Interleukin-32θ inhibits tumor-promoting effects of macrophage-secreted CCL18 in breast cancer.

BACKGROUND: Tumor-associated macrophages can promote breast cancer metastasis by secreting cytokines and growth factors. Interleukin (IL)-32θ, a newly identified IL-32 isoform, was previously shown to down-regulate various proinflammatory factors of macrophages. Here, we report the presence of IL-32θ in breast cancer tissues and evaluate its effects on macrophage-regulated breast cancer metastasis. METHODS: RT-qPCR was used to analyze the mRNA expression of IL-32θ, Chemokine (C-C motif) ligand 18 (CCL18) in breast cancer tissues. In vitro cell-based experiments using IL-32θ-expressing MDA-MB-231 cells were conducted to examine the effects of IL-32θ on metastasis and its molecular signaling. In vivo xenograft, immunohistochemistry, and optical imaging models were generated to support in vitro and clinical findings. RESULTS: The clinical data displayed opposite expression patterns of CCL18 and IL-32θ mRNA in macrophage-infiltrated breast tumor tissues compared with those in the other tissues tested. In MDA-MB-231 cells, IL-32θ overexpression attenuated migration, invasion, tumor-promoting factors, and increased epithelial markers levels upon treatment with conditioned media from THP-1-derived macrophages. Additionally, IL-32θ expression in a xenograft model led to a remarkable decrease in tumor size and macrophage-stimulated tumor promotion. This inhibition was mediated through a direct interaction with protein kinase C-δ (PKCδ), subsequently eliminating the downstream factors STAT3 and NF-κB. Blocking CCL18 during co-culture of macrophages and breast cancer cells reduced the levels of breast cancer progression-related factors and PKCδ downstream signaling suggesting CCL18 as the main macrophage-secreted factors triggering the signaling pathway inhibited by IL-32θ. CONCLUSIONS: Our findings demonstrate a novel role of IL-32θ as an intracellular modulator to suppress macrophage-promoted breast cancer progression by targeting CCL18-dependent signaling.

Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and reactive oxygen species generation in A549 and NCI-H1299 non-small cell lung cancer cells.

BACKGROUND: Calotropis gigantea (CG) is a tall and waxy flower that is used as a traditional remedy for fever, indigestion, rheumatism, leprosy, and leukoderma. However, the precise mechanisms of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells. In this study, we investigated whether CG extract exerted an apoptotic effect in A549 and NCI-H1299 NSCLC cells. METHODS: The ethanol extract of CG was prepared, and its apoptotic effects on A549 and NCI-H1299 NSCLC cells were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining, cell cycle analysis, real-time polymerase chain reaction (RT-PCR), western blotting, JC-1 staining, and ROS detection assay. RESULTS: The CG extract induced apoptosis through the stimulation of intrinsic and extrinsic signaling pathways in A549 and NCI-H1299 lung cancer cells. Cell cycle arrest was induced by the CG extract in both cell lines. Reactive oxygen species (ROS), which can induce cell death, were also generated in the CG-treated A549 and NCI-H1299 cells. CONCLUSIONS: These data confirmed that CG caused apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cells. Thus, CG can be suggested as a potential agent for lung cancer therapy.

Epimagnolin A inhibits IL-6 production by inhibiting p38/NF-κB and AP-1 signaling pathways in PMA-stimulated THP-1 cells.

