Autophagy as a Therapeutic Target in Breast Tumors: The Cancer stem cell perspective.

Breast cancer is a heterogeneous disease, with a subpopulation of tumor cells known as breast cancer stem cells (BCSCs) with self-renewal and differentiation abilities that play a critical role in tumor initiation, progression, and therapy resistance. The tumor microenvironment (TME) is a complex area where diverse cancer cells reside creating a highly interactive environment with secreted factors, and the extracellular matrix. Autophagy, a cellular self-digestion process, influences dynamic cellular processes in the tumor TME integrating diverse signals that regulate tumor development and heterogeneity. Autophagy acts as a double-edged sword in the breast TME, with both tumor-promoting and tumor-suppressing roles. Autophagy promotes breast tumorigenesis by regulating tumor cell survival, migration and invasion, metabolic reprogramming, and epithelial-mesenchymal transition (EMT). BCSCs harness autophagy to maintain stemness properties, evade immune surveillance, and resist therapeutic interventions. Conversely, excessive, or dysregulated autophagy may lead to BCSC differentiation or cell death, offering a potential avenue for therapeutic exploration. The molecular mechanisms that regulate autophagy in BCSCs including the mammalian target of rapamycin (mTOR), AMPK, and Beclin-1 signaling pathways may be potential targets for pharmacological intervention in breast cancer. This review provides a comprehensive overview of the relationship between autophagy and BCSCs, highlighting recent advancements in our understanding of their interplay. We also discuss the current state of autophagy-targeting agents and their preclinical and clinical development in BCSCs.

Elevated senescence in the bone marrow mesenchymal stem cells of acquired aplastic anemia patients: A possible implication of DNA damage responses and telomere attrition.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSC) are an integral part of the BM niche that is essential to maintain hematopoietic homeostasis. In aplastic anemia (AA), a few studies have reported phenotypic defects in the BM-MSC, such as reduced proliferation, imbalanced differentiation, and apoptosis; however, the alterations at the molecular level need to be better characterized. Therefore, the current study aims to identify the causative factors underlying the compromised functions of AA BM-MSC that might eventually be contributing to the AA pathobiology. METHODS: We performed RNA sequencing (RNA-Seq) using the Illumina platform to comprehend the distinction between the transcriptional landscape of AA and control BM-MSC. Further, we validated the alterations observed in senescence by Senescence- associated beta-galactosidase (SA -ß-gal) assay, DNA damage by γH2AX staining, and telomere attrition by relative telomere length assessment and telomerase activity assay. We used qRT-PCR to analyze changes in some of the genes associated with these molecular mechanisms. RESULTS: The transcriptome profiling revealed enrichment of senescence-associated genes and pathways in AA BM-MSC. The senescent phenotype of AA BM-MSC was accompanied by enhanced SA -ß-gal activity and elevated expression of senescence associated genes TP53, PARP1, and CDKN1A. Further, we observed increased γH2AX foci indicating DNA damage, reduced telomere length, and diminished telomerase activity in the AA BM-MSC. CONCLUSION: Our results highlight that AA BM-MSC have a senescent phenotype accompanied by other cellular defects like DNA damage and telomere attrition, which are most likely driving the senescent phenotype of AA BM-MSC thus hampering their hematopoiesis supporting properties as observed in AA.

Escin-sorafenib synergy up-regulates LC3-II and p62 to induce apoptosis in hepatocellular carcinoma cells.

