Apoptosis In NIH3T3 Cells Research Articles

Design, synthesis, and evaluation of pirfenidone-NSAIDs conjugates for the treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal, chronic and progressive lung disease that threaten public health like many cancers. In this study, targeting the significant driving factor, inflammatory response, of the IPF, several conjugates of pirfenidone (PFD) with non-steroidal anti-inflammatory drugs (NSAIDs), along with their derivatives, were designed and synthesized to enhance the anti-IPF potency of PFD. Among these compounds, the (S)-ibuprofen-PFD conjugate 5b exhibited the most potent anti-proliferation activity against NIH3T3 cells, demonstrating up to a 343-fold improvement compared to PFD (IC50 = 0.04 mM vs IC50 = 13.72 mM). Notably, 5b exhibited superior activity in inhibiting the migration of macrophages induced by TGF-β compared to PFD. Additionally, 5b demonstrated significant suppression of TGF-β-induced migration of NIH3T3 cells and induction of apoptosis in NIH3T3 cells. Mechanistic studies revealed that 5b reduced the expression of collagen I and α-SMA by inhibiting the TGF-β/SMAD3 pathway. In a bleomycin-induced pulmonary fibrosis model, treatment with 5b (40 mg/kg/day, orally) exhibited a more pronounced effect on reducing the degree of histopathological changes in lung tissue and alleviating collagen deposition compared to PFD (100 mg/kg/day, orally). Moreover, 5b could block the expression of collagen I, α-SMA, fibronectin, and pro-inflammatory factors (IL-6, IFN-γ, and TNF-α) compared to PFD, while demonstrating low toxicity in vivo. These preliminary results indicated that the hybridization of PFD with NSAIDs represented an effective modification approach to improve the anti-IPF potency of PFD. Consequently, 5b emerged as a promising candidate for the further development of new anti-IPF agents.

Naringin, a Natural Flavonoid, Modulates UVB Radiation-Induced DNA Damage and Photoaging by Modulating NER Repair and MMPS Expression in Mouse Embryonic Fibroblast Cells.

We have proven that naringin, a phytonutrient, diminishes oxidative damage and inflammatory responses by modulating PPAR-γ expressions in ultraviolet-B radiation (UVB)-induced NIH-3T3 cells. However, the role of naringin against DNA damage, photoaging, and apoptosis in NIH-3T3 cells has yet to be studied, necessitating investigation. We show that Naringin pretreatment significantly reduces UVB-induced alkaline DNA damage and potentially modulates NER gene (XPC, TFIIH, XPE, ERCC1, and GAPDH) expression, thereby augmenting DNA repair. We determined experimentally that naringin pretreatment prevents UVB-induced nuclear fragmentation in NIH-3T3 cells, as well as altering UVB-induced apoptotic marker (Bax, BCl-2, Caspase-9, and Caspase-3) expression in them. In addition, naringin pretreatment inhibits UVB-stimulated matrix metalloproteinase (MMP-2, MMP-9 and MMP-13) expression in these 3T3 cells. Therefore, we report that naringin can effectively avert UVB-mediated DNA damage, photoaging, and apoptosis in NIH-3T3 cells.

Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell Inhibits Ultraviolet B-induced Apoptosis in Fibroblasts

