Mitochondrial Transfer of Wharton's Jelly Mesenchymal Stem Cells Eliminates Mutation Burden and Rescues Mitochondrial Bioenergetics in Rotenone-Stressed MELAS Fibroblasts.

Wharton's jelly mesenchymal stem cells (WJMSCs) transfer healthy mitochondria to cells harboring a mitochondrial DNA (mtDNA) defect. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major subgroups of mitochondrial diseases, caused by the mt.3243A>G point mutation in the mitochondrial tRNALeu(UUR) gene. The specific aim of the study is to investigate whether WJMSCs exert therapeutic effect for mitochondrial dysfunction in cells of MELAS patient through donating healthy mitochondria. We herein demonstrate that WJMSCs transfer healthy mitochondria into rotenone-stressed fibroblasts of a MELAS patient, thereby eliminating mutation burden and rescuing mitochondrial functions. In the coculture system in vitro study, WJMSCs transferred healthy mitochondria to rotenone-stressed MELAS fibroblasts. By inhibiting actin polymerization to block tunneling nanotubes (TNTs), the WJMSC-conducted mitochondrial transfer was abrogated. After mitochondrial transfer, the mt.3243A>G mutation burden of MELAS fibroblasts was reduced to an undetectable level, with long-term retention. Sequencing results confirmed that the transferred mitochondria were donated from WJMSCs. Furthermore, mitochondrial transfer of WJMSCs to MELAS fibroblasts improves mitochondrial functions and cellular performance, including protein translation of respiratory complexes, ROS overexpression, mitochondrial membrane potential, mitochondrial morphology and bioenergetics, cell proliferation, mitochondrion-dependent viability, and apoptotic resistance. This study demonstrates that WJMSCs exert bioenergetic therapeutic effects through mitochondrial transfer. This finding paves the way for the development of innovative treatments for MELAS and other mitochondrial diseases.

Mitochondrial Transfer from Wharton's Jelly Mesenchymal Stem Cell to MERRF Cybrid Reduces Oxidative Stress and Improves Mitochondrial Bioenergetics.

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial disease affecting neuromuscular functions. Mt.8344A>G mutation in mitochondrial DNA (mtDNA) is the most common cause of MERRF syndrome and has been linked to an increase in reactive oxygen species (ROS) level and oxidative stress, as well as impaired mitochondrial bioenergetics. Here, we tested whether WJMSC has therapeutic potential for the treatment of MERRF syndrome through the transfer of mitochondria. The MERRF cybrid cells exhibited a high mt.8344A>G mutation ratio, enhanced ROS level and oxidative damage, impaired mitochondrial bioenergetics, defected mitochondria-dependent viability, exhibited an imbalance of mitochondrial dynamics, and are susceptible to apoptotic stress. Coculture experiments revealed that mitochondria were intercellularly conducted from the WJMSC to the MERRF cybrid. Furthermore, WJMSC transferred mitochondria exclusively to cells with defective mitochondria but not to cells with normal mitochondria. MERRF cybrid following WJMSC coculture (MF+WJ) demonstrated improvement of mt.8344A>G mutation ratio, ROS level, oxidative damage, mitochondrial bioenergetics, mitochondria-dependent viability, balance of mitochondrial dynamics, and resistance against apoptotic stress. WJMSC-derived mitochondrial transfer and its therapeutic effect were noted to be blocked by F-actin depolymerizing agent cytochalasin B. Collectively, the WJMSC ability to rescue cells with defective mitochondrial function through donating healthy mitochondria may lead to new insights into the development of more efficient strategies to treat diseases related to mitochondrial dysfunction.

Phyllanthus urinaria's Inhibition of Human Osteosarcoma Xenografts Growth in Mice is Associated with Modulation of Mitochondrial Fission/Fusion Machinery.