INTRODUCTION: Hepatocellular carcinoma (HCC) is a common solid cancer and the leading cause of cancer deaths worldwide. Sorafenib is the first drug used to treat HCC but its effectiveness needs to be improved, and it is important to find ways to treat cancer that combine sorafenib with other drugs. Synergistic therapies lower effective drug doses and side effects while enhancing the anticancer effect. PURPOSE: In the present study, the therapeutic potential of sorafenib in combination with escin and its underlying mechanism in targeting liver cancer has been established. STUDY DESIGN/METHODS: The IC50 of sorafenib and escin against HepG2, PLC/PRF5 and Huh7 cell lines were determined using MTT assay. The combination index, dose reduction index, isobologram and concentrations producing synergy were evaluated using the Chou-Talaly algorithm. The sub-effective concentration of sorafenib and escin was selected to analyze cytotoxic synergistic potential. Cellular ROS, mitochondrial membrane potential, annexin V and cell cycle were evaluated using a flow-cytometer, and autophagy biomarkers were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role. A DEN-induced liver cancer rat model was developed to check the synergy of sorafenib and escin. RESULTS: Different concentrations of escin reduced the IC50 of sorafenib in HepG2, PLC/PRF5 and Huh7 cell lines. Chou-Talaly algorithm determined cytotoxic synergistic concentrations of sorafenib and escin in these cell lines. Mechanistically, this combination over-expressed p62 and LC-II, reflecting autophagy block and induced late apoptosis, further reconfirmed by ATG5 knockdown. Sorafenib and escin combination  reduced HCC serum biomarker α-feto protein (α-FP) by 1.5 folds. This combination restricted liver weight, tumor number and size, also, conserved morphological features of liver cells. The combination selectively targeted the G0 /G1 phase of cancer cells. CONCLUSION: Escin and sorafenib combination potentially up-regulates p62 to block autophagy to induce late apoptosis in liver cancer cells.

Molecular regulation of autophagy and suppression of protein kinases by aescin, a triterpenoid saponin impedes lung cancer progression.

Lung cancer is the most common and lethal cancer worldwide, yet there are no adequate and novel medications to control this illness. Previous reports suggested the potential of protein kinases to target lung cancer by regulating autophagy. This study establishes the role of aescin, a triterpenoid saponin, in targeting protein kinases responsible for lung cancer proliferation and mobility. The experimental data revealed that aescin significantly impedes lung cancer cell proliferation by downregulating protein kinases such as AKT, mTOR, MEK, and ERK. Downregulation of AKT-mTOR may promote a string of events inducing cytotoxic autophagy-mediated apoptosis in the presence of aescin. Besides, aescin decreases mobility and invasion by downregulating HIF-1α and VEGF gene expressions. Moreover, it successfully monitors EGFR gene expression, improves lung histology, and regulates biochemical parameters in a pre-clinical DEN-induced lung cancer model. Aescin was observed to be safe and non-toxic in both in silico toxicity predictions and ex vivo erythrocyte fragility assays. Hence, this study elucidates the molecular mechanism of aescin in targeting protein kinases and suggests that it could be a safer and more viable therapeutic agent for lung cancer treatment.

Gossypol synergises antiproliferative effect of sorafenib in metastatic lung cancer cells following Chou-Talalay algorithm.

Combination therapy has been proposed as a promising approach for lung cancer treatment, as it can enhance anticancer efficacy, and reduce dosages and adverse effects. This study aimed to explore the therapeutic potential of gossypol, a natural polyphenolic compound with sorafenib for treating lung cancer cells and elucidating its mechanism of action. The MTT assay was utilized to determine the IC50 of sorafenib and gossypol against A549 and NCI H460 cell lines. The Chou-Talaly algorithm was employed to determine the combination index (CI). A sub-effective concentration of sorafenib and gossypol was chosen to investigate the possibility of cytotoxic synergy. Autophagy biomarkers were identified using Western blotting, and the function of autophagy was determined using ATG5 siRNA. Results show that IC50 of sorafenib significantly reduced in A549 and NCI H460 cells when co-treated with gossypol. The combination treatment showed a synergistic cytotoxic effect against tested cell lines. The Chou-Talaly algorithm confirmed sorafenib's dose reduction index (DRI) up to 3.86. In A549 cells, combination treatment down-regulated p62 and up-regulated LC3-II, indicating the initiation of autophagy-dependent cytotoxicity. This was further confirmed by siRNA ATG5 knockdown. Additionally, the combination treatment exclusively targeted G0/G1 phase cancer cells. In conclusion, the combination of gossypol and sorafenib shows a synergistic increase in the cytotoxic effect by promoting autophagy and apoptosis.

Escin enhanced the efficacy of sorafenib by autophagy-mediated apoptosis in lung cancer cells.