Epimagnolin A is a lignan obtained from the flower buds of Magnolia fargesii, which is traditionally used in Asian medicine for treating headache and nasal congestion. A herbal compound fargesin obtained from M. fargesii, has exerted anti-inflammatory effects in human monocytic THP-1 cells in the previous study. The anti-inflammatory effects of epimagnolin A, however, have been not elucidated yet. In this study, it was demonstrated that epimagnolin A reduced phorbol-12-myristate-13-acetate (PMA)-induced IL-6 promoter activity and IL-6 production in human monocytic THP-1 cells. Furthermore, it was investigated the modulating effects of epimagnolin A on mitogen-activated protein kinase, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) activities. Phosphorylation of p38 and nuclear translocation of p50 and c-Jun were down-regulated by epimagnolin A in the PMA-stimulated THP-1 cell. The results revealed that epimagnolin A attenuated the binding affinity of NF-κB and AP-1 transcription factors to IL-6 promoter and IL-6 production through p38/NF-kB and AP-1 signaling pathways in the PMA-stimulated THP-1 cells. These results suggest that epimagnolin A can be a useful drug for the treatment of inflammatory diseases.

Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association.

Interleukin (IL)-32θ, a newly identified IL-32 isoform, has been reported to exert pro-inflammatory effects through the association with protein kinase C delta (PKCδ). In this study, we further examined the effects of IL-32θ on IL-13 and IL-13Rα2 expression and the related mechanism in THP-1 cells. Upon stimulating IL-32θ-expressing and non-expressing cells with phorbol 12-myristate 13-acetate (PMA), the previous microarray analysis showed that IL-13Rα2 and IL-13 mRNA expression were significantly decreased by IL-32θ. The protein expression of these factors was also confirmed to be down-regulated. The nuclear translocation of transcription factors STAT3 and STAT6, which are necessary for IL-13Rα2 and IL-13 promoter activities, was suppressed by IL-32θ. Additionally, a direct association was found between IL-32θ, PKCδ, and signal transducer and activator of transcription 3 (STAT3), but not STAT6, revealing that IL-32θ might act mainly through STAT3 and indirectly affect STAT6. Moreover, the interaction of IL-32θ with STAT3 requires PKCδ, since blocking PKCδ activity eliminated the interaction and consequently limited the inhibitory effect of IL-32θ on STAT3 activity. Interfering with STAT3 or STAT6 binding by decoy oligodeoxynucleotides (ODNs) identified that IL-32θ had additive effects with the STAT3 decoy ODN to suppress IL-13 and IL-13Rα2 mRNA expression. Taken together, our data demonstrate the intracellular interaction of IL-32θ, PKCδ, and STAT3 to regulate IL-13 and IL-13Rα2 synthesis, supporting the role of IL-32θ as an inflammatory modulator.

7-Methoxy-luteolin-8-C-ß-6-deoxy-xylo-pyranos-3-uloside exactly (mLU8C-PU) isolated from Arthraxon hispidus inhibits migratory and invasive responses mediated via downregulation of MMP-9 and IL-8 expression in MCF-7 breast cancer cells.

7-Methoxy-luteolin-8-C-ß-6-deoxy-xylo-pyranos-3-uloside (mLU8C-PU) is a glycosylflavone of luteolin isolated from Arthraxon hispidus (Thunb.). Luteolin is known to exert anti-migratory and anti-invasive effects on tumor cells. However, there are no reports on the effects of mLU8C-PU on tumor invasiveness and associated signaling pathways. In this study, we demonstrated the anti-migratory and anti-invasive effects of mLU8C-PU in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 breast cancer cells. We also investigated the effect of mLU8C-PU on invasion- related signal transducers, including protein kinase Cα (PKCα), c-Jun N terminal kinase (JNK), activator protein-1 (AP-1), and nuclear factor-kappa B (NF-ĸB). TPA-induced membrane translocation of PKCα, phosphorylation of JNK, and the nuclear translocations of AP-1 and NF-κB were downregulated by mLU8C-PU in MCF-7 cells. In addition, mLU8C-PU also inhibited matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) expression. These results indicate that mLU8C-PU inhibits migratory and invasive responses in MCF-7 breast cancer cells by suppressing MMP-9 and IL-8 expression through mitigating TPA-induced PKCα, JNK activation, and the nuclear translocation of AP-1 and NF-κB. These results suggest that mLU8C-PU may be used as an anti-metastatic agent.

