TCF19 and p53 regulate transcription of TIGAR and SCO2 in HCC for mitochondrial energy metabolism and stress adaptation.

Alteration in glucose homeostasis during cancer metabolism is an important phenomenon. Though several important transcription factors have been well studied in the context of the regulation of metabolic gene expression, the role of epigenetic readers in this regard remains still elusive. Epigenetic reader protein transcription factor 19 (TCF19) has been recently identified as a novel glucose and insulin-responsive factor that modulates histone posttranslational modifications to regulate glucose homeostasis in hepatocytes. Here we report that TCF19 interacts with a non-histone, well-known tumor suppressor protein 53 (p53) and co-regulates a wide array of metabolic genes. Among these, the p53-responsive carbohydrate metabolic genes Tp53-induced glycolysis and apoptosis regulator (TIGAR) and Cytochrome C Oxidase assembly protein 2 (SCO2), which are the key regulators of glycolysis and oxidative phosphorylation respectively, are under direct regulation of TCF19. Remarkably, TCF19 can form different transcription activation/repression complexes which show substantial overlap with that of p53, depending on glucose-mediated variant stress situations as obtained from IP/MS studies. Interestingly, we observed that TCF19/p53 complexes either have CBP or HDAC1 to epigenetically program the expression of TIGAR and SCO2 genes depending on short-term high glucose or prolonged high glucose conditions. TCF19 or p53 knockdown significantly altered the cellular lactate production and led to increased extracellular acidification rate. Similarly, OCR and cellular ATP production were reduced and mitochondrial membrane potential was compromised upon depletion of TCF19 or p53. Subsequently, through RNA-Seq analysis from patients with hepatocellular carcinoma, we observed that TCF19/p53-mediated metabolic regulation is fundamental for sustenance of cancer cells. Together the study proposes that TCF19/p53 complexes can regulate metabolic gene expression programs responsible for mitochondrial energy homeostasis and stress adaptation.

Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation.

Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.

Herbometallic nano-drug inducing metastatic growth inhibition in breast cancer through intracellular energy depletion.

Cancer cells need extensive energy supply for their uncontrolled cell division and metastasis which is exclusively dependent on neighboring cells, especially adipocytes. Herein, we have introduced a novel herbometallic nano-drug, Heerak Bhasma nanoparticle (HBNP) from natural resources showing high potential in the reduction of energy supply thereby promoting cell death in breast cancer cells. Inductively coupled plasma optical emission spectra (ICP-OES), atomic absorption spectra (AAS), Raman spectra, X-ray diffraction analyses confirmed the physicochemical properties of HBNP. The differential light scattering (DLS) and field emission scanning electron microscope (FESEM) analyzed the cell-permeable size of HBNP, whereas, cell viability assay confirmed the non-toxic effect. Seahorse energy efflux assay, apoptotic cell quantification, ROS, mitochondrial membrane potential, in vivo oxidative stress etc. were measured using standard protocol. The notable changes in cancer energy metabolism investigated by cellular Mito and Glyco-stress analyses confirmed the HBNP induced intracellular energy depletion. Also, a significant reduction in mitochondrial membrane potential and subsequently, extensive reactive oxygen species (ROS) generations were observed in presence of HBNP followed by the induction of cell apoptosis. The cell invasion and wound healing assay followed by reduced expression both protein (MMP 2, MMP 9) and cytokine (IL6, IL10) had signified the effectiveness of HBNP against cancer metastasis. In addition, HBNP also showed an excellent antitumor activity in vivo followed by developing healing characteristics due to oxidative stress. All these findings strongly suggest that HBNP has the potential to be the new cancer therapeutic. A schematic phenomenon represents the overall HBNP mediated anticancer activity via limitation of both fatty acid uptake and energy metabolism.

Stemness and chemoresistance are imparted to the OC cells through TGFß1 driven EMT.

