Chondrogenic differentiation of mesenchymal stem cells through cartilage matrix-inspired surface coatings.

The stem cell niche comprises soluble molecules and extracellular matrix components which provide chemical and mechanical cues that determine the differentiation of stem cells. Here, the effect of polyelectrolyte multilayer (PEM) composition and terminal layer fabricated with layer-by-layer technique (LBL) pairing either hyaluronan [in its native (nHA) and oxidized form (oHA)] or chondroitin sulfate (CS) with type I collagen (Col I) is investigated on chondrogenic differentiation of human umbilical mesenchymal stem cells (hUC-MSCs). Physical studies performed to investigate the establishment and structure of the surface coatings show that PEM composed of HA and Col I show a dominance of nHA or oHA with considerably lesser organization of Col I fibrils. In contrast, distinguished fibrilized Col I is found in nCS-containing PEM. Generally, Col I-terminated PEM promote the adhesion, migration, and growth of hUC-MSCs more than GAG-terminated surfaces due to the presence of fibrillar Col I but show a lower degree of differentiation towards the chondrogenic lineage. Notably, the Col I/nHA PEM not only supports adhesion and growth of hUC-MSCs but also significantly promotes cartilage-associated gene and protein expression as found by histochemical and molecular biology studies, which is not seen on the Col I/oHA PEM. This is related to ligation of HA to the cell receptor CD44 followed by activation of ERK/Sox9 and noncanonical TGF-ß signaling-p38 pathways that depends on the molecular weight of HA as found by immune histochemical and western blotting. Hence, surface coatings on scaffolds and other implants by PEM composed of nHA and Col I may be useful for programming MSC towards cartilage regeneration.

Regulation of Mitochondrial Homeostasis and Nrf2 in Kidney Disease: Timing Is Critical.

Abnormal regulation of mitochondrial homeostasis plays a critical role in the progression of renal disease. Recent studies have shown that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) has time-dependent protective effects, which can be explained by the differing regulation of mitochondrial homeostasis during the various stages of kidney disease. In this review, we summarize the mechanisms whereby mitochondrial homeostasis is regulated and the nature of the dysregulation of mitochondrial homeostasis in renal disease. In addition, we summarize the dual roles of Nrf2 in kidney disease by discussing the studies that have shown the importance of the timing of its activation in the regulation of mitochondrial homeostasis. This should provide a theoretical basis for therapeutic strategies aimed at activating Nrf2 in kidney disease.

Activation of Transcription Factor EB Alleviates Tubular Epithelial Cell Injury via Restoring Lysosomal Homeostasis in Diabetic Nephropathy.

Disruption of lysosomal homeostasis contributes to the tubulopathy of diabetic nephropathy; however, its underlying mechanisms remain unclear. Herein, we report that decreased activity of transcription factor EB (TFEB) is responsible for the disturbed lysosome biogenesis and clearance in this pathological process. This was confirmed by the findings that insufficient lysosomal replenishment and damaged lysosomal clearance coincided with TFEB inactivation, which was mediated by mTOR hyperactivation in the renal tubular epithelial cells (TECs) of diabetic nephropathy. Furthermore, either TFEB overexpression or pharmacological activation of TFEB enhanced lysosomal clearance via promoting lysosomal biogenesis and protected TECs by reducing apoptosis in vitro. In addition, pharmacological activation of TFEB attenuated renal tubule injury, apoptosis, and inflammation in db/db mice. In conclusion, diabetes-induced mTOR activation represses TFEB function, thereby perturbing lysosomal homeostasis through impairing lysosomal biogenesis and clearance in TECs. Moreover, TFEB activation protects TECs from diabetic injuries via restoring lysosomal homeostasis.

SMAD3 promotes autophagy dysregulation by triggering lysosome depletion in tubular epithelial cells in diabetic nephropathy.

