Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells.

Bladder cancer (BC) is the sixth most common cancer in men. Moreover, chemotherapy for BC leads to various side effects. Metformin is known to induce apoptosis in vitro in many types of cancer. Furthermore, it has feasibility as a drug repositioning used for the treatment of cancer. The molecular mechanism of metformin mediating apoptosis in BC is still unclear. In this study, we showed that metformin stimulated the caspase-dependent apoptotic signaling pathway in T24 cells, a human BC cell line. Moreover, the induced apoptosis was partially inhibited by a general caspase inhibitor, z-VAD-fmk, which suggested that metformin-induced apoptosis in T24 cells is partially caspase-independent. Notably, we observed the nuclear translocation of apoptosis-inducing factors (AIFs) in metformin-promoted apoptosis, which is a typical characteristic of the caspase-independent apoptotic pathway. In addition, we found that metformin-mediated apoptosis occurred via degradation of the cellular FADD-like interleukin-1ß-converting enzyme inhibitory protein (c-FLIP) by facilitating ubiquitin/proteasome-mediated c-FLIPL degradation. Furthermore, treatment with the reactive oxygen species scavenger N-acetylcysteine, failed to suppress metformin-induced apoptosis and c-FLIPL protein degradation in metformin-treated T24 cells. In conclusion, these results indicate that metformin-induced apoptosis was mediated through AIF-promoted caspase-independent pathways as well as caspase-dependent pathways in T24 cells. As such, metformin could be used as a possible apoptotic agent for the treatment of BC.

Neferine-induced apoptosis is dependent on the suppression of Bcl-2 expression via downregulation of p65 in renal cancer cells.

Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor. Treatments with neferine dose-dependently downregulated B cell lymphoma-2 (Bcl-2) expression at the transcriptional level determined by reverse transcriptase-polymerase chain reaction. The forced expression of Bcl-2 and p65 attenuated the neferine-mediated apoptosis in Caki-1 cells. In addition, neferine induced apoptosis by downregulating Bcl-2 and p65 expression in the other two kidney cancer cell lines determined by flow cytometry and western blot analysis. Finally, we observed that treatment with neferine induced apoptosis by inhibiting the NF-κB pathway through caspase-mediated cleavage of the p65 protein by western blot analysis. Collectively, this study demonstrated that neferine-induced apoptosis is mediated by the downregulation of Bcl-2 expression via repression of the NF-κB pathway in renal cancer cells.

MiR-1208 Increases the Sensitivity to Cisplatin by Targeting TBCK in Renal Cancer Cells.

MicroRNAs (miRNAs) can be used to target a variety of human malignancies by targeting their oncogenes or tumor suppressor genes. Recent evidence has shown that miRNA-1208 (miR-1208) was rarely expressed in a variety of cancer cells, suggesting the possibility that miR-1208 functions as a tumor suppressor gene. Herein, ectopic expression of miR-1208 induced the accumulation of sub-G1 populations and the cleavage of procaspase-3 and PARP, which could be prevented by pre-treatment with the pan-caspase inhibitor, Z-VAD. In addition, miR-1208 increased the susceptibility to cisplatin and TRAIL in Caki-1 cells. Luciferase reporter assay results showed that miR-1208 negatively regulates TBC1 domain containing kinase (TBCK) expression by binding to the miR-1208 binding sites in the 3'-untranslated region of TBCK. In addition, miR-1208 specifically repressed TBCK expression at the transcriptional level. In contrast, inhibition of endogenous miR-1208 by anti-miRs resulted in an increase in TBCK expression. Downregulation of TBCK induced by TBCK-specific siRNAs increased susceptibility to cisplatin and TRAIL. These findings suggest that miR-1208 acts as a tumor suppressor and targets TBCK directly, thus possessing great potential for use in renal cancer therapy.

Mesenchymal stem cells attenuate adriamycin-induced nephropathy by diminishing oxidative stress and inflammation via downregulation of the NF-kB.

