Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells.

BACKGROUND: The fruit of Kochia scoparia Scharder is widely used as a medicinal ingredient for the treatment of dysuria and skin diseases in China, Japan and Korea. Especially, K. scoparia had been used for breast masses and chest and flank pain. OBJECTIVE: To investigate the anti-cancer effect of K. scoparia on breast cancer. MATERIALS AND METHODS: We investigated the anti-cancer effects of K. scoparia, methanol extract (MEKS) in vitro. We examined the effects of MEKS on the proliferation rate, cell cycle arrest, reactive oxygen species (ROS) generation and activation of apoptosis-associated proteins in MDA-MB-231, human breast cancer cells. RESULTS: MTT assay results demonstrated that MEKS decreased the proliferation rates of MDA-MB-231 cells in a dose-dependent manner with an IC50 value of 36.2 µg/ml. MEKS at 25 µg/ml significantly increased the sub-G1 DNA contents of MDA-MB-231 cells to 44.7%, versus untreated cells. In addition, MEKS induced apoptosis by increasing the levels of apoptosis-associated proteins such as cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 and cleaved Poly (ADP-ribose) polymerase (PARP). CONCLUSION: These results suggest that MEKS inhibits cell proliferation and induces apoptosis in breast cancer cells and that MEKS may have potential chemotherapeutic value for the treatment of human breast cancer.

Peptidoglycan enhances secretion of monocyte chemoattractants via multiple signaling pathways.

Peptidoglycan (PG) is detected in a high proportion in inflammatory cell-rich regions of human atheromatous plaques. In the present study, we determined the cellular factors involved in PG-mediated chemokine expression in mononuclear cells in order to understand the molecular mechanisms of inflammatory responses to bacterial pathogen-associated molecular patterns in the diseased artery. Exposure of human monocytic leukemia THP-1 cells to PG resulted in not only enhanced secretion of CCL2 and CCL4 but also profound induction of their gene transcripts, which were abrogated by oxidized 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine, an inhibitor of Toll-like receptors (TLRs)-2/4, but not by polymyxin B. PG enhanced phosphorylation of Akt and mitogen-activated protein kinases and activated protein kinase C. Pharmacological inhibitors such as SB202190, SP6001250, U0126, Akt inhibitor IV, rapamycin, and RO318220 significantly attenuated PG-mediated up-regulation of CCL2 and CCL4. We propose that PG contributes to vascular inflammation in atherosclerotic plaques by upregulating expression of mononuclear cell chemoattractants via TLR-2, protein kinase C, Akt, mTOR, and mitogen-activated protein kinases.

Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells.

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y(1), P2Y(6), and P2Y(11) receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD(+), an agonist of the human P2Y(11) receptor, and NADP(+) as well as ATP, an agonist for P2Y(1) and P2Y(11) receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD(+) and NADP(+) was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD(+) and NADP(+) activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

Cellular factors involved in CXCL8 expression induced by glycated serum albumin in vascular smooth muscle cells.

Glycated serum albumin (GSA) promotes vascular complications in diabetes. The aim of this study was to determine if GSA induces chemokine, particularly CXCL8 (IL-8), and to determine intracellular signaling pathways activated by GSA in vascular smooth muscle cells (VSMCs). GSA increased IL-8 transcription via promoter activation and enhanced CXCL8 release from VSMCs. GSA-induced promoter activation of the IL-8 gene was suppressed by dominant-negative mutants of TLR-4, MyD88, and TRIF, but not by a dominant-negative form of TLR-2. In addition, IL-8 up-regulation in response to GSA was inhibited by resveratrol, curcumin, diphenyleneiodium, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed GSA-induced promoter activation. Moreover, gene delivery of IkappaB suppressed CXCL8 release. This study suggests that GSA induces expression of IL-8 in VSMCs and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and NADPH oxidase are involved in that process.

TLR-4 agonistic lipopolysaccharide upregulates interleukin-8 at the transcriptional and post-translational level in vascular smooth muscle cells.