Macroautophagy/autophagy dysregulation has been noted in diabetic nephropathy; however, the regulatory mechanisms controlling this process remain unclear. In this study, we showed that SMAD3 (SMAD family member 3), the key effector of TGFB (transforming growth factor beta)-SMAD signaling, induces lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis. The pharmacological inhibition or genetic deletion of SMAD3 restored lysosome biogenesis activity by alleviating the suppression of TFEB, thereby protecting lysosomes from depletion and improving autophagic flux in renal tubular epithelial cells in diabetic nephropathy. Mechanistically, we found that SMAD3 directly binds to the 3'-UTR of TFEB and inhibits its transcription. Silencing TFEB suppressed lysosome biogenesis and resulted in a loss of the protective effects of SMAD3 inactivation on lysosome depletion under diabetic conditions. In conclusion, SMAD3 promotes lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis; this may be an important mechanism underlying autophagy dysregulation in the progression of diabetic nephropathy.Abbreviations: AGEs: advanced glycation end products; ATP6V1H: ATPase H+ transporting V1 subunit H; CTSB: cathepsin B; ChIP: chromatin immunoprecipitation; Co-BSA: control bovine serum albumin; DN: diabetic nephropathy; ELISA: enzyme-linked immunosorbent assay; FN1: fibronectin 1; HAVCR1/TIM1/KIM-1: hepatitis A virus cellular receptor 1; LAMP1: lysosomal associated membrane protein 1; LMP: lysosome membrane permeabilization; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NC: negative control; SIS3: specific inhibitor of SMAD3; SMAD3: SMAD family member 3; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TECs: tubular epithelial cells; TFEB: transcription factor EB; TGFB1: transforming growth factor beta 1; TGFBR1: transforming growth factor beta receptor 1; UTR: untranslated region; VPS11: VPS11 core subunit of CORVET and HOPS complexes.

Mechanisms and Functions of Mitophagy and Potential Roles in Renal Disease.

Mitophagy is an evolutionarily conserved process to selectively remove damaged or unnecessary mitochondria via the autophagic machinery. In this review, we focus on recent advances in the molecular mechanisms of mitophagy and how mitophagy contributes to cellular homeostasis in physiological and pathological contexts. We also briefly review and discuss the crosstalk between mitophagy and renal disease, highlighting its modulation as a potentially effective therapeutic strategy to treat kidney diseases such as acute kidney injury (AKI), diabetic kidney disease (DKD), and lupus nephritis (LN).

Nuclear factor erythroid-2 related factor 2 inhibits human disc nucleus pulpous cells apoptosis induced by excessive hydrogen peroxide.

OBJECTIVE Nuclear factor erythroid-2 related factor 2 (Nrf2)/ antioxidant response element (ARE) is a novel defensive pathway involved in the oxidative and chemical stress of cells. The aim of the study was to explore the role of Nrf2 on the apoptosis of human disc nucleus pulpous cells induced by hydrogen peroxide (H2O2). METHODS The degeneration model of human intervertebral disc nucleus pulpous cells was established. The expression of Nrf2 was interfered with using sulforaphane (SFN); for that end, three groups were established: a blank group (H2O2-/SFN-), control group (H2O2+/SFN-), and an experimental group (H2O2+/SFN+). CCK8, Hoechst 33258 living cell staining was used to detect reactive oxygen species (ROS) content. RESULTS The apoptotic rates of the three groups were [(0.40±0.46)%], [(25.98±11.28)%], and [(3.83±2.06)%, respectively. The difference was statistically significant (p<0.05). The relative content of ROS in the three groups was [(100±7)%], [(1538±91)%], and [(818±63)%]; the difference was statistically significant (p<0.05). In Western blotting, Nrf2 content in the experimental group was higher than that in the control group. CONCLUSION Nrf2 exists in the nucleus pulpous cells of human intervertebral discs, which is related to the degeneration of the intervertebral disc. It has negative feedback regulation and can prevent the degeneration of the intervertebral disc by inhibiting the apoptosis of nucleus pulpous cells of human intervertebral discs caused by excessive ROS, which provides a new intervention strategy for the prevention and treatment of the degeneration of intervertebral discs.

Autophagy Inhibition Sensitizes Renal Tubular Epithelial Cell to G1 Arrest Induced by Transforming Growth Factor beta (TGF-ß).

BACKGROUND Cell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-ß-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-ß has not been explored well. MATERIAL AND METHODS The effects of autophagy inhibition on TGF-ß-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-ß with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf). RESULTS Autophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-ß-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-ß treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-ß treated HK-2 cells. Western blot analysis showed that TGF-ß-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors. CONCLUSIONS Inhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-ß that contributes to the induction of tubulointerstitial fibrosis.

