Viability Of NIH3T3 Cells Research Articles

Insight into the synergistic antibacterial mechanism of bio ZnO nanoparticles synthesized from Sophora japonica flower buds extract

Developing green nanotechnology to produce safe and powerful nano-antimicrobials has become a hot direction for addressing the challenge of antibiotic resistance. In this study, to investigate the enhancement effect of bio-capping agents on the bioactivities of plant-mediated zinc oxide nanoparticles (ZnO NPs), we designed a facile one-step green method to obtain bio-functioned ZnO NPs using flavonoids-rich extract of S. japonica flower buds (ESJ). The characterization results revealed that crystalline ZnO NPs with sizes smaller than 15 nm were obtained and the resultant bio-NP consists of ZnO core capped by ESJ-derived biomolecules. The biomolecules promoted the ZnO NPs to adhere to the bacterial cells and penetrate into cells by trapping bacteria and altering membrane permeability, which enhanced the bioactivities of the green-synthesized bio ZnO NPs and reduced its cytotoxicity. And the bio ZnO NPs showed minimum inhibitory concentrations (MIC) of 0.5 and 0.125 mg/mL toward Staphylococci aureus and Escherichia coli, respectively, and had good ⋅OH and DPPH radical scavenging activities with IC50 of 0.31 and 1.55 mg/mL, respectively. In addition, the bio ZnO NPs could maintain 80% viability of NIH3T3 cells at 2*MIC (1 mg/mL). This study provides a thought for the design of highly effective antibacterial nanomaterials, which may kill bacteria by membrane-disruption action to evade drug-resistant antimicrobial mechanisms under the synergistic actions of plant-derived biomolecules.

PH dependent biocompatible room temperature covalent organic polymers for selective chemotherapeutic drug delivery

Covalent organic polymers (COP) are a special class of two-dimensional and three-dimensional cross-linked polymeric networks with repeating organic units. They are ideally suited for drug delivery applications because of their large surface area, porous nature, and tunable properties. Furan-2,5-dicarbohydrazide and 1,3,5-triformylbenzene were used to synthesize pH-responsive hydrazone-linked furan-based spherical COP(FRT-COP) at room temperature. The formulated FRT-COP were later stabilized using hyaluronic acid (HA) and then encapsulated anticancer drug doxorubicin (DOX), utilizing the porous structure of the synthesized COP. The newly developed FRT-COP have a pore size of 10 nm, and FRT-COP-DOX-HA exhibit drug loading capacity of 34.4 % and an encapsulation efficiency of 52.32 %. The 80 % viability of NIH 3T3 cells and 82.1 % viability of the CT2A cells, even at a higher concentration of 100 μg/mL of the carrier system, proves the outstanding biocompatibility of the carrier system for chemotherapeutic drug delivery administration. The FRT-COP-DOX-HA-treated NIH-3T3 normal cell line exhibits a viability of 80.59 % at a material concentration of 25 μg/mL. Furthermore, CT2A cancer cell lines show a viability of 33.1 % at a material concentration of 25 μg/mL. The in vivo results obtained from the experiments performed on male BALB/c nude mice also unequivocally demonstrated the exceptional suitability of the proposed drug delivery carrier system for antitumor chemotherapeutic drug administration.

Cepharanthine Inhibits Doxorubicin-Induced Cellular Senescence by Activating Autophagy via the mTOR Signaling Pathway.