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-ras(G12V) and SK-HEP-1-H-ras(G12V) 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-ras(G12V) 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.

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

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.

Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBx overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression.

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.

Intracellular interaction of interleukin (IL)-32α with protein kinase Cε (PKCε ) and STAT3 protein augments IL-6 production in THP-1 promonocytic cells.

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.

A1E inhibits proliferation and induces apoptosis in NCI-H460 lung cancer cells via extrinsic and intrinsic pathways.

It has been reported that extracts from Asian traditional/medical herbs possess therapeutic agents against cancers, metabolic diseases, inflammatory diseases, and other intractable diseases. In this study, we assessed the molecular mechanisms involved in the anticancer effects of A1E, the extract of Korean medicinal herbs. We examined the role of the cytotoxic and apoptotic pathways in the cancer chemopreventive activity in non-small-cell lung cancer (NSCLC) cell lines NCI-H460 and NCI-H1299. A1E inhibited the proliferation of NCI-H460 more efficiently than NCI-H1299 (p53(-/-)) cells. The apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blot, RT-PCR, and measurement of mitochondrial membrane potential. A1E induced cellular morphological changes and nuclear condensation at 24 h in a dose-dependent manner. A1E also perturbed cell cycle progression at the sub-G1 stage and altered cell cycle regulatory factors in NCI-H460 cells. Furthermore, A1E inhibited the PI3K/Akt and NF-κB survival pathways, and it activated apoptotic intrinsic and extrinsic pathways. A1E increased the expression levels of members of the extrinsic death receptor complex FasL and FADD. In addition, A1E treatment induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), whereas the expression levels of Bcl-2 and Bcl-xl were downregulated. A1E induced mitochondrial membrane potential collapse and cytochrome C release. Our results suggest that A1E induces apoptosis via activation of both extrinsic and intrinsic pathways and inhibition of PI3K/Akt survival signaling pathways in NCI-H460 cells. In conclusion, these data demonstrate the potential of A1E as a novel chemotherapeutic agent in NSCLC.

2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) induces apoptosis via modulating E7 expression and inhibition of PI3K/Akt pathway in SiHa human cervical cancer cells.

The Maillard reaction is a chemical reaction occurring between an amino acid and a reducing sugar, usually requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects, and although 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. We found that HPB242 treatment modulated expression of cyclins and tumor suppressor genes in SiHa human cervical cancer cell lines: cyclins and phospho-pRB were downregulated, whereas the expression of CDK inhibitors and p53 was enhanced. HPB242 induced apoptosis dose-dependently by suppressing E7 expression and leading to sub-G1 cell-cycle arrest in SiHa cell lines; treatment also led to the proteolytic cleavage of caspase-3, -9, and poly (ADP-ribose) polymerase. Moreover, HPB242 upregulated Fas expression, altered expressions of pro- and antiapoptotic factors, and also inhibited nuclear translocation of nuclear factor κB and phosphorylation of IκB. HPB242 treatment decreased phosphatidyl inositol-3 kinase and p-Akt expression levels, demonstrating that this survival pathway may also be inhibited by HPB242. Cumulatively, HPB242 promotes apoptosis by influencing E7 expression, inducing cell-cycle arrest at sub-G1 phase, and promoting both intrinsic (mitochondrial) and extrinsic (Fas-dependent) apoptosis in SiHa human cervical cancer cells.

The cadin-2-en-1ß-ol-1ß-D-glucuronopyranoside suppresses TPA-mediated matrix metalloproteinase-9 expression through the ERK signaling pathway in MCF-7 human breast adenocarcinoma cells.