Despite extensive studies on cellular responses to activation of Toll-like receptor-4 (TLR-4), it is not evident weather its activation affects gene expression of interleukin-8 (IL-8) in vascular smooth muscle cells (VSMCs). Therefore, this study has investigated whether and how TLR-4 influences IL-8 expression in VSMCs. Exposure of aortic smooth muscle cells to TLR-4 agonistic lipopolysaccharide (LPS) not only enhanced release of IL-8 protein but also induced IL-8 gene transcript via promoter activation. The LPS-induced activation of IL-8 promoter was attenuated by dominant-negative MKK1, but not by dominant-negative MKK3. The promoter activation was also impaired by dominant negative CCAAT/enhancer binding protein (C/EBP), IkappaB, and dominant negative c-Jun. In comparison with the mutation of the AP-1 binding site, the mutation of NF-kappaB site and C/EBP binding site in the IL-8 promoter region more significantly impaired the promoter activation. Moreover, both promoter activity and release of IL-8 were inhibited by U0126 and curcumin, but not by SB202190, epigallocatechin 3-gallate and resveratrol. The present study reports that TLR-4-agonistic LPS upregulates IL-8 at the transcriptional and post-translational level in VSMCs, and that ERK1/2, NF-kappaB, and C/EBP play major roles in the upregulation of IL-8.

7-Ketocholesterol upregulates interleukin-6 via mechanisms that are distinct from those of tumor necrosis factor-alpha, in vascular smooth muscle cells.

This study investigated the effects of 7-ketocholesterol on interleukin (IL)-6 expression in vascular smooth muscle cells (VSMC). Among the 7 IL examined, only IL-6 transcript was increased by 7-ketocholesterol treatment in human aorta smooth muscle cells. IL-6 transcripts increased up to 24 h after treatment with 7-ketocholesterol, and this effect was profoundly repressed by treatment with p38 MAPK inhibitors and to a lesser extent JNK inhibitors. 7alpha-Hydroxycholesterol, 27-hydroxycholesterol or cholesterol, however, did not induce IL-6 expression. Mechanisms of IL-6 induction by 7-ketocholesterol were investigated in comparison with tumor necrosis factor (TNF)-alpha. Whereas TNF-alpha activated IL-6 promoter, which was impaired by p38 MAPK inhibitors or by mutation in the NF-kappaB-binding site within the promoter region, 7-ketocholesterol did not affect IL-6 promoter activity. Instead, this oxysterol slowed degradation of IL-6 mRNA and increased the amount of cytoplasmic HuR. 7-ketocholesterol significantly increased the amount of intracellular IL-6 protein in the presence of brefeldin A. 7-Ketocholesterol also enhanced IL-6 release from VSMC. IL-6 release by 7-ketocholesterol, although significant, was not as remarkable as that induced by TNF-alpha. These data suggest that 7-ketocholesterol upregulates IL-6 via mechanisms distinct from TNF-alpha and contributes to the intra- and extracellular IL-6 deposits within the vasculature.

Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells.

Toll-like receptor (TLR)-4 signaling promotes cytokine synthesis in vascular smooth muscle cells (VSMC). However, it is unknown how TLR-4 regulates interleukin-6 (IL-6) in VSMC. Therefore, the present study investigated cellular factors involved in TLR-4-mediated IL-6 in VSMC in terms of MAPK and transcription elements. Exposure of aortic smooth muscle cells to TLR4-specific lipopolysaccharide (LPS) not only enhanced IL-6 release but also induced IL-6 transcript via promoter activation. The promoter activation was attenuated by dominant-negative MKK1 and to a lesser extent by dominant-negative MKK3, but not by dominant-negative MKK4. IL-6 promoter activity was diminished by U0126 or SB202190, but not by SP600125. Co-transfection with dominant negative CCAAT/enhancer binding protein or with IkappaB suppressed LPS-induced promoter activation, whereas the promoter activity was not influenced by dominant negative c-Jun. Mutation in the IL-6 promoter region at the binding site of NF-kappaB or C/EBP impaired promoter activation in response to LPS. Further impairment occurred when both NF-kappaB- and C/EBP-binding sites were mutated. LPS-induced IL-6 promoter activation was also prevented by pretreatment with epigallocatechin 3-gallate, curcumin, and resveratrol. The present study reports that TLR4-agonistic LPS induces IL-6 through transcriptional activation in VSMC and ERK1/2, p38 MAPK, NF-kappaB, and C/EBP play active roles in that process.