AIM: This study aimed to evaluate the molecular mechanism mitigating progress of chronic nephropathy by mesenchymal stem cells (MSCs). METHODS: Rats were divided into normal control (Normal), adriamycin (ADR)+vehicle (CON), and ADR+MSC (MSC) groups. Nephropathy was induced by ADR (4 mg/kg) and MSCs (2 × 106 ) were injected. Rats were euthanized 1 or 6 weeks after ADR injection. NF-kB, MAPKs, inflammation, oxidative stress, profibrotic molecules, and nephrin expression were evaluated. Electron and light microscopy were used for structural analysis. MSCs were co-cultured with renal tubular epithelial cells or splenocytes to evaluate relation with oxidative stress and inflammatory molecules RESULTS: Adriamycin treatment upregulated inflammation, oxidative stress, and profibrotic molecules; this was mitigated by MSCs. Glomerulosclerosis and interstitial fibrosis were observed in ADR-treated groups, and were more prominent in the CON group than in the MSC group. Fusion of foot processes and loss of slit diaphragms were also more prominent in the CON group than in the MSC group. In vitro, MSCs reduced oxidative stress related molecules, inflammatory cytokines, and NF-kB transcription. MSC- or ADR-induced regulation of NF-kB transcriptional activity was confirmed by a luciferase reporter assay. CONCLUSIONS: Mesenchymal stem cells attenuate ADR-induced nephropathy by diminishing oxidative stress and inflammation via downregulation of NF-kB.

Methylglyoxal-induced apoptosis is dependent on the suppression of c-FLIPL expression via down-regulation of p65 in endothelial cells.

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIPL . In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIPL , and the forced expression of c-FLIPL attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIPL mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIPL down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells.

TLR2 deficiency attenuates skeletal muscle atrophy in mice.

Oxidative stress and inflammation are associated with skeletal muscle atrophy. Because the activation of toll-like receptor (TLR) 2 induces oxidative stress and inflammation, TLR2 may be directly linked to skeletal muscle atrophy. This study examined the role of TLR2 in skeletal muscle atrophy in wild-type (WT) and TLR2 knockout (KO) mice. Immobilization for 2 weeks increased the expression of cytokine genes and the levels of carbonylated proteins and nitrotyrosine in the skeletal muscle, but these increases were lower in the TLR2 KO mice. Muscle weight loss and a reduction in treadmill running times induced by immobilization were also attenuated in TLR2 KO mice. Furthermore, immobilization increased the protein levels of forkhead box O 1/3, atrogin-1 and muscle ring finger 1 in the WT mice, which was attenuated in TLR2 KO mice. In addition, immobilization-associated increases in ubiquitinated protein levels were lower in the TLR2 KO mice. Immobilization increased the phosphorylation of Akt and p70S6K similarly in WT and KO mice. Furthermore, cardiotoxin injection into the skeletal muscle increased the protein levels of atrogin-1, interleukin-6, and nitrotyrosine and increased the levels of ubiquitinated proteins, although these levels were increased to a lesser extent in TLR2 KO mice. These results suggest that TLR2 is involved in skeletal muscle atrophy, and the inhibition of TLR2 offers a potential target for preventing skeletal muscle atrophy.

Age-related NADPH Oxidase (arNOX) Activity Correlated with Cartilage Degradation and Bony Changes in Age-related Osteoarthritis.

The purpose of this study was to investigate the age-related NADPH oxidase (arNOX) activity in patients with age-related knee osteoarthritis (OA). Serum and cartilage arNOX activities were determined using an oxidized ferricytochrome C reduction assay. Full-thickness knee joint cartilages obtained through total knee replacement surgery were graded according to the Outerbridge (OB) classification. Radiographic severity of OA was determined on Knee X-rays according to the Kellgren-Lawrence (K/L) grading system. Cartilage ß-galactosidase, HIF-1α, and GLUT-1 expression levels were evaluated as markers for tissue senescence, hypoxia, and glycolysis. Higher arNOX activities occurred with higher levels of cartilage ß-galactosidase, HIF-1α, and GLUT-1 (P = 0.002). arNOX activity in cartilages with surface defects (OB grade II, III) was higher than in those without the defects (OB grade 0, I) (P = 0.012). Cartilage arNOX activity showed a positive correlation with serum arNOX activity (r = -0.577, P = 0.023). Serum arNOX activity was significantly higher in the OA subgroup with bilateral ROA than in the OA with no or unilateral ROA (2.449 ± 0.81, 2.022 ± 0.251 nM/mL, respectively, P = 0.019). The results of this study demonstrate that OA itself is not a cause to increase arNOX activities, however, arNOX hyperactivity is related to a high degree of cartilage degradation, and a high grade and extent of ROA in age-related OA.

Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

Suppression of TBCK enhances TRAIL-mediated apoptosis by causing the inactivation of the akt signaling pathway in human renal carcinoma Caki-1 cells.