A sesquiterpene glycoside, cadin-2-en-1ß-ol-1ß-D-glucuronopyranoside (known as CR4-1), was isolated from Catharanthus roseus (Apocynaceae) hairy root cultures. C. roseus is widely used as an ornamental and medicinal plant and is cultivated mainly for its alkaloids. C. roseus has been reported to have pharmacologic properties such as anti-cancer, enzymatic anti-oxidant, and anti-diabetic effects. In this study, we demonstrated that CR4-1 significantly inhibited the in vitro invasion of MCF-7 human breast adenocarcinoma cells induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Matrix metalloproteinases (MMPs) are known to be involved in cancer invasion and metastasis. Zymographic analysis showed that CR4-1 suppressed TPA-induced MMP-9 activity in a dose-dependent manner. We further demonstrated that CR4-1 suppressed the phosphorylation of extracellular signal-regulated protein kinase, but not p38 kinase or c-Jun N-terminal kinase (JNK). Moreover, CR4-1 attenuated TPA-induced degradation of κBα inhibitor (IκB-α). These results suggest that CR4-1 reduces the invasiveness of human cancer cells by suppressing MMP-9 expression through inhibition of the NF-κB signaling pathways.

STK899704 inhibits stemness of cancer stem cells and migration via the FAK-MEK-ERK pathway in HT29 cells.

Colon cancer is one of the most lethal and common malignancies worldwide. STK899704, a novel synthetic agent, has been reported to exhibit anticancer effects towards numerous cancer cells. However, the effect of STK899704 on the biological properties of colon cancer, including cancer cell migration and cancer stem cells (CSCs), remains unknown. Here, we examined the inhibitory effect of STK899704 on cell migration and CSC stemness. In the wound healing assay, STK899704 significantly inhibited the motility of colon cancer cells. Furthermore, STK899704 downregulated the mRNA expression levels of the cell migration mediator focal adhesion kinase (FAK). STK899704 also suppressed mitogen-activated protein kinase kinase and extracellular signal-regulated kinase, which are downstream signaling molecules of FAK. Additionally, STK899704 inhibited stemness gene expression and sphere formation in colon cancer stem cells. These results suggest that STK899704 can be used to treat human colon cancer. [BMB Reports 2018; 51(11): 596-601].

Orientin inhibits invasion by suppressing MMP-9 and IL-8 expression via the PKCα/ ERK/AP-1/STAT3-mediated signaling pathways in TPA-treated MCF-7 breast cancer cells.

BACKGROUND: Orientin (luteolin 8-C-ß-D-glucopyranoside), a glycosyl dietary flavonoid, has therapeutic effects such as anti-inflammation and antiadipogenesis. However, there is little known about the antimigratory and anti-invasive effects of orientin. Thus, we demonstrate the anti-invasive effects of orientin compared with well-known anticancer flavonoid, luteolin and luteolin 8-C-ß-fucopyranoside (LU8C-FP). PURPOSE: We investigated whether orientin would inhibit the migration and invasion of 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced MCF-7 breast cancer cells. METHODS: We investigated the anti-invasive mechanism of orientin by using wound-healing assay, Matrigel invasion assay, gelatin zymography, qRT-PCR, ELISA, western blotting, nuclear, membrane and cytosolic fractionations, and immunofluorescence staining in MCF-7 cell line. RESULTS: We demonstrated the antimigratory and anti-invasive effects of orientin in TPA-treated MCF-7 cells. TPA-induced membrane translocation of protein kinase C alpha (PKCα), phosphorylation of extracellular signal regulated kinase (ERK), and nuclear translocations of activator protein-1 (AP-1) and signal transducer and activator of transcription 3 (STAT3) were downregulated by orientin. In addition, orientin also inhibited matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) expression. CONCLUSION: Orientin inhibits migratory and invasive responses by suppressing MMP-9 and IL-8 expression through mitigation of TPA-induced PKCα and ERK activation, as well as the nuclear translocation of AP-1 and STAT3. Therefore, orientin prevents tumor invasion and could be applied as a possible therapeutic agent for the treatment of cancer metastasis.

BCI induces apoptosis via generation of reactive oxygen species and activation of intrinsic mitochondrial pathway in H1299 lung cancer cells.

