Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Interplay of gut microbiome, fatty acids, and the endocannabinoid system in regulating development, progression, immunomodulation, and chemoresistance of cancer.

The roles of gut microorganisms in cancer are diverse. Studies on metagenomics and bioinformatics have documented diverse microbial etiology in different tumors. Evidence supports that a commensal microbiome could provide a promising strategy to treat and prevent cancer through interference in several biologic processes, such as host cell survival and death, host immune function, inflammation, oncogenic signaling, and several hormone receptor signaling and detoxification pathways. The cumulative evidence recommends that metabolites of commensal gut microorganisms (e.g., short-chain fatty acids, omega-3 and -6 fatty acids) play an important role in cancer prevention, with a robust antiproliferative effect of omega-3 fatty acids. Intriguingly, the endocannabinoid system (omega-3 and -6 fatty acid-derived neurotransmitter of the body) shows diverse effects on cancer prevention and oncogenesis depending on the context of the tumor microenvironment. Thus, an interplay of gut microorganisms with their fatty acid metabolites and the endocannabinoid system play an important role in the development, progression, immunomodulation, and chemoresistance of cancer. In this review, we highlight aspects of the current knowledge of and interactions between the microbiome with fatty acids and the host endocannabinoid system. We also document their effect on host immunomodulation and chemoresistance, and discuss how these insights might translate into future development of microbiome-targeted therapeutic interventions.

Kaempferol attenuates viability of ex-vivo cultured post-NACT breast tumor explants through downregulation of p53 induced stemness, inflammation and apoptosis evasion pathways.

Early onset of chemotherapy evasion is a therapeutic challenge. Chemotherapy-induced upregulation of stem cell markers imparts invasiveness and metastatic property to the resident tumor. The efficacy of Kaempferol in attenuating epithelial to mesenchymal transition has earlier been established in the breast cancer cell. In our study population, progression-free survival was observed to be statistically more significant in post-NACT low-grade tumors than the high-grade tumors. Further, in post-NACT TNBCs, high-grade tumors showed a preponderance of strong nuclear p53 expression and very low expression of Caspase 3, indicating that, altered p53 expression predisposes these tumors to apoptosis escape and up-regulation of stemness markers. Herein, we report the robust efficacy of Kaempferol on ex-vivo grown breast tumors, derived from post-NACT TNBC patients, through downregulation of nuclear p53, CD44, ALDH1, NANOG, MDR1, Ki67, BCL2 and upregulation of Caspase 3. Such tumors also showed concurrent deregulated RNA and protein expression of CD44, NANOG, ALDH1 and MDR1 with upregulation of Caspase 3 and cleaved Caspase 3, upon Kaempferol treatment. Validation of efficacy of the treatment dosage of Kaempferol through immunophenotyping on MDA-MB-231, suggested that Kaempferol at its IC-50 dosage was effective against CD44 and CD326 positive breast cancer through deregulating their expression. Protein-protein interaction network through STRING pathway analysis and co-expression study of candidate proteins showed the highest degree of co-expression of p53 and KI-67, CD44, NF- kappaB, ALDH1, NANOG, MDR1, and BCL2. Thus, potentially targetable oncogenic protein markers, that are susceptible to downregulation by Kaempferol, provides insight into biomarker-driven therapeutic approaches with it.

Deregulation of the CD44-NANOG-MDR1 associated chemoresistance pathways of breast cancer stem cells potentiates the anti-cancer effect of Kaempferol in synergism with Verapamil.

Chemoresistance is an imminent therapeutic challenge for breast cancer. Previous evidence suggests that breast cancer stem cells (BCSC) develop resistance through upregulation of stemness and chemo-evasion markers viz. SOX2, OCT4, NANOG, MDR1 and CD44, following anticancer chemotherapeutic treatments. Early studies suggest an inhibitory role of Kaempferol in BCSC propagation through downregulation of epithelial to mesenchymal transition. We hypothesized that the pathway involved in chemoresistance could be effectively addressed through Kaempferol (K), alone or in combination with Verapamil (V), which is an inhibitor of MDR1. We used K in combination with V, in multiple assays to determine if there was an inhibitory effect on BCSC. Both K and KV attenuated pH-dependent mammosphere formation in primary BCSC and MDA-MB-231 cells. RNA and protein (immunocytochemistry, western blot) expression of candidate markers viz. SOX2, OCT4, NANOG, MDR1 and CD44 were carried out in the presence or absence of candidate drugs in ex-vivo grown primary BCSC and MDA-MB-231 cell line. Immunoprecipitation assay, cell cycle analysis was carried out in MDA-MB-231. Our candidate drugs were not only anti-proliferative, but also downregulated candidate genes expression at RNA and protein level in both settings, with more robust efficacy in KV treatment than K; induced G2/M dependent cell cycle arrest, and interrupted physical association of CD44 with NANOG as well as MDR1 in MDA-MB-231. In primary tumor explant but not in adjacent normal tissue, our candidate drugs K and KV induced robust γH2AX expression. Thus, our candidate drugs are effective in attenuating BCSC survival.

