Regulation of Ras homolog family member G by microRNA-124 regulates proliferation and migration of human retinal pigment epithelial cells.

Uncontrolled retinal pigment epithelial (RPE) cell proliferation/migration contribute to the pathological tractional membrane development in proliferative vitreoretinopathy. Recent studies reported that microRNA (miR)-124 controls various cellular functions via the direct targeting of small Ras homolog family member G (RHOG). Therefore, we investigated the role of the neuron-specific miR-124 and RHOG in RPE cell proliferation/migration. Alterations in miR-124 and RhoG expression, as per cell confluence were evaluated through quantitative real-time PCR and western blotting, respectively. After transfection with miR-124, we quantified RPE cell viability and migration and observed cell polarization and lamellipodia protrusions. We evaluated the expression of RHOG/RAC1 pathway molecules in miR-124-transfected RPE cells. Endogenous miR-124 expression increased proportionally to RPE cell density, but decreased after 100% confluence. Overexpression of miR-124 decreased cell viability and migration, BrdU incorporation, and Ki-67 expression. Inhibition of endogenous miR-124 expression promoted RPE cell migration. Transfection with miR-124 reduced cell polarization, lamellipodia protrusion, and RHOG mRNA 3' untranslated region luciferase activity. Like miR-124 overexpression, RhoG knockdown decreased RPE cell viability, wound healing, and migration, and altered the expression of cell cycle regulators. These results suggest that miR-124 could be a therapeutic target to alleviate fibrovascular proliferation in retinal diseases by regulating RPE proliferation/migration via RHOG.

Cyclosporine A eyedrops with self-nanoemulsifying drug delivery systems have improved physicochemical properties and efficacy against dry eye disease in a murine dry eye model.

PURPOSE: We aimed to compare the physicochemical properties and in vivo efficacy of commercially available nanoemulsion cyclosporine A (CsA) eyedrops in benzalkonium chloride (BAC)-induced dry eye disease (DED). METHODS: Particle size analysis was performed on conventional 0.05% CsA (Restasis, C-CsA) and two new types of 0.05% CsA eyedrops based on a self-nanoemulsifying drug delivery system (SNEDDS, SNEDDS-N and -T). Turbidometry, pH measurements and instability indices of each CsA solution were measured. DED was induced with BAC, and animals were treated with vehicle or CsA preparations. Tear volume and fluorescein staining scores were evaluated on days 7 and 14. Eyes were enucleated and subjected to IHC, TUNEL staining, periodic acid-Schiff (PAS) staining, real-time PCR and western blotting. RESULTS: Both SNEDDSs had lower and more uniform particle size distribution than C-CsA, and a similar optical density to phosphate-buffered saline and stable pH, in contrast to the high turbidity and unstable pH of C-CsA. Aqueous tear volume and fluorescein staining scores were improved in C-CsA- and SNEDDS-treated mice. Numbers of PAS-positive goblet cells and levels of inflammatory mediators were decreased by both C-CsA and SNEDDS, although SNEDDS resolved inflammation more effectively than C-CsA. CONCLUSIONS: Cyclosporine A eyedrops with SNEDDS have improved physicochemical properties and treatment efficacy in BAC-induced DED.

Hsp90 inhibitor SY-016 induces G2/M arrest and apoptosis in paclitaxel-resistant human ovarian cancer cells.

The aim of the present study was to evaluate the in vitro effect of a heat shock protein (Hsp)90 inhibitor, SY-016, on the paclitaxel (PTX)-resistant human ovarian cancer cell line OVCAR-3PTX, and explore its mechanism of apoptosis. In the present study, SY-016 was used in combination with PTX to determine its effect on the cell proliferation and apoptosis of OVCAR-3PTX cells. The drug-resistant tumor cells were established in vitro by stepwise sequential exposure to increasing concentrations of PTX. The cell viability and cell cycle distribution were measured by MTT assay and flow cytometric analysis, respectively. The induction of apoptosis was measured by caspase-3 activity, DNA fragmentation and western blot analyses. The cell viability significantly decreased following treatment with PTX and SY-016 as compared with either drug alone. The DNA fragmentation assay revealed an induction of apoptosis. The results from the flow cytometric analysis revealed an increase in the percentage of cells in the G2/M phase. Downregulation of B-cell lymphoma (Bcl)-2, X-linked inhibitor of apoptosis protein, survivin, Akt, nuclear factor-κB and cyclin-dependent kinase 4, as well as upregulation of Bcl-2-associated X protein, were observed. SY-016 may contribute to the induction of apoptosis in OVCAR-3PTX cells. These results suggest that SY-016 in combination with PTX may be a beneficial chemotherapeutic strategy, particularly in patients with tumors refractory to PTX.

