Sojung Lee

Activation of the interleukin-32 pro-inflammatory pathway in response to human papillomavirus infection and over-expressionof interleukin-32 controls the expression of the humanpapillomavirus oncogene

High‐risk variants of human papillomavirus (HPV) induce cervical cancer by persistent infection, and are regarded as the principal aetiological factor in this malignancy. The pro‐inflammatory cytokine interleukin‐32 (IL‐32) is present at substantial levels in cervical cancer tissues and in HPV‐positive cervical cancer cells. In this study, we identified the mechanism by which the high‐risk HPV‐16 E7 oncogene induces IL‐32 expression in cervical cancer cells. We used antisense transfection, over‐expression, or knock‐down of IL‐32 to assess the effects of the HPV‐16 E7 oncogene on IL‐32 expression in cervical cancer cells. Cyclo‐oxygenase 2 (COX‐2) inhibitor treatment was conducted, and the expression levels, as well as the promoter activities, of IL‐32 and COX‐2 were evaluated in human HPV‐positive cervical cancer cell lines. E7 antisense treatment reduced the expression levels and promoter activities of COX‐2, which is constitutively expressed in HPV‐infected cells. Constitutively expressed IL‐32 was also inhibited by E7 antisense treatment. Moreover, IL‐32 expression was blocked by the application of the selective COX‐2 inhibitor, NS398, whereas COX‐2 over‐expression resulted in increased IL‐32 levels. These results show that the high‐risk variant of HPV induces IL‐32 expression via E7‐mediated COX‐2 stimulation. However, E7 and COX‐2 were down‐regulated in the IL‐32γ over‐expressing cells and recovered by IL‐32 small interfering RNA, indicating that E7 and COX‐2 were feedback‐inhibited by IL‐32γ in cervical cancer cells.

The Biflavonoid Amentoflavone Induces Apoptosis via Suppressing E7 Expression, Cell Cycle Arrest at Sub-G1 Phase, and Mitochondria-Emanated Intrinsic Pathways in Human Cervical Cancer Cells

Amentoflavone, a biflavonoid from Selaginella tamariscina, is known to possess several bioactivities such as antitumor, anti-inflammatory, and antifungal effects. However, the mechanism of the anticancer effects of amentoflavone on human cervical cancer cells has not been studied in detail. In this study, we demonstrated that amentoflavone induces apoptosis in SiHa and CaSki cervical cancer cells by suppressing human papillomavirus protein E7 expression. The cyclins and tumor suppressors were modulated by amentoflavone in SiHa and CaSki human cervical cancer cells: cyclin and hyperphosphorylated retinoblastoma (p-pRb) were down-regulated, whereas cyclin-dependent kinase inhibitors and p53 were enhanced. Amentoflavone up-regulated peroxisome proliferator-activated receptor γ (PPARγ) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression levels while inhibiting E7-mediated cyclooxygenase-2 (COX-2)/interleukin-32 (IL-32) expressions were downregulated, and Akt phosphorlylation was decreased in an amentoflavone-induced apoptotic process, suggesting that amentoflavone may be a PPARγ activator. Additionally, the expression of the anti-apoptotic factor Bcl-2 was decreased, whereas that of the well-known apoptotic factor Bax was increased, thereby releasing cytochrome c into cytosol in amentoflavone-treated cervical cancer cells. Furthermore, amentoflavone treatment led to the activation of caspase-3 and -9 and proteolytic cleavage of poly(ADP-ribose) polymerase. The expression level of the extrinsic death receptor Fas (CD95) was not altered by amentoflavone treatment. When these findings are taken together, the biflavonoid amentoflavone activates PPARγ/PTEN expressions and induces apoptosis via suppressing E7 expression, cell cycle arrest at sub-G₁ phase, and mitochondria-emanated intrinsic pathways in SiHa and CaSki human cervical cancer cells. These findings suggest that amentoflavone has potential for development as a therapeutic agent for human cervical cancer.

Epiregulin expression by Ets-1 and ERK signaling pathway in Ki-ras-transformed cells.

