Yong Kee Kim

Norepinephrine induces VEGF expression and angiogenesis by a hypoxia-inducible factor-1α protein-dependent mechanism.

A growing number of studies have demonstrated that physiological factors can influence the progression of several cancers via cellular immune function, angiogenesis and metastasis. Recently, stress‐induced catecholamines have been shown to increase the expression of various cancer progressive factors, including vascular endothelial growth factor (VEGF), matrix metalloproteinases and interleukins. However, a detailed mechanism remains to be identified. In this study, we investigated the role of adrenergic receptors and hypoxia‐inducible factor (HIF)‐1α protein in catecholamine‐induced VEGF expression and angiogenesis. Treatment of the cells with norepinephrine (NE) or isoproterenol induced VEGF expression and HIF‐1α protein amount in a dose‐dependent manner. Induction of VEGF expression by NE was abrogated when the cells were transfected with HIF‐1α–specific siRNA. Similarly, adenylate cyclase activator forskolin and cyclic AMP‐dependent protein kinase A inhibitor H‐89 enhanced and decreased HIF‐1α protein amount, respectively. More importantly, conditioned medium of NE‐stimulated cancer cells induced angiogenesis in a HIF‐1α protein–dependent manner. In addition, pretreatment of cells with propranolol, a β‐adrenergic receptor (AR) blocker, completely abolished induction of VEGF expression and HIF‐1α protein amount by NE in all of the tested cancer cells. However, treatment with the α1‐AR blocker prazosin inhibited NE‐induced HIF‐1α protein amount and angiogenesis in SK‐Hep1 and PC‐3 but not MDA‐MB‐231 cells. Collectively, our results suggest that ARs and HIF‐1α protein have critical roles in NE‐induced VEGF expression in cancer cells, leading to stimulation of angiogenesis. These findings will help to understand the mechanism of cancer progression by stress‐induced catecholamines and design therapeutic strategies for cancer angiogenesis.

Activation of Hypoxia-Inducible Factor-1α Is Necessary for Lysophosphatidic Acid–Induced Vascular Endothelial Growth Factor Expression

Purpose: Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). In the present study, we investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF. Experimental Design: Immunoblotting, reverse transcription-PCR, ELISA, immunofluorescence, and chromatin immunoprecipitation assay were used to examine the expression of VEGF and HIF-1α in various cancer cells. Specific HIF-1α small interfering RNA was transfected to various cancer cells to determine the role of HIF-1α in LPA-induced VEGF expression. Results: LPA induced expressions of VEGF and HIF-1α in OVCAR-3, CAOV-3, PC-3, and SK-Hep1 cells but not in SKOV-3 and Hep-3B cells. In OVCAR-3 and PC-3 cells, the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin/p70S6K and p42/p44 mitogen-activated protein kinase pathways were required for LPA-induced HIF-1α and VEGF expressions, whereas only the phosphoinositide 3-kinase/mammalian target of rapamycin/p70S6K pathway was important in SK-Hep1 cells. Immunofluorescence microscopy assay showed translocation of HIF-1α to nucleus by LPA, and chromatin immunoprecipitation assay revealed the binding of HIF-1α to the promoter of VEGF by LPA. Importantly, we found that small interfering RNA–induced reduction of HIF-1α expression significantly attenuated VEGF expression by LPA. Conclusions: Our results show for the first time that LPA induces VEGF via HIF-1α activation and reveal a critical role of HIF-1α in LPA-induced cancer cell proliferation and angiogenesis.

The Rho/ROCK pathway for lysophosphatidic acid-induced proteolytic enzyme expression and ovarian cancer cell invasion

Lysophosphatidic acid (LPA) is a biolipid that has diverse biological activities implicated in ovarian cancer initiation and progression. Previous studies have shown the critical role of the Rho/Rho-associated kinase (ROCK) pathway in LPA-induced ovarian cancer progression. However, detailed underlying mechanism by which the Rho/ROCK pathway induces ovarian cancer cell invasion is still incompletely understood. In the present study, we observed that the Rho/ROCK pathway is implicated in the production of proteolytic enzymes, leading to LPA-induced ovarian cancer cell invasion. LPA induced matrix metalloproteinase (MMP)-9 expression in CAOV-3 and PA-1 cells and urokinase-type plasminogen activator (uPA) expression in SKOV-3 cells. LPA-induced proteolytic enzyme expression was required for the invasion of ovarian cancer cells expressing corresponding enzymes. Pretreatment of cells with a pharmacological inhibitor of Rho/ROCK (Y-27632) or overexpression of a dominant-negative mutant of Rho (Rho N19) profoundly inhibited LPA-induced proteolytic enzyme expression as well as the invasive potential of ovarian cancer cells. In addition, transfection with dominant-negative Ras (Ras N17) significantly inhibited LPA-induced Rho activation as well as MMP-9 and uPA expression. Consistently, Y-27632 reduced LPA-induced nuclear factor (NF)-κB activation that is critical for proteolytic enzyme expression and cellular invasion. Collectively, we demonstrate a mechanism by which LPA promotes ovarian cancer progression through coordinate activation of a Ras/Rho/ROCK/NF-κB signaling pathway and the proteolytic enzyme secretion, providing novel biomarkers and promising therapeutic targets for ovarian cancer cell progression.

Lysophosphatidic acid induces STAT3 phosphorylation and ovarian cancer cell motility: their inhibition by curcumin.

Lysophosphatidic acid (LPA) is a biolipid that stimulates tumor cell invasion and metastasis. In this report, we determined the role of signal transducers and activators of transcription 3 (STAT3) and the effect of a chemopreventive agent, curcumin, on LPA-induced ovarian cancer cell motility. LPA phosphorylated STAT3 in a dose-dependent manner. Treatment of cells with a JAK/STAT inhibitor, AG490, inhibited LPA-induced cell motility. In contrast, transfection of a constitutively active form of STAT3 induced ovarian cancer cell motility. LPA also stimulated interleukin (IL)-6 and IL-8 secretion, which results in STAT3 phosphorylation. Treatment of the cells with curcumin inhibited LPA-induced IL-6 and IL-8 secretion and STAT3 phosphorylation, leading to blocked ovarian cancer cell motility. Collectively, the present study shows the critical role of STAT3 in ovarian cancer cell motility and that this process can be prevented by curcumin.

Expression of p21 WAF1/Cip1 through Sp1 sites by histone deacetylase inhibitor apicidin requires PI 3-kinase-PKCe signaling pathway

We previously reported that the activation of p21WAF1/Cip1 transcription by histone deacetylase inhibitor apicidin was mediated through Sp1 sites and pointed to the possible participation of protein kinase C (PKC). In this study, we investigated the role and identity of the specific isoforms of PKC involved and identified phosphatidylinositol 3-kinase (PI 3-kinase) as an upstream effector in HeLa cells. Using an isoform-specific pharmacological inhibitor of PKC, a PKCɛ dominant-negative mutant, and antisense oligonucleotide to inhibit PKCɛ specifically, we found that among PKC isoforms, PKCɛ was required for the p21WAF1/Cip1 expression by apicidin. In addition to PKCɛ, PI 3-kinase appeared to participate in the activation of p21WAF1/Cip1 promoter by apicidin, since inactivation of PI 3-kinase either by transient expression of dominant-negative mutant of PI 3-kinase or its specific inhibitors, LY294002 and wortmannin, attenuated the activation of p21WAF1/Cip1 promoter and p21WAF1/Cip1 protein expression by apicidin. Furthermore, membrane translocation of PKCɛ in response to apicidin was blocked by the PI 3-kinase inhibitor, indicating the role of PI 3-kinase as an upstream molecule of PKCɛ in the p21WAF1/Cip1 promoter activation by apicidin. However, the p21WAF1/Cip1 expression by apicidin appeared to be independent of the histone hyperacetylation, since apicidin-induced histone hyperacetylation of p21WAF1/Cip1 promoter region was not affected by inhibition of PI 3-kinase and PKC, suggesting that the chromatin remodeling through the histone hyperacetylation alone might not be sufficient for the expression of p21WAF1/Cip1 by apicidin. Taken together, these results suggest that the PI 3-kinase–PKCɛ signaling pathway plays a pivotal role in the expression of the p21WAF1/Cip1 by apicidin.

Sargaquinoic acid and sargahydroquinoic acid from Sargassum yezoense stimulate adipocyte differentiation through PPARα/γ activation in 3T3-L1 cells

We screened active compounds from natural marine products able to increase PPARα/γ transcriptional activity. Sargaquinoic acid (SQA) and sargahydroquinoic acid (SHQA) from Sargassum yezoense were identified as novel PPARα/γ dual agonists. The binding affinity of SQA with PPARγ was higher than that of the specific PPARγ agonist troglitazone, leading to an activation of PPARγ transcriptional activity. In parallel, treatment of 3T3‐L1 cells with SQA and SHQA led to an increase in adipocyte differentiation and increased expression of adipogenic marker genes such as aP2, PPARγ, resistin, adiponectin, C/EBPα and Glut4. Collectively, our data suggest that SQA and SHQA are novel PPARα/γ dual agonists and may be beneficial for reducing insulin resistance through regulation of adipogenesis.

Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-κB

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner, without alteration of the catalytic activity of COX‐2 itself. Ergolide decreased the level of iNOS and COX‐2 protein, and iNOS mRNA caused by stimulation of LPS/IFN‐γ in a concentration‐dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor‐κB (NF‐κB) activation, a transcription factor necessary for iNOS and COX‐2 expression in response to LPS/IFN‐γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF‐κB as well as IκB‐α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF‐κB itself or an upstream molecule of NF‐κB. Ergolide also directly inhibited the DNA‐binding activity of active NF‐κB in LPS/IFN‐γ‐pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF‐κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF‐κB resulted from blockade of the degradation of IκB and the direct modification of active NF‐κB, leading to the suppression of the expression of iNOS and COX‐2, which play important roles in inflammatory signalling pathway.

Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA-induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.

Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-κB activation

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.

Autotaxin promotes motility via G protein-coupled phosphoinositide 3-kinase γ in human melanoma cells

Autotaxin (ATX), an exo‐nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub‐nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein‐coupled phosphoinositide 3‐kinase γ (PI3Kγ) in ATX‐mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX‐induced motility. ATX increased the PI3K activity in p110γ, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kγ, strongly indicating the crucial role of PI3Kγ for ATX‐mediated motility in human melanoma cells