an increase in the expression of histone demethylating enzyme JMJD3 and UTX. Additionally, many SET1/COMPASS family of proteins, including MLL1 (C), MLL2, and WDR5, were associated with either UTX or JMJD3 or both and such association was elevated in EC upon exposure to inflammatory stimuli. Dynamic enrichment of H3K4me3 and loss of H3K27me3 at Notch-associated gene promoters caused ADAM17 and Jagged-1 derepression and abrupt Notch activation. Conversely, either reducing H3K4me3 or increasing H3K27me3 in EC undergoing inflammation attenuated Notch activation, endothelial inflammation, and apoptosis. Together, these findings indicate that dynamic chromatin modifications may cause an inflammatory and apoptotic switch of EC and that epigenetic reprogramming can potentially improve outcomes in endothelial inflammation-associated cardiovascular diseases.

Verteporfin disrupts multiple steps of autophagy and regulates p53 to sensitize osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. The current treatment approach includes neoadjuvant and adjuvant chemotherapy; however, drug resistance often hinders therapy in OS patients. Also, the post-relapse survival of OS patients is as low as 20%. We therefore planned to understand the molecular cause for its poor prognosis and design an appropriate therapeutic strategy to combat the disease. METHODS: We analyzed OS patient dataset from Gene Expression Omnibus (GEO) and identified the differentially expressed genes and the top deregulated pathways in OS. Subsequently, drugs targeting the major de-regulated pathways were selected and the following assays were conducted- MTT assay to assess cytotoxicity of drugs in OS cells; immunoblotting and immunostaining to analyze key protein expression and localization after drug treatment; LysoTracker staining to monitor lysosomes; Acridine Orange to label acidic vesicles; and DCFDA to measure Reactive Oxygen Species (ROS). RESULTS: The differential gene expression analysis from OS patient dataset implicated the striking involvement of cellular processes linked to autophagy and protein processing in the development of OS. We therefore selected the FDA approved drugs, chloroquine (CQ) and verteporfin (VP) known for autophagy inhibitory and proteotoxic functions to explore against OS. Importantly, VP, but not CQ, showed an extensive dose-dependent cytotoxicity. It resulted in autophagy disruption at multiple steps extending from perturbation of early autophagic processes, inhibition of autophagic flux to induction of lysosomal instability. Interestingly, VP treated protein lysates showed a ROS-dependent high molecular weight (HMW) band when probed for P62 and P53 protein. Further, VP triggered accumulation of ubiquitinated proteins as well. Since VP had a pronounced disruptive effect on cellular protein homeostasis, we explored the possibility of simultaneous inhibition of the ubiquitin-proteasomal system (UPS) by MG-132 (MG). Addition of a proteasomal inhibitor significantly aggravated VP induced cytotoxicity. MG co-treatment also led to selective targeting of P53 to the lysosomes. CONCLUSION: Herein, we propose VP and MG induce regulation of autophagy and protein homeostasis which can be exploited as an effective therapeutic strategy against osteosarcoma.

Differential response of photosynthetic apparatus towards alkaline pH treatment in NIES-39 and PCC 7345 strains of Arthrospira platensis.

Alkaline stress is one of the severe abiotic stresses, which is not well studied so far, especially among cyanobacteria. To affirm the characteristics of alkaline stress and the subsequent adaptive responses in Arthrospira platensis NIES-39 and Arthrospira platensis PCC 7345, photosynthetic pigments, spectral properties of thylakoids, PSII and PSI activities, and pigment-protein profiles of thylakoids under different pH regimes were examined. The accessory pigments showed a pH-mediated sensitivity. The pigment-protein complexes of thylakoids are also affected, resulting in the altered fluorescence emission profile. At pH 11, a possible shift of the PBsome antenna complex from PSII to PSI is observed. PSII reaction center is found to be more susceptible to alkaline stress in comparison to the PSI. In Arthrospira platensis NIES-39 at pH 11, a drop of 68% in the oxygen evolution with a significant increase of PSI activity by 114% is recorded within 24 h of pH treatment. Alterations in the cellular ultrastructure of Arthrospira platensis NIES-39 at pH 11 were observed, along with the increased number of plastoglobules attached with the thylakoid membranes. Arthrospira platensis NIES-39 is more adaptable to pH variation than Arthrospira platensis PCC 7345.

