Mitochondrial energy metabolism in baby hamster kidney (BHK-21/C13) cells treated with karnozin extra® / Metabolismo energético mitocondrial en células de riñón de hámster bebé (BHK-21 / C13) tratadas con karnozin extra®

SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.

Ultrasmall CuS@BSA nanoparticles with mild photothermal conversion synergistically induce MSCs-differentiated fibroblast and improve skin regeneration.

Mesenchymal stem cell (MSC)-based therapies have been used in skin regeneration due to their ability to differentiate into many cells, promote cytokine secretion and participate in collagen deposition. In this study, we concluded that a CuS@BSA nanoparticles exhibited similar potential in inducing MSCs differentiation to fibroblasts as Cu ions for wound healing. Methods: First, we verified the photothermal efficiency of CuS@BSA in vivo and vitro and had no cytotoxicity for MSCs when the temperature was controlled at 42 °C by adjusting the power of irradiation at 980 nm. And then we detected the expression of vimentin in MSCs, which further directed the MSCs to fibroblasts through Western blotting and Immunofluorescence when treated with CuS@BSA or pre-heat at 42 °C. In addition, we implanted MSCs into the Matrigel or electrospun PLA nanofiber membrane in vitro to evaluating the effect of heating or CuS@BSA on the morphological change of MSCs by SEM. Finally, we evaluated improving skin regeneration by the combination of preheated-MSCs and CuS@BSA nanoparticles that were encapsulated in Matrigel. Results: The CuS@BSA nanoparticles have good photothermal conversion efficiency. Not only CuS nanoparticles itself or after irradiation at 980 nm stimulated the expressioin of vimentin in MSCs. Besides, the CuS@BSA can promote cell proliferation as Cu ion through the expression of ERK. The combination of the CuS@BSA nanoparticles and thermal treatment synergistically improved the closure of an injured wound in an injured wound model. Conclusions: MSCs combined with CuS@BSA are a promising wound dressing for the reconstruction of full-thickness skin injuries.

Rikkunshito attenuates induction of epithelial-mesenchymal switch via activation of Sirtuin1 in ovarian cancer cells.

Rikkunshito, a traditional Japanese herbal medicine, improves appetite via activation of gastrointestinal hormone ghrelin pathway. The function of ghrelin is mediated by growth hormone secretagogue receptor (GHSR1a), and ghrelin has been known to possess diverse physiological functions including growth suppression of some cancer cells. Considering that increased ghrelin signaling by Rikkunshito could enhance sirtuin1 (SIRT1) activity in nervous system, we aimed to investigate the effect of Rikkunshito in ovarian cancer cells. Ovarian cancer cell lines were treated with Rikkunshito, and cellular viability, gene expressions and epithelial-mesenchymal transition (EMT) status were investigated. To investigate the involvement of SIRT1 by Rikkunshito in SKOV3 cancer cells, endogenous expression of SIRT1 was depleted using small interfering RNA (siRNA). Treatment with Rikkunshito elevated ghrelin, GHSR1a and SIRT1, while cellular viability was decreased. The treatment of Rikkunshito also inhibited cellular migration and invasion status in a dose-dependent manner, and these effects were translated to the enhanced EMT status, although the role of SIRT1 was not determined. Our study revealed a novel function of Rikkunshito in enhancing EMT status of ovarian cancer cells. Therefore, we would like to propose that Rikkunshito may be used as a novel adjunctive therapy in chemotherapy of ovarian cancer because platinum-based chemotherapy frequently used for the treatment of ovarian cancer inevitably impairs appetite.

Anti-Tumor Effects of Paeoniflorin on Epithelial-To-Mesenchymal Transition in Human Colorectal Cancer Cells.

BACKGROUND Colorectal cancer is one of the leading causes of death in China, and the development of effective drugs is urgently needed. Here, we report on Paeoniflorin (PF), a product isolated from the roots of the peony plant, as a possible candidate because of its anti-tumor effects on epithelial-to-mesenchymal transition (EMT) of PF in human colorectal cancer (CRC). MATERIAL AND METHODS Cell proliferation, wound healing, and Transwell assays were used to analyze the effects of PF on in vitro cell migration and invasion of HCT116 and SW480, 2 colorectal cancer cell lines. The tumor xenograft model was used to verify the anti-metastasis effects of PF in vivo. The RNA and protein levels of epithelia-cadherin (E-cadherin), Vimentin, and histone deacetylase2 (HDAC2) were measured by qPCR and Western blot analysis to explore the mechanism involved. RESULTS Our results showed that PF inhibited colorectal cancer cell migration and invasion and suppressed the metastatic potential of the cancer cells in vivo. Moreover, PF significantly decreased the expression of HDAC2 and Vimentin, while increasing the expression of E-cadherin. CONCLUSIONS These results suggest that PF inhibits colorectal cancer cell migration and invasion ability and reverses the EMT process, through inhibiting the expression of HDAC2, and then affects the expression level of E-cadherin and Vimentin at the cell level. Our results were also verified in the tumor xenograft model. This indicates that PF may be a candidate for colorectal cancer treatment.

