ABSTRACT
BACKGROUND: Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. METHODS: A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. RESULTS: A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. CONCLUSIONS: Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.
Subject(s)
Human papillomavirus 16/drug effects , Neoplastic Stem Cells/drug effects , Papillomavirus Infections/drug therapy , Plant Extracts/therapeutic use , Uterine Cervical Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement/drug effects , Epithelial-Mesenchymal Transition/drug effects , Female , Humans , Plants, Medicinal/chemistryABSTRACT
H9 is an ethanol extract prepared from nine traditional/medicinal herbs. This study was focused on the anticancer effect of H9 in non-small-cell lung cancer cells. The effects of H9 on cell viability, apoptosis, mitochondrial membrane potential (MMP; Δφm), and apoptosis-related protein expression were investigated in A549 human lung cancer cells. In this study, H9-induced apoptosis was confirmed by propidium iodide staining, expression levels of mRNA were determined by reverse transcriptase polymerase chain reaction, protein expression levels were checked by western blot analysis, and MMP (Δφm) was measured by JC- 1 staining. Our results indicated that H9 decreased the viability of A549 cells and induced cell morphological changes in a dose-dependent manner. H9 also altered expression levels of molecules involved in the intrinsic signaling pathway. H9 inhibited Bcl-xL expression, whereas Bax expression was enhanced and cytochrome C was released. Furthermore, H9 treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of poly(ADPribose) polymerase; the MMP was collapsed by H9. However, the expression levels of extrinsic pathway molecules such as Fas/FasL, TRAIL/TRAIL-R, DR5, and Fas-associated death receptor were downregulated by H9. These results indicated that H9 inhibited proliferation and induced apoptosis by activating intrinsic pathways but not extrinsic pathways in human lung cancer cells. Our results suggest that H9 can be used as an alternative remedy for human non-small-cell lung cancer.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Plant Extracts/pharmacology , Plants, Medicinal/chemistry , Apoptosis/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Caspase 3/genetics , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , fas Receptor/genetics , fas Receptor/metabolismABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Biphenyl Compounds/pharmacology , Lignans/pharmacology , PPAR gamma/agonists , PPAR gamma/metabolism , Signal Transduction/drug effects , Antineoplastic Agents/pharmacology , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Female , Humans , Mitochondria/metabolism , PPAR gamma/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Uterine Cervical Neoplasms/drug therapyABSTRACT
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.
Subject(s)
Interleukin-1beta/biosynthesis , Interleukins/metabolism , Protein Kinase C-delta/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Enzyme Activation/drug effects , HEK293 Cells , Humans , Interleukin-1beta/genetics , Phorbol Esters/pharmacology , Phosphorylation/drug effects , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Transcriptional Activation/drug effectsABSTRACT
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.
Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/genetics , CD18 Antigens/biosynthesis , Interleukins/metabolism , Proto-Oncogene Proteins/biosynthesis , STAT3 Transcription Factor/metabolism , Trans-Activators/biosynthesis , Amino Acid Substitution , CCAAT-Enhancer-Binding Protein-alpha/biosynthesis , CD11 Antigens , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation , HEK293 Cells , Humans , Myeloid Cells/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Binding , STAT3 Transcription Factor/genetics , Signal Transduction/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transcription, GeneticABSTRACT
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.
Subject(s)
Interleukins/metabolism , HEK293 Cells , Humans , Protein Interaction Maps , Protein Isoforms/metabolism , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/metabolism , Interleukin-10/biosynthesis , Interleukins/metabolism , Protein Kinase C-delta/metabolism , Base Sequence , CCAAT-Enhancer-Binding Protein-alpha/chemistry , Enzyme Activation/drug effects , HEK293 Cells , Humans , Interleukin-10/genetics , Interleukins/chemistry , Molecular Sequence Data , Phosphorylation/drug effects , Phosphoserine/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Interaction Mapping , Protein Structure, Tertiary , Protein Transport/drug effects , Tetradecanoylphorbol Acetate/pharmacology , U937 CellsABSTRACT
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.
Subject(s)
Down-Regulation , Interleukin-10/metabolism , Interleukins/metabolism , HEK293 Cells , Humans , Interleukin-10/genetics , Interleukins/genetics , Nuclear Envelope/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C-delta/metabolism , U937 CellsABSTRACT
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.