Epiregulin belongs to the epidermal growth factor family, binds to the epidermal growth factor receptor, and its expression is upregulated in various cancer cells, but the regulatory mechanism is unclear. We investigated the regulatory mechanism of epiregulin expression in Ki-ras-transformed cancer cells. In 267B1/Ki-ras cells, the RAF/MEK/ERK pathway was constitutively activated, epiregulin was up-regulated, and the expression and phosphorylation of Ets-1 were augmented. The inhibition of ERK by PD98059 decreased epiregulin and Ets-1 expression and suppressed the growth of 267B1/Ki-ras cells. A chromatin immunoprecipitation assay demonstrated that Ets-1 was bound to human epiregulin promoter, and this binding was abolished by PD98059. Silencing of Ets-1 by RNA interference decreased cellular epiregulin transcript expression. We suggest that the Ki-ras mutation in 267B1 prostate cells constitutively activates the RAF/MEK/ERK pathway and induces the activation of the Ets-1 transcription factor, ultimately leading to the increased expression of epiregulin.

Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach.

The E6 and E7 oncoproteins of human papilloma virus (HPV) type 16 have been known to cooperatively induce the immortalization and transformation of primary keratinocytes. We established an E7 transgenic mouse model to screen HPV-related biomakers using the omics approach. The methods used to identify HPV-modulated factors were genomics analysis by microarray using the Affymetrix 430 2.0 array to screen E7-modulated genes, and proteomics analysis using nano-LC-ESI-MS/MS to screen E7-modulated proteins with the lung tissue of E7 transgenic mice. According to omics data, cyclin B1, cyclin E2, topoisomerase IIα, calnexin, activated leukocyte cell adhesion molecule CD166, actinin α1, diaphorase 1, gelsolin, platelet glycoprotein, and annexin A2 and A4 were up-regulated in the E7-Tg mice, while proteoglycan 4, sarcolipin, titin, vimentin, drep 1, troponin and cofilin-1 were down-regulated. We further confirmed the significance of differences between the expression levels of the selected factors in E7-Tg and non-Tg mice by real-time PCR. Genes related to cancer cell adhesion, cell cycle and migration, proliferation and apoptosis, as well as to the intermediate filament network and to endoplasmic reticulum proteins, were selected. Taken together, the results suggest that the E7 oncogene modulates the expression levels of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- and migration-related (actinin α1, CD166) factors, which may play important roles in cellular transformation in cancer. In addition, the solubilization of the rigid intermediate filament network by specific proteolysis mediated via up-regulating gelsolin and down-regulating cofilin-1, as well as increased levels of endoplasmic reticulum protein calnexin with chaperone functions, might also be involved in E7-lung epithelial cells.

Optimization of an enzyme-linked immunosorbent assay to screen ligand of Peroxisome proliferator-activated receptor alpha.

Peroxisome proliferator-activated receptors (PPARs) are transcription factor which directly modulate gene expression by binding to specific agonists. It has been reported that PPARα controls lipid metabolism, inflammation, and atherosclerosis. PPARα activation by PPARα agonist can ultimately reduce the progression of atherosclerosis and decrease the incidence of coronary heart disease. In this study, we optimized enzyme-linked immunosorbent assay (ELISA) systems in order to screen putative PPARα agonists. These methods are based on the activation mechanism of PPARα where the ligand binding to PPARα induces the interaction of the receptor with transcriptional co-activators. Among co-activators such as SRC-1, TIF-2, and p300, although ligand-unbound PPARα had more strong binding with p300 at a lower concentrations of PPARα, ligand-bound PPARα had more specific and strong binding with SRC-1. We optimized and developed a novel and useful ELISA system to screen PPARα agonists. Wy14,643 and linoleic acid, the well-known PPARα ligands, increased the binding between PPARα and co-activators in a ligand dose-dependent manner. In this ELISA method to screen PPARα ligands, the use of specific anti-PPARα N-terminus antibody, full-length recombinant protein of human PPARα but not ligand-binding domain (LBD) of human PPARα, and his-tagged PPARα recombinant proteins but not GST-fused PPARα recombinant proteins is the critical factors. Development of this screening system may be useful in the discovery of PPARα ligands from various candidates such as chemical library and phytochemicals.