The effects of di(2-ethylhexyl)phthalate exposure and selenium nutrition on sertoli cell vimentin structure and germ-cell apoptosis in rat testis.

This study aimed to investigate the effects of di(2-ethylhexyl)phthalate (DEHP) on Sertoli-cell vimentin filaments and germ-cell apoptosis in testes of pubertal rats at different selenium (Se) status. Se deficiency was produced in 3-weeks old Sprague-Dawley rats by feeding them ≤ 0.05 Se mg/kg diet for 5 weeks, Se supplementation group was on 1 mg Se/kg diet, and DEHP was applied at 1000 mg/kg dose by gavage during the last 10 days of the feeding period. The diet with excess Se did not cause any appreciable alteration in vimentin staining and apoptosis of germ cells, but Se deficiency caused a mild decrease in the intensity of vimentin immunoreactivity and enhanced germ-cell apoptosis significantly (approximately 3-fold, p <0.0033). DEHP exposure caused disruption and collapse of vimentin filaments and significantly induced apoptotic death of germ cells (approximately 8-fold, p <0.0033). In DEHP-exposed Se-deficient animals, compared with the control, collapse of vimentin filaments was more prominent; there was serious damage to the seminiferous epithelium; and a high increment (approximately 25-fold, p <0.0033) in apoptotic germ cells was observed. Thus, Se deficiency exacerbated the toxicity of DEHP on Sertoli cells and spermatogenesis, whereas Se supplementation provided protection. These results put forward the critical role of Se in the modulation of redox status of testicular cells and emphasize the importance of Se status for reproductive health.

A proteomic investigation of similarities between conventional and herbal antidepressant treatments.

Increasing clinical evidence for the effectiveness of herbal antidepressants has led to investigations at the molecular level. Using two-dimensional gel electrophoresis, this study investigated similarities in protein expression between clomipramine, St John's wort and a Chinese herbal formula, xiao-yao-san, often used in mood disorder treatment. HT22 cells, derived from a mouse hippocampal cell line, were treated for 24 h, and protein expression was compared with that of the untreated cells (n = 4/group). Forty-three protein spots were found to be significantly differentially expressed (P < 0.05) in more than one of the treatment groups. Twenty-nine of these were identified using mass spectrometry. The most affected proteins were those involved in the cytoskeleton and energy metabolism, and an up-regulation of vimentin by all three treatments was confirmed by Western blotting. This study provides preliminary evidence for multiple common molecular targets between conventional and alternative antidepressants, which appear to collectively affect neuronal plasticity.

Effects of base-metal casting alloys on cytoskeletal filaments in cultured human fibroblasts.

PURPOSE: The present study was designed to determine the cytotoxic effects of some widely used dental base-metal casting alloys (Ni-Cr and Co-Cr) on the cytoskeleton in cultured human fibroblasts, and to evaluate whether any structural alteration is associated with the application of these alloys. MATERIALS AND METHODS: Ten specimens from six different alloys were prepared as 5-mm disks. Five of ten samples from each group were polished; the remaining five samples were left sandblasted with 50-microm Al2O3. All samples were directly exposed to human fibroblasts in a 24-well cell culture dish for 120 hours. Then, cells were fixed and stained with antibodies against major cytoskeletal elements--actin, vimentin, and microtubules--by immunofluorescent staining methods. Cells were analyzed in 3-D to document the cytoskeletal alterations using a laser confocal microscope. RESULTS: Disintegration of actin filaments was observed in lamellipodia of fibroblasts by the effect of both polished and sandblasted Ni-Cr and Co-Cr samples, with the exception of the polished Co-Cr alloy (Wirocast). Moreover, intracytoplasmic actin-decorated stress fibers were found bent and occasionally tangled in the sandblasted Ni-Cr (Wiron 99) and Co-Cr alloys (Wirocast and Co-Cr Degussa). Vimentin, a mesenchymal cell intermediate filament protein normally showing an intracellular meshwork pattern, was not affected by any of the polished or sandblasted alloys. Microtubules mainly remained intact in all dental alloy-treated groups. CONCLUSION: Taken together, it is possible to postulate that Ni-Cr and Co-Cr dental alloys, especially sandblasted forms, may have detrimental effects on the actin-based cytoskeleton, at least tested in vitro.