Nuclear factor erythroid-2 related factor 2 inhibits human disc nucleus pulpous cells apoptosis induced by excessive hydrogen peroxide

SUMMARY OBJECTIVE Nuclear factor erythroid-2 related factor 2 (Nrf2)/ antioxidant response element (ARE) is a novel defensive pathway involved in the oxidative and chemical stress of cells. The aim of the study was to explore the role of Nrf2 on the apoptosis of human disc nucleus pulpous cells induced by hydrogen peroxide (H2O2). METHODS The degeneration model of human intervertebral disc nucleus pulpous cells was established. The expression of Nrf2 was interfered with using sulforaphane (SFN); for that end, three groups were established: a blank group (H2O2-/SFN-), control group (H2O2+/SFN-), and an experimental group (H2O2+/SFN+). CCK8, Hoechst 33258 living cell staining was used to detect reactive oxygen species (ROS) content. RESULTS The apoptotic rates of the three groups were [(0.40±0.46)%], [(25.98±11.28)%], and [(3.83±2.06)%, respectively. The difference was statistically significant (p<0.05). The relative content of ROS in the three groups was [(100±7)%], [(1538±91)%], and [(818±63)%]; the difference was statistically significant (p<0.05). In Western blotting, Nrf2 content in the experimental group was higher than that in the control group. CONCLUSION Nrf2 exists in the nucleus pulpous cells of human intervertebral discs, which is related to the degeneration of the intervertebral disc. It has negative feedback regulation and can prevent the degeneration of the intervertebral disc by inhibiting the apoptosis of nucleus pulpous cells of human intervertebral discs caused by excessive ROS, which provides a new intervention strategy for the prevention and treatment of the degeneration of intervertebral discs.

RESUMO OBJETIVO O fator 2 relacionado a NF-E2 (Nrf2)/elemento de resposta antioxidante (ARE) é uma nova via defensiva envolvida no estresse oxidativo e químico das células. O objetivo deste estudo foi explorar o papel do Nrf2 na apoptose das células do núcleo pulposo do disco humano induzida pelo peróxido de hidrogênio (H2O2). MÉTODOS O modelo de degeneração das células do núcleo pulposo do disco intervertebral humano foi estabelecido. A expressão do Nrf2 foi interferida utilizando-se sulforafano (SFN). Para isso foram estabelecidos três grupos: um grupo vazio (H2O2-/SFN-), um grupo de controle (H2O2+/SFN-), e um grupo experimental (H2O2+/SFN+). Utilizando CCK8 e Hoechst 33258, o conteúdo de espécies reativas de oxigênio (ERO) foi detectado. RESULTADOS As taxas de apoptose dos três grupos foram [(0,40 ± 0,46)%], [(25,98 ± 11,28%)] e [(3,83 ± 2,06)%], respectivamente. A diferença apresentou significância estatística (p < 0,05). O conteúdo relativo de ERO nos três grupos foi [(100±7)%], [(1538±91%)], e [(818±63%); a diferença foi estatisticamente significativa (p < 0,05). O método de Western blotting indicou um maior conteúdo de Nrf2 no grupo experimental do que no grupo de controle. CONCLUSÃO O Nrf2 existe em células do núcleo pulposo do disco intervertebral humano, que estão relacionadas à degeneração do disco intervertebral. Ele apresenta regulação por feedback negativo e pode evitar a degeneração do disco intervertebral inibindo a apoptose de células do núcleo pulposo do disco causada por excesso de ERO. Essa informação proporciona uma nova estratégia de intervenção para a prevenção e o tratamento da degeneração do disco intervertebral.

Aloin promotes osteogenesis of bone-marrow-derived mesenchymal stem cells via the ERK1/2-dependent Runx2 signaling pathway.

Osteoporosis is characterized by low bone mass and the degeneration of bone structure, conditions which increase the risk of fracture. Aloin has been shown to affect bone metabolism, but its role in osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) remains unclear. The aim of our study was to determine whether aloin promotes the proliferation and osteogenic differentiation of BMSCs and, if so, whether it acts via activation of the ERK1/2-Runx2 signaling pathway. We found that the different concentrations of aloin tested had no obvious cytotoxic effects on the viability of BMSCs. Under osteogenic induction conditions, aloin increased cellular alkaline phosphatase activity, promoted BMSC mineralization, and increased osteogenic-related gene expression. In addition, treating the BMSCs with the signal transduction inhibitor PD98059 (ERK1/2) effectively attenuated Runx2 activation in these cells and also suppressed osteoblastic differentiation. Overall, our study demonstrates that aloin promotes osteogenic differentiation of BMSCs through activation of the ERK1/2-Runx2 signaling pathway.

