We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro , ...without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 106 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
Nonalcoholic fatty liver disease (NAFLD) and its evolution to inflammatory steatohepatitis (NASH) are the most common causes of chronic liver damage and transplantation that are reaching epidemic ...proportions due to the upraising incidence of metabolic syndrome, obesity, and diabetes. Currently, there is no approved treatment for NASH. The mitochondrial citrate carrier, Slc25a1, has been proposed to play an important role in lipid metabolism, suggesting a potential role for this protein in the pathogenesis of this disease. Here, we show that Slc25a1 inhibition with a specific inhibitor compound, CTPI-2, halts salient alterations of NASH reverting steatosis, preventing the evolution to steatohepatitis, reducing inflammatory macrophage infiltration in the liver and adipose tissue, while starkly mitigating obesity induced by a high-fat diet. These effects are differentially recapitulated by a global ablation of one copy of the Slc25a1 gene or by a liver-targeted Slc25a1 knockout, which unravel dose-dependent and tissue-specific functions of this protein. Mechanistically, through citrate-dependent activities, Slc25a1 inhibition rewires the lipogenic program, blunts signaling from peroxisome proliferator-activated receptor gamma, a key regulator of glucose and lipid metabolism, and inhibits the expression of gluconeogenic genes. The combination of these activities leads not only to inhibition of lipid anabolic processes, but also to a normalization of hyperglycemia and glucose intolerance as well. In summary, our data show for the first time that Slc25a1 serves as an important player in the pathogenesis of fatty liver disease and thus, provides a potentially exploitable and novel therapeutic target.
The Hedgehog (Hh) pathway is activated in some human cancers, including medulloblastoma. The glioma-associated oncogene homolog (GLI) transcription factors are critical mediators of the activated Hh ...pathway, and their expression may be elevated in some tumors independent of upstream Hh signaling. Thus, therapies targeting GLI transcription factors may benefit a wide spectrum of patients with mutations at different nodal points of the Hh pathway. In this study, we present evidence that arsenic trioxide (ATO) suppresses human cancer cell growth and tumor development in mice by inhibiting GLI1. Mechanistically, ATO directly bound to GLI1 protein, inhibited its transcriptional activity, and decreased expression of endogenous GLI target genes. Consistent with this, ATO inhibited the growth of human cancer cell lines that depended on upregulated GLI expression in vitro and in vivo in a xenograft model of Ewing sarcoma. Furthermore, ATO improved survival of a clinically relevant spontaneous mouse model of medulloblastoma with activated Hh pathway signaling. Our results establish ATO as a Hh pathway inhibitor acting at the level of GLI1 both in vitro and in vivo. These results warrant the clinical investigation of ATO for tumors with activated Hh/GLI signaling, in particular patients who develop resistance to current therapies targeting the Hh pathway upstream of GLI.
Cancer is a heterogeneous disease with multifaceted drug resistance mechanisms (e.g., tumour microenvironment TME, tumour heterogeneity, and immune evasion). Natural products are interesting ...repository of bioactive molecules, especially those with anticancer activities. Prodigiosin, a red pigment produced by Serratia marcescens, possesses inherent anticancer characteristics, showing interesting antitumour activities in different cancers (e.g., breast, gastric) with low or without harmful effects on normal cells. The present review discusses the potential role of prodigiosin in modulating and reprogramming the metabolism of the various immune cells in the TME, such as T and B lymphocytes, tumour-associated macrophages (TAMs), natural killer (NK) cells, and tumour-associated dendritic cells (TADCs), and myeloid-derived suppressor cells (MDSCs) which in turn might introduce as an immunomodulator in cancer therapy.
