Triple-negative breast cancer (TNBC) harbors a high percentage of breast cancer stem-like cells (BCSCs) that significantly contribute to poor prognosis, metastasis, and relapse of the disease. Thus, targeting BCSCs could be a promising approach to combat TNBC. In this context, we investigated nimbolide (Nim), a limonoid triterpenoid that has potent anticancer properties, but poor pharmacokinetics and low bioavailability limit its therapeutic application. So, to enhance the therapeutic potential of Nim, Nim-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Nim NPs) were formulated and the anticancer stem cell (CSC) effects evaluated in vitro and in vivo. In vitro studies suggested that Nim NPs significantly inhibited several inherent characteristics of BCSCs, such as stemness, self-renewability, chemoresistance, epithelial-to-mesenchymal transition (EMT), and migration in comparison to native Nim. Next, the mechanism behind the anti-CSC effect of Nim was explored. Mechanistically, we found that Nim epigenetically restores tumor suppressor gene secreted frizzled-related protein 1 (SFRP1) expression by downregulating DNA methyltransferases (DNMTs), leading to Wnt/ß-catenin signaling inhibition. Further, in vivo results demonstrated that Nim NPs showed enhanced anti-tumor and anti-metastatic effects compared to native Nim in two preclinical models without any systemic toxicity. Overall, these findings provide proof of concept that Nim-based phytonanomedicine can inhibit BCSCs by epigenetic reprogramming of the DNMTs-SFRP1-Wnt/ß-catenin signaling axis.
In vitro cell line model systems are essential in supporting the research community due to their low cost, uniform culturing conditions, homogeneous biological resources, and easy experimental design to study the cause and effect of a gene or a molecule. Human leukemia 60 (HL60) is an in-vitro hematopoietic model system that has been used for decades to study normal myeloid differentiation and leukemia biology. Here, we show that IMDM supplemented with 20% FBS is an optimal culturing condition and induces effective myeloid differentiation compared with RPMI supplemented with 10% FBS when HL60 is induced with 1α,25-dihydroxyvitamin D3 (Vit D3) and all-trans retinoic acid (ATRA). The chromatin organization is compacted, and the repressive epigenetic mark H3K27me3 is enhanced upon HL60-mediated terminal differentiation. Differential gene expression analysis obtained from RNA sequencing in HL60 cells during myeloid differentiation showed the induction of pathways involved in epigenetic regulation, myeloid differentiation, and immune regulation. Using high-throughput transcriptomic data (GSE74246), we show the similarities (genes that did not satisfy |log2FC|>1 and FDR<0.05) and differences (FDR <0.05 and |log2FC|>1) between granulocyte-monocyte progenitor vs HL60 cells, Vit D3 induced monocytes (vMono) in HL60 cells vs primary monocytes (pMono), and HL60 cells vs leukemic blasts at the transcriptomic level. We found striking similarities in biological pathways between these comparisons, suggesting that the HL60 model system can be effectively used for studying myeloid differentiation and leukemic aberrations. The differences obtained could be attributed to the fact that the cellular programs of the leukemic cell line and primary cells are different. We validated several gene expression patterns for different comparisons with CD34+ cells derived from cord blood for myeloid differentiation and AML patients. In addition to the current knowledge, our study further reveals the significance of using HL60 cells as in vitro model system under optimal conditions to understand its potential as normal myeloid differentiation model as well as leukemic model at the molecular level.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has emerged as a global pandemic worldwide. In this study, we used ARTIC primers-based amplicon sequencing to profile 225 SARS-CoV-2 genomes from India. Phylogenetic analysis of 202 high-quality assemblies identified the presence of all the five reported clades 19A, 19B, 20A, 20B, and 20C in the population. The analyses revealed Europe and Southeast Asia as two major routes for introduction of the disease in India followed by local transmission. Interestingly, the19B clade was found to be more prevalent in our sequenced genomes (17%) compared to other genomes reported so far from India. Haplotype network analysis showed evolution of 19A and 19B clades in parallel from predominantly Gujarat state in India, suggesting it to be one of the major routes of disease transmission in India during the months of March and April, whereas 20B and 20C appeared to evolve from 20A. At the same time, 20A and 20B clades depicted prevalence of four common mutations 241 C > T in 5' UTR, P4715L, F942F along with D614G in the Spike protein. D614G mutation has been reported to increase virus shedding and infectivity. Our molecular modeling and docking analysis identified that D614G mutation resulted in enhanced affinity of Spike S1-S2 hinge region with TMPRSS2 protease, possibly the reason for increased shedding of S1 domain in G614 as compared to D614. Moreover, we also observed an increased concordance of G614 mutation with the viral load, as evident from decreased Ct value of Spike and the ORF1ab gene.
Acute myeloid leukemia (AML) is a common and aggressive hematological malignancy. Acquisition of heterogeneous genetic aberrations and epigenetic dysregulation lead to the transformation of hematopoietic stem cells (HSC) into leukemic stem cells (LSC), which subsequently gives rise to immature blast cells and a leukemic phenotype. LSCs are responsible for disease relapse as current chemotherapeutic regimens are not able to completely eradicate these cellular sub-populations. Therefore, it is critical to improve upon the existing knowledge of LSC specific markers, which would allow for specific targeting of these cells more effectively allowing for their sustained eradication from the cellular milieu. Although significant milestones in decoding the aberrant transcriptional network of various cancers, including leukemia, have been achieved, studies on the involvement of post-transcriptional gene regulation (PTGR) in disease progression are beginning to unfold. RNA binding proteins (RBPs) are key players in mediating PTGR and they regulate the intracellular fate of individual transcripts, from their biogenesis to RNA metabolism, via interactions with RNA binding domains (RBDs). In this study, we have used an integrative approach to systematically profile RBP expression and identify key regulatory RBPs involved in normal myeloid development and AML. We have analyzed RNA-seq datasets (GSE74246) of HSCs, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), monocytes, LSCs, and blasts. We observed that normal and leukemic cells can be distinguished on the basis of RBP expression, which is indicative of their ability to define cellular identity, similar to transcription factors. We identified that distinctly co-expressing modules of RBPs and their subclasses were enriched in hematopoietic stem/progenitor (HSPCs) and differentiated monocytes. We detected expression of DZIP3, an E3 ubiquitin ligase, in HSPCs, knockdown of which promotes monocytic differentiation in cell line model. We identified co-expression modules of RBP genes in LSCs and among these, distinct modules of RBP genes with high and low expression. The expression of several AML-specific RBPs were also validated by quantitative polymerase chain reaction. Network analysis identified densely connected hubs of ribosomal RBP genes (rRBPs) with low expression in LSCs, suggesting the dependency of LSCs on altered ribosome dynamics. In conclusion, our systematic analysis elucidates the RBP transcriptomic landscape in normal and malignant myelopoiesis, and highlights the functional consequences that may result from perturbation of RBP gene expression in these cellular landscapes.