Use of growth factors to improve muscle healing after strain injury.

Muscle injuries represent a large number of professional and recreational sports injuries. Muscle strains habitually occur after an eccentric contraction, which often leads to an injury located in the myotendinous junction. Treatment varies widely, depending on the severity of the trauma, but has remained limited mostly to rest, ice, compression, elevation, antiinflammatory drugs, and mobilization. The authors' research group aims to develop new biologic approaches to improve muscle healing after injuries, including muscle strains. To achieve this goal, the authors investigated several parameters that will lead to the development of new strategies to enhance muscle healing. The authors first evaluated natural muscle healing after strain injuries and showed that muscle regeneration occurs in the early phase of healing but becomes impaired with time by the development of tissue fibrosis. Several growth factors capable of improving muscle regeneration were investigated; basic fibroblast growth factor, insulin-like growth factor, and nerve growth factors were identified as substances capable of enhancing muscle regeneration and improving muscle force in the strained injured muscle. The current study should aid in the development of strategies to promote efficient muscle healing and complete recovery after strain injury.

High-dose diltiazem prevents migration and proliferation of vascular smooth muscle cells in various in-vitro models of human coronary restenosis.

BACKGROUND: Restenosis after coronary angioplasty is considered to be caused mainly by increased migration and proliferation of smooth muscle cells (SMC). The concept of local, site-specific delivery of pharmacologic therapies has opened the door for new, high-dose drug regimes. METHODS AND RESULTS: SMC were isolated by enzymatic disaggregation with collagenase/elastase from human coronary plaque tissue of 29 patients (pSMC) and post mortem from the coronary media of 33 corpses (mSMC). Endothelial cells were isolated from human umbilical veins by enzymatic disaggregation with collagenase/dispase. By positive reaction with antibodies against smooth muscle alpha-actin and von Willebrand factor cells were identified as SMC or endothelial cells. In proliferation studies 5-150 micrograms/ml diltiazem was added to the culture media of pSMC, mSMC and endothelial cells. After 5 days there was a significant dose-dependent inhibition of cell proliferation (for pSMC with > 50 micrograms/ml, for mSMC with > 25 micrograms/ml, and for endothelial cells with > 5 micrograms/ml). In migration studies the effect of 5-150 micrograms/ml diltiazem on the velocity of migration of pSMC was investigated over a period of 48 h. Administration of diltiazem at concentrations of 100 and 150 micrograms/ml caused a significant inhibition of the migration of pSMC. The cytoskeletal components smooth muscle alpha-actin, vimentin, and alpha-tubulin of pSMC and the expression of von Willebrand factor of endothelial cells were investigated after an incubation period of 5 days with 50 and 150 micrograms/ml diltiazem. In the transfilter coculture model the effect of 50 micrograms/ml diltiazem on mSMC was investigated after mechanical injury of cocultured endothelial cells. Administration of diltiazem at a concentration of 50 micrograms/ml inhibited the development of a neointimal proliferate in the transfilter coculture model significantly (P < 0.001). CONCLUSIONS: A high dose of diltiazem inhibited the migratory and proliferative activities of coronary SMC significantly. In further experimental studies the effect of locally applied high doses of diltiazem on postangioplasty restenosis should be elucidated.

A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis.

BACKGROUND: Recent advances in the understanding of the biology of restenosis indicate that it is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. The aim of this study was to investigate the in vitro effect of five potential antiproliferative agents on smooth muscle cells from human atherosclerotic femoral arteries. METHODS AND RESULTS: Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. Dalteparin sodium (0.001-100 anti-Xa units/ml), cyclosporine A (0.005-500 micrograms/ml), colchicine (0.00004-4 pg/ml), etoposide (0.002-200 micrograms/ml), and doxorubicin (0.0005-50 micrograms/ml) were added to the cultures. Six days after seeding, cells were trypsinized and cell number was measured by a cell counter. All five agents tested exhibited a significant inhibition of smooth muscle cell proliferation (P < 0.001). After an incubation time of 48 h, the cytoskeletal components, alpha-actin, vimentin, and microtubules were investigated. At peak concentrations, all five tested agents except dalteparin sodium caused severe damage to the cytoskeleton. CONCLUSIONS: All five potential antiproliferative agents exhibited a significant inhibition of smooth muscle cell proliferation. The development of new intravascular delivery systems may open the way for local antiproliferative treatment strategies in interventional cardiology.

Induction of vimentin modification and vimentin-HSP72 association by withangulatin A in 9L rat brain tumor cells.

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 microM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 microM and a completely upshift mobility was observed after 5 h treatment at 50 microM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.