Dysregulation of the pathways that preserve mitochondrial integrity hallmarks many human diseases including diabetes, neurodegeration, aging and cancer. The mitochondrial citrate transporter gene, ...SLC25A1 or CIC, maps on chromosome 22q11.21, a region amplified in some tumors and deleted in developmental disorders known as velo-cardio-facial- and DiGeorge syndromes. We report here that in tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence promoting proliferation. CIC levels are increased in human cancers and its inhibition has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial homeostasis in metabolically active, high proliferating tissues and imply that this protein is a therapeutic target in cancer and likely, in other human diseases.
Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management ...by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti‐inflammatory signaling and membrane‐stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF‐κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live‐imaging and pathology through both preventive and post‐onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.
Through studying the molecular mechanisms of glucocorticoids in Duchenne Muscular Dystrophy, the authors developed VBP15 as a novel oral drug with great therapeutic potentials in mdx mice.VBP15 acts like steroids, yet lacks their side effects.
Abstract
Background
Recently, there is increasing awareness focused on the identification of naturally occurring anticancer agents derived from natural products. Manuka honey (MH) has been recognized ...for its biological properties as antimicrobial, antioxidant, and anticancer properties. However, its antiproliferative mechanism in hepatocellular carcinoma is not investigated. The current study focused mainly on investigating the molecular mechanism and synergistic effect of anticancer properties of MH on Doxorubicin (DOX)-mediated apoptotic cell death, using two different p53 statuses (HepG2 and Hep3B) and one non-tumorigenic immortalized liver cell line.
Results
MH treatment showed a proliferative inhibitory effect on tested cells in a dose-dependent manner with IC
50
concentration of (6.92 ± 0.005%) and (18.62 ± 0.07%) for HepG2 and Hep3B cells, respectively, and induced dramatic morphological changes of Hep-G2 cells, which considered as characteristics feature of apoptosis induction after 48 h of treatment. Our results showed that MH or combined treatments induced higher cytotoxicity in p53-wild type, HepG2, than in p53-null, Hep3B, cells. Cytotoxicity was not observed in normal liver cells. Furthermore, the synergistic effect of MH and Dox on apoptosis was evidenced by increased annexin-V-positive cells and Sub-G1 cells in both tested cell lines with a significant increase in the percentage of Hep-G2 cells at late apoptosis as confirmed by the flow cytometric analysis. Consistently, the proteolytic activities of caspase-3 and the degradation of poly (ADP-ribose) polymerase were also higher in the combined treatment which in turn accompanied by significant inhibitory effects of pERK1/2, mTOR, S6K, oncogenic β-catenin, and cyclin D1 after 48 h. In contrast, the MH or combined treatment-induced apoptosis was accompanied by significantly upregulated expression of proapoptotic Bax protein and downregulated expression of anti-apoptotic Bcl-2 protein after 48 h.
Conclusions
Our data showed a synergistic inhibitory effect of MH on DOX-mediated apoptotic cell death in HCC cells. To our knowledge, the present study provides the first report on the anticancer activity of MH and its combined treatment with DOX on HCC cell lines, introducing MH as a promising natural and nontoxic anticancer compound.
Impaired reproductive health is a worldwide problem that affects the psychological well-being of a society. Despite the technological developments to treat infertility, the global infertility rate is ...increasing significantly. Many infertility conditions are currently treated using various advanced clinical approaches such as intrauterine semination (IUI), in vitro fertilization (IVF), and intracytoplasmic injection (ICSI). Nonetheless, clinical management of some conditions such as dysfunctional endometrium, premature ovarian failure, and ovarian physiological aging still pose significant challenges. Stem cells based therapeutic strategies have a long-standing history to treat many infertility conditions, but ethical restrictions do not allow the broad-scale utilization of adult mesenchymal stromal/stem cells (MSCs). Easily accessible, placental derived or amniotic stem cells present an invaluable alternative source of non-immunogenic and non-tumorigenic stem cells that possess multilineage potential. Given these characteristics, placental or amniotic stem cells (ASCs) have been investigated for therapeutic purposes to address infertility in the last decade. This study aims to summarize the current standing and progress of human amniotic epithelial stem cells (hAECs), amniotic mesenchymal stem cells (hAMSCs), and amniotic fluid stem cells (hAFSCs) in the field of reproductive medicine. The therapeutic potential of these cells to restore or enhance normal ovarian function and pregnancy outcomes are highlighted in this study.