BACKGROUND: TBC1 domain-containing kinase (TBCK) protein functions as a growth suppressor in certain cell types and as a tumor promoter in others. Although TBCK knockdown increases the responsiveness of cancer cells to anticancer drugs, the detailed mechanisms by which TBCK knockdown increases susceptibility to anticancer drugs remain unknown. OBJECTIVE: This study analyzed the role of TBCK in sensitivities to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and doxorubicin in human renal cancer cells. METHODS: Flow cytometry was employed to evaluate the extent of apoptosis. Western blotting, transient transfection, and lentiviral infection techniques were conducted to investigate the impact of TBCK on apoptosis-related protein expression and mitogen-activated protein kinase (MAPK). RESULTS: TBCK knockdown in renal cancer cells inhibits ERK and Akt signaling pathways and increases TRAIL and doxorubicin sensitivity. In TBCK-knockdown Caki-1 cells, ERK and Akt phosphorylation was suppressed compared to control cell lines, and TRAIL and doxorubicin sensitivities were increased in these cells. In addition, the phosphorylation of PDK1 was suppressed in TBCK-suppressed cells, indicating that TBCK may be involved in the PDK1 and Akt signaling pathways. The introduction of dominantly active Akt into TBCK-suppressed cells restored their sensitivity to TRAIL. In addition, TBCK downregulation enhanced TRAIL sensitivity in different renal cancer cell lines. CONCLUSIONS: These data suggest that TBCK could potentially have a crucial function in influencing the effects of anti-cancer drugs including TRAIL by modulating the signaling pathway involving Akt and PDK1 in human renal cancer cells.

CHOP down-regulates cFLIP(L) expression by promoting ubiquitin/proteasome-mediated cFLIP(L) degradation.

The transcription factor CHOP/GADD153 is induced during the unfolded protein response and is related to the induction of ER stress-mediated apoptosis. However, how CHOP is organized between the pro-survival and pro-apoptotic roles of ER stress remains largely undefined. In this study, we identified the apoptosis regulating protein suppressed by CHOP. We found that treatment of Caki cells with CHOP-inducing drugs including withaferin A, thapsigargin, brefeldin A, and silybin led to a strong reduction in cFLIP(L) protein levels together with a concomitant increase in the CHOP protein. Interestingly, Wit A down-regulated cFLIP(L) expression via both suppressing mRNA transcription and increasing cFLIPL protein instability. We also found that forced expression of CHOP dose-dependently led to a decrease of cFLIP(L) protein expression but did not alter cFLIP(L) mRNA levels. Additionally, we observed that siRNA-mediated CHOP silencing recovered the cFLIP(L) expression decreased by CHOP-inducing agents in Caki cells. Finally, we showed that CHOP facilitates ubiquitin/proteasome-mediated cFLIP(L) degradation, leading to down-regulation of cFLIP(L). Finally, cFLIP(L) over-expression reduced cell death induced by treatment with brefeldin A, thapsigargin, and silybin. Taken together, our results provide novel evidence that cFLIP(L) is a CHOP control target and that CHOP-induced down-regulation of cFLIP(L) is due to activation of the ubiquitin/proteasome pathways.

Dapagliflozin induces apoptosis by downregulating cFILPL and increasing cFILPS instability in Caki-1 cells.

Dapagliflozin is a sodium/glucose cotransporter 2 inhibitor used recently to treat patients with type 2 diabetes. A recent study has demonstrated that dapagliflozin induces apoptosis in human renal and breast tumor cells. However, to the best of our knowledge, the molecular mechanism underlying dapagliflozin-mediated apoptosis in Caki-1 human renal carcinoma cells has not been elucidated. The present study demonstrated that the dapagliflozin treatment dose-dependently increased cell death in Caki-1 cells. Dapagliflozin treatment also induced apoptosis as confirmed by FITC-conjugated Annexin V/PI staining. Additionally, treatment with dapagliflozin reduced the expression levels of anti-apoptotic proteins, cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein (cFLIP)L and cFLIPS in Caki-1 cells. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone inhibited dapagliflozin-induced apoptosis, implying that dapagliflozin-induced apoptosis is regulated by a caspase-dependent pathway. By contrast, N-acetylcysteine had no effect on dapagliflozin-induced apoptosis and downregulation of cFLIPL and cFLIPS expression. Furthermore, overexpression of cFLIPL, but not cFLIPS, partially inhibited apoptosis induced by dapagliflozin. cFLIPL and cFLIPS mRNA levels remained constant in Caki-1 cells after treatment with 0, 20, 40, 60, 80 and 100 µM dapagliflozin. Notably, it was confirmed that cFLIPS protein levels were reduced due to the increased cFLIPS instability in dapagliflozin-treated Caki-1 cells. The present study also demonstrated that dapagliflozin had no effect on HK-2 normal human kidney cells. Taken together, the present study revealed that dapagliflozin induced apoptosis via the downregulation of cFLIPL and an increase in cFLIPS instability, suggesting that dapagliflozin may be a feasible drug candidate for the treatment of human renal cancer.