Doxorubicin (Dox) is a clinical first-line broad-spectrum anticancer agent. A dose-dependent cardiotoxic and myelosuppressive response limits the clinical use of Dox. Recent research indicates that Dox-induced cardiotoxicity is associated with senescent cell accumulation and that antiaging therapy can alleviate aging-related disorders. Cepharanthine (Cep) is commonly used to treat various acute and chronic illnesses, including leukopenia, snakebites, dry mouth, and hair loss. Whether Cep alleviates Dox-induced senescence is unknown. The expression of genes and proteins associated with aging was examined using NIH3T3 cell lines. The experiments were divided into a control group, a Dox group, and a Cep group on different days. NIH3T3 senescent cells were detected by senescence-β-galactosidase (SA-β-Gal) staining, and Western blotting was used to detect the protein levels of p16, p53, AMP-activated protein kinase (AMPK), mammalian target of the rapamycin (mTOR), p62, and Light Chain 3 (LC3). Fluorescence was used to detect the expression of monomeric red fluorescence protein-green fluorescence protein-Light Chain 3 (mRFP-GFP-LC3) and LC3 puncta in NIH3T3 cells. Real-time quantitative reverse transcription polymerase chain reaction (RT‒qPCR) was used to test the expression of senescence-associated secretory phenotypes (SASP: Interleukin 6 (IL-6), Interleukin 1 beta (IL-1β), and Interleukin 8 (IL-8)). Cell Counting Kit-8 (CCK-8) was used to assess NIH3T3 cell viability. Here, we reported that Cep reversed the Dox-induced increase in the proportion of SA-β-Gal-positive cells and the high expression of aging-related proteins (p53, p < 0.05; p16, p < 0.05) and aging-related genes (IL-6, p < 0.05; IL-1β, p < 0.05; IL-8, p < 0.05) on the 3rd day. Mechanistically, Cep reduced the increase in the levels of phospho-mTOR (p < 0.05) on Days 1 and 3 and p62 protein (p < 0.05) caused by Dox on Day 1 and reversed the decline in LC3II/LC3I levels (p < 0.05) caused by Dox on Day 3, which is associated with the regulation of senescence. Additionally, the viability of NIH3T3 cells was significantly increased in the concentration range of 0.5-5 μM Cep (p < 0.05). We first found that Cep could suppress SA-β-Gal activity (p < 0.05) and the development of SASP. Additionally, in Cep-treated cells, Cep could restore autophagy dysfunction and suppress the mTOR signaling pathway. This research provides a new view on the mechanics of aging and autophagy and aids in developing novel antiaging drugs.

Improving the Antibacterial Property of Chitosan Hydrogel Wound Dressing with Licorice Polysaccharide

A series of hydrogels with different ratios of chitosan and licorice polysaccharide (LP) were prepared by crosslinking to different concentrations of genipin (gp). They were characterized by FTIR (Fourier transform infrared spectroscopy), SEM (Scanning electron microscope), swelling ratio, rheological measurements, degradation with time, cytotoxicity, and antibacterial efficacy. Results show that the hydrogels have porous structures. With an increase in LP content, the swelling rate grows in the early stage of immersion in buffer and drops later. The swelling ratio ranged from 986% to 1677%, and stiffness varied from 777 Pa to 1792 Pa. The addition of LP reduced the mechanical strength and delayed gelation and degradation of the hydrogels. However, the most important discovery was that gp increases the viability of NIH 3T3 cells from 94% to 137%, and LP raises the bacteriostatic efficacy from 51% to 78%. Hydrogels synthesized from 1% genipin, 3% chitosan, and 4% licorice polysaccharide showed the best antibacterial and fibroblast proliferation promoting activities. They exhibited moderate swelling and degradation rates over time, while being more suitable to affect healing of chronic wound infections. These results provide a new strategy to improve the antibacterial effectiveness and cyto-compatibility of chitosan hydrogels with water soluble active LPs from Glycyrrhiza that derive from traditional Chinese medicine.

Biocidal and bioresorbable chitosan/triclosan/collagen matrixes

Biomedical application of biomaterials has increased in recent years. Some preferred characteristics of these materials are biocompatibility, biodegradability and antimicrobial effect. We are facing a constant search for antimicrobial materials to be used instead of antibiotics therapy to reduce the possibility of dental surgery contamination. In this study, biocidal, bioresorbable and non-toxic matrixes were synthesized from chitosan, triclosan and collagen. Three experimental groups received different chitosan/collagen combinations, (0.5:1, 0.75:1 and 1:1), all with the same dose of triclosan (0.1%). Antimicrobial effect was measured by the inhibition of S. aureus growth. Moreover, matrixes were placed in a PBS-collagenase solution to measure degradation over time; matrix residues were evaluated at 1, 4, and 7 days. Finally, cell toxicity of each matrix was analyzed on NIH-3T3 fibroblast cells. As a result, inhibition of S. aureus growth was similar in the three established experimental groups of matrixes vs vancomycin antibiotic as control. These data suggest potent antimicrobial effect of chitosan/triclosan/collagen matrixes. Degradation over time showed that 80% of the matrix was degraded after 4 days, thus suggesting that chitosan/triclosan/collagen matrixes are bioresorbable material. On the other hand, viability of NIH-3T3 cells was between 60% to 74% after 24 h and prior to matrix exposition to culture cells. These data indicate light toxicity in the 0.5:1 and 0.75:1 matrix groups and non-toxic effect in the 1:1 matrix group. By taking together these data, we propose the application of chitosan/triclosan/collagen matrixes to prevent bacterial contamination after dental surgery.

