Fargesin exerts anti-inflammatory effects in THP-1 monocytes by suppressing PKC-dependent AP-1 and NF-ĸB signaling.

BACKGROUND: Fargesin is a lignan from Magnolia fargesii, an oriental medicine used in the treatment of nasal congestion and sinusitis. The anti-inflammatory properties of this compound have not been fully elucidated yet. PURPOSE: This study focused on assessing the anti-inflammatory effects of fargesin on phorbal ester (PMA)-stimulated THP-1 human monocytes, and the molecular mechanisms underlying them. METHODS: Cell viability was evaluated by MTS assay. Protein expression levels of inflammatory mediators were analyzed by Western blotting, ELISA, Immunofluorescence assay. mRNA levels were measured by Real-time PCR. Promoter activities were elucidated by Luciferase assay. RESULTS: It was found that pre-treatment with fargesin attenuated significantly the expression of two major inflammatory mediators, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Fargesin also inhibited the production of pro-inflammation cytokines (IL-1ß, TNF-α) and chemokine (CCL-5). Besides, nuclear translocation of transcription factors nuclear factor-kappa B (NF-ĸB) and activator protein-1 (AP-1), which regulate multiple pro-inflammatory genes, was suppressed by fargesin in a PKC-dependent manner. Furthermore, among the mitogen-activated protein kinases (MAPKs), only c-Jun N-terminal kinase (JNK) was downregulated by fargesin in a PKC-dependent manner, and this reduction was involved in PMA-induced AP-1 and NF-ĸB nuclear translocation attenuation, demonstrated using a specific JNK inhibitor. CONCLUSION: Taken together, our results found that fargesin exhibits anti-inflammation effects on THP-1 cells via suppression of PKC pathway including downstream JNK, nuclear factors AP-1 and NF-ĸB. These results suggest that fargesin has anti-inflammatory properties with potential applications in drug development against inflammatory disorders.

Trifolin induces apoptosis via extrinsic and intrinsic pathways in the NCI-H460 human non-small cell lung-cancer cell line.

BACKGROUND: Trifolin (kaempferol-3-O-galactoside), which is a galactose-conjugated flavonol, exhibits antifungal and anticancer effects. However, the mechanisms underlying its anticancer activities have not yet been examined. PURPOSE: In this study, the anticancer effects of trifolin were examined in human lung cancer cells. METHODS: Cytotoxicity was determined by evaluating cell viability. Apoptosis was analyzed through flow cytometry and western blotting analysis. Death receptors and inhibitors of apoptosis were evaluated through RT-PCR. RESULTS: Trifolin induced apoptosis in NCI-H460 human non-small cell lung cancer (NSCLC) cells by inhibiting the survival pathway and inducing the intrinsic and extrinsic apoptosis pathways. Trifolin decreased levels of Akt/p-Akt, whereas levels of expression of phosphatidylinositide 3-kinase (PI3K), cyclin D1, cyclin E, and cyclin A were not altered. Trifolin initiated cytochrome c release by inducing mitochondrial outer membrane permeabilization (MOMP). Trifolin increased Bcl-2-associated X protein (Bax) levels and decreased b-cell lymphoma 2 (Bcl-2) levels, while the levels of Bcl-xL were not altered. In addition, trifolin increased the levels of the death receptor involving the Fas/Fas ligand (FasL) and Fas-associated protein with the death domain (FADD), which consequently activated caspase-8, caspase-9, caspase-3, and the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). CONCLUSION: These results suggested that trifolin induced apoptosis via death receptor-dependent and mitochondria-dependent pathways and that trifolin can be used as a therapeutic agent in human lung cancer.

IL-32θ gene expression in acute myeloid leukemia suppresses TNF-α production.

