Shu-Chen Hsieh

Allyl Sulfides Inhibit Cell Growth of Skin Cancer Cells through Induction of DNA Damage Mediated G2/M Arrest and Apoptosis

Diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), extracted from crushed garlic by steam-distillation, have been reported to provide the anticancer activity in several cancer types. However, their mechanisms of effects on skin cancer cells remain unclear. Therefore, we used human melanoma A375 cells and basal cell carcinoma cells as the models to elucidate the effects of these three allyl sulfides. Basal cell carcinoma (BCC) is known to be the most prevalent type of skin cancer, and melanoma is the most lethal form. We found that DATS revealed better growth inhibition of A375 and BCC cells than DADS and DAS did. We further demonstrated that DATS increased intracellular reactive oxygen species (ROS) generation, induced cytosolic Ca(2+) mobilization, and decreased mitochondrial membrane potential (DeltaPsim). Western blot results showed the concordance for the expression of molecules involved in G(2)/M arrest and apoptosis observed by cell cycle and cell viability analysis. Moreover, we detected the activation of p53 pathway in response to the oxidative DNA damage. DATS also displayed selective target of growth inhibition between skin cancer cells and normal keratinocyte HaCaT cells. Taken together, these results suggest that DATS is a potential anticancer compound for skin cancer.

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE(-/-)) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.

Curcumin protects against thioacetamide-induced hepatic fibrosis by attenuating the inflammatory response and inducing apoptosis of damaged hepatocytes

Inflammation and hepatic stellate cell (HSC) activation are the most crucial steps in the formation of hepatic fibrosis. Hepatocytes damaged by viral or bacterial infection, alcohol or toxic chemicals initiate an inflammatory response that activates collagen production by HSCs. Recent studies indicate curcumin has liver-protective effects due to its anti-inflammatory, antioxidant and anticancer activities; however, the mechanisms are not well understood. In this study, we show that curcumin protected against hepatic fibrosis in BALB/c mice in vivo by inhibiting HSC activation, inflammatory responses and inducing apoptosis of damaged hepatocytes. Using the thioacetamide (TAA)-induced hepatic fibrosis animal model, we found that curcumin treatment up-regulated P53 protein expression and Bax messenger RNA (mRNA) expression and down-regulated Bcl-2 mRNA expression. Together, these responses increased hepatocyte sensitivity to TAA-induced cytotoxicity and forced the damaged cells to undergo apoptosis. Enhancing the tendency of damaged hepatocytes to undergo apoptosis may be the protective mechanism whereby curcumin suppresses inflammatory responses and hepatic fibrogenesis. These results provide a novel insight into the cause of hepatic fibrosis and the cytoprotective effects curcumin has on hepatic fibrosis suppression.

Transcriptional upregulation of DDR2 by ATF4 facilitates osteoblastic differentiation through p38 MAPK-mediated Runx2 activation.

Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at −1150 bp in the DDR2 promoter was required for ATF4‐mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast‐specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2‐induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

p53 downstream target DDA3 is a novel microtubule-associated protein that interacts with end-binding protein EB3 and activates β -catenin pathway

We have previously identified mouse DDA3 as a p53-inducible gene. To explore the functional role of DDA3, we screened a mouse brain cDNA library by the yeast two-hybrid assay, and identified the microtubule plus-end binding protein EB3 as a DDA3-interacting protein. Binding of DDA3 to EB3 was verified by glutathione S-transferase (GST) pull-down assay and subcellular colocalization; co-immunoprecipitation further indicated that interaction of these two proteins within cells required intact microtubules. Domains of DDA3-EB3 interaction were mapped by GST pull-down assay to amino acids 118–241 and 242–329 of DDA3 and the N- and C-termini of EB3. Immunofluorescence analysis revealed colocalization of DDA3 with microtubules in various cell phases, and regions encompassing aa 118–241 and 242–329 contained microtubule-interacting and bundling activities. In vitro microtubule-binding assay showed that DDA3 and EB3 associated directly with microtubules, and cooperated with each other for microtubule binding. In addition, DDA3 bound to the EB3 interacting partner adenomatous polyposis coli 2 (APC2), a homolog of the tumor suppressor APC, which is a component of the β-catenin destruction complex. Ectopic expression of DDA3 and EB3 enhanced β-catenin-dependent transactivation and cyclin D1 production, whereas knockdown of endogenous DDA3 or EB3 inhibited β-catenin-mediated transactivation and the ability of cells to form colonies. Together, our results identify DDA3 as a novel microtubule-associated protein that binds to EB3, and implicate DDA3 and EB3 in the β-catenin-mediated growth signaling.

The therapeutic potential and mechanisms of action of quercetin in relation to lipopolysaccharide-induced sepsis in vitro and in vivo.

Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1β in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1β levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.