Ovarian cancer (OC) is the fourth most common gynecological malignancy due to its highly aggressive, recurrent, and drug-resistant nature. The last two features are rendered by the presence of cancer stem cells (CSCs). Factors like TGFß1 and their downstream signaling pathways are upregulated in most cancers and are known to induce EMT and stemness, but the exact mechanisms underlying the process remain unelucidated. In our study, TGFß1 induced enhanced stem-like properties like high expression of the pluripotent markers SOX2, OCT4a, and NANOG, along with CD44, and CD117 in the OC cells. In addition, increased activity of the aldehyde dehydrogenase enzyme, formation of compact spheroids, and a quiescent phenotype were observed. In deciphering the mechanism behind it, our data propose ZEB1 transcription factor to play a substantial role in inducing the EMT-mediated stemness and chemoresistance. Further, in our study, we elucidated the significant contribution of both Smad and non-Smad pathways like ERK, JNK, and P38 MAPK pathways in the induction of stem-like characteristics. The novelty of the study also resides with the fact in the expression of different lineage-specific markers, like CD31, CD45, and CD117 along with CD44 in the TGFß1-induced epithelial ovarian cancer spheroids. This suggests a tendency of the spheroidal cells towards differentiating into heterogenic populations, which is a distinctive feature of a stem cell. Taken together, the present study provides an insight to the molecular cues involved in the acquisition of stemness and chemoresistance along with tumor heterogeneity in TGFß1-induced OC cells.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Metallo-protease Peptidase M84 from Bacillusaltitudinis induces ROS-dependent apoptosis in ovarian cancer cells by targeting PAR-1.

We have purified Peptidase M84 from Bacillus altitudinis in an effort to isolate anticancer proteases from environmental microbial isolates. This metallo-protease had no discernible impact on normal cell survival, but it specifically induced apoptosis in ovarian cancer cells. PAR-1, a GPCR which is reported to be overexpressed in ovarian cancer cells, was identified as a target of Peptidase M84. We observed that Peptidase M84 induced PAR-1 overexpression along with activating its downstream signaling effectors NF-κB and MAPK to promote excessive reactive oxygen species (ROS) generation. This evoked apoptotic death of the ovarian cancer cells through the intrinsic route. In in vivo set-up, weekly intraperitoneal administration of Peptidase M84 in syngeneic mice significantly diminished ascites accumulation, increasing murine survival rates by 60%. Collectively, our findings suggested that Peptidase M84 triggered PAR-1-mediated oxidative stress to act as an apoptosis inducer. This established Peptidase M84 as a drug candidate for receptor mediated targeted-therapy of ovarian cancer.

Mesenchymal stromal cells, metabolism, and mitochondrial transfer in bone marrow normal and malignant hematopoiesis.

Hematopoietic stem cell (HSC) transplantation-based treatments are in different phases of clinical development, ranging from current therapies to a promise in the repair and regeneration of diseased tissues and organs. Mesenchymal stromal/stem cells (MSCs), which are fibroblast-like heterogeneous progenitors with multilineage differentiation (osteogenic, chondrogenic, and adipogenic) and self-renewal potential, and exist in the bone marrow (BM), adipose, and synovium, among other tissues, represent one of the most widely used sources of stem cells in regenerative medicine. MSCs derived from bone marrow (BM-MSCs) exhibit a variety of traits, including the potential to drive HSC fate and anti-inflammatory and immunosuppressive capabilities via paracrine activities and interactions with the innate and adaptive immune systems. The role of BM-MSC-derived adipocytes is more controversial and may act as positive or negative regulators of benign or malignant hematopoiesis based on their anatomical location and functional crosstalk with surrounding cells in the BM microenvironment. This review highlights the most recent clinical and pre-clinical findings on how BM-MSCs interact with the surrounding HSCs, progenitors, and immune cells, and address some recent insights on the mechanisms that mediate MSCs and adipocyte metabolic control through a metabolic crosstalk between BM microenvironment cells and intercellular mitochondrial transfer in normal and malignant hematopoiesis.

Synthesis, Characterization, and Biological Evaluation of Radiolabeled Glutamine Conjugated Polymeric Nanoparticles: A Simple Approach for Tumor Imaging.

Application of nanoradiopharmaceuticals for molecular imaging has gained worldwide importance for their multifaceted potentials focusing on providing a safe and cost-effective approach. Biodistribution studies on such species are capable of bringing nanomedicine to patients. Current therapeutically available labeling strategies suffer from different limitations, including off-target cytotoxicity and radiolabel release over time. Poly(lactic-co-glycolic acid)(PLGA) nanoparticles are biodegradable carriers for a variety of contrast agents that can be employed in medicine with high loading capacity for multimodal imaging agents. Here, glutamine-conjugated PLGA polymers were used to construct polymeric nanoparticles (G-PNP) similar to unconjugated PLGA nanoparticles (PNP)s formulated for ex vivo cell labeling and in vivo tumor scintigraphy studies. G-PNP/PNP, characterized by Fourier-transform infrared, atomic-force-microscopy, particle-size, and zeta-potential studies, were biocompatible as evaluated by MTT assay. G-PNPs were radiolabeled with 99mtechnetium (99mTc) by borohydrite reduction. G-PNPs demonstrated higher cellular uptake than PNPs, with no major cytotoxicity. Radiochemical purity indicated that 99mTc labeled G-PNP (99mTc-G-PNP) can form a stable complex with substantial stability in serum with respect to time. Imaging studies showed that 99mTc-G-PNP significantly accumulated at the C6 glioma cell induced tumor-site in rats. Thus, 99mTc-G-PNP demonstrated favorable characteristics and imaging potential which may make it a promising tumor imaging nanoprobe as a nanoradiopharmaceutical.