Combining anti-cancer drugs has been exploited as promising treatment strategy to target lung cancer. Synergistic chemotherapies increase anti-cancer effect and reduce effective drug doses and side effects. In this study, therapeutic potential of escin in combination with sorafenib has been explored. 3-(4,5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide assay was used to calculate IC50 values. The synergy was evaluated using Chou-Talaly algorithm. Cellular reactive oxygen species, mitochondrial membrane potential, annexin V, and cell-cycle studies were done by flow-cytometer, and autophagy biomarkers expression were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role, diethylnitrosamine-induced lung cancer model was used to check the synergy of sorafenib/escin. Escin significantly reduced the IC50 of sorafenib in A549 and NCIH460 cells. The combination of sorafenib/escin produced a 2.95 and 5.45 dose reduction index for sorafenib in A549 and NCI-H460 cells. The combination of over-expressed p62 and LC3-II reflects autophagy block-mediated late apoptosis. This phenomenon was reconfirmed by ATG5 knockdown. This combination also selectively targeted G0/G1 phase of cancer cells. In in vivo study, the combination reduced tumour load and lower elevated serum biochemical parameters. The combination of sorafenib/escin synergistically inhibits autophagy to induce late apoptosis in lung cancer cells' G0/G1 phase.

Autophagy: A Cellular Guardian against Hepatic Lipotoxicity.

Lipotoxicity is a phenomenon of lipid-induced cellular injury in nonadipose tissue. Excess of free saturated fatty acids (SFAs) contributes to hepatic injury in nonalcoholic fatty liver disease (NAFLD), which has been growing at an unprecedented rate in recent years. SFAs and their derivatives such as ceramides and membrane phospholipids have been shown to induce intrahepatic oxidative damage and ER stress. Autophagy represents a cellular housekeeping mechanism to counter the perturbation in organelle function and activation of stress signals within the cell. Several aspects of autophagy, including lipid droplet assembly, lipophagy, mitophagy, redox signaling and ER-phagy, play a critical role in mounting a strong defense against lipotoxic lipid species within the hepatic cells. This review provides a succinct overview of our current understanding of autophagy-lipotoxicity interaction and its pharmacological and nonpharmacological modulation in treating NAFLD.

Molecular signaling network and therapeutic developments in breast cancer brain metastasis.

Breast cancer (BC) is one of the most frequently diagnosed cancers in women worldwide. It has surpassed lung cancer as the leading cause of cancer-related death. Breast cancer brain metastasis (BCBM) is becoming a major clinical concern that is commonly associated with ER-ve and HER2+ve subtypes of BC patients. Metastatic lesions in the brain originate when the cancer cells detach from a primary breast tumor and establish metastatic lesions and infiltrate near and distant organs via systemic blood circulation by traversing the BBB. The colonization of BC cells in the brain involves a complex interplay in the tumor microenvironment (TME), metastatic cells, and brain cells like endothelial cells, microglia, and astrocytes. BCBM is a significant cause of morbidity and mortality and presents a challenge to developing successful cancer therapy. In this review, we discuss the molecular mechanism of BCBM and novel therapeutic strategies for patients with brain metastatic BC.

Purpurin ameliorates alcohol-induced hepatotoxicity by reducing ROS generation and promoting Nrf2 expression.