The proinflammatory cytokine TNF-α is highly expressed in patients with acute myeloid leukemia (AML) and has been demonstrated to induce rapid proliferation of leukemic blasts. Thus suppressing the production of TNF-α is important because TNF-α can auto-regulate own expression through activation of NF-κB and p38 mitogen-activated protein kinase (MAPK). In this study, we focused on the inhibitory effect of IL-32θ on TNF-α production in acute myeloid leukemia. Approximately 38% of patients with AML express endogenous IL-32θ, which is not expressed in healthy individuals. Furthermore, plasma samples were classified into groups with or without IL-32θ; then, we measured proinflammatory cytokine TNF-α, IL-1ß, and IL-6 levels. TNF-α production was not increased in patients with IL-32θ expression than that in the no-IL-32θ group. Using an IL-32θ stable expression system in leukemia cell lines, we found that IL-32θ attenuated phorbol 12-myristate 13-acetate (PMA)-induced TNF-α production. IL-32θ inhibited phosphorylation of p38 MAPK, inhibitor of κB (IκB), and nuclear factor κB (NF-κB), which are key positive regulators of TNF-α expression, and inhibited nuclear translocation of NF-κB. Moreover, the presence of IL-32θ attenuated TNF-α promoter activity and the binding of NF-κB with the TNF-α promoter. In addition, IL-32γ-induced TNF-α production has no correlation with inhibition of TNF-α via IL-32θ expression. Thus, IL-32θ may serve as a potent inhibitor of TNF-α in patients with AML.

6-O-Veratroyl catalpol suppresses pro-inflammatory cytokines via regulation of extracellular signal-regulated kinase and nuclear factor-κB in human monocytic cells.

The compound 6-O-veratroyl catalpol (6-O) is a bioactive iridoid glucoside that was originally isolated from Pseudolysimachion rotundum var. subintegrum. It has been demonstrated that catapol derivative iridoid glucosides including 6-O, possess anti-inflammatory activity in carragenan-induced paw edema mouse model as well as bronchoalveolar lavage fluid of ovalbumin-induced mouse model. In the present study, we investigated whether 6-O modulates inflammatory responses in THP-1 monocytic cells stimulated with phorbol12-myristate-13-acetate (PMA). Our data showed that 6-O inhibited PMA induced interleukin (IL)-1ß and tumor necrosis factor (TNF)-α expression in THP-1 monocytic cells. Mechanistic studies revealed that 6-O suppressed the activity of protein kinase C (PKC), which further resulted in downstream inactivation of extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) inflammatory pathway. The results implied that 6-O may protect against inflammatory responses that could be a potential compound in treating inflammatory diseases.

Luteolin 8-C-ß-fucopyranoside downregulates IL-6 expression by inhibiting MAPKs and the NF-κB signaling pathway in human monocytic cells.

Numerous studies have been suggested that derivatives can improve the effects of original substances. Therefore, we made luteolin derivative luteolin 8-C-ß-fucopyranoside (LU8C-FP) for better anti-inflammatory and anti-cancer effects. In a previous study, we demonstrated that LU8C-FP inhibits invasion of human breast cancer cells via suppression of matrix metalloproteinase 9 and IL-8, which play major roles in tumor progression and cancer cell invasion. Various stimuli trigger inflammatory responses by inducing pro-inflammatory cytokines and chemokines in THP-1 cells. IL-6 induces inflammation via inducing various cytokines and appears to be a potential mediator of inflammatory diseases. Here, we investigated the precise mechanism by which LU8C-FP inhibited phorbol 12-myristate 13-acetate-induced IL-6 mRNA and protein expression. We showed LU8C-FP downregulated IL-6 expression by inhibiting mitogen-activated protein kinases and the nuclear factor-kappaB signaling pathway in human monocytic cells. Furthermore, LU8C-FP exerts less cytotoxicity than luteolin and also it has specific inhibitory effect on IL-6 expression. However, luteolin has a variety of inhibitory effects on pro-inflammatory cytokines and chemokines. Our in vitro studies may provide valuable information leading to the use of LU8C-FP to treat inflammatory diseases caused by IL-6.

IL-32θ inhibits monocytic differentiation of leukemia cells by attenuating expression of transcription factor PU.1.

