Role of mitogen-activated protein kinase in Zn-BC-AM PDT-induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn-BC-AM was found to effectively induce apoptosis in a welldifferentiated nasopharyngeal carcinoma (NPC) HK-1 cell line. Sustained activation of p38 mitogen-activated protein kinase (MAPK) and cjun N-terminal kinase (JNK) as well as a transient increase in activation of extracellular signal-regulated kinase (ERK) were observed immediately after Zn-BC-AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDTinduced apoptosis of HK-1 cells. PD169316 also prevented the loss of Bcl-2 and Bcl-xL in PDT-treated HK-1 cells. However, inhibition of JNK with SP600125 had no effect on Zn-BC-AM PDT-induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn-BC-AM PDT-induced apoptosis. Further study showed that knockdown of the p38b isoform with siRNA also increased Zn-BC-AM PDT-induced apoptosis, indicating that the anti-apoptotic effect of PD169316 in PDT-treated HK-1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38b and ERK may enhance the therapeutic efficacy of Zn-BC-AM PDTon NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.postprin

Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma

<p>Abstract</p> <p>Background</p> <p>Most patients with ovarian cancer are diagnosed with advanced stage disease (<it>i.e</it>., stage III-IV), which is associated with a poor prognosis. Differentially expressed genes (DEGs) in stage III serous ovarian carcinoma compared to normal tissue were screened by a new differential display method, the annealing control primer (ACP) system. The potential targets for markers that could be used for diagnosis and prognosis, for stage III serous ovarian cancer, were found by cluster and survival analysis.</p> <p>Methods</p> <p>The ACP-based reverse transcriptase polymerase chain reaction (RT PCR) technique was used to identify DEGs in patients with stage III serous ovarian carcinoma. The DEGs identified by the ACP system were confirmed by quantitative real-time PCR. Cluster analysis was performed on the basis of the expression profile produced by quantitative real-time PCR and survival analysis was carried out by the Kaplan-Meier method and Cox proportional hazards multivariate model; the results of gene expression were compared between chemo-resistant and chemo-sensitive groups.</p> <p>Results</p> <p>A total of 114 DEGs were identified by the ACP-based RT PCR technique among patients with stage III serous ovarian carcinoma. The DEGs associated with an apoptosis inhibitory process tended to be up-regulated clones while the DEGs associated with immune response tended to be down-regulated clones. Cluster analysis of the gene expression profile obtained by quantitative real-time PCR revealed two contrasting groups of DEGs. That is, a group of genes including: <it>SSBP1</it>, <it>IFI6 DDT</it>, <it>IFI27</it>, <it>C11orf92</it>, <it>NFKBIA</it>, <it>TNXB</it>, <it>NEAT1 </it>and <it>TFG </it>were up-regulated while another group of genes consisting of: <it>LAMB2</it>, <it>XRCC6</it>, <it>MEF2C</it>, <it>RBM5</it>, <it>FOXP1</it>, <it>NUDCP2</it>, <it>LGALS3</it>, <it>TMEM185A</it>, and <it>C1S </it>were down-regulated in most patients. Survival analysis revealed that the up-regulated genes such as <it>DDAH2, RNase K and TCEAL2 </it>might be associated with a poor prognosis. Furthermore, the prognosis of patients with chemo-resistance was predicted to be very poor when genes such as <it>RNase K, FOXP1</it>, <it>LAMB2 </it>and <it>MRVI1 </it>were up-regulated.</p> <p>Conclusion</p> <p>The DEGs in patients with stage III serous ovarian cancer were successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in this study might help predict the prognosis of patients with stage III serous ovarian cancer as well as suggest targets for the development of new treatment regimens.</p

Role of ER Stress Response in Photodynamic Therapy: ROS Generated in Different Subcellular Compartments Trigger Diverse Cell Death Pathways

We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca2+ homeostasis manifested by Ca2+ rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway

Photodynamic therapy induced production of cytokines by latent Epstein Barr virus infected epithelial tumor cells

Photodynamic therapy (PDT) is a method to treat cancer or non-cancer diseases by activation of the light-sensitive photosensitizers. Epstein Barr virus (EBV) has been implicated in the development of certain cancers such as nasopharyngeal carcinoma and B cell lymphoma. This study aims to examine the effects of EBV infection on the production of pro-inflammatory cytokines and chemokines in cells after the photosensitizer Zn-BC-AM PDT treatment. Epithelial tumor cell lines HONE-1 and latent EBV-infected HONE-1 (EBV-HONE-1) cells were used in this study. Cells were treated with the photosensitizer Zn-BC-AM for 24 hours before light irradiation. RT-PCR and quantitative ELISA methods were used for the evaluation of mRNA expression and production of cytokines, respectively. Results show that Zn-BC-AM PDT increases the production of IL-1α and IL-1β in EBV-HONE-1. Over a 10-fold increase in the production of H-6 was observed in the culture supernatant of Zn-BC-AM PDTtreated HONE-1 cells. PDT-induced IL-6 production was observed in HONE-1 cells. EBV-HONE-1 has a higher background level of IL-8 production than the HONE-1. The production of IL-8 was suppressed in EBV-HONE-1 cells after Zn-BC-AM PDT. Our results indicate that the response of HONE-1 cells to Zn-BC-AM PDT depends on the presence of latent EBV infection. Since IL-8 is a cytokine with angiogenic activity, Zn-BC-AM PDT may exert an anti-angiogenic effect through the suppression of IL-8 production by the EBV-infected cells.link_to_subscribed_fulltex