Bortezomib inhibits proliferation, migration, and TGF-ß1-induced epithelial-mesenchymal transition of RPE cells.

Purpose: Nuclear factor kappa B (NF-κB) plays an important role in the epithelial-mesenchymal transition (EMT) of RPE cells. We investigated the effects of a proteasome inhibitor, bortezomib, on the EMT in RPE cells. In addition, we assessed the influence of bortezomib on regulation of the NF-κB pathway during this process. Methods: After treatment with various concentrations of bortezomib, cell viability was analyzed with the water-soluble tetrazolium salt-8 assay, cell-cycle regulation was evaluated with flow cytometry, and cell migration was monitored with in vitro wound healing and Transwell migration assays. To induce fibroblastoid transformation, the RPE cells were treated with recombinant human transforming growth factor (TGF)-ß1 (10 ng/ml), and western blot and immunocytochemical analyses were performed to evaluate altered expression of EMT markers after treatment with bortezomib. To verify the effect of bortezomib on shrinkage by myofibroblastic transformation, a contraction assay of the RPE-collagen gel lattice was performed. Results: Treatment with bortezomib decreased RPE viability in a dose-dependent manner, and flow cytometry revealed that these effects were due to arrest of the G2/M phase cell-cycle. In the in vitro wound healing and Transwell migration assays, treatment with 20 nM bortezomib significantly impeded RPE migration. Treatment with bortezomib also significantly inhibited TGF-ß1-induced transdifferentiation of the RPE cells. The effects on proliferation, migration, and the EMT were mediated by regulation of the NF-κB signaling pathway. In addition, bortezomib inhibited contraction of the RPE-collagen gel lattices. Conclusions: Bortezomib inhibits myofibroblastic transformation of RPE cells by downregulating NF-κB expression and prevents contraction of the RPE-collagen gel matrix. Thus, bortezomib represents a candidate putative therapeutic agent for management of retinal fibrotic diseases.

Comparison of serum trefoil factor 3 with the pepsinogen test for the screening of diffuse-type gastric cancer.

Emerging data show that serum trefoil factor 3 (TFF3) alone and combined with the serum pepsinogen (PG) test can increase the diagnostic yield of gastric cancer. We aimed to evaluate the diagnostic value of serum TFF3 for the screening of gastric cancer in Korean patients, especially for the screening of the diffuse type of gastric cancer, and compared TFF3 to the serum PG test. We enrolled 25 healthy controls and 79 subjects with gastric cancer who underwent endoscopic resection or surgery from June 2006 to June 2015. Data about age, sex, histological type according to the Lauren classification, stage of gastric cancer, and status of H. pylori were collected. Serum levels of PG I and PG II were measured by the latex-enhanced turbidimetric immunoassay, and serum TFF3 levels were measured by enzyme-linked immunosorbent assay. The optimal cutoff value of serum TFF3 was ≥8.9 ng/mL to diagnose gastric cancer, with 73.4 % sensitivity and 92.0 % specificity, which were higher than those of the serum PG I/II ratio, with 69.6 % sensitivity and 68.0 % specificity. The optimal sensitivity and specificity of serum TFF3 for the diagnosis of diffuse-type gastric cancer were 68.0 and 92.0 %, respectively, which were lower than those for the diagnosis of intestinal-type gastric cancer (75.6 and 100 %, respectively). Serum TFF3 is a more stable and useful marker than the serum PG test for the screening of gastric cancer in Korean patients. Serum TFF3 showed good diagnostic power in detecting both intestinal- and diffuse-type gastric cancer although it showed decreased power in diffuse type.

Salinomycin reduces stemness and induces apoptosis on human ovarian cancer stem cell.