Inhibition of extracellular vesicle-associated MMP2 abrogates intercellular hepatic miR-122 transfer to liver macrophages and curtails inflammation.

Hepatic miRNA, miR-122, plays an important role in controlling metabolic homeostasis in mammalian liver. Intercellular transfer of miR-122 was found to play a role in controlling tissue inflammation. miR-122, as part of extracellular vesicles released by lipid-exposed hepatic cells, are taken up by tissue macrophages to activate them and produce inflammatory cytokines. Matrix metalloprotease 2 or MMP2 was found to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. MMP2 was found to increase the movement of the extracellular vesicles along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries, and by targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. MMP2 thus could be a useful target to restrict high-fat-diet-induced obesity-related metaflammation.

Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells.

miRNAs are 20-22 nt long post-transcriptional regulators in metazoan cells that repress protein expression from their target mRNAs. These tiny regulatory RNAs follow tissue and cell-type specific expression pattern, aberrations of which are associated with various diseases. miR-122 is a liver-specific anti-proliferative miRNA that, we found, can be transferred via exosomes between human hepatoma cells, Huh7 and HepG2, grown in co-culture. Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells. Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells. Our observations suggest existence of a reciprocal interaction between two different hepatic cells with distinct miR-122 expression profiles. This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal. According to our data, human hepatoma cells use IGF1 to prevent intercellular exosomal transfer of miR-122 to ensure its own proliferation by preventing expression of growth retarding miR-122 in neighbouring cells.

Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex.

The RNA import complex (RIC) from the mitochondrion of the kinetoplastid protozoan Leishmania tropica contains two subunits that directly bind to import signals on two distinct subsets of tRNA and interact with each other allosterically. What happens to the tRNA subsequent to its loading on the complex is unknown. A third subunit-RIC9-has intrinsic affinity for both types of tRNA and is essential for import in vivo. Here we show that antibody against RIC9 inhibited the import of both types of tRNA into mitoplasts in vitro, but failed to inhibit the binding of these tRNAs to their respective receptors, indicating that RIC9 acts in a subsequent step. Using photoaffinity crosslinking-immunoprecipitation to detect translocation intermediates, it was observed that tRNA was transferred from its cognate receptor to RIC9, followed by translocation across the membrane and release as free tRNA in the inner compartment. Transfer required elevated temperatures and ATP, but ATP was substituted by acid pH. These tRNA movements were sensitive to uncouplers and inhibitors, suggesting distinct roles of the electrical and chemical components of the proton motive force generated by vectorial proton translocation accompanying ATP hydrolysis. By analysis of partially assembled complexes in L. tropica depleted of various subunits, and in vitro assembly assays, RIC9 was shown to make stable contacts with RIC8A, a tRNA receptor and RIC6, a membrane-embedded component. The results have implications for the mechanism of tRNA import.

Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion.

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex. The complex contains three mitochondrion- and eight nuclear-encoded subunits; six of the latter are necessary and sufficient for import. Antisense-mediated knockdown of essential subunits resulted in the depletion of mitochondrial tRNAs and inhibition of organellar translation. Functional complexes were reconstituted with recombinant subunits expressed in Escherichia coli. Several essential RIC subunits are identical to specific subunits of respiratory complexes. These findings provide new information on the evolution of tRNA import and the foundation for detailed structural and mechanistic studies.

Mitochondrial differentiation in kinetoplastid protozoa: a plethora of RNA controls.

Differentiation of kinetoplastid protozoa during their complex life cycles is accompanied by stepwise changes in mitochondrial functions. Recent studies have begun to reveal multilevel post-transcriptional regulatory mechanisms by which the expression of the nuclear and mitochondrially encoded components of respiratory enzymes is coordinated, as well as the identities of some general and gene-specific factors controlling mitochondrial differentiation.

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria.

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA(Tyr) (Type I) transiently stimulates the rate of entry of tRNA(Ile) (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA(Ile). Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA(Tyr) to the D domain, and those of tRNA(Ile) to the variable region-T domain (V-T region), indicating that both activities originate from a single RNA-receptor interaction. A third tRNA, human tRNA(Lys), is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.