BACKGROUND: Ultraviolet (UV)B irradiated-skin cells may respond either by activating protective or apoptotic mechanisms. Several treatments has been reported to prevent apoptosis of the skin cell. To date, despite of the cell, secretome of mesenchymal stem cell (MSC) has been progressively explored for various treatment. Current research was conducted to find out the possible effect of MSC's secretome to protect normal cells from the UVB irradiation.METHODS: For preparation of the conditioned media human umbilical cord blood mesenchymal stem cell (CM-hUCB-MSC), hUCB was collected and separated to collect mononuclear cell (MNC). MNC was cultured in MSC medium until passage 4, then checked for MSC biomarkers. Conditioned media was then produced from the cultured MSC. For induction of apoptosis, NIH3T3 cells were pretreated with/without CM-hUCB-MSC, prior to the UVB irradiation for 5-60 minutes. After 24 hours, apoptosis evaluation was conducted with Sub-G1 assay with hypotonic fluorochrome solution using flow cytometer.RESULTS: The average of apoptotic cells irradiated with UVB for 30 minutes was 94.2%. The 30-minutes-UVB irradiation significantly induced apoptosis in NIH3T3 cells (p=0.000). The averages of apoptotic cells by pretreatment of 0, 5, 10 and 20% CM-hUCB-MSC prior to UVB irradiation were 94.2, 46.8, 31.8 and 31.5 %, respectively. The pretreatment of 5, 10 and 20% CM-hUCB-MSC prior to UVB irradiation could significantly decrease the percentage of apoptosis caused by UVB (p=0.001).CONCLUSION: Taken together, CM-hUCB-MSC inhibited UVB-induced apoptosis in NIH3T3 cells significantly, suggesting that CM-hUCB-MSC might be a potential anti-UVB-induced apoptosis factor. Hence, further research should be explored to disclose its specific intracellular mechanism.KEYWORDS: UVB, stem cell, conditioned media, fibroblast, apoptosis, aging, secretome, NIH3T3 cell

Protective properties of Huperzine A through activation Nrf2/ARE-mediated transcriptional response in X-rays radiation-induced NIH3T3 cells.

Huperzine A (HupA), derived from Huperzia Serrata, has exhibited a variety of biological actions, in particular neuroprotective effect. However, the protective activities of HupA on murine embryonic fibroblast NIH3T3 cells after X-rays radiation have not been fully elucidated. Herein, HupA treatment dramatically promoted cell viability, abated a G0/G1 peak accumulation, and ameliorated increase of cell apoptosis in NIH3T3 cells after X-rays radiation. Simultaneously, HupA notably enhanced activities of anti-oxidant enzymes, inhibited activity of lipid peroxide, and efficiently eliminated production of reactive oxygen species in NIH3T3 cells after X-rays radiation. Dose-dependent increase of antioxidant genes by HupA were associated with up-regulated Nrf2 and down-regulated Keap-1 expression, which was confirmed by increasing nuclear accumulation, and inhibiting of degradation of Nrf2. Notably, augmented luciferase activity of ARE may explained Nrf2/ARE-mediated signaling pathways behind HupA protective properties. Moreover, expression of Nrf2 HupA-mediated was significant attenuated by AKT inhibitor (LY294002), p38 MAPK inhibitor (SB202190) and ERK inhibitor (PD98059). Besides, HupA-mediated cell viability, and ROS production were dramatically bated by LY294002, SB202190, and PD98059. Taken together, HupA effectively ameliorated X-rays radiation-induced damage Nrf2-ARE-mediated transcriptional response via activation AKT, p38, and ERK signaling in NIH3T3 cells.

Evaluation of the anticancer effects of Aloe vera and aloe emodin on B16F10 murine melanoma and NIH3T3 mouse embryogenic fibroblast cells

Aloe vera (L.) Burm. f. is well known for its beneficial effects on the skin. Moreover, the antioxidant, immunostimulant, and anticancer effects of the plant leaf extracts have been reported in scientific research. This study was conducted to demonstrate the cytotoxic effects of several leaf extracts and aloe emodin (AE) on a type of skin cancer. A. vera aqueous and methanolic extracts of fresh leaves, methanolic extract of dried leaves, and leaf gel extract (AVG) were prepared separately. Cytotoxicity was assessed using the MTT test. Apoptosis and necrosis were detected by flow cytometry using Annexin V/PI. All the extracts exhibited a selective cytotoxic effect on the cells. The mechanism of AVG cytotoxicity on B16F10 murine melanoma cells was found to be apoptosis, whereas that of AE was necrosis. The observation that treatment with AVG delayed the apoptosis in NIH3T3 cells, while it exerted an apoptotic activity on B16F10 cells, provides some scientific evidence for the folkloric and alternative uses of A. vera gel as a protective and skin healer. Therefore, A. vera gel and aloe emodin can be used as potential targets for anticancer drug research.

Rrp15 affects cell cycle, proliferation, and apoptosis in NIH3T3 cells.