Effects of Nanoporous and Microgrooved Substrates on Cell Morphology and Cell Migration of Hepatoma Cells

Understanding the mechanism of how surface topography influences mammalian cells is important for the development of medical implants and tissue engineering. Although variety of cell types have been used in cell-substrate studies for different purposes, hepatocytes have been paid relatively less attention. In this study, we investigated the influences of nanopores and microgrooves on the morphology and migration of hepatoma cell line, BEL-7402 cells. The cells were cultured on different synthetic substrates including flat cell culture plate, flat silicon wafer sputtered with alumina, nanoporous (140 nm in diameter) anodized aluminum membrane (AAM) surface, flat polydimethylsiloxane (PDMS), and PDMS patterned with microgrooves of various widths (10 μm, 30 μm and 50 μm in width, and 2 μm in depth). Cellular behaviour on these surfaces was studied using fluorescent microscopy, time lapse microscopy and scanning electron microscopy (SEM)

Effects of Nanoporous and Microgrooved Substrates on Cell Morphology and Cell Migration of Hepatoma Cells

Understanding the mechanism of how surface topography influences mammalian cells is important for the development of medical implants and tissue engineering. Although variety of cell types have been used in cell-substrate studies for different purposes, hepatocytes have been paid relatively less attention. In this study, we investigated the influences of nanopores and microgrooves on the morphology and migration of hepatoma cell line, BEL-7402 cells. The cells were cultured on different synthetic substrates including flat cell culture plate, flat silicon wafer sputtered with alumina, nanoporous (140 nm in diameter) anodized aluminum membrane (AAM) surface, flat polydimethylsiloxane (PDMS), and PDMS patterned with microgrooves of various widths (10 μm, 30 μm and 50 μm in width, and 2 μm in depth). Cellular behaviour on these surfaces was studied using fluorescent microscopy, time lapse microscopy and scanning electron microscopy (SEM)

Biocompatibility of PDMS microgrooves and nanoporous anodized aluminum membrane to Bel-7402 cells and their effects on cell adhesion, cell spreading and cell motility

Poster no. NANO-10

Quantitative Analysis of Hepatic Cell Morphology and Migration in Response to Nanoporous and Microgrooved Surface Structures

Material surface topography is an important factor for regulating cellular behaviour. Understanding the mechanism of how surface topography influences mammalian cells is critical for the development of medical implants and tissue engineering. In this study, we investigated the influences of nanoporous and microgrooved substrates on the morphology and migration of hepatic cell line, BEL-7402 cells. Cells were cultured on nanoporous (140 nm in diameter) anodized alumina membrane (AAM), nanoporous (140 nm in diameter) polydimethylsiloxane (PDMS), and microgrooves (10 μm, 30 μm, and 50 μm in width, and 2 μm in depth) patterned PDMS, then imaged by fluorescent microscopy, time-lapse microscopy, and scanning electron microscopy (SEM). Cell morphology and migration were investigated through image analysis. The results suggest that the nanoporous and microgrooved surface structure induced totally different changes on BEL-7402 cells. Compared to the well-spread cells on the flat surface plate, the cells formed spheroids on the nanoporous AAM surface and nanoporous PDMS surface with no elongation and alignment, while the cells grew with elongated and aligned morphology along the microgrooves on the PDMS substrates. The BEL-7402 cell migration speed was significantly higher on the nanoporous substrates than on the flat surfaces. On the microgrooved PDMS substrates, the cells migrated along the groove direction and showed relatively small difference of the overall velocity compared to the cells on the flat PDMS surface. Our findings provide insights into the control of cell morphological features and migratory behaviour by using artificial nanoporous or microgrooved substrates, which can benefit the research on hepatocellular carcinoma metastasis, tissue engineering, and medical implant design.Department of Applied Physic

Comparative in vitro Cytotoxicity Study on Uncoated Magnetic Nanoparticles: Effects on Cell Viability, Cell Morphology, and Cellular Uptake

Magnetic iron oxide nanoparticles (MIONPs) must be biocompatible, and a thorough knowledge on their potential cytotoxicity is crucial for their biomedical applications. However, the detailed study about the effects of iron oxide nanoparticles on cell viability, cell morphology, and cellular uptake of different mammalian cells is still insufficient. In this paper, comparative cytotoxicity study of uncoated magnetite nanoparticles at different concentrations was performed on human cervical cancer cell line (HeLa) and immortalized normal human retinal pigment epithelial cell line (RPE). The size, structure, and magnetic behavior of the MIONPs were characterized using transmission electron microscopy (TEM), X-ray diffractometry (XRD), and vibrating sample magnetometry (VSM) respectively. After 24-hour incubation with the MIONPs, the cell viability was determined by live/dead assay, the cell morphology at high magnification was observed under scanning electron microscopy (SEM), and the cellular uptake of MIONPs was measured under TEM and verified by energydispersive X-ray spectroscopy (EDX) analysis. Our results indicate that the uncoated MIONPs at a high concentration (0.40 mg/ml) were toxic to both HeLa and RPE cells. However, the cytotoxicity of uncoated MIONPs at low concentrations was cell-type specific, and RPE cells were more susceptible to these MIONPs than HeLa cells. The effects of the MIONPs on cell morphology and the nanoparticles uptake also showed different features between these two cell lines. Hence cell type should be taken into consideration in the in vitro cytotoxicity study of uncoated MIONPs. Additionally, it should be noticed that the cell morphological changes and the uptake of nanoparticles can take place even though no toxic effect of these MIONPs at low concentrations was reflected in the traditional cell viability assay.Department of Applied Physic