A genome-wide expression profile of noncoding RNAs in human osteosarcoma cells as they acquire resistance to cisplatin.

BACKGROUND: Recurrence after cisplatin therapy is one of the major hindrances in the management of cancer. This necessitates a deeper understanding of the molecular signatures marking the acquisition of resistance. We therefore modeled the response of osteosarcoma (OS) cells to the first-line chemotherapeutic drug cisplatin. A small population of nondividing cells survived acute cisplatin shock (persisters; OS-P). These cells regained proliferative potential over time re-instating the population again (extended persisters; OS-EP). RESULT: In this study, we present the expression profile of noncoding RNAs in untreated OS cells (chemo-naive), OS-P, OS-EP and drug-resistant (OS-R) cells derived from the latter. RNA sequencing was carried out, and thereafter, differential expression (log2-fold ± 1.5; p value ≤ 0.05) of microRNAs (miRNAs) was analyzed in each set. The core set of miRNAs that were uniquely or differentially expressed in each group was identified. Interestingly, we observed that most of each group had their own distinctive set of miRNAs. The miRNAs showing an inverse correlation in expression pattern with mRNAs were further selected, and the key pathways regulated by them were delineated for each group. We observed that pathways such as TNF signaling, autophagy and mitophagy were implicated in multiple groups. CONCLUSION: To the best of our knowledge, this is the first study that provides critical information on the variation in the expression pattern of ncRNAs in osteosarcoma cells and the pathways that they might tightly regulate as cells acquire resistance.

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.

Common and Unique microRNAs in Multiple Carcinomas Regulate Similar Network of Pathways to Mediate Cancer Progression.

Cancer is a complex disease with a fatal outcome. Early detection of cancer, by monitoring appropriate molecular markers is very important for its therapeutic management. In this regard, the short non-coding RNA molecules, microRNAs (miRNAs) have shown great promise due to their availability in circulating fluids facilitating non-invasive detection of cancer. In this study, an in silico comparative analysis was performed to identify specific signature miRNAs dysregulated across multiple carcinomas and simultaneously identify unique miRNAs for each cancer type as well. The miRNA-seq data of cancer patient was obtained from GDC portal and their differential expressions along with the pathways regulated by both common and unique miRNAs were analyzed. Our studies show twelve miRNAs commonly dysregulated across seven different cancer types. Interestingly, four of those miRNAs (hsa-mir-210, hsa-mir-19a, hsa-mir-7 and hsa-mir-3662) are already reported as circulatory miRNAs (circRNAs); while, the miR-183 cluster along with hsa-mir-93 have been found to be incorporated in exosomes signifying the importance of the identified miRNAs for their use as prospective, non-invasive biomarkers. Further, the target mRNAs and pathways regulated by both common and unique miRNAs were analyzed, which interestingly had significant commonality. This suggests that miRNAs that are commonly de-regulated and specifically altered in multiple cancers might regulate similar pathways to promote cancer. Our data is of significance because we not only identify a set of common and unique miRNAs for multiple cancers but also highlight the pathways regulated by them, which might facilitate the development of future non-invasive biomarkers conducive for early detection of cancers.

Transcriptomic analysis associated with reversal of cisplatin sensitivity in drug resistant osteosarcoma cells after a drug holiday.

