Yi-Ju Lee

L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines

Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy.

Increased PD-1 and decreased CD28 expression in chronic hepatitis B patients with advanced hepatocellular carcinoma.

Background/aims: Hepatitis B infection is a well‐known cause of hepatocellular carcinoma (HCC). This study aims to investigate the role that the co‐stimulatory molecule CD28 and co‐inhibitory molecule programmed death‐1 (PD‐1) play in compromising the function of tumour‐infiltrating lymphocytes (TIL) in hepatitis B virus (HBV)‐related HCC.

ZAK inhibits human lung cancer cell growth via ERK and JNK activation in an AP-1-dependent manner.

Novel mixed‐lineage kinase protein zipper sterile‐α‐motif kinase (ZAK) was first cloned by our laboratory. Lung cancer is the leading cause of cancer‐related death in the world, including in Taiwan. Here, we wanted to investigate whether ZAK plays a potential role in lung cancer development. First, Western blot analysis results demonstrated that four cell lines expressed high levels of ZAK from among a panel of 10 lung cancer cell lines, and two of three normal lung cells expressed ZAK. ZAK gene expressions were down‐regulated in lung cancers by real‐time PCR analysis. Overexpression of ZAK suppressed cell proliferation in parallel with increased phosphorylated levels of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK). In contrast, ZAK silencing cells inhibited the expressions of phosphorylated ERK and JNK without affecting the expression of phosphorylated p38. The effect of the decreased cell growth rate was significantly but incompletely reversed when ZAK‐overexpressing cells were treated with a specific ERK or JNK inhibitor. Moreover, c‐Fos and c‐Jun, the major downstream components of MAPKs, were up‐regulated by ERK and JNK, respectively. When ZAK‐overexpressing cells introduced with c‐Jun RNA interference (RNAi), the activator protein‐1 (AP‐1) transcription activity detected by a secreted alkaline phosphatase (SEAP) assay was suppressed and the decreased cell number was reversed compared with the control RNAi‐treated group. More importantly, ZAK significantly depressed tumor growth in in vivo study. Taken together, results from both in vitro and in vivo studies indicated that the decrease of lung cancer cell proliferation by ZAK may involve the ERK and JNK pathways via an AP‐1 transcription factor. (Cancer Sci 2010)

Hepatitis delta virus epigenetically enhances clusterin expression via histone acetylation in human hepatocellular carcinoma cells.

Both isoforms of the hepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) are highly associated with virus proliferation and may act as co-activators of cellular gene expression. Human hepatocellular carcinoma (HCC) cell line Huh7, which stably expresses HDAgs, was differentially screened and the results showed that clusterin gene expression was enhanced. The mechanisms for HDAg-mediated clusterin gene upregulation were investigated. Expression of HDAgs was associated with enhanced histone H3 acetylation within the clusterin promoter in a chromatin immunoprecipitation assay. Transient transfection of HDAg-expressing plasmids into Huh7 cells also enhanced clusterin expression and histone acetylation. Furthermore, HDV replication was associated with histone hyperacetylation and clusterin induction. The effect of increased clusterin expression was determined by a chemosensitivity assay with adriamycin treatment. These data indicated that HDV-induced clusterin protein increases cell survival potential. Thus, it is possible that epigenetic regulation by HDV contributes to a pathological outcome of hepatitis D/hepatitis B viral hepatitis and HCC.

Curcumin Protects against UVB-Induced Skin Cancers in SKH-1 Hairless Mouse: Analysis of Early Molecular Markers in Carcinogenesis

Curcumin (CUR) has been shown to possess a preventive effect against various cancers and interfere with multiple-cell signaling pathways. We evaluated the protective effects of CUR in regression of UVB-induced skin tumor formation in SKH-1 hairless mice and its underlying early molecular biomarkers associated with carcinogenesis. Mice irradiated with UVB at 180 mJ/cm2 twice per week elicited 100% tumor incidence at 20 weeks. Topical application of CUR prior to UVB irradiation caused delay in tumor appearance, multiplicity, and size. Topical application of CUR prior to and immediately after a single UVB irradiation (180 mJ/cm2) resulted in a significant decrease in UVB-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase in p53 and p21/Cip1-positive cell population in epidermis. Simultaneously, CUR also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels. The results suggest that the protective effect of CUR against photocarcinogenesis is accompanied by downregulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-κB, and of inflammatory responses involving COX-2, PGE2, and NO, while upregulation of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.

Extracellular Matrix Controls Insulin Signaling in Mammary Epithelial Cells Through the RhoA/Rok Pathway

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin‐stimulated tyrosine phosphorylation of insulin receptor substrate‐1 (IRS‐1) and association of PI3K with IRS‐1, whereas cells cultured on stromal ECM are inefficient in transducing these post‐receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS‐1 are greater in these cells, leading to serine phosphorylation of IRS‐1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS‐1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum‐mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin‐mediated cellular differentiation and survival would be defective. J. Cell. Physiol. 220: 476–484, 2009.