PCL/sulfonated keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation.

Heparin-like polymers have a good anticoagulant effect due to some groups similar to heparin. Herein, sulfonated keratin(SK) was prepared by chain transfer radical polymerization of 3-sulfopropyl methacrylate(SPMA) to improve blood coagulation nature of keratin. Poly(ε-caprolactone)(PCL)/SK nanofibrous mats with the ratio of 7 and 3 were then fabricated by electrospinning. In vitro cytotoxicity and blood compatibility tests were performed to assess the biocompatibility. Viability of NIH 3T3 cells on PCL/SK mats was higher than that on the pristine PCL mats, indicating their good cytocompatibility. These sulfonated keratin-containing mats enhanced endothelial cell growth, while inhibited smooth muscle cell proliferation and reduced platelet adhesion in the presence of GSH and GSNO, as a result of NO generation. Furthermore, the biocomposite mats prolonged the activated partial thromboplastin time(APTT) effectively without hemolysis. Taken together, PCL/SK mats are potential for applications in vascular tissue engineering.

Design of biomimetic collagen matrices by reagent-free electron beam induced crosslinking: Structure-property relationships and cellular response

Novel strategies to mimic mammalian extracellular matrix (ECM) in vitro are desirable to study cell behavior, diseases and new agents in drug delivery. Even though collagen represents the major constituent of mammalian ECM, artificial collagen hydrogels with characteristic tissue properties such as network size and stiffness are difficult to design without application of chemicals which might be even cytotoxic. In our study we investigate how high energy electron induced crosslinking can be utilized to precisely tune collagen properties for ECM model systems. Constituting a minimally invasive approach, collagen residues remain intact in the course of high energy electron treatment. Quantification of the 3D pore size of the collagen network as a function of irradiation dose shows an increase in density leading to decreased pore size. Rheological measurements indicate elevated storage and loss moduli correlating with an increase in crosslinking density. In addition, cell tests show well maintained viability of NIH 3T3 cells for irradiated collagen gels indicating excellent cellular acceptance. With this, our investigations demonstrate that electron beam crosslinked collagen matrices have a high potential as precisely tunable ECM-mimetic systems with excellent cytocompatibility.

Comparative Studies on the Anti-Inflammatory and Uvioresistant Activity of Phycocyanin and Phycoerythrin in Nostoc sphaeroides Kutzing in Vitro

Objective:Comparative studies on the anti-inflammatory and uvioresistant activity of phycocyanin and phycoerythrin in Nostoc sphaeroides Kutzing were developed in vitro. Methods:Raw264.7 cells induced by LPS were used to evaluate the anti-inflammatory activity through detecting NO and TNF-α secretion. NIH-3T3 cell damaged by UVB was used to evaluate the uvioresistant activity. Results:Raw264.7 cells induced by LPS secreted NO and TNF-α extremely significantly(P<0.01),and phycocyanin and phycoerythrin extremely significantly reduced the level of NO and TNF-α secretion(P<0.01). Phycoerythrin was more effective than phycocyanin on the ability of NO secretion inhibition. And phycocyanin was more effective than phycoerythrin on the ability of TNF-α secretion inhibition.UVB 4000 mJ/cm2inhibited the viability of NIH-3T3 cells successfully,but phycocyanin and phycoerythrin showed no protection efficacy on UVB induced cells damage.Conclusion:Phycocyanin and phycoerythrin shows significant anti-inflammatory activity,but there is no protection efficacy on UVB induced cells damage.

Manipulation of molecular pathways and senescence hallmarks by natural compounds in fibroblast cells.