BACKGROUND: Resistance to chemotherapy is one of the major hurdles in current cancer therapy. With the increasing occurrence of drug resistance, a paradigm shift in treatment strategy is required. Recently "medication vacation" has emerged as a unique, yet uncomplicated strategy in which withdrawal of drug pressure for certain duration allowed tumor cells to regain sensitivity to the drug. However, little is known about the molecular alterations associated with such an outcome. METHODS: In this study, human osteosarcoma (OS) cells resistant to the extensively used drug cisplatin, were withdrawn from drug pressure, and thereafter cytotoxic response of the cells to the drug was evaluated. We further performed next-generation RNA sequencing and compared transcriptome between parental (OS), resistant (OS-R) and the drug withdrawn (OS-DW) cells. Differentially expressed transcripts were identified, and biological association network (BAN), gene ontology (GO) and pathway enrichment analysis of the differentially regulated transcripts were performed to identify key events associated with withdrawal of drug pressure. RESULTS: Following drug withdrawal, the sensitivity of the cells to the drug was found to be regained. Analysis of the expression profile showed that key genes like, IRAK3, IL6ST, RELA, AKT1, FKBP1A and ADIPOQ went significantly down in OS-DW cells when compared to OS-R. Also, genes involved in Wnt signaling, PI3K-Akt, Notch signaling, and ABC transporters were drastically down-regulated in OS-DW cells compared to OS-R. Although, a very small subset of genes maintained similar expression pattern between OS, OS-R and OS-DW, nonetheless majority of the transcriptomic pattern of OS-DW was distinctively different and unique in comparison to either the drug sensitive OS or drug resistant OS-R cells. CONCLUSION: Our data suggests that though drug withdrawal causes reversal of sensitivity, the transcriptomic pattern does not necessarily show significant match with resistant or parental control cells. We strongly believe that exploration of the molecular basis of drug holiday might facilitate additional potential alternative treatment options for aggressive and resistant cancers.

Drug Tolerant Cells: An Emerging Target With Unique Transcriptomic Features.

Long-term outcome of cancer therapy is often severely perturbed by the acquisition of drug resistance. Recent evidence point toward the survival of a subpopulation of tumor cells under acute drug stress that over time can re-populate the tumor. These transiently existing, weakly proliferative, drug-tolerant cells facilitate tumor cell survival until more stable resistance mechanisms are acquired. From a therapeutic perspective, understanding the molecular features of the tolerant cells is critical to attenuation of resistance. In this article, we discuss the transcriptomic features of drug-tolerant osteosarcoma cells that survive a high dose of cisplatin shock. We present the unique transcriptome of the minimally dividing tolerant cells in comparison with the proliferative persisters or resistant cells derived from the tolerant cells. Targeting the tolerant cells can represent an efficient therapeutic strategy impeding tumor recurrence.

Analysis of seven putative Na+/H+ antiporters of Arthrospira platensis NIES-39 using transcription profiling and in silico studies: an indication towards alkaline pH acclimation.

Na+/H+ antiporters mediated pH regulation is one of the known mechanism(s), which advocates a possible role of the antiporters in the alkaline pH tolerance of Arthrospira platensis NIES-39. Seven putative Na+/H+ antiporters have been reported in A. platensis NIES-39. Based upon the in silico analysis, the seven putative antiporters were characterized into two different superfamilies, where A1, Q2, L2, and L6 belonged to the CPA1 family whereas C5, D5 and O6 belonged to CPA2 family. The orientation of functionally important residues in both CPA1 and CPA2 subfamily are conserved in modeled Q2 and C5 antiporters. Conserved domain analysis of the seven putative antiporters indicated the presence of nine different kinds of domains. Out of these nine domains, six domains function as monovalent cation-proton antiporters and two as the universal stress protein (Usp) category. Transcription profile of these seven antiporters was also generated at three different pH (7, 9 and 11) and time frames which showed a significant difference in the mRNA levels along with a temporal pattern of the expression profile. The in silico and the real-time PCR analysis put together, suggest the active participation of these seven putative Na+/H+ antiporters in alkaline pH homeostasis of this cyanobacterial strain where CPA1 subfamily antiporters play a major role.

Dysregulated expression but redundant function of the long non-coding RNA HOTAIR in diabetic kidney disease.