Conditional loss of PTEN leads to skeletal abnormalities and lipoma formation

To understand the role of tumor suppressor PTEN in cartilage development, we have generated chondrocyte specific PTEN deletion mice using Col2a1Cre and PTENloxp/loxp mice. PTEN mutant mice are viable and fertile, nonetheless, develop kyphosis over time. Histological analyses show mutant vertebrae and intervertebral discs are larger and therefore the spines are longer than in control mice. In addition, the growth plates are thicker, invading trabecular bone areas are deeper, and marrow adipocyte populations are higher in PTEN mutant mice. Furthermore, the growth plates, not normally fused in mouse long bones, are fused in PTEN mutants. Intriguingly, PTEN mice develop lipomas and show abnormal accumulation of fat tissues along spines. Cell tracking assays have confirmed that lipomas and a portion of fat tissues were derived from Col2a1Cre PTENloxp/loxp cells. Further analyses have suggested that the phenotypes of PTEN mutant likely attribute to PTENs negatively regulating role in PI3K/Akt pathway.

Human DDA3 is an oncoprotein down-regulated by p53 and DNA damage.

Mouse DDA3 (mDDA3) is a microtubule-associated protein that promotes cell growth. mDDA3 contains an intronic p53 binding motif that is absent in human DDA3 (hDDA3), and is transcriptionally activated during DNA damage in a p53-dependent way. We now report that hDDA3 mRNA and protein levels were suppressed by p53, as well as in DNA damaged cells harboring wild type, but not mutant-p53 expression. We have located three consensus El-Deiry decamers at -1478/-1403 of the hDDA3 gene, and shown by chromatin immunoprecipitation that p53 bound to the region. Luciferase analysis showed that the hDDA3 promoter containing the putative p53 binding motif was responsible for p53-mediated repression. Expression of hDDA3 decreased the cells requirement for serum, furthermore, overexpression of hDDA3 mRNA was detected in hepatoma tissues. Together our results show that hDDA3 is a p53- and DNA-damage down-regulated target that exhibits oncogenic characteristics.

Diallyl Trisulfide Induces Apoptosis of Human Basal Cell Carcinoma Cells via Endoplasmic Reticulum Stress and the Mitochondrial Pathway

Diallyl trisulfide (DATS), an active component of garlic oil, has attracted much attention because of its anticancer effect on several types of cancers. However, the mechanism of DATS-induced apoptosis of basal cell carcinoma (BCC) is not fully understood. In the present study, we revealed that DATS-mediated dose-dependent induction of apoptosis in BCC cells was associated with intracellular reactive oxygen species accumulation and disrupted mitochondrial membrane potential. Western analysis demonstrated concordant expression of molecules involved in mitochondrial apoptosis, including DATS-associated increases in phospho-p53, proapoptotic Bax, and decreases in antiapoptotic Bcl-2 and Bcl-xl in BCC cells. Moreover, DATS induced the release of cytochrome c, apoptosis-inducing factor, and HtrA2/Omi into the cytoplasm, and activated factors downstream of caspase-dependent and caspase-independent apoptosis, including nuclear translocation of apoptotic-inducing factor and endonuclease G and the caspase cascade. These results were confirmed by pretreatment with the antioxidant N-acetyl-L-cysteine and the caspase inhibitor (z-VAD-fmk), the latter of which did not completely enhance the viability of DATS-treated BBC cells. Exposure to DATS additionally induced endogenous endoplasmic reticulum stress markers and intracellular Ca2+ mobilization, upregulation of Bip/GRP78 and CHOP/GADD153, and activation of caspase-4. Our findings suggest that DATS exerts chemopreventive potential via ER stress and the mitochondrial pathway in BCC cells.

Mouse DDA3 gene is a direct transcriptional target of p53 and p73

The p53 tumor suppressor is a transcription factor that activates the expression of many target genes. We have previously reported the identification of a p53-regulated mouse gene DDA3. The 5′ upstream genomic region of the mouse DDA3 was cloned, and sequence analysis revealed the presence of a potential p53 response element (RE2) residing at nucleotides +390∼+409 relative to the first translation start site. When fused upstream to a luciferase reporter gene, 5′ genomic regions of the DDA3 gene containing RE2 were shown to be responsive to the wild-type, but not mutant p53, in a transient transfection assay. RE2 was sufficient to confer the transactivation responsiveness to p53. Furthermore, gel mobility shift analysis showed that RE2 formed specific complexes with wild-type p53. Induction of DDA3 was found in adriamycin treated normal mouse embryonic fibroblast cells (MEF), but not in p53 knockout (p53−/−) MEF. Overexpression of p73 induced DDA3 mRNA expression, and luciferase reporter analysis indicated that RE2 was responsive to transactivation by members of the p73 family proteins. Consistent with these findings, elevated expression of p73 protein and DDA3 mRNA were observed concomitantly in the p53−/− MEF cells treated with cisplatin. These results together demonstrated that DDA3 is a transcriptional target gene of p53 and its related-protein p73.