OBJECTIVE: Cancer stem cells (CSCs) represent a subpopulation of undifferentiated tumorigenic cells thought to be responsible for tumor initiation, maintenance, drug resistance, and metastasis. The role of CSCs in drug resistance and relapse of cancers could significantly affect outcomes of ovarian cancer patient. Therefore, therapies that target CSCs could be a promising approach for ovarian cancer treatment. The antibiotic salinomycin has recently been shown to deplete CSCs. In this study, we evaluated the effect of salinomycin on ovarian cancer stem cells (OCSCs), both alone and in combination with paclitaxel (PTX). METHODS: The CD44⁺CD117⁺CSCs were obtained from the ascitic fluid of patients with epithelial ovarian cancer by using an immune magnetic-activated cell sorting system. OCSCs were treated with PTX and salinomycin either singly or in combination. Cell viability and apoptosis assays were performed and spheroid-forming ability was measured. The expression of sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 3/4 (OCT3/4) mRNA was determined using reverse transcription polymerase chain reaction, and protein expression was observed using western blot analysis. RESULTS: Treatment with salinomycin alone reduced the stemness marker expression and spheroid-forming ability of OCSCs. Treatment with PTX alone did not decrease the viability of OCSCs. Treatment with a combination of salinomycin decreased the viability of OCSCs and promoted cell apoptosis. The enhancement of combination treatment was achieved through the apoptosis as determined by annexin V/propidium iodide (PI) staining, caspase-3 activity, and DNA fragmentation assay. CONCLUSION: Based on our findings, combining salinomycin with other anti-cancer therapeutic agents holds promise as an ovarian cancer treatment approach that can target OCSCs.

Salinomycin inhibited cell proliferation and induced apoptosis in human uterine leiomyoma cells.

OBJECTIVE: The aim of this study was to investigate the anti-proliferative effect of the salinomycin in cell proliferation and apoptosis in primary cultured human uterine leiomyoma cells. METHODS: Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caspase-3 activity assay and DNA fragmentation assay were performed to determine the effect of apoptosis. The expression of apoptosis regulatory-related proteins was evaluated by western blot. RESULTS: The cell viability and proliferation of uterine leiomyoma cells were significantly reduced by salinomycin treatment in a dose-dependent manner. DNA fragmentation assay results showed apoptotic cell death after salinomycin incubation. Salinomycin activated caspase-3, -8, and -9, causing apoptosis in uterine leiomyoma cells. Down-regulation of Bcl-2, XIAP, and FLIP with a concomitant increase in Bax, Fas, and DR5 were observed. CONCLUSION: These results provided the first evidence that salinomycin induce both intrinsic and extrinsic apoptosis. Therefore, salinomycin may be a promising chemopreventive and therapeutic agent against human uterine leiomyoma.

Antitumor effects of flavopiridol on human uterine leiomyoma in vitro and in a xenograft model.

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21(cip/wafl) and p27(kip1) in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma.

Salinomycin induces apoptosis via death receptor-5 up-regulation in cisplatin-resistant ovarian cancer cells.

BACKGROUND: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to effective disease treatment. Recently, salinomycin was proven to be highly-effective for the elimination of cancer stem cells both in vitro and in vivo. The objective of the present study was to evaluate the anticancer properties of salinomycin in cisplatin-resistant ovarian cancer cells (A2780cis). MATERIALS AND METHODS: The tetrazolium dye (MTT) assay was used to determine cell viability. Flow cytometric analysis was performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis-related proteins was evaluated by western blot analysis. RESULTS: Cell viability was significantly reduced by salinomycin treatment in a dose-dependent manner. Flow cytometry showed an increase in sub-G1 phase cells. Salinomycin increased the expression of death receptor-5 (DR5), caspase-8 and Fas-associated protein with death domain (FADD). A decline in the expression of FLICE-like inhibitory protein (FLIP), activation of caspase-3 and increased poly ADP-ribose polymerase (PARP) cleavage, triggered apoptosis. Furthermore, annexin-V staining also revealed the apoptotic induction. CONCLUSION: These findings provide important insights regarding the activation of caspase-8 and DR5, to our knowledge, for the first time in salinomycin-treated cisplatin-resistant ovarian cancer and demonstrate that salinomycin could be a prominent anticancer agent.

The synergistic apoptotic interaction of Indole-3-Carbinol and Genistein with TRAIL on endometrial cancer cells.