PU.1 is a key transcription factor regulating the myeloid differentiation. PU.1-induced monocytic differentiation into macrophage is also important for blood cancer development. Therefore, we chose THP-1 monocytic leukemia cells to investigate the function of a recently discovered IL-32θ. Genetic analyses identified differences in the sequences of IL-32θ and IL-32ß. Using previously established cell lines that stably express IL-32θ and IL-32ß and cell lines transiently expressing IL-32θ, we observed that expression of IL-32θ inhibited phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation in both THP-1 and HL-60 cells. IL-32θ also suppressed expression of the macrophage cell surface markers, CD11b, CD18, and CD36. Interestingly, expression of IL-32ß or IL-32θ had no effect on the expression levels of cell cycle related factors. As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest. IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment. Transient expression of PU.1 in THP-1/IL-32θ cells rescued the observed differentiation defect. Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect. These observations indicate that intracellular IL-32θ inhibits the differentiation of monocytes into macrophages by attenuating PU.1 expression.

15-Hydroxyeicosatetraenoic Acid Inhibits Phorbol-12-Myristate-13-Acetate-Induced MUC5AC Expression in NCI-H292 Respiratory Epithelial Cells.

It has been reported that overexpression of MUC5AC induced by excessive inflammation leads to airway obstruction in respiratory diseases such as chronic obstructive pulmonary disease and asthma. 15-Hydroxyeicosatetraenoic acid (15-HETE) has been reported to have anti-inflammatory effects, but the role of 15-HETE in respiratory inflammation has not been determined. Therefore, the aim of this study was to investigate the effects of 15-HETE on MUC5AC expression and related pathways. In this study, phorbol-12-myristate-13-acetate (PMA) was used to stimulate NCI-H292 bronchial epithelial cells in order to examine the effects of 15-HETE. 15-HETE inhibited PMA-induced expression of MUC5AC mRNA and secretion of MUC5AC protein. Also, 15-HETE regulated matrix metallopeptidase 9 (MMP-9), mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK). In addition, 15-HETE decreased translocation of specificity protein-1 (Sp-1) transcription factor and nuclear factor kappaB (NF-κB) into nuclear. Furthermore, 15-HETE enhanced transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) as a PPARγ agonist. This activity reduced phosphorylation of protein kinase B (PΚB/Akt) by increasing the expression of phosphatase and tensin homolog (PTEN). In conclusion, 15-HETE regulated MUC5AC expression via modulating MMP-9, MEK/ERK/Sp-1 and PPARγ/PTEN/ Akt signaling pathways in PMA-treated respiratory epithelial cells.

Peroxisome proliferator-activated receptor-gamma agonist 4-O-methylhonokiol induces apoptosis by triggering the intrinsic apoptosis pathway and inhibiting the PI3K/Akt survival pathway in SiHa human cervical cancer cells.

4-O-Methylhonokiol (MH), a bioactive compound derived from Magnolia officinalis, is known to exhibit antitumor effects in various cancer cells. However, the precise mechanism of its anticancer activity in cervical cancer cells has not yet been studied. In this study, we demonstrated that MH induces apoptosis in SiHa cervical cancer cells by enhancing peroxisome proliferator-activated receptor-gamma (PPARγ) activation, followed by inhibition of the PI3K/Akt pathway and intrinsic pathway induction. MH upregulated PPARγ and PTEN expression levels while it decreased p-Akt in the MH-induced apoptotic process, thereby supporting the fact that MH is a PPARγ activator. Additionally, MH decreased the expression of Bcl-2 and Bcl-XL, inducing the intrinsic pathway in MH-treated SiHa cells. Furthermore, MH treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of polyADP ribose polymerase. The expression levels of Fas (CD95) and E6/E7 oncogenes were not altered by MH treatment. Taken together, MH activates PPARγ/PTEN expression and induces apoptosis via suppression of the PI3K/Akt pathway and mitochondria-dependent pathways in SiHa cells. These findings suggest that MH has potential for development as a therapeutic agent for human cervical cancer.

Water-extracted Ampelopsis brevipedunculata downregulates IL-1ß,CCL5, and COX-2 expression via inhibition of PKC-mediated JNK/NF-κB signaling pathways in human monocytic cells.

