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.

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.

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.

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.

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.

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.

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.

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.

Effect of water glass coating of tricalcium phosphate granules on in vivo bone formation.

BACKGROUND: Recently, some authors introduced a water glass (WG, sodium-silicate glass; Na2O·SiO2·nH2O) coating over tricalcium phosphate (TCP) bioceramic to modulate its resorption rate and enhance the bone cell behaviors. In this study, four different types of granular samples were prepared to evaluate the ability of new bone formation in vivo using micro-computed tomography and histology. METHODS: Four types sample groups: group A (pure HA as a negative resorption control); group B (pure TCP as a positive resorption control); group C (WG-coated TCP as an early resorption model); and group D (same as group C but heat-treated at 500°C as a delayed resorption model). Cylindrical tube-type carriers with holes were fabricated with HA by extrusion and sintering. Each carrier was filled densely with each granular sample. Four types of tubes were implanted into the medial femoral condyle and medial tibial condyle of New Zealand White rabbits. RESULTS: The HA group (A) showed the lowest amount of new bone formation. All the TCP sample groups (B, C, and D) showed more new bone formation. On the other hand, among the TCP groups, group C (early resorption model) showed slightly more bone formation. The amount of residual bioceramics was most abundant in the HA group (A). All the TCP sample groups showed less residual bioceramics than group A. Among the TCP groups, group C showed slightly more residual bioceramics. Group B showed the lowest amount of residual bioceramics. CONCLUSIONS: The WG-coated TCP sample (group C) is the best bone substitute candidate because of its proper biodegradation rate and the Si ions release because the WG-coated layer reduces the material resorption and enhances the new bone formation. That is, the WG-coated TCP is believed to be the best material for the application of an artificial bone substitute material.

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.

Effect of water glass coating of tricalcium phosphate on in vitro cellular proliferation and osteogenic differentiation.

Background In this study, the properties of the water glass (WG, sodium-silicate glass) were utilized to control the biodegradability of the beta tricalcium phosphate materials by the WG coating on the tricalcium phosphate disc surface with various coating thickness, chemistry, and heat-treatment. Methods Four types of disc specimens were prepared. A sample group A consisted of pure hydroxyapatite (HA) as a negative resorption control; a sample group B consisted of pure beta tricalcium phosphate as a positive resorption control; a sample group C consisted of beta tricalcium phosphate coated with WG as an early resorption model; and a sample group D consisted beta tricalcium phosphate coated with WG and heat-treated at 500°C as a delayed resorption model. Using human bone marrow-derived mesenchymal stem cells, for the analysis of cellular attachment and proliferative activity, 4-6-Diamidino-2-Phenylindole fluorescence technique was used. For the analysis of osteteogenic differentiation, alkaline phospastase (ALP) activity was measured. Results The mean z-scores of four groups (A, B, C, and D) in cellular attachment at 4 h after seeding were -1.21, -0.15, 0.42, and 0.94, respectively, and statistically significantly different in all groups respectively. Seven days after seeding, the mean z-scores of cellular proliferation were 1.97, 0.71, 1.48, and 1.83 in the four groups, respectively. The mean z-scores of the ALP activity per the mean z-scores of cell numbers of respective groups on the seventh day were 0.40, -1.51, 0.12, and 0.06, respectively, in four groups. Conclusion Initial cellular attachment is better on beta tricalcium phosphate than on HA and is enhanced by WG coating, especially with sintering at the high temperature. Cellular proliferation is considered to be increased by maintaining its attachment site through reduced dissolution of beta tricalcium phosphate by WG coating. Osteogenic differentiation in in-vitro study on the WG-coated beta tricalcium phosphate is thought to be as the result of increased silicon ion release from the WG.

Clusterin deficiency induces lipid accumulation and tissue damage in kidney.

Clusterin is a secretory glycoprotein that is involved in multiple physiopathological processes, including lipid metabolism. Previous studies have shown that clusterin prevents hepatic lipid accumulation via suppression of sterol regulatory element-binding protein (SREBP) 1. In this study, we examined the role of clusterin in renal lipid accumulation in clusterin-knockout mice and NRK52e tubular epithelial cells. Clusterin deficiency increased the expression of SREBP1 and its target genes and decreased malonyl-CoA decarboxylase protein levels in the kidney. Expression of the endocytic receptor, megalin, and scavenger receptor class A was increased in clusterin-deficient mice. Functional analysis of lipid metabolism also revealed that lipid uptake and triglyceride synthesis were increased and fatty acid oxidation was reduced, leading to increased lipid accumulation in clusterin-deficient mice. These phenomena were accompanied by mesangial expansion, fibrosis and increased urinary protein-to-creatinine ratio. High-fat feeding aggravated these clusterin deficiency-induced pathological changes. Clusterin knockdown in NRK52e cells increased lipogenic gene expression and lipid levels, whereas overexpression of clusterin by treatment with adenovirus or recombinant clusterin protein suppressed lipogenic gene expression and lipid levels. Transforming growth factor-beta 1 (TGFB1) expression increased in the kidney of clusterin-deficient mice and suppression of TGFB1 in NRK52e cells suppressed lipid accumulation. These results suggest that clusterin deficiency induces renal lipid accumulation by dysregulating the expression of lipid metabolism-related factors and TGFB1, thereby leading to chronic kidney disease. Hence, clusterin may serve as a therapeutic target for lipid-induced chronic kidney disease.