Induction of apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. The purpose of this study was to determine whether Indole-3-Carbinol (I3C) and Genistein in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis in endometrial cancer cell (Ishikawa) and to assess apoptotic mechanism. The MTT assay and flow cytometry were performed to determine cell viability and cell cycle. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay, annexin V binding assay and western blot analysis. There was no effect in cell growth inhibition and cell cycle progression alone or in two-combination. However, the treatment of I3C and Genistein followed by TRAIL showed significant cell death and marked increase in sub-G1 arrest. Three-combination treatment revealed elevated expression of DR4, DR5 and cleaved forms of caspase-3, caspase-8, PARP. The Flip was found down regulated. Moreover, increase in caspase-3 activity and DNA fragmentation indicated the induction of apoptosis. The results indicate that I3C and Genistein with TRAIL synergistically induced apoptosis via death receptor dependent pathway. Our findings might provide a new insight into the development of novel combination therapies against endometrial cancer.

Salinomycin inhibits Akt/NF-κB and induces apoptosis in cisplatin resistant ovarian cancer cells.

BACKGROUND: Despite advances in treatment, ovarian cancer is the most lethal gynecologic malignancy. Therefore significant efforts are being made to develop novel strategies for the treatment of ovarian cancer. Salinomycin has been shown to be highly effective in the elimination of cancer stem cells both in vitro and in vivo. The present study focused on investigating important cell signaling molecules such as Akt and NF-κB during salinomycin-induced apoptosis in cisplatin resistant ovarian cancer cells (A2780cis). METHODS: MTT assay was performed to determine cell viability. Flow cytometry and DNA fragmentation assay were performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis related proteins was evaluated by Western blot analysis. RESULTS: The cell viability was significantly reduced by salinomycin treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Salinomycin inhibited the nuclear transportation of NF-κB, and downregulated Akt expression. Declined Bcl-2, activation of caspase-3 and increased PARP cleavage triggered apoptosis. Moreover, DNA fragmentation assay also revealed apoptotic induction. CONCLUSION: The result suggested that salinomycin-induced apoptosis in A2780cis was associated with inhibition of Akt/NF-κB. It may become a potential chemotherapeutic agent for the cisplatin resistant ovarian cancer therapy.

Perfluorooctane sulfonate-induced apoptosis of cerebellar granule cells is mediated by ERK 1/2 pathway.

Perfluorooctane sulfonate (PFOS), a ubiquitous environmental pollutant, is considered as a neurotoxicant to mammalian species. However, the underlying mechanism of its neurotoxicity is largely unknown. In the present study, we examined roles of mitogen-activated protein kinases (MAPKs) in PFOS-induced apoptosis of neuronal cells to elucidate the molecular mechanism. Cerebellar granule cells were isolated from 7-d old rats and maintained in culture for additional 7 d. Cells were exposed to PFOS and caspase-3 activity and nuclear morphology were evaluated by enzyme activity assay and Hoechst 33342 staining, respectively, to determine its effects on apoptosis. The treatment with PFOS resulted in caspase-3 activation and nuclear condensation and fragmentation. PFOS exposure selectively increased activation of ERK that remained above control over 6 h. The inhibitor of ERK pathway, PD98059, substantially blocked caspase-3 activation induced by PFOS, whereas inhibitors of JNK and p38 MAPK, SP600125 and SB203580, respectively, had no effect. PKC inhibitors, bisindolylmaleimide I and Gö6976, dampened caspase-3 activity and ERK activation induced by PFOS. Collectively, it is suggested that PKC and ERK play proapoptotic roles in PFOS-induced apoptosis of cerebellar granule cells and PKC act as an upstream regulator of ERK activation.

Regulatory role of osteopontin in malignant transformation of endometrial cancer.

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.

Perfluorooctane sulfonate induces apoptosis of cerebellar granule cells via a ROS-dependent protein kinase C signaling pathway.