The fungal metabolite, citrinin, inhibits lipopolysaccharide/interferon-γ-induced nitric oxide production in glomerular mesangial cells.

The mycotoxin, citrinin (CTN), is a secondary metabolite of the fermented products of Monascus. The mycotoxin can either suppress or stimulate immune responses. In the present study, the immunomodulatory role of CTN in nitric oxide (NO) production, a proinflammatory mediator in the process of inflammation, was investigated. NO is well known as a mediator of immune responses. Overproduction of NO catalyzed by inducible nitric oxide synthase (iNOS) protects host cells against microbial invasion, while aberrant iNOS induction is associated with the pathophysiology of inflammatory events. Herein, we report that CTN significantly suppressed lipopolysaccharide (LPS)/interferon (IFN)-γ-induced NO production in MES-13 cells, a glomerular mesangial cell line. The percentage of NO reduction caused by CTN was far greater than that of the decline in cell viability. CTN decreased iNOS gene and protein expressions in concentration-dependent manners. CTN caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation. Furthermore, LPS/IFN-γs induction of interferon response factor-1 (IRF-1) mRNA expression was inhibited by CTN. Moreover, CTN attenuated IκB-α phosphorylation and reduced NF-κBs translocation to the nuclear fraction. Taken together, our data indicated that CTN significantly suppressed NO and iNOS expressions in MES-13 cells via inhibition of the JAK/STAT-1α and NF-κB signaling pathways.

Effects of human parvovirus B19 VP1 unique region protein on macrophage responses

BackgroundActivity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage.MethodsTo investigate the roles of B19-VP1u in response to macrophage, phospholipase A2 activity, cell migration assay, phagocytosis activity, metalloproteinase assay, RT-PCR and immunoblotting were performed.ResultsIn the present study, we report that migration, phagocytosis, IL-6, IL-1β mRNA, and MMP9 activity are significantly increased in RAW264.7 cells by B19-VP1u protein with sPLA2 activity, but not by B19-VP1uD175A protein that is mutated and lacks sPLA2 activity. Additionally, significant increases of phosphorylated ERK1/2 and JNK proteins were detected in macrophages that were treated with B19-VP1u protein, but not when they were treated with B19-VP1uD175A protein.ConclusionTaken together, our experimental results suggest that B19-VP1u with sPLA2 activity affects production of IL-6, IL-1β mRNA, and MMP9 activity, possibly through the involvement of ERK1/2 and JNK signaling pathways. These findings could provide clues in understanding the role of B19-VP1u and its sPLA2 enzymatic activity in B19 infection and B19-related diseases.

TGF‐β inhibits prolactin‐induced expression of β‐casein by a Smad3‐dependent mechanism

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor, affecting cell proliferation, apoptosis, and extracellular matrix homeostasis. It also plays critical roles in mammary gland development, one of which involves inhibition of the expression of milk proteins, such as β‐casein, during pregnancy. Here we further explore the underlying signaling mechanism for it. Our results show that TGF‐β suppresses prolactin‐induced expression of β‐casein mRNA and protein in primary mouse mammary epithelial cells, but its effect on protein expression is more evident. We also find out that this inhibition is not due to the effect of TGF‐β on cell apoptosis. Furthermore, inhibition of TGF‐β type I receptor kinase activity by a pharmacological inhibitor SB431542 or overexpression of dominant negative Smad3 substantially restores β‐casein expression. By contrast, inhibition of p38 and Erk that are known to be activated by TGF‐β does not alleviate the inhibitory effect of TGF‐β. These results are consistent with our other observation that Smad but not MAPK pathway is activated by TGF‐β in mammary epithelial cells. Lastly, we show that prolactin‐induced tyrosine phosphorylation of Jak2 and Stat5 as well as serine/threonine phosphorylation of p70S6K and S6 ribosomal protein are downregulated by TGF‐β, although the former event requires considerably long exposure to TGF‐β. We speculate that these events might be involved in repressing transcription and translation of β‐casein gene, respectively. Taken together, our results demonstrate that TGF‐β abrogates prolactin‐stimulated β‐casein gene expression in mammary epithelial cells through, at least in part, a Smad3‐dependent mechanism. J. Cell. Biochem. 104: 1647–1659, 2008.

Transglutaminase 2 contributes to apoptosis induction in jurkat t cells by modulating Ca2+ homeostasis via cross-linking RAP1GDS1

Background Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet. Results Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca2+ uptake. Ca2+-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca2+ release from the endoplasmic reticulum via both Ins3P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca2+uptake. Conclusions Our data indicate that TG2 might act as a Ca2+ sensor to amplify endoplasmic reticulum-derived Ca2+ signals to enhance mitochondria Ca2+ uptake. Since enhanced mitochondrial Ca2+ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca2+ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca2+ homeostasis.