The anti-inflammatory and anti-hepatotoxic effects of Ampelopsis brevipedunculata (A.bre) have been well known in folk medicine. An ethanol-extract of A.bre has been reported to inhibit carbon tetrachloric acid induced hepatic injury, suggesting that extracted components from A.bre could potentially treat inflammatory disease. To test this hypothesis, in this study, we extracted polysaccharide components from leaves of A.bre and investigated the anti-inflammatory effects in PMA stimulated THP-1 cells. THP-1 cells activated by PMA in the presence or absence of A.bre demonstrated that a water-extract of A.bre inhibited the expression of pro-inflammatory cytokine IL-1ß and chemokine CCL-5 in a dose-dependent manner. In addition, A.bre suppressed production of cyclooxygenase (COX)-2 in THP-1 cells activated by PMA. Moreover, A.bre markedly down-regulated the expression of p-JNK1/3, whereas it did not inhibit production of the phosphorylated form of p38 and extracellular signal-regulated kinase in THP-1 cells treated by PMA. Particularly, A.bre inhibited the translocation of transcription factor NF-κB from the cytosol into the nucleus in PMA-stimulated THP-1 cells. Collectively, our data showed that water-extracted A.bre inhibited the protein kinase C-JNKs/NF-κB signaling pathways, resulting in the suppression of IL-1ß, CCL-5, and COX-2 expression. This study suggests that water extracted A.bre may be a therapeutic agent against inflammatory disease.

IL-32θ downregulates CCL5 expression through its interaction with PKCδ and STAT3.

Interleukin-32 (IL-32) exists in several isoforms and plays an important role in inflammatory response. Recently, we identified a new isoform, IL-32θ, and performed a microarray analysis to identify IL-32θ-regulated genes in THP-1 myelomonocytic cells. Upon stimulating IL-32θ-expressing THP-1 cells with phorbol myristate acetate (PMA), we found that the CCL5 transcript level was significantly reduced. We confirmed the downregulation of CCL5 protein expression by using an enzyme-linked immunosorbent assay (ELISA). Because STAT3 phosphorylation on Ser727 by PKCδ is reported to suppress CCL5 protein expression, we examined whether IL-32θ-mediated STAT3 Ser727 phosphorylation occurs through an interaction with PKCδ. In this study, we first demonstrate that IL-32θ interacts with PKCδ and STAT3 using co-immunoprecipitation (Co-IP) and pulldown assay. Moreover, STAT3 was rarely phosphorylated on Ser727 in the absence of IL-32θ, leading to the binding of STAT3 to the CCL5 promoter. These results indicate that IL-32θ, through its interaction with PKCδ, downregulates CCL5 expression by mediating the phosphorylation of STAT3 on Ser727 to render it transcriptionally inactive. Therefore, similar to what we have reported for IL-32α and IL-32ß, our data from this study suggests that the newly identified IL-32θ isoform also acts as an intracellular modulator of inflammation.

Luteolin induces cell cycle arrest and apoptosis through extrinsic and intrinsic signaling pathways in MCF-7 breast cancer cells.

Luteolin is a common flavonoid that exists in medicinal herbs, fruits, and vegetables. Luteolin has biochemical functions including anti-allergy, anti-inflammation, and anti-cancer functions. However, its efficacy and precise mode of action against breast cancer are still under study. To elucidate whether luteolin exhibits an anticancer effect in breast cancer, MCF-7 breast cancer cells were incubated with luteolin, and apoptosis was assessed by observing nuclear morphological changes and by performing cell viability assay, cell cycle analysis, annexin V-FITC/PI double staining, western blotting, RT-PCR, and mitochondrial membrane potential measurements. Luteolin inhibited growth through perturbation of cell cycle progression at the sub-G1 and G1 phases in MCF-7 cells. Furthermore, luteolin enhanced the expression of death receptors, such as DR5, and activated caspase cascades. It enhanced the activities of caspase-8/-9/-3 in a dose-dependent manner, followed by inactivation of PARP. Activation of caspase-8 and caspase-9 induced caspase-3 activity, respectively, in apoptosis of extrinsic and intrinsic pathways. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and increased Bax expression by inhibiting expression of Bcl-2. Taken together, these results suggest that luteolin provokes cell cycle arrest and induces apoptosis by activating the extrinsic and intrinsic pathways.

Interleukin-32α modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase Cɛ-dependent sumoylation.