AIMS/HYPOTHESIS: Long non-coding RNAs (lncRNAs) are garnering increasing attention for their putative roles in the pathogenesis of chronic diseases, including diabetic kidney disease (DKD). However, much about in vivo lncRNA functionality in the adult organism remains unclear. To better understand lncRNA regulation and function in DKD, we explored the effects of the modular scaffold lncRNA HOTAIR (HOX antisense intergenic RNA), which approximates chromatin modifying complexes to their target sites on the genome. METHODS: Experiments were performed in human kidney tissue, in mice with streptozotocin-induced diabetes, the db/db mouse model of type 2 diabetes, podocyte-specific Hotair knockout mice and conditionally immortalised mouse podocytes. RESULTS: HOTAIR was observed to be expressed by several kidney cell-types, including glomerular podocytes, in both human and mouse kidneys. However, knockout of Hotair from podocytes had almost no effect on kidney structure, function or ultrastructure. Glomerular HOTAIR expression was found to be increased in human DKD, in the kidneys of mice with streptozotocin-induced diabetes and in the kidneys of db/db mice. Likewise, exposure of cultured mouse podocytes to high glucose caused upregulation of Hotair expression, which occurred in a p65-dependent manner. Although HOTAIR expression was upregulated in DKD and in high glucose-exposed podocytes, its knockout did not alter the development of kidney damage in diabetic mice. Rather, in a bioinformatic analysis of human kidney tissue, HOTAIR expression closely paralleled the expression of its genic neighbour, HOXC11, which is important to developmental patterning but which has an uncertain role in the adult kidney. CONCLUSIONS/INTERPRETATION: Many lncRNAs have been found to bind to the same chromatin modifying complexes. Thus, there is likely to exist sufficient redundancy in the system that the biological effects of dysregulated lncRNAs in kidney disease may often be inconsequential. The example of the archetypal scaffold lncRNA, HOTAIR, illustrates how lncRNA dysregulation may be a bystander in DKD without necessarily contributing to the pathogenesis of the condition. In the absence of in vivo validation, caution should be taken before ascribing major functional roles to single lncRNAs in the pathogenesis of chronic diseases.

Refined template selection and combination algorithm significantly improves template-based modeling accuracy.

In contrast to ab-initio protein modeling methodologies, comparative modeling is considered as the most popular and reliable algorithm to model protein structure. However, the selection of the best set of templates is still a major challenge. An effective template-ranking algorithm is developed to efficiently select only the reliable hits for predicting the protein structures. The algorithm employs the pairwise as well as multiple sequence alignments of template hits to rank and select the best possible set of templates. It captures several key sequences and structural information of template hits and converts into scores to effectively rank them. This selected set of templates is used to model a target. Modeling accuracy of the algorithm is tested and evaluated on TBM-HA domain containing CASP8, CASP9 and CASP10 targets. On an average, this template ranking and selection algorithm improves GDT-TS, GDT-HA and TM_Score by 3.531, 4.814 and 0.022, respectively. Further, it has been shown that the inclusion of structurally similar templates with ample conformational diversity is crucial for the modeling algorithm to maximally as well as reliably span the target sequence and construct its near-native model. The optimal model sampling also holds the key to predict the best possible target structure.

Evolution of endonuclease IV protein family: an in silico analysis.

DNA repair is one of the key cellular events which balances between evolvability and integrity of the genome. Endonuclease IV enzymes are class II AP endonucleases under base excision repair pathway which act on abasic site and break the phosphodiester bond at the 5' side. The role and activity of endonuclease IV proteins vary among different organisms; even it is absent in higher eukaryotes. The evolution of this protein family was studied by analyzing all homologs of the endonuclease IV protein family through different in silico techniques including phylogenetic tree generation and model building. The sequence analysis revealed four consensus sequence motifs within the AP2EC domain which are functionally important and conserved throughout the evolution process. It was also observed that the species and endonuclease IV gene evolution shape up differently in most of the organisms. Presence of the mitochondria-targeted signal peptides in fungal species Saccharomyces and Coccidioides suggest a possible endosymbiotic transfer of endonuclease IV genes to lower eukaryotes. Evolutionary changes among various clades in the protein-based phylogenetic tree have been investigated by comparison of homology models which suggests the conservation of overall fold of endonuclease IV proteins except for few alterations in loop orientation in few clades.