Rodent Model Preclinical Assessment of PEGylated Block Copolymer Targeting Cognition and Oxidative Stress Insults of Alzheimer's Disease.

Misfolded peptide amyloid beta (Aß42), neurofibrillary tangles of hyper-phosphorylated tau, oxidative damage to the brain, and neuroinflammation are distinguished determinants of Alzheimer's disease (AD) responsible for disease progression. This multifaceted neurodegenerative disease is challenging to cure under a single treatment regime until the key disease determinants are traced for their sequential occurrence in disease progression. In an early report, a novel side-chain tripeptide containing PEGylated block copolymer has been tested thoroughly in vitro and in silico for the early inhibition of Aß42 aggregation as well as degradation of preformed Aß42 fibril deposits. The present study demonstrates a preclinical assessment of the PEGylated block copolymer in colchicine-induced AD-mimicking rodent model. The colchicine-induced Wistar rats receiving an intranasal delivery of the block copolymer at a daily dosage of 100 µg/kg and 200 µg/kg body weights, respectively, for 14 days manifested a notable attenuation of behavioral deficit pattern, oxidative stress, and neurotransmitters' deficiency as compared to the untreated ones. The current study also reports the ameliorative property of the PEGylated compound for progressive neuroinflammation and decreased mitochondrial bioenergetics in astrocytoma cell line, viz., U87. A closer look into the drug mechanism of action of a compact 3D PEGylated block copolymer confirmed its disintegrative interaction with Aß42 fibril via in silico simulation. The results obtained from this study signify the potential of the novel PEGylated block copolymer to ameliorate the cognitive decline and progressive oxidative insults in AD and may envision a successful clinical phase trial. The amelioration of disease condition of colchicine-induced AD rat. Initially the rat has given colchicine via stereotaxic surgery which led to a mimicking condition of AD including neuronal death in hippocampal CA1 region. After recovery from the surgery, the rat was treated with the PEGylated block copolymer through intranasal delivery, and this has led to the decrease in neuronal death in hippocampal CA1 region. The mechanism of drug action has shown by the separation of monomer chains of Aß42.

SIRT6 promotes mitochondrial fission and subsequent cellular invasion in ovarian cancer.

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a "metabolic disorder," is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1-SIRT7) belong to the group of NAD+ deacetylase and ADP-ribosylation enzymes that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non-malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2-driven activatory phosphorylation of DRP1 at serine-616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA-mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.

Glutamine regulates ovarian cancer cell migration and invasion through ETS1.

Cancer cells are dependent on glutamine for their metabolism and growth. Despite being the most abundant amino acid in the blood, glutamine deprivation occurs in the core of the tumor rendering less access to glutamine to the nearby tumor cells. Tumor cells mostly use the glutamine for mitochondrial oxidative phosphorylation (OXPHOS) to produce energy and the ingredients of the biomass required for the highly proliferating and metastatic ovarian cancer cells. But there is a lack of reports on the regulation of glutamine starvation on metastatic behavior and epithelial to mesenchymal transition (EMT) of ovarian cancer cells. We found that glutamine starvation reduced the migration and invasion properties of the ovarian cancer cells, PA1 and SKOV3. The expression of the invasion-inducing proteins, like matrix metalloproteinases (MMP2 and MMP9), were downregulated upon glutamine starvation. MMP genes are mostly regulated by the ETS1 oncogenic transcription factor in invasive tumor cells. Here we demonstrated the significant involvement of ETS1 on EMT and invasion in glutamine-deprived cells. We have further shown that the regulation of ETS1 expression and nuclear localization upon glutamine starvation is controlled in a cell type-specific manner. In PA1 cells, glutamine-induced ETS1 over-expression is HIF1α-dependent, while in SKOV3, its translocation to the nucleus is regulated through the mTOR pathway. Considering all, our study suggests that glutamine plays a very significant role in migration and invasion in ovarian cancer cells and ETS1 plays a key role in inducing such oncogenic parameters.