Interleukin-32 (IL-32) is a proinflammatory cytokine. However, there is growing evidence that IL-32 also plays a mediatory role intracellularly. In this study, we present evidence that IL-32α modifies and inhibits promyelocytic leukemia zinc finger (PLZF), a sequence-specific transcriptional regulator that regulates the expression of a subset of interferon (IFN)-stimulated genes (ISGs). We screened IL-32α-interacting proteins in a human spleen cDNA library using the yeast two-hybrid assay, and investigated the functional relevance of the interaction between IL-32α and PLZF. We demonstrated that IL-32α interacts with protein kinase C (PKC)δ and PKCɛ in a phorbol 12-myristate 13-acetate (PMA) dependent way, and that PKCɛ regulates the interaction of IL-32α with PLZF. We verified the involvement of PKCɛ in the interaction between these proteins by using various PKC inhibitors. PLZF is known to be modified by small ubiquitin-like modifier (SUMO)-1, but it is unclear whether SUMO-2 conjugation of PLZF occurs. We showed that IL-32α inhibited SUMO-2-conjugation of PLZF. Further, we demonstrated that sumoylated PLZF decreased when IL-32α was co-expressed. PKCɛ affected the sumoylation of PLZF only in the presence of IL-32α because PKC inhibitor treatment did not reduce PLZF sumoylation in the absence of IL-32α. We finally investigated whether IL-32α-mediated inhibition of PLZF sumoylation affected the transcriptional activity of PLZF, and demonstrated that the inhibition of sumoylation of PLZF by IL-32α down-regulated ISGs induced by PLZF. Together, our data suggest that IL-32α associates with PLZF and PKCɛ, and then inhibits PLZF sumoylation, resulting in suppression of the transcriptional activity of PLZF.

Interleukin-32α downregulates the activity of the B-cell CLL/lymphoma 6 protein by inhibiting protein kinase Cε-dependent SUMO-2 modification.

A proinflammatory cytokine IL-32 acts as an intracellular mediator. IL-32α interacts with many intracellular molecules, but there are no reports of interaction with a transcriptional repressor BCL6. In this study, we showed that PMA induces an interaction between IL-32α, PKCε, and BCL6, forming a trimer. To identify the mechanism of the interaction, we treated cells with various inhibitors. In HEK293 and THP-1 cell lines, treatment with a pan-PKC inhibitor, PKCε inhibitor, and PKCδ inhibitor decreased BCL6 and IL-32α protein expression. MAPK inhibitors and classical PKC inhibitor did not decrease PMA-induced BCL6 and IL-32α protein expression. Further, the pan-PKC inhibitor and PKCε inhibitor disrupted PMA-induced interaction between IL-32α and BCL6. These data demonstrate that the intracellular interaction between IL-32α and BCL6 is induced by PMA-activated PKCε. PMA induces post-translational modification of BCL6 by conjugation to SUMO-2, while IL-32α inhibits. PKCε inhibition eliminated PMA-induced SUMOylation of BCL6. Inhibition of BCL6 SUMOylation by IL-32α affected the cellular function and activity of the transcriptional repressor BCL6 in THP-1 cells. Thus, we showed that IL-32α is a negative regulator of the transcriptional repressor BCL6. IL-32α inhibits BCL6 SUMOylation by activating PKCε, resulting in the modulation of BCL6 target genes and cellular functions of BCL6.

IL-32θ negatively regulates IL-1ß production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation.

It has been well known that IL-32 exerts pro-inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL-32θ, a new isoform of IL-32, decreased the phorbol 12-myristate 13-acetate (PMA)-induced IL-1ß expression in THP-1 human myelomonocyte. We previously reported that the IL-32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL-32θ interacted with PKCδ, and consequently inhibited PKCδ-mediated phosphorylation of PU.1. Moreover, IL-32θ attenuated the localization of PU.1 into the IL-1ß promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1ß production.

Luteolin induces intrinsic apoptosis via inhibition of E6/E7 oncogenes and activation of extrinsic and intrinsic signaling pathways in HPV-18-associated cells.