Osteosarcoma is an aggressive bone cancer arising from primitive transformed cells of mesenchymal origin to form malignant osteoid. Phyllanthus urinaria [Formula: see text]P. urinaria[Formula: see text] is a widely used folk medicine in cancer treatment, however the mechanism of P. urinaria inhibited human osteosarcoma is unclear. The present study was aimed at investigating the antitumoral effects of an aqueous P. urinaria on human osteosarcoma in vivo and the related underlying mechanisms, mainly focusing on mitochondrial dynamic dysfunction. Our results showed that oral administration of P. urinaria to mice led to significant inhibition of tumor development without substantial changes to body weight or major organs. Histological examinations with H&E, Giemsa, and Masson trichrome stains confirmed inhibition of tumor growth by the P. urinaria treatment. Immunohistochemical staining of proliferation markers antigen KI-67 (Ki67) and proliferating cell nuclear antigen (PCNA), as well as a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated a decrease of tumor proliferation and an increase of apoptosis, which was associated with the modulation of B-cell lymphoma 2 (Bcl-2) family activating the caspase cascade in the P. urinaria-treated mice. The neovascularization marker cluster of differentiation 31 (CD31) was inhibited in P. urinaria-treated xenografts, implicating the potential anti-angiogenic effect of P. urinaria. P. urinaria treatment resulted in a significant decrease in the mitochondrial fusion proteins, including mitofusin 1/2 (Mfn1/2) and optic atrophy type 1 (Opa1), as well as an increase in the fission protein dynamin-related protein 1 (Drp1). The results of this study suggest mitochondrial dysfunction is associated with dynamic change that is involved in the apoptosis and anti-angiogenesis elicited by P. urinaria.

MDA5 complements TLR3 in suppression of neuroblastoma.

Toll-like receptor3 (TLR3) has been confirmed to be differentially expressed in neuroblastoma (NB), and predicts a favorable prognosis with a high expression in tumor tissues. Treatment with TLR3 agonist--polyinosinic-polycytidylic acid [poly(I:C)] could induce significant but limited apoptosis in TLR3-expressing NB cells, suggesting that other viral RNA sensors, including melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) in the cytosolic compartment might also be implicated in poly(I:C)-induced NB cell death. MDA5 and RIG-I were induced by poly(I:C) to express in two of six NB cell lines, SK-N-AS (AS) and SK-N-FI, which were associated with up-regulation of caspase9 and active caspase3. While knockdown of either MDA5 or RIG-I alone is ineffective to decrease caspase9 and active caspase3, simultaneously targeting MDA5 and TLR3 showed the best effect to rescue poly(I:C) induced up-regulation of mitochondrial antiviral signaling protein (MAVS), caspase9, active caspase3, and apoptosis in AS cells. Over-expression of MDA5 in FaDu cells resulted in significantly less colony formation and more poly(I:C)-induced cell death. Further studies in human NB tissue samples revealed that MDA5 expression in NB tissues predicted a favorable prognosis synergistically with TLR3. Our findings indicate that MDA5 may serve as a complementary role in the TLR3 activated suppression of NB.

Phyllanthus urinaria induces mitochondrial dysfunction in human osteosarcoma 143B cells associated with modulation of mitochondrial fission/fusion proteins.

Phyllanthus urinaria (P. urinaria), a widely used herbal medicine, has been reported to possess various biological characteristics including anti-inflammation, anti-virus, anti-bacteria, anti-hepatotoxicity and anti-cancer. This study provides molecular evidence associated with the dynamics and organization of mitochondria in osteosarcoma 143B cells resulted from P urinaria. Herein, P. urinaria-induced cytotoxicity and ROS associated with the inhibition of mitochondrial membrane potential were reversed by N-acetylcysteine (NAC). The endogenous antioxidant enzymes such as manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX1) were activated by P. urinaria, but not correlated to catalase. P. urinaria decreased mitochondrial respiration activity as well as respiratory chain enzymes and HIF-1α in osteosarcoma 143B cells. Additionally, both adenosine triphosphate (ATP) synthase activation and ATP production were suppressed by P. urinaria. We further investigated changes of mitochondrial dynamic in osteosarcoma 143B cells. P. urinaria indeed fragmented the mitochondrial network of osteosarcoma 143B cells. We found a significant decrease in optic atrophy type 1 (Opa1) and mitofusin 1 (Mfn1) related to fusion proteins as well as increase mitochondrial fission 1 protein (Fis1) related to fission protein. It indicated that P. urinaria modulated the mitochondrial dynamics via fusion and fission machinery. Altogether, this study offers the evidence that mitochondrial dysfunction with dynamic change is essential components for the anti-cancer mechanism elicited by P. urinaria.

Phyllanthus urinaria Induces Apoptosis in Human Osteosarcoma 143B Cells via Activation of Fas/FasL- and Mitochondria-Mediated Pathways.

