The effect of levamisole in the treatment of recalcitrant recurrent erythema multiforme major: An observational study.

BACKGROUND: Erythema multiforme major (EMM) is an immune-mediated mucocutaneous eruption mostly triggered by herpes simplex virus (HSV) infection. A vicious circle of recurrence may be developed due to HSV reactivation and prolonged use of systemic corticosteroids to control EMM. Levamisole is an immunomodulator and has been applied to prevent relapses of recurrent HSV infection. OBJECTIVE: To evaluate the clinical efficacy and safety of levamisole in patients with recalcitrant recurrent EMM. METHODS: We enrolled 23 patients with recurrent EMM treated with levamisole and 24 controls, and analyzed the demographics, treatments and outcomes. RESULTS: Patients with recurrent EMM for years (mean 3.99 ± 2.71) showed significantly reduced recurrences after various durations of levamisole treatment (recurrences after and before treatment: 3.98 ± 1.04 vs 6.75 ± 1.45 times per year, p = 1.33x10-8). The recurrences of EMM also significantly reduced after levamisole treatment comparing to that of patients without levamisole treatment (p = 3.77x10-9). No patient was reported to have severe side effects during or after levamisole treatment. CONCLUSIONS: Levamisole was effective in reducing recurrences of recalcitrant recurrent EMM and can thus be considered an alternative or add-on therapy for this disorder.

Prurigo pigmentosa: a clinicopathological study and analysis of associated factors.

BACKGROUND: Prurigo pigmentosa (PP), a rare inflammatory disease of the skin, is mostly reported in the ethnic Japanese population. Its pathogenesis remains unclear. The chronic and recurrent nature of PP implies a possible role of viral infection in the pathogenesis. The anti-inflammatory mechanism of doxycycline, which is well documented as a good treatment for PP, is related to the suppression of interleukin expression. METHODS: We identified and retrospectively analyzed 16 biopsy-proven and criteria-matched patients over a seven year period at a single medical center. DNA extracted from formalin-fixed, paraffin-embedded specimens was analyzed for herpes simplex virus-1 (HSV-1), HSV-2, and human herpes virus-6 (HHV-6) DNA by polymerase chain reaction. Immunohistochemistry was performed to determine the interleukin-6 (IL-6) and interleukin-8 (IL-8) expression in PP skin lesions. RESULTS: Clinicopathological findings in the ethnic Chinese population are similar to those reported in Japanese studies. All patients had a good response to doxycycline treatment, with a mean duration of use of 2.4 weeks. However, recurrence was noted in six patients. HSV-1, HSV-2, and HHV-6 DNA in PP skin lesions were negative. Immunohistochemistry showed IL-6 (P = 0.035) to be more strongly expressed in PP skin lesions. There was no statistical significance of elevated IL-8 expression in PP (P = 0.123). CONCLUSIONS: Prurigo pigmentosa is not uncommon in the ethnic Chinese population. There was no evidence of herpes virus DNA in PP skin lesions. Increased expression of IL-6 in PP skin lesions may explain the effects of doxycycline in terms of its anti-inflammatory properties.

The forkhead transcription factor FOXO3a increases phosphoinositide-3 kinase/Akt activity in drug-resistant leukemic cells through induction of PIK3CA expression.

The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110alpha through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110alpha gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110alpha was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110alpha expression and hyperactivation of the PI3K/Akt pathway.

Doxorubicin activates FOXO3a to induce the expression of multidrug resistance gene ABCB1 (MDR1) in K562 leukemic cells.

Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression.

FOXO3a induces differentiation of Bcr-Abl-transformed cells through transcriptional down-regulation of Id1.