Differentiation Model Establishment and Differentiation-Related Protein Screening in Primary Cultured Human Sebocytes.

Sebocyte differentiation is a continuous process, but its potential molecular mechanism remains unclear. We aimed to establish a novel sebocyte differentiation model using human primary sebocytes and to identify the expression profiles of differentiation-associated proteins. Primary human sebocytes were cultured on Sebomed medium supplemented with 2% serum for 7 days. Flow cytometry showed that S phase cells were decreased time-dependently, while G1 and subG1 (apoptosis) phase cells increased under serum starvation. Transmission electron microscopy and Oil Red O staining revealed a gradual increase of intracellular lipid accumulation. Expression of proliferation marker was diminished, while expression of differentiation, apoptosis, and lipogenic markers elevated gradually during 7-day culture. iTRAQ analysis identified 3582 expressed proteins in this differentiation model. Compared with day 0, number of differentially expressed proteins was 132, 54, 321, and 96 at days 1, 3, 5, and 7, respectively. Two overexpressed proteins (S100 calcium binding protein P and ferredoxin reductase) and 2 downexpressed proteins (adenosine deaminase and keratin 10) were further confirmed by Western blot and immunohistochemistry.

Massive Proteinuria-Induced Injury of Tubular Epithelial Cells in Nephrotic Syndrome is Not Exacerbated by Furosemide.

BACKGROUND/AIMS: Massive proteinuria, a significant sign of nephrotic syndrome (NS), has the potential to injure tubular epithelial cells (TECs). Furosemide is widely used for the treatment of edema, a common manifestation of NS. However, whether furosemide treatment affects massive proteinuria-induced TEC injury in patients with NS is unknown. METHODS: The effect of furosemide on TEC damage was investigated in vitro. In addition, a clinical study was conducted to study whether the short-term treatment of nephrotic edema with furosemide could exacerbate TEC injury. RESULTS: The proliferation of in vitro human kidney-2 (HK-2) cells exposed to massive urinary protein (8 mg/mL) significantly decreased (P<0.05), while the levels of kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin (NGAL) in the supernatants significantly increased (P<0.05). Importantly, furosemide treatment did not further increase the expression of Kim-1 and NGAL in HK-2 cells upregulated by massive proteinuria. For the clinical study, 26 patients with NS, all prescribed the recommended dosage of prednisone (1 mg/kg/day), were randomly assigned to two groups. One group (n=13) received furosemide (60-120 mg/day, intravenously) for 1 week; the remaining participants (control group) did not receive furosemide or any other diuretics. The results showed that the 24-h urine volume in the furosemide-treated group was slightly, but not significantly, higher than that in the control group (P>0.05). In addition, serum levels of BUN, Scr, Cys C, and urinary Kim-1 and NGAL were not significantly different between the two groups (all P>0.05). Twenty-three patients underwent a renal biopsy. Of these, 22 patients exhibited vacuolar degeneration of the TECs; 8 patients showed brush border membrane shedding of the TECs; and 12 patients showed protein casts. However, there were no significant differences between the two groups (all P>0.05). CONCLUSION: In summary, massive proteinuria induced the injury of TECs in patients with NS, and furosemide treatment did not aggravate this injury.

ω3-polyunsaturated fatty acids induce cell death through apoptosis and autophagy in glioblastoma cells: In vitro and in vivo.