Phyllanthus urinaria (P. urinaria), in this study, was used for the treatment of human osteosarcoma cells, which is one of the tough malignancies with few therapeutic modalities. Herein, we demonstrated that P. urinaria inhibited human osteosarcoma 143B cells growth through an apoptotic extrinsic pathway to activate Fas receptor/ligand expression. Both intracellular and mitochondrial reactive oxygen species were increased to lead to alterations of mitochondrial membrane permeability and Bcl-2 family including upregulation of Bid, tBid, and Bax and downregulation of Bcl-2. P. urinaria triggered an intrinsic pathway and amplified the caspase cascade to induce apoptosis of 143B cells. However, upregulation of both intracellular and mitochondrial reactive oxygen species and the sequential membrane potential change were less pronounced in the mitochondrial respiratory-defective 143Bρ(0) cells compared with the 143B cells. This study offers the evidence that mitochondria are essential for the anticancer mechanism induced by P. urinaria through both extrinsic and intrinsic pathways.

Liver hepcidin and stainable iron expression in biliary atresia.

Hepcidin is a proposed mammalian host defense peptide that was identified on the basis of its antimicrobial activity, but it was later shown to be a crucial regulator of iron homeostasis and a mediator of the anemia of chronic inflammation. Hepcidin and stainable iron expression in biliary atresia (BA) were investigated in this study. Fresh liver tissues were obtained from 10 patients in the early stage of BA when they underwent Kasai's procedure, 9 in the late stage of BA when they received liver transplantation and 5 controls receiving liver resection for benign lesions other than cholestasis or fibrosis. Real-time quantitative reverse-transcription PCR (QRT-PCR), immunohistochemical staining and ELISA were performed to gauge hepcidin mRNA and protein expression in liver and plasma. Archival liver specimens from patients in the early and late stages of BA were treated with Perls' acid ferrocyanide technique for hepatic stainable iron. The results demonstrated that liver hepcidin mRNA expression was 100-fold lower in late-stage BA than in the early stage by QRT-PCR. Significantly weaker liver hepcidin immunostaining and lower plasma hepcidin levels were found in late-stage BA than in the early stage. There was also significantly lower stainable iron in the liver of late-stage BA. The major site of stainable iron was in Kupffer cells. The results support a role for hepcidin as a key regulator of mammalian iron metabolism and chronic inflammation, whose expression correlates with the degree of stainable iron in BA.

Small bowel intussusception in symptomatic pediatric patients: experiences with 19 surgically proven cases.

Nineteen cases of surgically proven symptomatic pediatric small bowel intussusceptions (SBI) were retrospectively reviewed. Clinical presentations included vomiting (89.5%), abdominal pain and/or irritable crying (89.5%), fever (52.6%), bloody stools (26.3%), palpable abdominal masses (15.8%), hematemesis (10.5%), jaundice (5.3%), and seizures (5.3%). The duration between symptom onset and hospitalization ranged between 20 and 336 hours (average 75.8 hours). Two patients with suspected appendicitis and small bowel obstruction were operated on promptly. Sonograms revealed target lesions (average diameter 2.9 cm) suggestive of intussusception in 13 out of 17 patients, with 10 lesions located in the paraumbilical or left abdominal regions. Barium enemas in 12 of these 13 patients demonstrated no colonic lesions. Diagnosis and surgery were delayed in 16 patients (average delay = 32 hours). The remaining 1 patient with positive sonographic findings underwent early surgery after computed tomographic (CT) confirmation of SBI. Surgery revealed ileoileal intussusceptions in 11 patients, jejunojejunal in 4, jejunoileal in 3, and duodenojejunal in 1. Eight patients had lead points. Bowel complications (ischemia, necrosis, or perforation) occurred in 8 patients. The duration between symptom onset and surgery in patients with bowel complications was significantly longer than for patients without complications (p = 0.0026). In conclusion, delayed diagnosis and surgical treatment in symptomatic pediatric patients with SBI were common, leading to a high rate (42%) of bowel complications. Sonographic demonstration of a 2-3 cm target lesion, especially if paraumbilical or left abdominal, is suggestive of SBI and may obviate the need for a barium enema; however, CT is helpful for confirming SBI. In symptomatic SBI, once diagnosed, early surgical referral is strongly recommended.