Riken 2810430M08 (hereinafter referred to as Rrp15) is a newly identified and reported gene from the mouse genome. In our previous work, we found that the gene had a relationship with the proliferation and activation of T cells. Rrp15 protein is highly homologous with RRP15 (budding yeast), which has an important role in ribosomal RNA processing. We explored the potential function of Rrp15 in apoptosis, cell proliferation, and its involvement with RNA in the nucleus. We constructed a knockdown of the Rrp15 gene in NIH3T3 cells and then performed real‐time PCR, western blotting, flow cytometry, and immunofluorescence to determine the function of the Rrp15 gene. Knockdown of the Rrp15 gene suppresses proliferation and induces apoptosis. We also found that the Rrp15 protein was normally distributed in the nucleus and bound to RNA or pre‐RNA in the nucleus. Additionally, Rrp15 altered the activity of the 20S proteasome. Rrp15 promotes proliferation and inhibits apoptosis in NIH3T3 cells and may have a relationship with RNA in the nucleus.

Composite wound dressings of pectin and gelatin with aloe vera and curcumin as bioactive agents

Aloe vera and curcumin loaded oxidized pectin–gelatin (OP-Gel) matrices were used as antimicrobial finishes on nonwoven cotton fabrics to produce composite wound care devices. The drug release characteristics of the biocomposite dressings indicated that curcumin is released through a biphasic mechanism – erosion of the polymeric matrix, followed by diffusion, while aloe vera is released upon leaching of the polymeric matrix. A 50/50 composition of aloe vera/curcumin was used to fabricate OP-Gel-Aloe Curcumin dressings. However, contrary to our expectations, OP-Gel-Aloe Curcumin dressings exhibited lesser antimicrobial activity compared to OP-Gel-Aloe and OP-Gel-Curcumin dressings. The cytocompatibility of the fabricated dressings was evaluated using NIH3T3 mouse fibroblast cells. OP-Gel-Aloe treated fibroblasts had the highest viability, with the matrices providing a substrate for good cell attachment and proliferation. On the other hand, OP-Gel-Curcumin and OP-Gel-Aloe Curcumin seemed to have induced apoptosis in NIH3T3 cells. In vivo wound healing analysis was carried out using an excisional splint wound model on C57BL/6J mice. OP-Gel-Aloe treated wounds exhibited very rapid healing with 80% of the wound healing in just 8 days. Furthermore, aloe vera exerted a strong anti-inflammatory effect and prominent scar prevention. Histological examination revealed that an ordered collagen formation and neovascularization could be observed along with migration of nuclei. Therefore, OP-Gel-Aloe biocomposite dressings are proposed as viable materials for effective wound management.

Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation.

Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.

Abstract B13: R-spondin3 overexpression induces transformation and deregulates expression of genes controlling cell proliferation and apoptosis in NIH3T3 cells

Abstract It has been reported that R-spondin3 (Rspo3) is a common insertion site for the viral genome in mouse mammary tumor virus (MMTV) induced-mammary tumors and that HC11 cells over-expressing this protein become tumorigenic when transplanted into mice. In addition, we have found that Rspo-3 over-expression is associated with loss of hormone regulation of initially pregnancy-dependent MMTV-induced mouse mammary tumors. In order to determine whether this gene may behave as an oncogen in culture, even in cells that do not derive from mammary tissue, NIH3T3 cells were stably transfected with mouse Rspo3 full-length cDNA under the control of the cytomegalovirus (CMV) promoter (3T3-Rspo3 cells). We intend this cell line to provide a tool that might allow the study of signaling pathways altered by Rspo-3 expression We found that 3T3-Rspo3 cells showed the capacity to form foci when cultured on top of a lawn of normal NIH3T3 cells attached to plastic dishes and to grow as colonies when seeded in soft-agar. To determine signaling transduction pathways that might be altered by Rspo3 over-expression, we analyzed the level of expression of phosphorylated proteins critical in the regulation of cell growth or death and found that transfected cells showed higher levels of phosphorylated AKT and p38 MAPK, while no significant differences were found when the levels of other phosphorylated MAPKs (as Erk1/2) were analyzed. Besides, crystal violet staining assays revealed that Rspo3 transfected cells were less dependent on serum concentration to grow in culture. Interestingly, survival of 3T3-Rspo3 cells was reduced by treatment with a potent and selective phosphatidylinositol-3 kinase (PI-3K) pharmacological inhibitor, LY # 294002, a well-known regulator of signaling by AKT. In addition, expression of Cyclin D1, a protein associated with cell proliferation, was significantly increased and correlated with Rspo3 overexpression. To determine other genes that might be involved in 3T3-Rspo3 cell survival, we studied the level of expression of Bcl family members. We found that the cells over-expressing Rspo3 showed diminished expression of pro-apoptotic proteins BAD and BAX, comparing to control cells. On the other hand, we observed higher expression of the anti-apoptotic (long) Bcl-X form. Besides, preliminary results indicate that LY # 294002 treatments alter the expression pattern of Bcl family members at the protein level, suggesting that AKT phosphorylation might be involved in regulation of the balance between pro- and anti-apoptotic proteins. In summary, we have developed a tool that can be used to dissect signaling pathways that may be responsible for the transforming effects triggered by over-expression of Rspo3. Our results, added to the reported in vivo studies, provide evidences that Rspo3 can be fully characterized as an oncogene. In addition, we have determined that activation of the PI-3K/AKT pathway might be involved in such an activity. Citation Information: Cancer Res 2009;69(23 Suppl):B13.