INTRODUCTION AND AIM: Purpurin, a naturally occurring anthraquinone isolated from the roots of Rubia cordifolia, exhibits anti-cancer, anti-genotoxic, anti-microbial, neuromodulatory and photodynamic activity. However, purpurin's in vivo and in vitro antioxidant mechanism remains unexplored. The present study explores the anti-oxidative mechanism of purpurin under the influence of alcohol using in vivo and in vitro test systems. METHODS: Mice hepatocytes and alcohol-induced liver toxicity model were used to evaluate the effect of purpurin. The non-enzymatic and enzymatic oxidative stress markers were estimated by the colorimetric method. The reactive oxygen species (ROS) were quantified in mitochondria and cells using flow cytometer. Real-time PCR and western blotting were used to quantify cytochrome 450 subtype 2E1 (CYP2E1) and Nrf2 expression in the liver tissue of mice. In silico studies were performed through receptor-ligand binding interaction. KEY FINDINGS: Purpurin effectively reduced total cellular and mitochondrial ROS in primary hepatocytes and WRL-68 cells. It prevented alcohol-induced ROS-dependent biochemical and cellular insults observed by analysing the serum glutamic pyruvic transaminase (SGPT), glutamic-oxaloacetic transaminase (SGOT) levels and CYP2E1 expression in liver tissue of alcohol-administered mice. Moreover, it also restored the activity of antioxidant enzymes. Its antioxidant effect was established by glutathione and ROS-dependent mechanisms using buthionine sulfoximine and N-acetyl cysteine. Along with alcohol, purpurin up-regulated Nrf2 expression in hepatocytes. SIGNIFICANCE: This work confirmed the ameliorative effect of purpurin for alcohol-induced hepatotoxicity by drabbing free radicals and curbing oxidative stress via activation of antioxidant signalling pathways.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Lipoic acid blocks autophagic flux and impairs cellular bioenergetics in breast cancer and reduces stemness.

Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cancer cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We show that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment, which may lead to cell death in breast cancer cells/BCSCs.

ULK1 Signaling in the Liver: Autophagy Dependent and Independent Actions.

Liver is the primary organ for energy metabolism and detoxification in the human body. Not surprisingly, a derangement in liver function leads to several metabolic diseases. Autophagy is a cellular process, which primarily deals with providing molecules for energy production, and maintains cellular health. Autophagy in the liver has been implicated in several hepatic metabolic processes, such as, lipolysis, glycogenolysis, and gluconeogenesis. Autophagy also provides protection against drugs and pathogens. Deregulation of autophagy is associated with the development of non-alcoholic fatty liver disease (NAFLD) acute-liver injury, and cancer. The process of autophagy is synchronized by the action of autophagy family genes or autophagy (Atg) genes that perform key functions at different steps. The uncoordinated-51-like kinases 1 (ULK1) is a proximal kinase member of the Atg family that plays a crucial role in autophagy. Interestingly, ULK1 actions on hepatic cells may also involve some autophagy-independent signaling. In this review, we provide a comprehensive update of ULK1 mediated hepatic action involving lipotoxicity, acute liver injury, cholesterol synthesis, and hepatocellular carcinoma, including both its autophagic and non-autophagic functions.

Multifaceted role of chemokines in solid tumors: From biology to therapy.

Chemokines are small secretory chemotactic cytokines that control the directed migration of immune cells. Chemokines are involved in both anti-and pro-tumorigenic immune responses. Accumulating evidence suggests that the balance between these responses is influenced by several factors such as the stage of tumorigenesis, immune cell activation, recruitment of immune activating or immunosuppressive cells in the tumor microenvironment (TME), and chemokine receptor expression on effector and regulatory target cells. Cancer cells engage in a complex network with their TME components via several factors including growth factors, cytokines and chemokines that are critical for the growth of primary tumor and metastasis. However, chemokines show a multifaceted role in tumor progression including maintenance of stem-like properties, tumor cell proliferation/survival/senescence, angiogenesis, and metastasis. The heterogeneity of solid tumors in primary and metastatic cancers presents a challenge to the development of successful cancer therapy. Despite extensive research on how solid tumors escape immune cell-mediated anti-tumor response, finding an effective therapy for metastatic cancer still remains a challenge. This review discusses the multifarious roles of chemokines in solid tumors including various chemokine signaling pathways such as CXCL8-CXCR1/2, CXCL9, 10, 11-CXCR3, CXCR4-CXCL12, CCL(X)-CCR(X) in primary and metastatic cancers. We further discuss the novel therapeutic approaches that have been developed by major breakthroughs in chemokine research to treat cancer patients by the strategic blockade/activation of these signaling axes alone or in combination with immunotherapies.

Caffeine prevents restenosis and inhibits vascular smooth muscle cell proliferation through the induction of autophagy.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Thyroid Hormone Receptor α Regulates Autophagy, Mitochondrial Biogenesis, and Fatty Acid Use in Skeletal Muscle.

Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone α (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor α (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis, and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions. Two genetic mouse models were used in this study: TRα1PV/+ mice, which express the mutant Thra1PV gene ubiquitously, and SM-TRα1L400R/+ mice, which express TRα1L400R in a muscle-specific manner. Gastrocnemius muscle from TRα1PV/+, SM-TRα1L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (lysosomal-associated membrane protein [LAMP]-1 and LAMP-2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and estrogen-related receptor α (ERRα), key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins, were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired and was associated with decreased acylcarnitines and tricarboxylic acid cycle intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired.

Moderate/subclinical calcium deficiency attenuates trabecular mass, microarchitecture and bone growth in growing rats.

Adequate dietary calcium (Ca) intake is essential for bone accretion, peak bone mass (PBM) attainment, bone quality and strength during the mammalian growth period. Severe Ca deficiency during growing age results in secondary hyperparathyroidism (SHPT) and poor bone quality and strength. However, the impact of moderate Ca deficiency during rats early growth period on bone health and the reversibility with supplementing calcium later in adult life remains unclear. Female Sprague-Dawley (SD) rats (postnatal 28th day, P28) were initiated either with a moderate calcium-deficient diet (MCD, 0.25% w/w Ca) or a control diet (0.8% w/w Ca, control group) till P70. Thereafter, MCD rats were continued either with MCD diet or supplemented with calcium diet (0.8% w/w Ca, calcium supplemented group, CaS) till P150. Another group (control rats) were fed 0.8% w/w Ca containing diet from P28 till P150. MCD group, as compared to the control group, had significantly reduced serum ionized Ca and procollagen type 1 N-terminal propeptide (P1NP) at P70 while no significant change was observed in serum corrected Ca, inorganic phosphate (P), alkaline phosphatase (ALP), 25-hydroxy vitamin D [25(OH)D], intact parathyroid hormone (iPTH), and urinary C-terminal telopeptide of collagen 1 (CTX-1), Ca, and P. Femoral and tibial metaphysis in MCD rats had significantly reduced linear growth, cortical and trabecular volumetric BMD (vBMD), trabecular microarchitecture (BV/TV%, trabecular thickness, separation and number, structural model index and connectivity density), cortical thickness, and bone stiffness despite the absence of secondary hyperparathyroidism (SHPT). Continued MCD at P70-P150 results in persistence of compromised bone strength while calcium supplementation (CaS group) improved all the parameters related to bone strength and microarchitecture. Our results indicate that uncorrected moderate/subclinical calcium deficiency in growing rats can result in poor bone quality and strength despite the absence of SHPT. This finding could have relevance in children with poor calcium intake in childhood and adolescence.

Calcium repletion to rats with calcipenic rickets fails to recover bone quality: A calcipenic "memory".

Calcipenic rickets is prevalent in underprivileged children in developing countries. Calcipenic rickets resulting from dietary calcium (Ca) deficiency decreases bone mass and deteriorates bone microstructure in humans. The effect of dietary Ca replenishment (CaR) on rachitic bones in animal models depends on the amount, critical period and duration of replenishment, however, the extent of recovery in various bone parameters including bone quality remains unclear. We investigated the effect of CaR in rat skeleton after inducing calcipenic rickets. Female SD rats (postnatal 28 days/P28) were rendered calcipenic by feeding calcium deficient (CaD) diet (0.1% Ca) till P70 while control SD rats were fed Ca sufficient diet (0.8% Ca). At P70, calcipenic rats were switched to 0.8% Ca diet till P150 for one group and P210 for another group (endpoint). The CaD groups received 0.1% Ca diet throughout the study (P210). In the CaD groups, serum Ca and phosphate, and bone mineral density (BMD) were significantly decreased whereas serum alkaline phosphatase (ALP), iPTH and CTX-1 were increased compared to age-matched controls. Moreover, at the endpoint, the CaD group had reduced bone mass, surface referent bone formation parameters, tissue mineralization and strength accompanied by the increased osteoid thickness and microarchitectural decay (measured by trabecular geometric parameters) with poor crystal packing. The CaR group showed complete recovery in serum Ca, iPTH, ALP and CTX-1, and BMD, however, the bone quality parameters including bone strength, microarchitectural decay, tissue mineralization, and crystallinity were incompletely restored. Decreased surface referent bone formation and increased unmineralized bones (osteoid) indicative of osteomalacia were also observed in the CaR group at P210 compared with control despite prolonged replenishment. We conclude that a prolonged Ca repletion following the induction of calcipenic rickets in rats although shows the recovery of biochemical measures of bone metabolism and bone mass, however, the bone quality remains compromised. This suggests that a "memory" of calcipenia occurring at the early growth stage persists in the skeleton of adult rats despite a prolonged Ca replenishment.