Aging contributes to an increased risk of developing a number of neurodegenerative and chronic disorders, predominantly related to oxidative stress (OS) and defects in the antioxidant balance. This study focused on the antisenescence effect of four plant species (Falcaria vulgaris, Ixiolirion tataricum, Ajuga chamaecistus, and Scabiosa flavida) on H2 O2 -induced premature senescence in rat NIH3T3 fibroblasts, which were found to be rich in effective phytochemicals with traditional ethnobotanical backgrounds. Plant materials were collected, identified, and extracted. To determine the viability of NIH3T3 cells, an MTT assay was conducted. The levels of OS markers and the senescence-associated ß-galactosidase (SA-ß-GAL) activity were analyzed by the Elisa reader. The cell cycle pattern was evaluated by flow cytometry. The expression of senescence-related inflammatory cytokines and the molecules involved in aging signaling pathways were investigated using the real-time reverse transcription polymerase chain reaction (RT-PCR). H2 O2 treatment decreased cell viability and increased lipid peroxidation (LPO) and the reactive oxygen species (ROS) in NIH3T3s. However, S. flavida exhibited low cytotoxicity, reduced OS and SA-ß-GAL activities in NIH3T3 cells compared with the H2 O2 -treated group. I. tataricum was the second best plant, although it was more toxic to NIHT3T cells. S. flavida decreased G0/G1 arrest and facilitated the G2/M transition of NIH3T3s, also downregulated the expression of p38, p53, p16, and the related inflammatory mediators. S. flavida potentially modulated senescence-associated hallmarks in fibroblasts exposed to H2 O2 , thus it may inhibit the aging process via controlling the OS. Therefore it is a promising candidate for future antiaging explorations.

Dual functional NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ core–shell nanoparticles for cell temperature sensing and imaging

Lanthanide-doped up-conversion nanoparticles (UCNPs) provide a remote temperature sensing approach to monitoring biological microenvironments. In this research, the UCNPs of NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ with hexagonal (β)-phase were synthesized and applied in cell temperature sensing as well as imaging after surface modification with meso-2, 3-dimercaptosuccinic acid. In the core–shell UCNPs, Yb3+ ions were introduced as energy transfer media between sensitizers of Nd3+ and activators of Er3+ to improve Er3+emission and prevent their quenching behavior due to multiple energy levels of Nd3+. Under the excitations of 808 nm and 980 nm lasers, the NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ nanoparticles exhibited an efficient green band with two emission peaks at 525 nm and 545 nm, respectively, which originated from the transitions of 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 for Er3+ ions. We demonstrate that an occurrence of good logarithmic linearity exists between the intensity ratio of these two emission peaks and the reciprocal of the inside or outside temperature of NIH-3T3 cells. A better thermal stability is proved through temperature-dependent spectra with a heating–cooling cycle. The obtained viability of NIH-3T3 cells is greater than 90% after incubations of about 12 and 24 (h), and they possess a lower cytotoxicity of UCNPs. This work provides a method for monitoring the cell temperature and its living state from multiple dimensions including temperature response, cell images and visual up-conversion fluorescent color.

Poly(carboxybetaine methacrylate)-functionalized magnetic composite particles: A biofriendly support for lipase immobilization

In this work, poly(carboxybetaine methacrylate) as an extremely hydrophilic polymer was modified on superparamagnetic Fe3O4 nanoparticles (pCB-Fe3O4), which were employed to immobilize porcine pancreatic lipase. The properties of immobilized lipase were investigated in comparison with the free enzyme counterpart. Enzymatic stability, reusability, and activity of the immobilized lipase were found significantly superior to that of the free lipase. In particular, at an elevated temperature of 60°C, the immobilized lipase retained 50% of its initial activity after 150min, while the free enzyme displayed only one-third activity of the immobilized enzyme. Besides, the immobilized lipase retained >60% of its initial activity after 7 cycles. Furthermore, the value of Kcat/Km indicated the catalytic efficiency of the immobilized lipase was increased by 50% compared to that of the free one. The pCB-Fe3O4 particles displayed non-cytotoxicity, while the naked Fe3O4 particles caused only 50% viability of NIH 3T3 cells. These results showed that pCB-Fe3O4 composite particles had higher efficiency and improved stability for lipase immobilization, which are more promising for industrial scale up of biocatalytic systems with excellent biocompatibility.