Among brain tumors, glioblastoma (GBM) is the most aggressive type and is associated with the lowest patient survival rate. Numerous lines of evidence have established that omega-3-polyunsaturated fatty acids (ω3-PUFAs) have potential for the prevention and therapy of several types of cancers. Docosahexaenoic acid (DHA), an ω3-PUFA, was reported to inhibit growth and induce apoptotic and autophagic cell death in several cancer cell lines; however, its effects on GBM cells are still unknown. in the present study, we examined the cytotoxic effect of DHA on the GBM cell lines, D54MG, U87MG, U251MG and GL261. Treatment of GBM cells with DHA induced PARP cleavage, increased the population of sub-G1 cells, and increased the number of TUNEL-positive cells, which are all indicative of apoptosis. Furthermore, treatment of GBM cells with DHA resulted in a significant increase in autophagic activity, as revealed by increased LC3-II levels, GFP-LC3 puncta, and autophagic flux activation, accompanied by activation of 5'-AMP-activated protein kinase (AMPK) and decreases in phosphorylated Akt (p-AktSer473) levels and mTOR activity. In vivo, endogenous expression of Caenorhabditis elegans ω3-desaturase, which converts ω6-PUFAs to ω3-PUFAs, in fat-1 transgenic mice yielded a significant decrease in tumor volume following subcutaneous injection of mouse glioma cells (GL261), when compared with wild-type mice. TUNEL-positive cell numbers and LC3-II levels were elevated in tumor tissue from the fat-1 transgenic mice compared with tumor tissue from the wild-type mice. In addition, p-Akt levels were decreased and p-AMPK levels were increased in tumor tissue from the fat-1 transgenic mice. These results indicate that ω3-PUFAs induce cell death through apoptosis and autophagy in GBM cells; thus, it may be possible to use ω3-PUFAs as chemopreventive and therapeutic agents for GBM.

Insulin Promotes the Proliferation of Human Umbilical Cord Matrix-Derived Mesenchymal Stem Cells by Activating the Akt-Cyclin D1 Axis.

Background. The functions of insulin in mesenchymal stem cells (MSC) remain poorly understood. Methods. MSC from human umbilical cord matrix (UCM) cultured in serum-free media (SFM) with or without insulin were subjected to various molecular biological analyses to determine their proliferation and growth states, expression levels of Akt-cyclin D1 signaling molecules, and in vitro differentiation capacities. Results. Insulin accelerated the G1-S cell cycle progression of UCM-MSC and significantly stimulated their proliferation and growth in SFM. The pro-proliferative action of insulin was associated with augmented cyclin D1 and phosphorylated Akt expression levels. Akt inactivation remarkably abrogated insulin-induced increases in cyclin D1 expression and cell proliferation, indicating that insulin enhances the proliferation of UCM-MSC via acceleration of the G1-S transition mediated by the Akt-cyclin D1 pathway. Additionally, the UCM-MSC propagated in SFM supplemented with insulin exhibited similar specific surface antigen profiles and differentiation capacities as those generated in conventional media containing fetal bovine serum. Conclusions. These findings suggest that insulin acts solely to promote UCM-MSC proliferation without affecting their immunophenotype and differentiation potentials and thus have important implications for utilizing insulin to expand clinical-grade MSC in vitro.

Toxoplasma gondii induces autophagy and apoptosis in human umbilical cord mesenchymal stem cells via downregulation of Mcl-1.

Autophagy and apoptosis are critical for controlling Toxoplasma gondii (T. gondii) infection. T. gondii infection during pregnancy can damage the fetus and cause birth defects; however, the molecular mechanisms of this process are poorly understood. This study aims to determine the activities of autophagy and apoptosis as well as their regulatory mechanisms during T. gondii infection by using human umbilical cord mesenchymal stem cells (hUC-MSCs) as a model of congenital diseases. LC3B, a hallmark protein of autophagy was incrementally upregulated with the infection duration, whereas p62 was downregulated in T. gondii-infected hUC-MSCs. Concurrent to this result, the invasion of T. gondii into hUC-MSCs increased in a time-dependent manner. The expression levels of Bcl-2 family proteins including Bcl-2, Bcl-xL, Bim, Bax, Bid and Bak were not altered; however, Mcl-1 levels in hUC-MSCs were dramatically decreased upon T. gondii infection. In addition, at 24 h post-infection, cleaved PARP and cleaved caspase-3 protein levels were elevated in hUC-MSCs. Importantly, Mcl-1 overexpression reduced the levels of autophagy- and apoptosis-related proteins in T. gondii-infected hUC-MSCs. Mcl-1 proteins were primarily expressed in the fraction containing mitochondria and strongly interacted with Beclin-1 under normal conditions; however, these interactions were remarkably attenuated by T. gondii infection. These results suggest that mitochondrial Mcl-1 is an essential signaling mediator regulating the activation of autophagy and apoptosis during T. gondii infection.

Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition.

The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

Induction of Angiogenesis by Matrigel Coating of VEGF-Loaded PEG/PCL-Based Hydrogel Scaffolds for hBMSC Transplantation.

hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(ε-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, integrin1ß, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.

