Yesol Bak

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.

Intracellular Interaction of Interleukin (IL)-32α with Protein Kinase Cϵ (PKCϵ) and STAT3 Protein Augments IL-6 Production in THP-1 Promonocytic Cells

Background: IL-32α is known to interact with FAK1, and IL-32α overexpression in chronic myeloid leukemia cells increases natural killer cell-mediated killing. Results: IL-32α interacted with PKCϵ and STAT3, mediated STAT3 phosphorylation, and thereby augmented IL-6 production. Conclusion: IL-32α elevated IL-6 production through interaction with PKCϵ and STAT3. Significance: The interaction of IL-32α with PKCϵ and STAT3 reveals a new intracellular mediatory role of IL-32α. IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCϵ inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCϵ, and this interaction was totally inhibited by the PKCϵ inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCϵ and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCϵ and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.

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-32δ interacts with IL-32β and inhibits IL-32β-mediated IL-10 production

There is growing evidence for multifunctional properties of IL‐32. We previously demonstrated that IL‐32β 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‐32β binding to PKCδ by interacting with IL‐32β. The inhibitory effect of IL‐32δ on IL‐32β association with PKCδ was further verified by immuno‐fluorescence staining. The co‐localization of IL‐32β and PKCδ around the nuclear membrane was disrupted by IL‐32δ. Our data therefore indicate that IL‐32δ plays an inhibitory role against IL‐32β function, which also suggests that IL‐32 may be regulated by its own isoform.

Interleukin (IL)-32β-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

Background: IL-32β promotes IL-10 production in myeloid cells. Results: IL-32β-mediated C/EBPα serine 21 phosphorylation by PKCδ induced the dissociation of C/EBPα from IL-10 promoter, thereby promoting IL-10 production. Conclusion: IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 production by mediating C/EBPα serine 21 phosphorylation by PKCδ. Significance: Our data suggest that IL-32β functions as an intracellular regulator of IL-10 production. We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β 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-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β 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-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.

Peroxiredoxin II Is Essential for Maintaining Stemness by Redox Regulation in Liver Cancer Cells.

Redox regulation in cancer stem cells (CSCs) is viewed as a good target for cancer therapy because redox status plays an important role in cancer stem‐cell maintenance. Here, we investigated the role of Peroxiredoxin II (Prx II), an antioxidant enzyme, in association with maintenance of liver CSCs. Our study demonstrates that Prx II overexpressed in liver cancer cells has high potential for self‐renewal activity. Prx II expression significantly corelated with expression of epithelial‐cell adhesion molecules (EpCAM) and cytokerain 19 in liver cancer tissues of hepatocellular carcinoma (HCC) patients. Downregulation of Prx II in Huh7 cells with treatment of siRNA reduced expression of EpCAM and CD133 as well as Sox2 in accordance with increased ROS and apoptosis, which were reversed in Huh7‐hPrx II cells. Huh7‐hPrx II cells exhibited strong sphere‐formation activity compared with mock cells. Vascular endothelial growth factor (VEGF) exposure enhanced sphere formation, cell‐surface expression of EpCAM and CD133, and pSTAT3 along with activation of VEGF receptor 2 in Huh7‐hPrx II cells. The result also emerged in Huh7‐H‐rasG12V and SK‐HEP‐1‐H‐rasG12V cells with high‐level expression of Prx II. Prx II was involved in regulation of VEGF driving cancer stem cells through VEGFR‐2/STAT3 signaling to upregulate Bmi1 and Sox2. In addition, knockdown of Prx II in Huh7‐H‐rasG12V cells showed significant reduction in cell migration in vitro and in tumorigenic potential in vivo. Taken together, all the results demonstrated that Prx II plays a key role in the CSC self‐renewal of HCC cells through redox regulation. Stem Cells 2016;34:1188–1197

IL-32θ inhibits stemness and epithelial-mesenchymal transition of cancer stem cells via the STAT3 pathway in colon cancer

Interleukin (IL)-32 is a well-known cytokine associated with inflammation, virus infections and cancer. IL-32θ is a newly identified isoform of IL-32, whose function has yet to be elucidated. In this study, we investigated IL-32θ function in colon cancer stem cells. Using samples from colon cancer patients, we found that the expression of IL-32θ mRNAs was significantly suppressed in tumor regions. We investigated the effects of IL-32θ on colon cancer. Ectopic expression of IL-32θ attenuated invasion, migration in vitro and in vivo tumorigenicity of colon cancer cells. IL-32θ inhibited epithelial-mesenchymal transition (EMT), resulting in the suppression of their migratory and invasive capabilities of HT29 colon cancer cells. In addition, IL-32θ altered various properties of CSCs, including sphere formation and expression of stemness related genes. IL-32θ directly bound to STAT3 and inhibited its nuclear translocation, leading to inhibited transcription of downstream factors, including Bmi1 and ZEB1. We showed that IL-32θ inhibited the STAT3-ZEB1 pathway and consequently inhibited key factors of stemness and EMT. Taken together, our findings reveal that IL-32θ can be a tumor suppressor, indicating that IL-32θ could possibly be used in therapies for colon cancer.

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-1β, 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.

MicroRNA-128 suppresses paclitaxel-resistant lung cancer by inhibiting MUC1-C and BMI-1 in cancer stem cells

The existence of cancer stem cells (CSCs) is the main reason for failure of cancer treatment caused by drug resistance. Therefore, eradicating cancers by targeting CSCs remains a significant challenge. In the present study, because of the important role of BMI-1 proto-oncogene, polycomb ring finger (BMI-1) and C-terminal Mucin1 (MUC1-C) in tumor growth and maintenance of CSCs, we aimed to confirm that microRNA miR-128, as an inhibitor of BMI-1 and MUC1-C, could effectively suppress paclitaxel (PTX)-resistant lung cancer stem cells. We showed that CSCs have significantly higher expression levels of BMI-1, MUC1-C, stemness proteins, signaling factors, and higher malignancy compared with normal tumor cells. After transfection with miR-128, the BMI-1 and MUC1-C levels in CSCs were suppressed. When miR-128 was stably expressed in PTX-resistant lung cancer stem cells, the cells showed decreased proliferation, metastasis, self-renewal, migration, invasive ability, clonogenicity, and tumorigenicity in vitro and in vivo and increased apoptosis compared with miR-NC (negative control) CSCs. Furthermore, miR-128 effectively decreased the levels of β-catenin and intracellular signaling pathway-related factors in CSCs. MiR-128 also decreased the luciferase activity of MUC1 reporter constructs and reduced the levels of transmembrane MUC1-C and BMI-1. These results suggested miR-128 as an attractive therapeutic strategy for PTX-resistant lung cancer via inhibition of BMI-1 and MUC1-C.

IL-32θ negatively regulates IL-1β 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‐1β 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‐1β promoter region. These findings reveal that IL‐32θ reduces PKCδ‐mediated phosphorylation of PU.1, resulting in attenuation of IL‐1β production.