Perfluorinated chemicals (PFCs) have been widely used in a variety of industry and consumer products. Perfluorooctane sulfonate (PFOS), a prominent member of perfluoroalkyls, is known as a neurotoxicant in developing brain and affects behavior and motor activity. However, mechanism of neurotoxicity still remains unknown. In this study, we attempted to analyze apoptotic effects of PFOS on developing neuron. Cerebellar granule cells derived from 7-day old SD rats and grown in culture for additional 7 days were used to mimic postnatal day (PND)-14 conditions. PFOS exposure increased ROS production, which was blocked by ROS inhibitor, N-acetylcysteine (NAC). PFOS selectively induced dose-dependent translocations of PKC-α, -ßII and -ɛ among PKC isozymes tested. The translocation of these specific PKC isozymes was blocked by NAC. A panel of different approaches was utilized to detect apoptotic effects. PFOS induced caspase-3 activity and nucleosomal DNA fragmentation in a dose-dependent manner, which were blocked by pretreatment of NAC. These apoptotic effects were further confirmed by TUNEL staining. Increases of caspase-3 activity and nucleosomal DNA fragmentation were dampened by the inhibition of PKC isozymes using siRNA technique. Taken together, our results suggest that PFOS may induce apoptosis of cerebellar granule cells via a ROS-mediated PKC signaling pathway. PKC signal transduction pathway is pivotal in learning and memory and apoptosis of neuronal cells is a critical event in neurotoxicity. Thus, this study may contribute to understand a new mechanistic aspect of PFOS-induced neurotoxicities.

PKC-δ mediates TCDD-induced apoptosis of chondrocyte in ROS-dependent manner.

Exposure to dioxin-like compounds is associated with arthritis in humans. A recent study reported that 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptosis in chondrocytes, which is a critical event in the pathogenesis of cartilage disease. In this study, protein kinase C (PKC) signaling pathway was investigated to determine the mechanism of TCDD-induced rabbit articular chondrocyte apoptosis. TCDD exposure induced glutathione-mediated ROS generation and the translocation of PKC isozymes. Among the PKC isozymes tested, PKC-δ showed the most sensitive translocation. The translocation was then blocked by ROS inhibitors (trolox and N-acetyl cysteine), a PKC-δ inhibitor (rottlerin), a caspase-3 inhibitor (z-DEVD-fmk) or an AhR blocker (α-naphthoflavone). TCDD increased caspase-3 activity, the activating enzyme for PKC-δ, and prior treatment with trolox blocked such an increase. These results suggest that the translocation of PKC-δ was mediated by ROS-dependent caspase-3 activity. Pretreatment with rottlerin or trolox dampened TCDD-induced apoptosis of chondrocyte, as determined by TUNEL staining and ELISA. Taken together, this study suggests that ROS generation is an upstream event for TCDD-induced chondrocyte apoptosis and PKC-δ mediates the apoptotic processes through ROS-dependent caspase-3 activation. This is a first finding demonstrating the role of PKC-δ in chondrocyte apoptosis stimulated by an environmental pollutant. The results may contribute to understanding the mechanism of joint disease associated with the exposure of dioxin-like compounds and identifying a target for the therapeutic interventions.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis of articular chondrocytes in culture.

Positive associations of halogenated aromatic hydrocarbons and arthritis have been reported in human populations. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent congener of its class, is associated with musculoskeletal dysfunctions in humans and animals, its role on arthritis remains unknown. Apoptosis of chondrocytes has become a focus of interest in the pathogenesis of arthritis. We investigated the potential of TCDD as an inducer of chondrocyte apoptosis and evaluated its mechanism of action. Rabbit chondrocytes in culture were exposed to TCDD. Responses of dioxin-responsive genes and enzyme activity were analyzed by RT-PCR and EROD assay, respectively. Generation of reactive oxygen species (ROS) and nitric oxide (NO) were also determined. A panel of different approaches including caspase-3 assay, ELISA, flow cytometry, and TUNEL staining was utilized to detect apoptotic effects. Dioxin induced mRNAs of dioxin-responsive genes and EROD activity in an AhR-dependent manner. Dose-dependent increases in ROS and NO production were observed. All apoptosis detection techniques used in this study revealed an increase of apoptotic effects in a dose-dependent manner. The increase of apoptosis was blocked by inhibitors of ROS or NO, suggesting that apoptotic effects may be mediated via ROS- and NO-dependent pathways. This is a first report to demonstrate the potential of TCDD to induce apoptosis in chondrocytes, which could be an initial process in cartilage degradation. This finding may shed a new light in studying the possible role of environmental pollutants in the etiology of arthritis.