Indole-3-carbinol as inhibitors of glucocorticoid-induced apoptosis in osteoblastic cells through blocking ROS-mediated Nrf2 pathway.

Apoptosis of osteoblasts induced by glucocorticoid (GC) has been identified as a main cause of osteoporosis, bone loss and fractures, and the oxidative stress was found as an important contributor. Therefore, natural or synthetic agents with antioxidant activities can antagonize GCs-induced apoptosis in osteoblasts, and thus demonstrate the potential application to reverse osteoporosis. In this study, we showed that, indole-3-carbinol (I3C), a natural product found in broadly consumed plants of the Brassica genus, could block the cytotoxic effects of dexamethasone (Dex), and elucidated the underlying molecular mechanisms. Firstly, we showed that, I3C could effectively suppress Dex-induced cytotoxicity and apoptotic cell death in osteoblastic cells, as evidenced by the decrease in Sub-G1 cell population. Treatment of the cells with Dex resulted in activation of caspase-3/-8/-9 and subsequent cleavage of PARP, which was also effectively blocked by co-incubation of I3C. Moreover, exposure to Dex triggered a rapid onset and time-dependent superoxide overproduction in osteoblastic cells, which was effectively suppressed by addition of I3C. Excess intracellular ROS induced by Dex significantly suppressed the expression levels of Nrf2 and the downstream effectors, HO1 and NQO1, but these changes could be reversed by I3C. Knockdown of Nrf2 using siRNA silencing technique significantly reversed the protective effects of I3C against Dex-induced apoptosis and ROS generation. Taken together, I3C can reverse cytotoxicity of Dex through blocking ROS overproduction and enhancement of Nrf2 expression. This study may provide a safe and good strategy for molecular intervention of GCs-induced osteoporosis by using natural products.

Docosahexaenoic acid-induced apoptosis is mediated by activation of mitogen-activated protein kinases in human cancer cells.

BACKGROUND: The role of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in cancer prevention has been demonstrated; however, the exact molecular mechanisms underlying the anticancer activity of ω3-PUFAs are not fully understood. Here, we investigated the relationship between the anticancer action of a specific ω3-PUFA docosahexaenoic acid (DHA), and the conventional mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38 whose dysregulation has been implicated in human cancers. METHODS: MTT assays were carried out to determine cell viability of cancer cell lines (PA-1, H1299, D54MG and SiHa) from different origins. Apoptosis was confirmed by TUNEL staining, DNA fragmentation analysis and caspase activity assays. Activities of the conventional MAPKs were monitored by their phosphorylation levels using immunoblotting and immunocytochemistry analysis. Reactive oxygen species (ROS) production was measured by flow cytometry and microscopy using fluorescent probes for general ROS and mitochondrial superoxide. RESULTS: DHA treatment decreased cell viability and induced apoptotic cell death in all four studied cell lines. DHA-induced apoptosis was coupled to the activation of the conventional MAPKs, and knockdown of ERK/JNK/p38 by small interfering RNAs reduced the apoptosis induced by DHA, indicating that the pro-apoptotic effect of DHA is mediated by MAPKs activation. Further study revealed that the DHA-induced MAPKs activation and apoptosis was associated with mitochondrial ROS overproduction and malfunction, and that ROS inhibition remarkably reversed these effects of DHA. CONCLUSION: Together, these results indicate that DHA-induced MAPKs activation is dependent on its capacity to provoke mitochondrial ROS generation, and accounts for its cytotoxic effect in human cancer cells.

Omega-3 polyunsaturated fatty acids and cancer.

While the anticancer effect of omega-3 polyunsaturated fatty acids (omega-3 fatty acids), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been the subject of intense study, our understanding regarding the underlying mechanisms of omega-3 fatty acids against cancer is still limited. Recent studies describing the cancer protective effect of EPA and DHA have sparked a renewed interest in using these fatty acids for cancer prevention and treatment. Here, we summarize the significance of omega- 3 fatty acids in the initiation and progression of cancer, and review the complex mechanisms by which EPA and DHA are thought to have anticancer activities during cancer development. It is concluded that omega-3 fatty acids may exert their anticancer actions by influencing multiple targets implicated in various stages of cancer development, including cell proliferation, cell survival, angiogenesis, inflammation, metastasis and epigenetic abnormalities that are crucial to the onset and progression of cancer.