Neferine increases sensitivities to multiple anticancer drugs via downregulation of Bcl-2 expression in renal cancer cells.

BACKGROUND: Neferine is the major alkaloid extracted from a seed embryo of Nelumbo nucifera and shows cytotoxic effects in various human cancer cells. However, no detailed studies have been reported on its antitumor efficacy of a combinational treatment in human renal cancer cells. OBJECTIVE: This study evaluated the antitumor effects of a combination therapy of neferine and various drugs on renal cancer Caki-1 cells. METHODS: Flow cytometry analysis was performed to evaluate the cell cycle analysis and apoptosis, respectively. Western blotting and reverse transcription polymerase chain reaction were performed to analyze the effect of neferine on the expression of apoptosis-related genes in Caki-1 cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry. RESULTS: Treatment with neferine dose-dependently induces apoptosis and Bcl-2 downregulation in Caki-1 cells. In addition, neferine triggers cell cycle arrest at the G2/M phase in Caki-1 cells. The neferine-induced apoptosis was mediated by ROS generation, and neferine-facilitated Bcl-2 downregulation was regulated at the transcriptional level through the suppression of p65 expression, resulting in inactivation of the NF-κB pathway in Caki-1 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), intensely reversed the effects of neferine on apoptosis and Bcl-2 downregulation. We determined that neferine markedly potentiates the antitumor effects of multiple anticancer drugs (cisplatin, silybin, and thapsigargin), and those effects can be reversed by Bcl-2 overexpression or NAC pretreatment in Caki-1 cells. CONCLUSION: These results suggest that neferine can increase chemosensitivities to anticancer drugs via downregulation of Bcl-2 expression through ROS-dependent suppression of the NF-κB signaling pathway in human renal cancer cells.

Optimal supplementation of dexamethasone for clinical purposed expansion of mesenchymal stem cells for bone repair.

BACKGROUND: Although mesenchymal stem cells (MSCs) are generally considered to represent a very promising tool for bone repair, no optimal protocol has yet been developed for the isolation and expansion of these cells for large-scale clinical applications. METHODS: Mesenchymal stem cells were supplemented with four different concentrations of dexamethasone: 0 M (Con), 0.2 × 10(-8) M (D0.2), 1.0 × 10(-8) M (D1.0) and 5.0 × 10(-8) M (D5.0); and analyzed every week for 5 weeks (P1-P5). Cells were analyzed via an alkaline phosphatase assay, DNA quantification, Oil Red stain, and flow cytometry for CD105 and CD90. Additionally, P3 and P5 cells were subcutaneously transplanted into nude mice after seeding in ceramic cubes. RESULTS: Proliferation of the cells was significantly higher in the D0.2 group. Alkaline phosphatase activities remained at low levels in the Con and D0.2 groups, but increased to high levels in the D1.0 and D5.0 groups as time elapsed. CD105 expression at P5 was lower than at P1, P2 and P3. Adipocyte differentiation was highest at P3. At the 8th week, in vivo bone formation was enhanced by the MSCs in a dexamethasone-supplemented culture for 3 or 5 weeks, and D0.2 was also higher than Con. CONCLUSIONS: The supplementation of MSCs under low-level rather than physiological concentrations (2 × 10(-9) M) of dexamethasone facilitates the culture expansion of these cells for osteogenic purposes by enhancing cell proliferation without diverse differentiation, and also promotes bone formation after in vivo transplantation.

Pioglitazone mediates apoptosis in Caki cells via downregulating c-FLIP(L) expression and reducing Bcl-2 protein stability.

Pioglitazone is an anti-diabetic agent used in the treatment of type 2 diabetes, which belongs to the thiazolidinediones (TZDs) group. TZDs target peroxisome proliferator-activated receptor γ (PPARγ), which functions as a transcription factor of the nuclear hormone receptor. Pioglitazone has antitumor effects in several cancer types and could be a tool for drug therapy in various cancer treatments. Nevertheless, the molecular basis for pioglitazone-induced anticancer effects in renal cancer (RC) has not yet been elucidated. Thus, the aim of the present study was to investigate the detailed signaling pathway underlying pioglitazone-induced apoptosis in Caki cells derived from human clear cell renal cell carcinoma. As a result, it was demonstrated by flow cytometry analysis and Annexin V-propidium iodide staining that pioglitazone treatment induced apoptotic cell death in a dose-dependent manner in Caki cells. The protein expression levels of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein (c-FLIP)(L) and Bcl-2, which were determined by western blotting, decreased after pioglitazone treatment in Caki cells. Flow cytometry and western blot analyses demonstrated that pioglitazone-mediated apoptosis was blocked following pretreatment with the pan-caspase inhibitor, z-VAD-fmk, indicating that pioglitazone-induced apoptosis was mediated via a caspase-dependent signaling pathway. However, the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), did not affect pioglitazone-mediated apoptosis and degradation of c-FLIP(L) and Bcl-2 protein. Of note, it was found by western blot analysis that Bcl-2 protein expression was downregulated by the decreased protein stability of Bcl-2 in pioglitazone-treated Caki cells. In conclusion, these findings indicated that pioglitazone-induced apoptosis is regulated through caspase-mediated degradation of FLIP(L) and reduction of Bcl-2 protein stability, suggesting that pioglitazone is a feasible apoptotic agent that could be used in the treatment of human RC.

Lactucin induces apoptosis through reactive oxygen species-mediated BCL-2 and CFLARL downregulation in Caki-1 cells.

BACKGROUND: Lactucin, a naturally occurring active sesquiterpene lactone, is abundantly found in chicory and romaine lettuce. A recent study reported that lactucin could induce apoptosis in leukemia cells. However, its cytotoxicity and potential molecular mechanisms underlying cancer cell death remain unclear. OBJECTIVE: Therefore, in this study, we aimed to investigate the direct effect and underlying mechanism of action of lactucin on renal cancer cells. METHODS: MTT assay and flow cytometry were performed to evaluate the rate of cell proliferation and apoptosis, respectively. Western blotting, reverse transcription polymerase chain reaction, and protein stability analyses were performed to analyze the effect of lactucin on the expression of apoptosis-related proteins such as B-cell lymphoma 2 (BCL-2) and CFLAR (CASP8 and FADD like apoptosis regulator) long isoform (CFLARL) in Caki-1 human renal cancer cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry. RESULTS: Lactucin treatment induced apoptosis in Caki-1 cells in a dose-dependent manner via activation of the caspase pathway. It downregulated BCL-2 and CFLARL expression levels by suppressing BCL-2 transcription and CFLARL protein stability, respectively. Pretreatment with N-acetyl-1-cysteine, a ROS scavenger, attenuated the lactucin-induced apoptosis and restored the BCL-2 and CFLARL expression to basal levels. Lactucin-facilitated BCL-2 downregulation was regulated at the transcriptional level through the inactivation of the NF-κB pathway. CONCLUSIONS: Our study is the first to demonstrate that lactucin-induced apoptosis is mediated by ROS production, which in turn activates the caspase-dependent apoptotic pathway by inhibiting BCL-2 and CFLARL expression in Caki-1 cells.

A novel method for large-scale immuno-SEM using protein G coupled polystyrene beads.

Polystyrene beads with a mean diameter of 0.76 µm were coupled with protein G and then anti-type II collagen IgG or anti-chondroitin-4-sulphate IgG were tagged to protein G. Antibody-tagged beads were applied to articular cartilage and labelled beads were counted in each sample. Antibody-tagged beads labelled significantly higher than IgG isotype control. We propose immuno-SEM using protein G coupled beads as a valuable method for micrometre range observation for specific protein distribution on surfaces of tissues or organs. This will provide information about structure as well as antigenicity on the surface at the same time.

Mesenchymal Stem Cells Decrease Oxidative Stress in the Bowels of Interleukin-10 Knockout Mice.