Luteolin, a flavonoid extracted from a number of plants with recognized anticancer, anti-inflammatory and anti-oxidative activities, inhibits angiogenic processes and modulates multidrug resistance. However, the efficacy and mechanisms of action of this flavonoid agent are still undergoing study. In order to elucidate whether luteolin exhibits an anticancer effect in cervical cancer cells, HeLa cells were incubated with luteolin and apoptosis was assessed by observing nuclear morphological changes, and performing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cell cycle analysis, western blotting, RT-PCR and mitochondrial membrane potential measurements were also carried out. Luteolin showed a significant dose-dependent cytotoxic effect only in human papillomavirus (HPV)-positive cervical cancer cells, when compared to its effect on HPV-negative cervical cancer C33A cells. Expression levels of human papilloma virus E6 and E7 oncogenes were suppressed, those of related factors pRb and p53 were recovered and E2F5 was increased by luteolin treatment. Furthermore, luteolin enhanced the expression of death receptors and death receptor downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. In particular, luteolin enhanced the activity of caspase-3 and -8 in a dose-dependent manner. Activation of caspase-3 induced caspase-8 activity and vice versa. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited Bcl-2 and Bcl-xL expression. In conclusion, luteolin exerts anticarcinogenic activity through inhibition of E6 and E7 expression and cross-activation of caspase-3 and -8. Taken together, these results suggest that luteolin induces inactivation of HPV-18 oncogene expression and apoptosis by activating the intrinsic and extrinsic pathways.

IL-32α down-regulates ß2 integrin (CD18) expression by suppressing PU.1 expression in myeloid cells.

Myeloid-specific CD18 associates with CD11 and plays a critical role in leukocyte adhesion to the endothelium. In this study, we observed that CD18 expression was decreased by IL-32α in THP-1 and K562 cells upon PMA stimulation, and investigated the mechanism by which IL-32α down-regulated CD18 expression. We found that IL-32α suppressed the expression of PU.1, a major transcription factor for CD18. Because we previously demonstrated that IL-32α mediated STAT3 S727 phosphorylation by PKCε, and STAT3 regulates PU.1 expression, we performed time-course analyses of STAT3 S727 phosphorylation and found that IL-32α induces prolonged phosphorylation of STAT3 S727 until 72h after PMA stimulation. The expression pattern of C/EBPα, another transcriptional regulator of PU.1, was not affected by IL-32α. In addition, we showed that STAT3 binding to the PU.1 promoter was suppressed by IL-32α. Thus, we examined the relatedness among these factors and found that IL-32α-mediated STAT3 S727 phosphorylation induced C/EBPα association. When STAT3 was mutated at S727 to proline (S727P), the mutant STAT3 S727P did not interact with C/EBPα. We further demonstrated that only the intact STAT3 interacted with the basic leucine zipper region of C/EBPα. The PU.1 promoter was activated by co-expression of STAT3 and IL-32α upon PMA stimulation. However, the promoter activity was inhibited with STAT3 and C/EBPα co-expression. Therefore, our data suggest that IL-32α-mediated STAT3 S727 phosphorylation induced C/EBPα association, which inhibited PU.1 expression, and then resulted in the down-regulation of CD18 expression.

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-32ß and inhibited IL-32ß-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.

Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells.

Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.

Interleukin (IL)-32ß-mediated CCAAT/enhancer-binding protein α (C/EBPα) phosphorylation by protein kinase Cδ (PKCδ) abrogates the inhibitory effect of C/EBPα on IL-10 production.

We previously reported that IL-32ß promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32ß abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32ß knockdown by siRNA and that IL-32ß shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32ß suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32ß may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32ß interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32ß suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32ß. Taken together, our results show that IL-32ß-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.

Interleukin-32δ interacts with IL-32ß and inhibits IL-32ß-mediated IL-10 production.

There is growing evidence for multifunctional properties of IL-32. We previously demonstrated that IL-32ß upregulates IL-10 production through the association with PKCδ. In this study, we examined the effects of other IL-32 isoforms on IL-10 production. We found that IL-32δ decreased IL-10 production and investigated the inhibitory mechanism of IL-32δ. We showed that IL-32δ suppressed IL-32ß binding to PKCδ by interacting with IL-32ß. The inhibitory effect of IL-32δ on IL-32ß association with PKCδ was further verified by immuno-fluorescence staining. The co-localization of IL-32ß and PKCδ around the nuclear membrane was disrupted by IL-32δ. Our data therefore indicate that IL-32δ plays an inhibitory role against IL-32ß function, which also suggests that IL-32 may be regulated by its own isoform.