Heejong Kim

A proinflammatory cytokine interleukin-32β promotes the production of an anti-inflammatory cytokine interleukin-10

A new proinflammatory cytokine interleukin‐32 (IL‐32) has six isoforms. Although IL‐32 can be detected in sera from patients suffering from Crohn’s disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL‐32β by generating K562‐IL‐32β stable cell lines. This report confirms, using IL‐32 small interfering RNA, that IL‐32β induces an anti‐inflammatory cytokine IL‐10 in K562‐IL‐32β cells and U937 promonocytic cells, which express endogenous IL‐32β upon phorbol 12‐myristate 13‐acetate (PMA) treatment, and monocyte‐derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin‐32β was induced in monocyte‐derived macrophages by LPS and in monocyte‐derived DC by LPS, poly(I:C), or anti‐CD40 antibody, but was not induced by PMA. We showed that IL‐32β expression was increased in a time‐dependent manner in monocyte‐derived DC upon LPS treatment and peaked at 24 hr. Production of IL‐10 was exactly coincident with IL‐32β expression, but IL‐1β and tumour necrosis factor‐α production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin‐12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL‐32β and IL‐10 were no longer increased. Knock‐down of IL‐32β by IL‐32 small interfering RNA led to the decrease of IL‐10, but the increase of IL‐12 in monocyte‐derived DC, which means that IL‐32β promotes IL‐10 production, but limits IL‐12 production. We also showed that IL‐10 neutralization increases IL‐12, IL‐1β and tumour necrosis factor‐α production, which implies that IL‐10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL‐32β upregulates the production of an anti‐inflammatory cytokine IL‐10, and then IL‐10 suppresses proinflammatory cytokines.

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.

Interaction network mapping among IL-32 isoforms.

IL-32 has been studied for its pleiotropic effects ranging from host immune responses to cell differentiation. Although several IL-32 isoforms have been characterized for their effects on cells, the roles of the others remain unclear. We previously reported that IL-32δ interacted with IL-32β and inhibited IL-32β-mediated IL-10 production. Thus, we performed comprehensive analyses to reveal more interactions between IL-32 isoforms in this study. We screened the interactions of 81 combinations of nine IL-32 isoforms by using a yeast two-hybrid assay, which identified 13 heterodimeric interactions. We verified these results by using reciprocal immunoprecipitation assays and reconfirmed 10 interactions, and presented the interaction network map between IL-32 isoforms. Our data suggest that IL-32 may have diverse intracellular effects through the interactions with its different isoforms.

The liquid Panax ginseng inhibits epidermal growth factor-induced metalloproteinase 9 and cyclooxygenase 2 expressions via inhibition of inhibitor factor kappa-B-alpha and extracellular signal-regulated kinase in NCI-H292 human airway epithelial cells.

Background Ginseng (Panax ginseng C.A. Meyer) has been used in Asian countries for the treatment of various diseases. However, the mechanisms of liquid Panax ginseng (LG) on allergic inflammatory response in epidermal growth factor (EGF)–stimulated human airway epithelial cells remain largely unclear. Methods MUC5AC, cyclooxygenase (COX) 2, and matrix metalloproteinase (MMP) 9 expressions were measured using reverse transcription-polymerase chain reaction, Western blotting, and gelatin zymogram analyses in NCI-H292 cells. Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) protein levels were analyzed by Western blotting. Results To gain insight into the antiallergy effects of LG, we examined its influence on epidermal growth factor (EGF)–induced MMP-9 and COX-2 productions in NCI-H292 cells. LG was treated for 1 hour and then followed by EGF treatment for 24 hours into NCI-H292 cells. The decrease of COX-2 production was correlated with the reduced levels of proteins and mRNAs of inducible MMP-9 and MUC5AC. LG blocked upstream signaling of NF-kappa-B activation via inhibition of phosphorylations of inhibitor factor-kappa- B-alpha (I-kappa-B-alpha) and ERK. These results suggest that LG protects NCI-H292 cells from EGF-induced damage by down-regulation of COX-2, MMP-9, and MUC5AC gene expressions by blocking NF-kappa-B and ERK. Conclusion LG modulates allergic inflammatory response in EGF-stimulated NCI-H292 human airway epithelial cells via inhibition of I-kappa-B-alpha and ERK.

Naringenin Derivative Diethyl (5,4'-dihydroxy flavanone-7-yl) Phosphate Inhibits Cell Growth and Induces Apoptosis in A549 Human Lung Cancer Cells

Anti-cancer effects of naringenin derivative diethyl (5,4′-dihydroxy flavanone-7-yl) phosphate were evaluated in human lung cancer cells. The effect of diethyl (5,4′-dihydroxy flavanone-7-yl) phosphate (dEdHF-7-p) on A549 cell viability was measured using MTS assay and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using Hoechst staining. The influence of dEdHF-7-p on cell cycle distribution was determined using propidium iodide (PI) staining, and protein expression was determined by Western blot analysis. A newly synthesized naringenin derivative dEdHF-7-p suppressed cell growth of A549 though mechanisms including inhibition of cell cycle and increased apoptosis. Apoptotic and cell cycle modulators were changed by dEdHF-7-p in A549 cells; cyclins, ppRB, and antiapoptotic factor Bcl-2 were down-regulated, whereas apoptotic factor Bax and cyclin-dependent kinase inhibitors p21 and p53 were enhanced, thereby releasing cytochrome c into the cytosol of dEdHF-7-p -treated-A549 cells. dEdHF-7-p treatment processed caspases-3/-8/-9 and cleavage of poly ADP-ribose polymerase. The dEdHF-7-p treatment enhanced Fas expression and decreased expression of cell survival factors such as PI3K and p-Akt in a dose-dependent manner. Taken together, dEdHF-7-p induces apoptosis by inhibiting the PI3K/Akt survival signaling pathway and modulating mitochondria-emanated intrinsic and Fas extrinsic pathways in A549 cells.