The anti-apoptotic activity associated with phosphatidylinositol transfer protein α activates the MAPK and Akt/PKB pathway

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein α (PI-TPα; SPIα cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555–1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIα>wtNIH3T3>SPIβ cells (i.e. wild type cells overexpressing PI-TPβ). Upon incubation of SPIβ cells with the PI-TPα-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-κB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIα cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIβ cells as visualized by confocal laser scanning microscopy.

The Possible Mechanism of Tetrachlorohydroquinone-Induced Apoptosis and Tumor Promotion in NIH3T3 Cells

ISEE-109 Objective: Pentachlorophenol (PCP) and its salt are used extensively as biocide and wood preservative. Because of improper disposal, PCP has become an environmental pollutant and is considered to be ubiquitous. Tetrachlorohydroquinone (TCHQ) is the major toxic metabolite of PCP. Recently, we found that TCHQ was a tumor promoter in a mouse skin carcinogenesis model. However, the detail mechanism of TCHQ-induced tumor promotion is still not clear. In this study, we intended to discover the role of Bcl-2 in TCHQ-induced apoptosis and its related possible signaling pathways in NIH3T3 cells. Material and Methods: DNA fragmentation assay was used to characterize apoptosis in NIH3T3 cells. Flow cytometry analysis was performed to quantify apoptotic cells and intracellular ROS levels. Western blotting analysis was applied to investigate the molecular basis for the effects of TCHQ in NIH3T3 cells. Results: Over-expression of Bcl-2 effectively suppresses TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphologic changes and DNA fragmentation. Although production of reactive oxygen species (ROS) contributes to TCHQ-induced apoptosis, Bcl-2 fails to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, over-expressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. Interestingly, TCHQ induced a significant up-regulation of Bcl-2 expression, and over-expressed Bcl-2 can inhibit TCHQ-induced apoptosis. We also found that TCHQ increased ATM protein expression, but did not induce downstream p53 accumulation and Bax up-regulation. In addition, TCHQ induced expression of both phospho-p38 and phospho-JNK, which belong to MAPK cascade pathway. Conclusions: These data indicate that Bcl-2 prevents TCHQ-induced apoptosis at the downstream of oxidative damage events. The possible signaling pathway of TCHQ-induced apoptosis may relate to MAPK pathway instead of ATM-dependent pathway. Taken together, we suggest that chronic exposure of TCHQ could facilitate the process of tumor promotion through induction of Bcl-2 expression and subsequent apoptosis inhibition.

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers. We have previously demonstrated that taxol induces calpain-mediated apoptosis in NIH3T3 cells [Piñeiro et al., Exp. Cell Res., 2007, 313:369–379]. In this study we found that translation was significantly inhibited during taxol-induced apoptosis in these cells. We have studied the phosphorylation status and expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are implicated in translation regulation. We found that taxol treatment did not induce changes in eIF2α phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels. MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or 4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together these findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition.