Acoustically evoked seizures (e.g., audiogenic seizures or AGS) are common in models of inherited epilepsy and occur in a variety of species including rat, mouse, and hamster. Two models that have ...been particularly well studied are the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR) strains. Acute and repeated AGS, as well as comorbid conditions, displays a close phenotypic overlap in these models. Whether these similarities arise from convergent or divergent structural changes in the brain remains unknown. Here, we examined the brain structure of Sprague Dawley (SD) and Wistar (WIS) rats, and quantified changes in the GEPR-3 and WAR, respectively. Brains from adult, male rats of each strain (n=8–10 per group) were collected, fixed, and embedded in agar and imaged using a 7 tesla Bruker MRI. Post-acquisition analysis included voxel-based morphometry (VBM), diffusion tensor imaging (DTI), and manual volumetric tracing. In the VBM analysis, GEPR-3 displayed volumetric changes in brainstem structures known to be engaged by AGS (e.g., superior and inferior colliculus, periaqueductal grey) and in forebrain structures (e.g., striatum, septum, nucleus accumbens). WAR displayed volumetric changes in superior colliculus, and a broader set of limbic regions (e.g., hippocampus, amygdala/piriform cortex). The only area of significant overlap in the two strains was the midline cerebellum: both GEPR-3 and WAR showed decreased volume compared to their control strains. In the DTI analysis, GEPR-3 displayed decreased fractional anisotropy (FA) in the corpus callosum, posterior commissure and commissure of the inferior colliculus (IC). WAR displayed increased FA only in the commissure of IC. These data provide a biological basis for further comparative and mechanistic studies in the GEPR-3 and WAR models, as well as provide additional insight into commonalities in the pathways underlying AGS susceptibility and behavioral comorbidity.
•GEPR-3 rats display volumetric abnormalities in brainstem components of the audiogenic seizure network.•WAR rats display volumetric abnormalities in forebrain/limbic structures.•WAR rats display ventriculomegaly.•Both GEPR-3 and WAR strains display volumetric decreases in midline cerebellum.
Human amniotic epithelial cells (hAECs) are non-immunogenic epithelial cells that can develop into cells of all three germline lineages. However, a refined clinically reliable method is required to ...optimize the preparation and banking procedures of hAECs for their successful translation into clinical studies. With the goal of establishing standardized clinically applicable hAECs cultured cells, we described the use of a powerful epithelial cell culture technique, termed
C
onditionally
R
eprogrammed Cells (CRC) for ex vivo expansion of hAECs. The well-established CRC culture method uses a Rho kinase inhibitor (Y-27632) and J2 mouse fibroblast feeder cells to drive the indefinite proliferation of all known epithelial cell types. In this study, we used an optimized CRC protocol to successfully culture hAECs in a CRC medium supplemented with xenogen-free human serum. We established that hAECs thrive under the CRC conditions for over 5 passages while still expressing pluripotent stem markers (OCT-4, SOX-2 and NANOG) and non-immunogenic markers (CD80, CD86 and HLA-G) suggesting that even late-passage hAECs retain their privileged phenotype. The hAECs-CRC cells were infected with a puromycin-selectable lentivirus expressing
luciferase
and
GFP (green fluorescent protein)
and stably selected with puromycin. The hAECs expressing GFP were injected subcutaneously into the flanks of Athymic and C57BL6 mice to check the tolerability and stability of cells against the immune system. Chemiluminescence imaging confirmed the presence and viability of cells at days 2, 5, and 42 without acute inflammation or any tumor formation. Collectively, these data indicate that the CRC approach offers a novel solution to expanding hAECs in humanized conditions for future clinical uses, while retaining their primary phenotype.
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