Faculty Bibliography

Klotho is an antiaging gene involved in the suppression of several age-related phenotypes, but few studies have examined the mechanism underlying the regulation of human Klotho gene expression. In this study, we investigated the transcriptional regulation of the Klotho gene by epidermal growth factor (EGF) in HEK293 human embryonic kidney cells. By using serial deletion constructs of the promoter, we identified a proximal 45 bp (-90 to -45) region responsible for EGF-induced promoter activity. The Egr-1-binding motif is located within this region. Forced expression of Egr-1 stimulated Klotho gene promoter activity. A point mutation in the Egr-1-binding motif abrogated promoter inducibility by EGF or ectopic Egr-1 expression. Knockdown of Egr-1 by expression of small interfering RNA (siRNA) attenuated EGF-induced Klotho promoter activity. Further analysis showed that the Ras/MEK/Erk signaling cascade is involved in EGF-induced activation of the Klotho promoter. We conclude that the Klotho gene is activated by EGF in HEK293 cells.

Assessments of skin irritation potentials are important aspects of the development of nanotechnology. Nanosilica is currently being widely used for commercial purposes, but little literature is available on its skin toxicity and irritation potential. This study was designed to determine whether nanosilica has the potential to cause acute cutaneous toxicity, using cultured HaCaT keratinocytes (CHK), a human skin equivalent model (HSEM), and invivo model. Nanosilica was characterized by scanning electron microscopy. We evaluated the cytotoxic effects of nanosilica on CHKs and the HSEM. In addition, we also investigated whether two commercially available nanosilicas with different sizes (7 and 10-20 nm) have different effects. To confirm invitro results, we evaluated the irritation potentials of nanosilicas on rabbit skin. Nanosilicas reduced the cell viabilities of CHKs in a dose-dependent manner. However, the HSEM revealed no irritation at 500 microg/ml of nanosilica. Furthermore, this result concurred with Draize skin irritation test findings. The present study data indicate that nanosilica does not cause acute cutaneous irritation. Furthermore, this study shows that the HSEM used provides more useful screening data than the conventional cell culture model on the relative toxicities of NPs.

Curcumin, a natural compound, is a well-known chemopreventive agent with potent anticarcinogenic activity in a wide variety of tumor cells. Curcumin inhibits cancer cell proliferation in part by suppressing cyclin D1 and inducing expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Both p53-dependent and p53-independent mechanisms regulate p21(Waf1/Cip1) expression, but the mechanism by which curcumin regulates p21(Waf1/Cip1) expression remains unknown. Here, we report that transcription of the p21(Waf1/Cip1) gene is activated by early growth response-1 (Egr-1) independently of p53 in response to curcumin treatment in U-87MG human glioblastoma cells. Egr-1 is a transcription factor that helps regulate differentiation, growth, and apoptosis in many cell types. Egr-1 expression is induced by curcumin through extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK), but not the p38, mitogen-activated protein kinase (MAPK) pathways, which mediate the transactivation of Elk-1. Transient expression of Egr-1 enhanced curcumin-induced p21(Waf1/Cip1) promoter activity, whereas suppression of Egr-1 expression by small interfering RNA abrogated the ability of curcumin to induce p21(Waf1/Cip1) promoter activity. In addition, stable knockdown of Egr-1 expression in U-87MG cells suppressed curcumin-induced p21 expression. Our results indicate that ERK and JNK MAPK/Elk-1/Egr-1 signal cascade is required for p53-independent transcriptional activation of p21(Waf1/Cip1) in response to curcumin in U-87MG human glioblastoma cells.

Curcumin, a constituent of turmeric, has anti-inflammatory, anti-carcinogenic, and chemopreventive effects in several animal tumor models. The expression of P-glycoprotein (P-gp), encoded by the mdr gene, is often associated with multidrug resistance (MDR) to unrelated chemotherapeutic drugs in cancer cells. Here, we demonstrate that curcumin down-regulates P-gp expression in multidrug-resistant L1210/Adr cells. Transfection with a series of 5'-deleted constructs of the mdr1b gene promoter indicated that a proximal region between -205 and +42 of the sequence was responsible for the suppression of promoter activity by curcumin. This response might be associated with the inhibition of the phosphatidyinositol 3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-kappa B) signaling pathway by curcumin. Moreover, curcumin reversed the MDR of the L1210/Adr cells. Thus, curcumin can contribute to the reversal of the MDR phenotype, probably due to the suppression of P-gp expression via the inhibition of the PI3K/Akt/NF-kappa B signaling pathway.

Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen compound that is widely used as an effective chemotherapeutic agent for treatment and prevention of breast cancer. Unfortunately, prolonged treatment with TAM causes TAM-responsive tumors to become TAM resistant through an as-yet-unknown mechanism. To develop novel anti-breast cancer agents that are therapeutically superior to TAM, we must first fully understand the biological effects of TAM. In this study, we found that TAM treatment of MDA-MB-361 breast cancer cells activated p21Waf1/Cip1 gene transcription independently of p53. Furthermore, TAM-induced p21Waf1/Cip1 promoter activity was enhanced by transient expression of the gene encoding Early Growth Response-1 (Egr-1) protein, a transcription factor that plays an important role in cell growth and differentiation. The TAM-induced p21Waf1/Cip1 promoter activity was blocked by the expression of small interfering RNA (siRNA) targeted to Egr-1 mRNA. In addition, induction of Egr-1 expression by TAM occurred at the transcriptional level via Ets-domain transcription factor Elk-1 through the JNK and p38 mitogen-activated protein (MAP) kinase pathways. Inhibition of the JNK and p38 MAP kinase signals inhibited Egr-1-mediated p21Waf1/Cip1 promoter activity. We conclude that TAM stimulation of p21Waf1/Cip1 gene transcription in MDA-MB-361 cells depends largely on Elk-1-mediated Egr-1 expression induced by activation of the JNK and p38 MAP kinase pathways.

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.

Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by overexpression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenothiazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduction of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter. Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients.