Curcumin enhances the anti‐cancer effects of Paris Saponin II in lung cancer cells

Objectives

To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer.

Materials and Methods

The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells.

Results

The combination displayed a synergistic anti‐cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF‐α to induce cancer cells apoptosis, and up‐regulated IGFBP‐1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI‐H446 cells. Meanwhile, the combination suppressed PCNA and NF‐κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI‐H460 and NCI‐H446 cells, enhanced the phosphorylation of JNK in NCI‐H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI‐H520 cells.

Conclusions

PSII combined with CUR had a synergistic anti‐cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

     

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid

Human lung neoplasms frequently express mutations that down‐regulate expression of various tumor suppressor molecules, including mitogen‐activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti‐tumor therapies. We now report that 4‐phenyl‐3‐butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non‐cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell‐cell communication. H2009 human lung carcinoma cells and ras‐transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras‐transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell–cell communication and connexin 43 phosphorylation in ras‐transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up‐regulation of p38 MAPK activity, altered connexin 43 expression, and down‐regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down‐regulated p38 MAPK activity and/or activated JNK and altered cell–cell communication. J. Cell. Biochem. 113: 269–281, 2012. © 2011 Wiley Periodicals, Inc.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Activation and induction of cytosolic phospholipase A2 by IL‐1β in human tracheal smooth muscle cells: Role of MAPKs/p300 and NF‐κB

Cytosolic phospholipase A₂ (cPLA₂) plays a pivotal role in mediating agonist‐induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL‐1β. However, the mechanisms underlying IL‐1β‐induced cPLA₂ expression and PGE₂ synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL‐1β‐induced cPLA₂ protein and mRNA expression, PGE₂ production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL‐1β‐induced cPLA₂ expression was also inhibited by pretreatment with a NF‐κB inhibitor, helenalin or transfection with siRNA of NIK, IKKα, or IKKβ. IL‐β‐induced NF‐κB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA₂ expression induced by IL‐1β. Moreover, p300 was associated with the cPLA₂ promoter, which was dynamically linked to histone H4 acetylation stimulated by IL‐1β. These results suggest that in HTSMCs, activation of MAPKs, NF‐κB, and p300 are essential for IL‐1β‐induced cPLA₂ expression and PGE₂ secretion. J. Cell. Biochem. 109: 1045–1056, 2010. © 2010 Wiley‐Liss, Inc.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.     

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion

Sjögren's syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögren's syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH₂‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10^?4 m . In vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögren's syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.     

MLK3 is a novel target of dehydroglyasperin D for the reduction in UVB‐induced COX‐2 expression in vitro and in vivo

Dehydroglyasperin D (DHGA‐D), a compound present in licorice, has been found to exhibit anti‐obesity, antioxidant and anti‐aldose reductase effects. However, the direct molecular mechanism and molecular targets of DHGA‐D during skin inflammation remain unknown. In the present study, we investigated the effect of DHGA‐D on inflammation and its mechanism of action on UVB‐induced skin inflammation in HaCaT human keratinocytes and SKH‐1 hairless mice. DHGA‐D treatment strongly suppressed UVB‐induced COX‐2 expression, PGE2 generation and AP‐1 transactivity in HaCaT cells without affecting cell viability. DHGA‐D also inhibited phosphorylation of the mitogen‐activated protein kinase kinase (MKK) 3/6/p38, MAPK/Elk‐1, MKK4/c‐Jun N‐terminal kinase (JNK) 1/2/c‐Jun/mitogen, and stress‐activated protein kinase (MSK), whereas phosphorylation of the mixed‐lineage kinase (MLK) 3 remained unaffected. Kinase and co‐precipitation assays with DHGA‐D Sepharose 4B beads showed that DHGA‐D significantly suppressed MLK3 activity through direct binding to MLK3. Knockdown of MLK3 suppressed COX‐2 expression as well as phosphorylation of MKK4/p38 and MKK3/6/JNK1/2 in HaCaT cells. Furthermore, Western blot assay and immunohistochemistry results showed that DHGA‐D pre‐treatment significantly inhibits UVB‐induced COX‐2 expression in vivo. Taken together, these results indicate that DHGA‐D may be a promising anti‐inflammatory agent that mediates suppression of both COX‐2 expression and the MLK3 signalling pathway through direct binding and inhibition of MLK3.     