Effects of mRg2, a Mixture of Ginsenosides Containing 60% Rg2, on the Ultraviolet B–Induced DNA Repair Synthesis and Apoptosis in NIH3T3 Cells

Ginseng has been used worldwidely as a traditional medicine of Asian countries for treatment of various diseases including cancer. The purpose of this study was to determine the effect of ginseng saponin mRg2, a mixture of ginsenosides containing 60% Rg2, on the repair and apoptosis of ultraviolet B (UVB)-exposed NIH3T3 cells. When cells were exposed to UVB and then incubated with normal growth medium for 48 h, cell viability, as determined by trypan blue exclusion assay decreased to about 25%. However, when mRg2 was included in the postincubation medium, the UVB-induced loss of cell viability was significantly reduced as compared with that postincubated in normal growth medium. 4,6-diamidino-2-phenylindole (DAPI) staining showed that postincubation of the UVB-exposed cells in medium containing mRg2 significantly reduced the apoptotic nuclear fragmentation. Interestingly, when cells were preincubated with mRg2 for 24 h and then exposed to various doses of UV, the amount of repair synthesis significantly increased as compared with those in cells exposed to UVB alone. Western blot analysis indicated that the mRg2 postincubation after UVB exposure potentiated the level of p53 and p21. The level of Triton nonextractable proliferating cell nuclear antigen (PCNA) also remained elevated by mRg2 postincubation. All these results suggest that mRg2 protects cells against UVB-induced genotoxicity by increasing DNA repair and decreasing apoptosis, in possible association with the modulation of protein levels involved in cell cycle arrest or progression.

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (ΔΨm) which could be responsible of Ca 2+ release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.

La3+‐promoted proliferation is interconnected with apoptosis in NIH 3T3 cells

Lanthanum ion (La(3+)) has been reported to affect proliferation or apoptosis of different cells. In the present study, La(3+) was confirmed to promote both proliferation and apoptosis of NIH 3T3 cells at the same concentrations. La(3+) was shown to promote proliferation by helping the cells to pass through the G1/S restriction point and enter S phase, however, the proliferating cells induced by incubation with La(3+) eventually underwent apoptosis. The proliferation and apoptosis of NIH 3T3 cells induced by La(3+) were well correlated with cell cycle alterations. La(3+) caused the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2; while inhibition of ERK phosphorylation by 2'-amino-3'-methoxyflavone (PD98059) suppressed both proliferation and apoptosis induced by La(3+). Based on the above experimental results, we postulated that La(3+)-promoted proliferation of NIH 3T3 cells could be interconnected with the cell apoptosis, possibly through cell cycle machinery. Our results thus support the recent hypothesis that proliferation and apoptosis of cell are intrinsically coordinated.

Phosphatidylinositol transfer protein α regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The conditioned medium (CM) from SPIalpha cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPIbeta cells (i.e., wild-type cells overexpressing PI-TPbeta). CM from SPIalpha cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPIalpha cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPIalpha cells tested on SPIbeta cells was inhibited by approximately 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPIalpha cells. The latter results imply that PI-TPalpha mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like receptor.

Bcl-2 overexpression inhibits tetrachlorohydroquinone-induced apoptosis in NIH3T3 cells: a possible mechanism for tumor promotion.

TCHQ is a major carcinogenic metabolite of the widely used wood preservative PCP. Recently, we found that TCHQ was a promoter in a mouse skin carcinogenesis model. However, the mechanism is still not clear. In this study, we showed that overexpression of Bcl-2 effectively suppressed TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphological changes and DNA fragmentation. Although production of ROS contributes to TCHQ-induced apoptosis, Bcl-2 failed to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, overexpressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. We also found that TCHQ induced a change in mitochondrial transmembrane potential, and that caspase-9 and subsequent caspase-3 can be activated during TCHQ-induced acute apoptosis. Interestingly, TCHQ induced a significant upregulation of Bcl-2 expression, and over-expressed Bcl-2 can dramatically inhibit the change of mitochondria membrane potential and activation of both caspase-9 and -3. Thus, our results suggest TCHQ-induced tumor promotion may be through a mechanism of upregulation of Bcl-2 protein and subsequent apoptosis inhibition.

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production.

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.

Laser-Scanning Coherent Anti-Stokes Raman Scattering Microscopy and Applications to Cell Biology

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.