Leukemic transformation often requires activation of protein kinase B (PKB/c-Akt) and is characterized by increased proliferation, decreased apoptosis, and a differentiation block. PKB phosphorylates and inactivates members of the FOXO subfamily of Forkhead transcription factors. It has been suggested that hyperactivation of PKB maintains the leukemic phenotype through actively repressing FOXO-mediated regulation of specific genes. We have found expression of the transcriptional repressor Id1 (inhibitor of DNA binding 1) to be abrogated by FOXO3a activation. Inhibition of PKB activation or growth factor deprivation also resulted in strong down-regulation of Id1 promoter activity, Id1 mRNA, and protein expression. Id1 is highly expressed in Bcr-Abl-transformed K562 cells, correlating with high PKB activation and FOXO3a phosphorylation. Inhibition of Bcr-Abl by the chemical inhibitor STI571 resulted in activation of FOXO3a and down-regulation of Id1 expression. By performing chromatin immunoprecipitation assays and promoter-mutation analysis, we demonstrate that FOXO3a acts as a transcriptional repressor by directly binding to the Id1 promoter. STI571 treatment, or expression of constitutively active FOXO3a, resulted in erythroid differentiation of K562 cells, which was inhibited by ectopic expression of Id1. Taken together our data strongly suggest that high expression of Id1, through PKB-mediated inhibition of FOXO3a, is critical for maintenance of the leukemic phenotype.

Chronic protein kinase B (PKB/c-akt) activation leads to apoptosis induced by oxidative stress-mediated Foxo3a transcriptional up-regulation.

Increased protein kinase B (PKB; c-Akt) activation is a hallmark of diverse neoplasias providing both proliferative and antiapoptotic survival signals. In this study, we investigated the effect of chronic PKB activation on cellular survival and proliferation using cytokine-dependent bone marrow-derived Ba/F3 cells, in which PKBalpha activation can be directly, and specifically, induced by addition of 4-hydroxytamoxifen (4-OHT). Direct activation of PKB rescued Ba/F3 cells from cytokine withdrawal-induced apoptosis; however, surprisingly, these antiapoptotic effects were short lived, cells only being protected for up to 48 hours. We observed that activation of PKB in survival factor-deprived cells led to a dramatic increase of Foxo3a on both the transcriptional and protein level leading to expression of its transcriptional targets Bim and p27(kip1). High levels of PKB activity result in increased aerobic glycolysis and mitochondrial activity resulting in overproduction of reactive oxygen species. To determine whether oxidative stress might itself be responsible for Foxo3a up-regulation, we utilized hydrogen peroxide (H(2)O(2)) as an artificial inducer of oxidative stress and N-acetylcysteine (NAC), a thiol-containing radical oxygen scavenger. Addition of NAC to the culture medium prolonged the life span of cells treated with 4-OHT and prevented the up-regulation of Foxo3a protein levels caused by PKB activation. Conversely, treatment of Ba/F3 cells with H(2)O(2) caused an increase of Foxo3a on both transcriptional and protein levels, suggesting that deregulated PKB activation leads to oxidative stress resulting in Foxo3a up-regulation and subsequently cell death. Taken together, our data provide novel insights into the molecular consequences of uncontrolled PKB activation.

Epstein-Barr virus represses the FoxO1 transcription factor through latent membrane protein 1 and latent membrane protein 2A.

Epstein-Barr virus (EBV) infection is associated with the development of many B-cell lymphomas, including Burkitt's lymphoma, Hodgkin's lymphoma, and posttransplant lymphoproliferative disease. The virus alters a diverse range of cellular molecules, which leads to B-cell growth and immortalization. This study was initiated to investigate the interplay between EBV and a proapoptotic transcription factor target, FoxO1. In this report, we show that EBV infection of B cells leads to the downregulation of FoxO1 expression by phosphatidylinositol 3-kinase-mediated nuclear export, by inhibition of FoxO1 mRNA expression, and by alteration of posttranslational modifications. This repression directly correlates with the expression of the FoxO1 target gene Bcl-6 and inversely correlates with the FoxO1-regulated gene Cyclin D2. Expression of the EBV genes for latent membrane protein 1 and latent membrane protein 2A decreases FoxO1 expression. Thus, our data elucidate distinct mechanisms for the regulation of the proapoptotic transcription factor FoxO1 by EBV.