Background/Aims: Inflammatory bowel disease (IBD) is an autoimmune disease characterized by chronic inflammation mainly in the large intestine. The interleukin-10 knockout (IL-10 KO) mouse is a well-known animal model of IBD that develops spontaneous intestinal inflammation resembling Crohn's disease. Oxidative stress is considered to be the leading cause of cell and tissue damage. Reactive oxygen species (ROS) can cause direct cell injury and/or indirect cell injury by inducing the secretion of cytokines from damaged cells. This study evaluated the effects of mesenchymal stem cell (MSC) on the progression of IBD. Methods: In this study, human bone marrow-derived MSCs were injected into IL-10 KO mice (MSC). Oxidative stress and inflammation levels were evaluated in the large intestine and compared with those in control IL-10 KO mice (CON) and normal wild-type control mice (Wild). Results: The levels of ROS (superoxide and hydrogen peroxidase) and a secondary end-product of lipid peroxidation (malondialdehyde) were considerably higher in the CON, while superoxide dismutase and catalase levels were lower in the MSC. Inflammation-related marker (interferon-γ, tumor necrosis factor-α, IL-4, and CD8) expression and inflammatory histological changes were much less pronounced in MSC than in CON. Conclusions: MSCs affect the redox balance, leading to the suppression of IBD.

Superficial course of the medial plantar nerve: case report.

The medial and lateral plantar nerves are branched from the tibial nerve and move to the tip of the toes. A variation of medial plantar nerve was found on the left side of a 78-year-old Korean male cadaver. The tibial nerve was divided into the lateral and medial plantar nerves beneath the plantar flexor. The medial plantar nerve passed deep to plantar aponeurosis and superficial to the flexor digitorum brevis. It gave off a common plantar digital nerve and then divided into three proper plantar digital nerves near the metatarsal bases. In this article, we report a superficial course of the medial plantar nerve and describe its unique morphology and discuss the clinical significance of this variation.

Upregulation of interleukin-8/CXCL8 in vascular smooth muscle cells from spontaneously hypertensive rats.

Chemokines promote vascular inflammation and play a pathogenic role in the development and maintenance of hypertension. In the present study, the expression of the chemokine interleukin-8/CXCL8 (IL-8/CXCL8) was investigated in cultured vascular smooth muscle cells (VSMC) obtained from the thoracic aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). IL-8/CXCL8 expression in thoracic aorta tissue and VSMC in SHR were significantly higher than in WKY. However, the expression of CXCR1 mRNA in VSMC from WKY was higher than that in VSMC from SHR. Angiotensin II (Ang II) induced a higher level of IL-8/CXCL8 mRNA expression in VSMC from SHR than in VSMC from WKY. The time course of Ang II-induced IL-8/CXCL8 expression in VSMC from SHR correlated with those of Ang II-induced CXCL1 and Ang II type 1 (AT1) receptor expression, and the expression of IL-8/CXCL8 by Ang II was inhibited by the AT1 receptor antagonist losartan. The effect of Ang II on IL-8/CXCL8 expression was not dependent on nuclear factor-kappaB (NF-kappaB) activation, but was mediated by an extracellular signal-regulated kinase (ERK) signaling pathway. Although Ang II directly induced IL-8/CXCL8 expression, expression of Ang II-induced IL-8/CXCL8 decreased in VSMC transfected with heme oxygenase-1. These results suggest that IL-8/CXCL8 plays an important role in the pathogenesis of Ang II-induced hypertension and vascular lesions in SHR.

Metformin-induced apoptosis facilitates degradation of the cellular caspase 8 (FLICE)-like inhibitory protein through a caspase-dependent pathway in human renal cell carcinoma A498 cells.

Renal cell carcinoma (RCC) is one of the most common types of cancer in adults. Previous studies have reported that the survival rate was significantly lower for renal cancer patients with diabetes than for those without diabetes. Metformin is a well-known anti-diabetic agent used for the treatment of type 2 diabetes mellitus (T2DM). It also inhibits cell proliferation and angiogenesis and is known to possess antitumor effects. However, the molecular mechanism for metformin-induced apoptosis in renal cell carcinoma is not understood. In the present study, treatment with metformin induced apoptosis in A498 cells in a dose-dependent manner. It was revealed that degradation of cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) and activation of procaspase-8 were associated with metformin-mediated apoptosis. By contrast, treatment with metformin did not affect the mRNA level of c-FLIPL in A498 cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk, a pan-caspase inhibitor) almost completely blocked metformin-induced apoptosis and degradation of c-FLIPL protein. However, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, did not inhibit metformin-mediated apoptosis in A498 cells. Taken together, the results of the present study demonstrated that metformin-induced apoptosis involved degradation of the c-FLIPL protein and activation of caspase-8 in human renal cell carcinoma A498 cells and suggested that metformin could be potentially used for the treatment of renal cancer.