Development of human dermal epithelial cell-based bioassay for the dioxins.

None of bioassays is complete for assessing biological impact in humans upon the xenobiotic exposure due to species and organ-specific responsiveness. Thus, it is speculated that the human cell-based bioassay may be more appropriate system because of its direct relevance to humans. Here, we have developed a human epidermal cell-based bioassay for the dioxins and related compounds. The AD12-SV40-immortalized human keratinocyte cell line was stably transfected with a recombinant expression vector which contains the luciferase gene under dioxin-inducible control of four DREs. The tansfectants showed a consistent dose-response of luciferase activity upon dioxin exposure even after 120 passages. The maximal half effective dose (EC50) was 200 pM with a maximum of 32-fold induction of luciferase activity at 5 nM. The minimum detection limit was 10 pM. Optimal exposure time for the assay was 24h. When cells were treated with aryl hydrocarbon receptor agonists of different toxic equivalent factor (TEF) values, the shape of the dose-response curve for each compound was parallel to that of TCDD and the maximum response was similar, indicating that this bioassay system can be applied to generate the total toxic equivalency (TEQ) estimate from the samples. When relative induction potency of luciferase activities for each compound was calculated, it was similar to WHO-TEF values within an order of magnitude. This human cell system can be used as an efficient screening tool to quantify the TEQ values of dioxin-like chemicals in the samples and may help understand the interspecies difference between humans and animals.

TCDD alters PKC signaling pathways in developing neuronal cells in culture.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce neurodevelopmental deficits such as poor cognitive development and motor dysfunction. However, the mechanism of TCDD-mediated neurotoxicity remains unclear. Since PKC signaling is one of the most pivotal events involved in neuronal function and development, we analyzed the effects of TCDD on the PKC signaling pathway in cerebellar granule cells derived from PND-7 rat brain. Immunoblot analysis revealed the presence of PKC-alpha, betaII, delta, epsilon, lambda and iota in both cytosol and membrane fractions of cerebellar granule cells, but PKC-gamma was below the detectable level. TCDD induced a significant translocation of PKC-alpha, -betaII and -epsilon from cytosol to membrane fraction (p<0.05) and a marginal translocation of PKC-delta at high dose only (p<0.1). It also increased RACK-1, an adaptor protein for PKC, in a dose-dependent manner. Exposure to TCDD induced a dose-dependent increase of both [3H] PDBu binding and the intracellular calcium level. The results suggest that the selective PKC isozymes and RACK-1 are involved in TCDD-mediated signaling pathway and these proteins may be possible molecular targets in neuronal cells for TCDD exposure. Our study provides basic data to understand mechanism of TCDD-induced neurotoxicity with respect to PKC signaling pathway and a scientific basis for improving the health risk assessment of neurotoxicants by identifying intracellular target molecules in neuronal cells.

Translocation of PKC-betaII is mediated via RACK-1 in the neuronal cells following dioxin exposure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce neurotoxic effects. However, the mechanism of TCDD-mediated signaling pathways and its possible molecular targets in neurons remains unknown. In this study, we analyzed effects of TCDD on neurofilament subunits, receptor for activated C kinase-1 (RACK-1), and PKC-betaII activity in developing neuronal cells. TCDD induced a significant increase of RACK-1, an adaptor protein for protein kinase C (PKC), in cerebellar granule cells in both dose- and time-dependent manner, indicating that RACK-1 is a sensitive molecular target in neuronal cells for TCDD exposure. TCDD induced a dose-dependent translocation of PKC-betaII from cytosol to membrane fractions. However, when RACK-1 induction was blocked by antisense oligonucleotide or alpha-naphthoflavone, Ah receptor (AhR) inhibitor, the translocation of PKC-betaII was inhibited. Our data suggests that TCDD activates PKC-betaII via RACK-1 in an AhR-dependent manner. This is the first report identifying RACK-1 as a target molecule involved in TCDD-mediated signaling pathways. TCDD exposure also increased the level of neurofilament-H mRNA. These results suggest that identification of target molecules may contribute to improve our understanding of TCDD-mediated signaling pathway and the risk assessment of TCDD-induced neurotoxicities.