S. aureus haemolysin A‐induced IL‐8 and IL‐6 release from human airway epithelial cells is mediated by activation of p38‐ and Erk‐MAP kinases and additional,cell type‐specific signalling mechanisms

Soluble virulence‐associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o‐, S9 or A549 cells with recombinant Hla (rHla). In a dose‐dependent manner, rHla induced secretion of IL‐8 in all three cell types, but IL‐6 release only in 16HBE14o‐ and S9 cells. rHla‐mediated secretion of IL‐8 and IL‐6 was suppressed by pre‐incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL‐8 release in all three cell types and for IL‐6 release in 16HBE14o‐ and S9 cells. The rHla‐mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca²⁺]_i, an early response in rHla‐treated cells. Inhibitors of calmodulin or calcium/calmodulin‐dependent kinase II attenuated rHla‐mediated release of IL‐8 in 16HBE14o‐ and A549 cells and of IL‐6 in 16HBE14o‐ cells. This indicates that rHla may mediate simultaneous activation of calmodulin‐dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin‐dependent signalling did not affect rHla‐induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

Proteomic differential display analysis identified upregulated astrocytic phosphoprotein PEA‐15 in human malignant pleural mesothelioma cell lines

We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2‐DE and LC‐MS/MS. The human MPM cell lines were NCI‐H28, NCI‐H2052 and NCI‐H2452, and the human pleural mesothelial cell line was MeT‐5A. Between MeT‐5A and NCI‐H2052, we found 38 protein spots whose expression levels were different, from the results of 2‐DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI‐H2052 than in MeT‐5A. These spots were analyzed by LC‐MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2‐DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA‐15, in all three MPM cell lines. Western blotting using specific antibodies against PEA‐15 confirmed the elevated expression level of PEA‐15 in all three MPM cell lines compared with MeT‐5A cells and normal pleura tissues from patients. PEA‐15 was knocked down in NCI‐H2052 cells, and the proliferation of PEA‐15‐silenced NCI‐H2052 cells was suppressed 7–15% compared with negative control cells. These results suggest that PEA‐15 expression is likely to be associated with the tumorigenesis of MPM.     

Macrophage-induced expression and release of matrix metalloproteinase 1 and 3 by human preadipocytes is mediated by IL-1β via activation of MAPK signaling

Obesity is associated with a chronic low‐grade inflammation and increased macrophage infiltration in adipose tissue. Matrix metalloproteinases (MMPs) are involved in adipose tissue remodeling and inflammatory responses in obesity. This study investigated whether macrophage‐derived factors modulate expression and secretion of MMP1 and MMP3 in human preadipocytes. The potential mediators and signaling pathways were also explored. MMP1 and MMP3 were primarily expressed and secreted by preadipocytes and dramatically reduced post‐differentiation. Preadipocytes were incubated with RPMI 1640 medium (control) or THP‐1 macrophage‐conditioned (MC) medium (25% and 100%) for 24 h. MC medium markedly increased mRNA levels of MMP1 (up to 122‐fold) and MMP3 (up to 59‐fold), as well as protein release of MMP1 (up to 378‐fold) and MMP3 (up to 10‐fold) in a dose‐dependent manner. Treatment with IL‐1β or TNFα, the major products of macrophages, also induced MMP1 and MMP3 secretion by preadipocytes. Neutralizing IL‐1β abolished the induction of MMP1 and MMP3 in preadipocytes by MC medium while the effects of TNFα neutralization were modest. Furthermore, MC medium or IL‐1β led to the phosphorylation of p38, ERK and JNK MAPKs. Inhibition of p38, ERK and JNK reversed the stimulatory effects of MC or IL‐1β on MMP1 and MMP3 production. MC medium and IL‐1β also activated NF‐κB p65 whereas reduced IκBα protein expression in preadipocytes. These results suggest that macrophage accumulation in adipose tissue has a central role in stimulating MMP1 and MMP3 production by preadipocytes, and this is partially mediated by IL‐1β via activation of the MAPK and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2869–2880, 2011. © 2011 Wiley‐Liss, Inc.     

Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.     

Cirsium japonicum var. maackii and apigenin block Hif‐2α‐induced osteoarthritic cartilage destruction

Although Hif‐2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif‐2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif‐2α‐induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL‐1β‐, IL‐6, IL‐17‐ and TNF‐α‐induced up‐regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX‐2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif‐2α, which directly up‐regulates MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif‐2α expression and inhibited Hif‐2α‐induced MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression in articular chondrocytes. IL‐1β induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif‐2α expression, was completely blocked by apigenin in a concentration‐dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif‐2α inhibitors.