A glucose-sensitive block glycopolymer hydrogel based on dynamic boronic ester bonds for insulin delivery

Hydrogels are good candidates to satisfy many needs for functional and tunable biomaterials. How to precisely control the gel structure and functions is crucial for the construction of sophisticated soft biomaterials comprising the hydrogels, which facilitates the impact of the surrounding environment on a unique biological function occurring. Here, glucose-responsive hydrogels comprised of 3-acrylamidophenyl boronic acid copolymerized with 2-lactobionamidoethyl methacrylate (p(APBA-b-LAMA)) were synthesized, and further evaluated as carriers for insulin delivery. The formation of (p(APBA-b-LAMA)) hydrogel was based on dynamic covalent bond using the association of boronic acid with diols. P(APBA-b-LAMA) hydrogel with the typical porous structure showed a rapid increase in equilibrium of swelling, which was up to 1856% after incubation with aqueous solution. Using insulin as a model protein therapeutic, p(APBA-b-LAMA) hydrogel exhibited high drug loading capability up to 15.6%, and also displayed glucose-dependent insulin release under physiological conditions. Additionally, the viability of NIH3T3 cells was more than 90% after treated with p(APBA-b-LAMA) hydrogel, indicating that the hydrogel had no cytotoxicity. Consequently, the novel p(APBA-b-LAMA) hydrogel has a practical application for diabetes treatment.

Metabolic-induced cytotoxicity of diosbulbin B in CYP3A4-expressing cells

As a candidate antitumor agent, diosbulbin B (DB) can induce serious liver toxicity and other adverse reactions. DB is mainly metabolized by CYP3A4 in vitro and in vivo, but the cytotoxicity and anti-tumor mechanisms of DB have yet to be clarified. This study aimed to determine whether the cytotoxicity and anti-tumor effects of DB are related to the metabolism-induced activation of CYP3A4 in various cell models, including CYP-free NIH3T3 cells, primary rat hepatocytes, HepG2 and L02 cells of high CYP3A4 expression and wild-type. Results showed that DB did not markedly decrease the viability of NIH3T3 cells. DB metabolites, obtained from the metabolism by mouse liver microsomes, did not elicit cytotoxicity on NIH3T3 cells either. By contrast, DB could induce significant cytotoxicity on primary rat hepatocytes. The DB induced cytotoxicity on HepG2 or L02 cells with high CYP3A4 expression were stronger than those on wild-type cells. As a metabolic biomarker, the metabolite conjugate (M31) of DB with GSH was detected in the incubation system. A higher amount of M31 was generated in the transfected HepG2 and L02 cells than in the wild-type cells at different time points. Ketoconazole, however, could restrain DB induced cytotoxicity on primary rat hepatocytes and in CYP3A4 transfected HepG2 and L02 cells. Therefore, the cytotoxicity of DB was closely related to CYP3A4-metabolized reactive DB metabolites.

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals.

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3–24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.

Effect of sunlight transformed by luminophore-containing materials on cell functions in vitro and in vivo.

Effect of sunlight transformed by luminophore-containing materials on cell viability and functional state of the retina was assessed using the photodamage model. Exposure to the luminescent component of light improved viability of NIH 3T3 cells and promoted recovery of electric activity in rabbit retina after photodamage.

Sheets of vertically aligned BaTiO3 nanotubes reduce cell proliferation but not viability of NIH-3T3 cells.

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants.

Activation of telomerase and cyclooxygenase‐2 in PDGF and FGF inhibition of C2‐ceramide‐induced apoptosis

In the present study, the roles of telomerase and prostaglandin E(2) (PGE(2)) in platelet-derived growth factor (PDGF's) and fibroblast growth factor-2 (FGF-2's) effects against C(2)-ceramide-induced cell death were investigated. C(2)-ceramide reduced the viability of NIH3T3 cells in a condition without calf serum (CS) in accordance with decreasing telomerase activity according to the TRAP assay. The addition of CS significantly protected cells from C(2)-ceramide-induced apoptosis through increased telomerase activity, and the phosphorylations of PDGF and the FGF-2-like receptor in NIH3T3 cells were detected. Adding PDGF and FGF-2 decreased the cytotoxic effect elicited by C(2)-ceramide through stimulating telomerase activity, which was blocked by adding a telomerase inhibitor (TI). Activations of ERKs and JNKs were detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities induced by PDGF and FGF were respectively inhibited by the addition of the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125. Accordingly, induction of cyclooxygenase-2 (COX-2) protein expression and PGE(2) production was detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities stimulated by PDGF and FGF were reduced by adding a specific COX-2 inhibitor, NS398, through a decrease in PGE(2) production. Incubation of cells with PGE(2) or the EP1 agonist, 17-PT, but not the EP2 agonist, sulprostone, the EP3 agonist, butaprost, or the EP4 agonist, PGE(1) alcohol, significantly enhanced the telomerase activity of NIH3T3 cells. PGE(2) protection of NIH3T3 cells against C(2)-ceramide-induced cell death was identified by the MTT and LDH-release assays, and it was inhibited by adding the EP1 antagonist, SC-19220. Ceramide metabolites including ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), and a standard control of exogenous ceramide C(2)-dihydroceramide show no effect on the telomerase activity and viability of NIH3T3 cells. The involvement of COX-2/PGE(2)-mediated telomerase activation by PDGF and FGF-2 against C(2)-ceramide-induced cell death is first demonstrated herein.