Amniotic fluid stem cells ameliorate cisplatin-induced acute renal failure through induction of autophagy and inhibition of apoptosis.

BACKGROUND: We have recently demonstrated that amniotic fluid stem cells (AFSC) express renal progenitor markers and can be differentiated in vitro into renal lineage cell types, viz, juxtaglomerular and renal proximal tubular epithelial-like cells. Here, we have evaluated the therapeutic efficacy of AFSC in a cisplatin-induced rat model of acute renal failure (ARF) and investigated the underlying mechanisms responsible for their renoprotective effects. METHODS: ARF was induced in Wistar rats by intra-peritoneal injection of cisplatin (7 mg/kg). Five days after cisplatin injection, rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On days 8 and 12 after cisplatin injection, the blood biochemical parameters, histopathological changes, apoptosis and expression of pro-apoptotic, anti-apoptotic, and autophagy-related proteins in renal tissues were studied in both groups of rats. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor, was administered by the intra-peritoneal route. RESULTS: Administration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins, viz, PUMA, Bax, cleaved caspase-3, and cleaved caspase-9, as compared to the saline-treated group. Furthermore, in the AFSC-treated group as compared to the saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1, and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin. CONCLUSION: AFSC led to amelioration of cisplatin-induced ARF which was mediated by inhibition of apoptosis and activation of autophagy. The protective effects of AFSC were blunted by chloroquine, an inhibitor of autophagy, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

Novel Transcriptional Mechanisms for Regulating Metabolism by Thyroid Hormone.

The thyroid hormone plays a key role in energy and nutrient metabolisms in many tissues and regulates the transcription of key genes in metabolic pathways. It has long been believed that thyroid hormones (THs) exerted their effects primarily by binding to nuclear TH receptors (THRs) that are associated with conserved thyroid hormone response elements (TREs) located on the promoters of target genes. However, recent transcriptome and ChIP-Seq studies have challenged this conventional view as discordance was observed between TH-responsive genes and THR binding to DNA. While THR association with other transcription factors bound to DNA, TH activation of THRs to mediate effects that do not involve DNA-binding, or TH binding to proteins other than THRs have been invoked as potential mechanisms to explain this discrepancy, it appears that additional novel mechanisms may enable TH to regulate the mRNA expression. These include activation of transcription factors by SIRT1 via metabolic actions by TH, the post-translational modification of THR, the THR co-regulation of transcription with other nuclear receptors and transcription factors, and the microRNA (miR) control of RNA transcript expression to encode proteins involved in the cellular metabolism. Together, these novel mechanisms enlarge and diversify the panoply of metabolic genes that can be regulated by TH.

Changes in Hepatic TRß Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model.

BACKGROUND: Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. METHODS: Adult male mice were treated with daily injections of T3 (20 µg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. RESULTS: Although TH receptor (TRα and TRß) mRNAs decreased slightly after chronic T3 treatment, only TRß protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRß protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. CONCLUSIONS: Decreased hepatic TRß protein expression occurred after chronic T3 exposure in adult male wild-type and Mct8-knockout mice. Gene array pathway and metabolomics analyses showed abnormalities in hepatic lipogenic gene expression and acylcarnitine levels, respectively, after withdrawal, despite normalization of serum TSH and TH levels. These findings may help explain the variable clinical presentations of some patients during hyperthyroidism and recovery, since TRß protein, target gene expression, and metabolic adaptive changes can occur in individual tissues without necessarily being reflected by circulating TH and TSH concentrations.