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.

miR-148a increases the sensitivity to cisplatin by targeting Rab14 in renal cancer cells.

MicroRNA (miR) can exert various biological functions by targeting oncogenes or tumor suppressor genes in numerous human malignancies. Recent evidence has shown that miR-148a increases the drug sensitivity of various cancer cells. Herein, we show that ectopic expression of miR-148a induces apoptosis, reduces clonogenicity, and increases the sensitivity to TRAIL and cisplatin in renal cancer cells. The luciferase reporter assay showed that miR-148a negatively regulated ras-related protein 14 (Rab14) expression by binding to the miR-148a binding site in the 3' untranslated region (3'UTR) of Rab14. Rab14-specific siRNA-induced downregulation of Rab14 increases the sensitivity to cisplatin, while forced expression of Rab14 lacking 3'-UTR abrogated the pro-apoptotic function of miR-148a in renal cancer cells. These findings suggest that miR-148a acts as a tumor suppressor and holds great potential for renal cancer therapy by directly targeting Rab14.

Response of human bone marrow-derived MSCs on triphasic Ca-P substrate with various HA/TCP ratio.

Calcium phosphates (Ca-P) are used commonly as artificial bone substitutes to control the biodegradation rate of an implant in the body fluid. This study examined the in vitro proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) on triphasic Ca-P samples. For this aspect, hydroxyapatite (HA), dicalcium phosphate dehydrate (DCPD), and calcium hydroxide (Ca(OH)2 ) were mixed at various ratios, cold compacted, and sintered at 1250°C in air. X-ray diffraction showed that the ß-tricalcium phosphate (TCP) to α-TCP phase transformation increased with increasing DCPD/HA ratio. The micro-hardness deceased with increasing TCP content, whereas the mean grain size and porosity increased with increasing TCP concentration. To evaluate the in vitro degree of adhesion and proliferation on the HA/TCP samples, human BMSCs were incubated on the HA/TCP samples and analyzed by a cells proliferation assay, expression of the extracellular matrix (ECM) genes, such as α-smooth muscle actin (α-SMA) and fibronectin (FN), and FITC-phalloidin fluorescent staining. In terms of the interactions of human BMSCs with the triphasic Ca-P samples, H50T50 (Ca/P = 1.59) markedly enhanced cell spreading, proliferation, FN, and α-SMA compared with H100T0 (Ca/P = 1.67). Interestingly, these results show that among the five HA/TCP samples, H50T50 is the optimal Ca-P composition for in vitro cell proliferation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 72-80, 2017.

Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation.

Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of ß- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs.

Inhibition of c-FLIPL expression by miRNA-708 increases the sensitivity of renal cancer cells to anti-cancer drugs.

Dysregulation of the anti-apoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been associated with tumorigenesis and chemoresistance in various human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anti-cancer drugs. However, whether miRNAs can suppress c-FLIPL expression in cancer cells is unclear. The aim of this study was to identify miRNAs that could inhibit the growth of renal cancer cells and induce cell death by inhibiting c-FLIPL expression. We found that MiRNA-708 and c-FLIPL expression were inversely correlated. While c-FLIPL expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to the miRNA-708 binding site in the 3' untranslated region (3'UTR) of c-FLIPL. Ectopic expression of miRNA-708 increased the accumulation of sub-G1 populations and cleavage of procaspase-3 and PARP, which could be prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Ectopic expression of miRNA-708 also increased the sensitivity to various apoptotic stimuli such as tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin (Dox), and thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIPL without any change in c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIPL protein expression. The expression of c-FLIPL was upregulated in renal cell carcinoma (RCC) tissues compared to normal tissues. In contrast, miRNA-708 expression was reduced in RCC tissues. Finally, miRNA-708 enhanced the tumor-suppressive effect of Dox in a xenograft model of human RCC. In conclusion, miRNA-708 acts as a tumor suppressor because it negatively regulates the anti-apoptotic protein c-FLIPL and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.