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As + 3 - and MMA + 3 -induced apoptosis through inhibition of telomerase activity via JNK activation

The effects of six arsenic compounds including As + 3 , MMA + 3 , DMA + 3 , As + 5 , MMA + 5 , and DMA + 5 on the viability of NIH3T3 cells were examined. As + 3 and MMA + 3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As + 3 and MMA + 3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As + 3 and MMA + 3 treatments. An increase in the intracellular peroxide level was examined in As + 3 - and MMA + 3 -treated NIH3T3 cells, and As + 3 - and MMA + 3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As + 3 - and MMA + 3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As + 3 - and MMA + 3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As + 3 - and MMA + 3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As + 3 or MMA + 3 . These data provide the first evidence to indicate that apoptosis induced by As + 3 and MMA + 3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

Doxazosin induces apoptosis in cardiomyocytes cultured in vitro by a mechanism that is independent of alpha1-adrenergic blockade.

The alpha1-adrenoceptor-blocking antihypertensive doxazosin has been associated with increased risk of heart failure and is known to induce prostate cell apoptosis. We hypothesized that it might also induce apoptosis in cardiomyocytes. Hoechst dye vital staining and flow cytometry provided evidence that doxazosin induced apoptosis time- and dose-dependently in cardiomyocytes of the HL-1 cell line. TUNEL assays and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability test confirmed that doxazosin induced DNA damage and cell death in these cells. MTT tests showed that doxazosin treatment decreased cell viability in primary cultures of neonatal rat cardiomyocytes, and Hoechst dye vital staining demonstrated doxazosin-induced apoptosis in primary cultures of human adult cardiomyocytes. The proapoptotic effect of doxazosin on cardiomyocytes seems not to depend on alpha1 blockade, because it was not modified by cotreatment with alpha- or beta-adrenergic agonists or with the irreversible alpha1-blocker phenoxybenzamine and because doxazosin also decreased the viability of NIH 3T3 cells, which lack alpha1-adrenoceptors. It also does not involve calcineurin, being unaffected by the presence of the calcineurin inhibitors cyclosporin A and FK506. Three other alpha1-blockers were also investigated; prazosin was proapoptotic, like doxazosin, but 5-methylurapidil and terazosin were not. The alpha1-blockers doxazosin and prazosin induce the apoptosis of cardiomyocytes cultured in vitro by a mechanism that is independent of alpha1 blockade and calcineurin.

R-Ras is regulated by activators and effectors distinct from those that control Ras function.

Like Ras, constitutively activated mutants of the Ras-related protein R-Ras cause tumorigenic transformation of NIH3T3 cells. However, since R-Ras causes a transformed phenotype distinct from that induced by Ras, it is likely that R-Ras controls signaling pathways and cellular processes distinct from those regulated by Ras. To address this possibility, we determined if R-Ras is regulated by activators and effectors distinct from those that regulate Ras function. We observed that Ras guanine nucleotide exchange factors failed to activate R-Ras in vivo, indicating that R-Ras is activated by distinct GEFs. Consistent with this, mutants of R-Ras with mutations analogous to the Ras(15A)/(17N) dominant negative proteins did not antagonize Ras GEF function and lacked the growth inhibitory activity seen with these mutant Ras proteins. Thus, R-Ras, but not Ras, is dispensable for the viability of NIH3T3 cells. Finally, whereas constitutively activated Ras can overcome the growth inhibitory action of the Ras(17N) dominant negative protein via Raf-dependent and -independent activities, transforming mutants of R-Ras failed to do so. This inability was consistent with our observation that Ras-, but not R-Ras-transformed, NIH3T3 cells possessed constitutively upregulated Raf kinase activities. Thus, R-Ras and Ras are regulators of distinct signaling pathways and cellular processes.