The compound (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.

Benzo[a]pyrene Alters the Expression of Genes in A549 Lung Cancer Cells and Cancer Stem Cells.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a principal component of cigarette smoke. B[a]P can cause lung carcinogenesis and plays a key role in lung cancer progression. The role of B[a]P has been reported in lung cancer, but its effects on lung cancer stem cells (CSCs) have not been investigated. Emerging evidence indicates that CSCs are associated with carcinogenesis, tumor initiation, relapse, and metastasis. Therefore, targeting CSCs to defeat cancer is a challenging issue in the clinic. This study explored whether B[a]P alters gene expression in lung cancer cells and CSCs. The lung adenocarcinoma A549 cell line was used to investigate the role of B[a]P on lung cancer cells and lung CSCs using microarray and quantitative PCR. B[a]P (1 µM) provoked gene expression changes in A549 cancer cells and CSCs by deregulating numerous genes. Gene pathway analysis was performed using GeneMANIA and GIANT. We identified genes that were coexpressed and showed physical interactions. These findings improve our understanding of the mechanism of B[a]P in lung cancer and cancer stem cells and can be an attractive therapeutic target.

(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol attenuates PMA-induced inflammatory responses in human monocytic cells through PKCδ/JNK/AP-1 pathways.

(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP), a new (E)-2,4-bis(p-hydroxyphenyl)-2 - butenal derivative, reportedly has therapeutic effects such as anti-arthritic properties. Although previous studies showed that MMPP has anti-arthritic effects on rheumatoid arthritis (RA), the anti-inflammation mechanism of MMPP remains unclear. In this study, phorbol-12-myristate 13-acetate (PMA) was used as an inflammatory stimulus to evaluate the detailed mechanism of the MMPP-mediated anti-inflammatory effect in human monocytic THP-1 cells. We investigated the effects of MMPP on inflammation-related pathways including protein kinase Cδ (PKCδ), mitogen-activated protein kinase, and activator protein-1 (AP-1). PMA induced the translocation of PKCs from the cytosol to the membrane and phosphorylated JNK. MMPP inhibited PMA-induced membrane translocation of PKCδ, phosphorylation of JNK, and nuclear translocation of AP-1, resulting in downregulation of cyclooxygenase-2 and chemokine ligand 5 production. These findings indicate that MMPP inhibits inflammatory responses in THP-1 cells by mitigating PMA-induced activation of PKCδ and JNK and nuclear translocation of AP-1. Therefore, MMPP may be useful as an anti-inflammatory drug.

The Novel Small Molecule STK899704 Promotes Senescence of the Human A549 NSCLC Cells by Inducing DNA Damage Responses and Cell Cycle Arrest.

The novel synthetic compound designated STK899704 (PubChem CID: 5455708) suppresses the proliferation of a broad range of cancer cell types. However, the details of its effect on lung cancer cells are unclear. We investigated the precise anticancer effect of STK899704 on senescence and growth arrest of A549 human non-small cell lung cancer (NSCLC) cells. STK899704 affected NSCLC cell cycle progression and decreased cell viability in a dose-dependent manner. Immunofluorescence staining revealed that STK899704 destabilized microtubules. Cell cycle analysis showed an increase in the population of NSCLC cells in the sub-G1 and G2/M phases, indicating that STK899704 might cause DNA damage via tubulin aggregation. Furthermore, we observed increased mitotic catastrophe in STK899704-treated cells. As STK899704 led to elevated levels of the p53 pathway-associated proteins, it would likely affect the core DNA damage response pathway. Moreover, STK899704 promoted senescence of NSCLC cells by inducing the p53-associated DNA damage response pathways. These findings suggest that the novel anti-proliferative small molecule STK899704 promotes cell death by inducing DNA damage response pathways and senescence after cell cycle arrest, being a potential drug for treating human lung cancers.