Dae Hwa Choi

Heat shock protein 27 is associated with irinotecan resistance in human colorectal cancer cells

Heat shock protein (Hsp) in tumor cells has been proposed to enhance their resistance to chemotherapeutic agents. In the present study, we investigated the influence of Hsp expression on the irinotecan resistance of human colorectal cancer cells. Among eight Hsp genes tested in this study, we confirmed that the expression of Hsp27 correlated with irinotecan resistance in colorectal cancer cells. Specific inhibition of Hsp27 expression using an antisense oliogodeoxynucleotide increased the irinotecan sensitivity. On the contrary, an overexpression of Hsp27 decreased the irinotecan sensitivity in colorectal cancer cells. Elevated expression of Hsp27 decreased caspase‐3 activity in colorectal cancer cells. The expression level of Hsp27 determined by immunohistochemical analysis correlated with the clinical response to irinotecan in colorectal cancer patients. Hsp27 expression levels of irinotecan‐non‐responder (mean staining score, 6.28; proportion of high staining score, 64.2%) were significantly higher compared to those of irinotecan‐responder (mean staining score, 3.16; proportion of high staining score, 33.3%) (P for t‐test = 0.045). These findings suggest that Hsp27 is involved in the irinotecan resistance of colorectal cancer cells possibly by reducing caspase‐3 activity.

Tristetraprolin regulates expression of VEGF and tumorigenesis in human colon cancer

Tristetraprolin (TTP) is an AU‐rich element‐binding protein that regulates mRNA stability. Here, we report that TTP suppress the growth of human colon cancer cells both in vivo and in vitro by regulating of the expression of vascular endothelial growth factor (VEGF). TTP protein expression in human colonic tissues was markedly decreased in colonic adenocarcinoma compared with in normal mucosa and adenoma. VEGF expression was higher in colonic adenocarcinoma than in normal mucosa and adenoma. Specific inhibition of TTP expression by RNA‐interference increased the expression of VEGF in cultured human colon cancer cells, and TTP overexpression markedly decreased it. In addition, elevated expression of TTP decreased the expression level of luciferase linked to a 3′ terminal AU‐rich element (ARE) of VEGF mRNA. Colo320/TTP cells overexpressing TTP grew slowly in vitro and became tumors small in size when xenografted s.c into nude mice. These findings demonstrate that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells, suggesting that it can be used as novel therapeutic agent to treat colon cancer.

Reactive oxygen species-dependent EndoG release mediates cisplatin-induced caspase-independent apoptosis in human head and neck squamous carcinoma cells

Cisplatin is a chemotherapeutic agent that is widely used to treat cancers such as head and neck squamous cell carcinoma (HNSCC). Previously, we have reported that cisplatin induced an early caspase‐dependent apoptosis (8 hr) in a HNSCC cell, HN4. In this study, we examined a late caspase‐independent apoptosis as well as an early caspase‐dependent apoptosis in cisplatin‐treated HN4 cells. While z‐VAD‐fmk, a pan‐caspase inhibitor, blocked the caspase activities and protected cells from the early apoptosis, it did not provide protection against delayed apoptosis occurring after extended exposure (16 hr) to cisplatin, suggesting that the delayed apoptotic response in the presence of z‐VAD‐fmk was caspase‐independent. Cisplatin treatment induced reactive oxygen species (ROS) generation, loss of the mitochondrial membrane potential (MMP) and nuclear translocation of endonuclease G (EndoG). Small interfering RNA mediated‐knockdown of EndoG significantly protected cells from the delayed apoptosis induced by cisplatin in the presence of z‐VAD‐fmk. Overexpression of Bcl‐2 in HN4 cells prevented loss of MMP, nuclear translocation of EndoG and protected cells from the delayed apoptosis induced by cisplatin in the presence of z‐VAD‐fmk. Pretreatment with N‐acetyl‐L‐cysteine (NAC), a ROS scavenger, prevented both ROS generation, loss of the MMP and nuclear translocation of EndoG. Together, our data indicate that cisplatin treatment induced ROS‐mediated loss of the MMP, and, then, the nuclear translocation of EndoG, which played a crucial role in caspase‐independent apoptosis of HN4 cells in the presence of z‐VAD‐fmk. This is the first report about the involvement of EndoG in cisplatin‐induced caspase‐independent apoptosis of cells.

Stability of the LATS2 Tumor Suppressor Gene Is Regulated by Tristetraprolin

LATS2 is a tumor suppressor gene implicated in the control of cell growth and the cell cycle. Here, we investigated the post-transcriptional regulation of LATS2 expression by tristetraprolin (TTP). Our results show that the expression level of LATS2 is inversely correlated with TTP expression in human cancer cell lines. Overexpression of TTP reduced the expression level of LATS2. Conversely, treatment with small interfering RNA against TTP increased the expression level of LATS2 through stabilization of LATS2 mRNA and suppressed the proliferation of A549 human lung cancer cells. LATS2 mRNA contains AU-rich elements (AREs) within the 3′-untranslated region, and TTP destabilized a luciferase mRNA containing LATS2 ARE. In addition, RNA electrophoretic mobility shift assay revealed that TTP directly bound to the ARE of LATS2 mRNA. These results establish LATS2 mRNA as a physiological target of TTP and suggest the possibility that TTP controls cell growth through regulation of LATS2 mRNA stability.

NF-κB activation is required for cisplatin-induced apoptosis in head and neck squamous carcinoma cells

This study demonstrates a requirement for NF‐κB activation in cis‐diamminedichloroplatinum (cisplatin)‐induced apoptosis in human head and neck squamous cell carcinoma (HNSCC) cell lines. This conclusion was supported by the following observations: cisplatin induced IκBα degradation and NF‐κB‐dependent transcriptional activation prior to cell death; pyrrolidine dithiocarbamate (PDTC), a chemical inhibitor of NF‐κB activation, prevented apoptosis; lactacystin, an inhibitor of IκBα degradation, also prevented apoptosis; and finally, the expression of a super‐repressor mutant IκBα blocked apoptosis. The expression of tumor necrosis factor α (TNFα) was promoted by cisplatin treatment and was suppressed by PDTC treatment. In addition, a neutralizing antibody against TNFα protected cells from cisplatin‐induced apoptosis. These findings suggest that NF‐κB activation is required for cisplatin‐induced apoptosis and TNFα may play an important role in NF‐κB‐mediated apoptosis in cisplatin‐treated HNSCC cell lines.

Butyrate response factor 1 enhances cisplatin sensitivity in human head and neck squamous cell carcinoma cell lines

Cisplatin is a widely used chemotherapeutic agent in head and neck squamous cell carcinoma (HNSCC). Resistance to cisplatin is a common feature of HNSCC. To identify genes that may regulate cisplatin sensitivity, we carried out a cDNA microarray analysis of gene expression in cisplatin‐sensitive and cisplatin‐resistant HNSCC‐derived cell lines. Among genes differentially expressed by cisplatin treatment, we have confirmed the elevated expression of butyrate responsive factor 1 (BRF1) in cisplatin‐sensitive HNSCC cells and have demonstrated that the expression level of BRF1 is associated with cisplatin‐sensitivity. Specific inhibition of BRF1 expression using an antisense oligodeoxynucleotide (ODN) decreased the cisplatin‐sensitivity and, on the contrary, overexpression of BRF1 increased cisplatin‐sensitivity in HNSCC cells. Elevated expression of BRF1 decreased the level of the human inhibitor of apoptosis protein‐2 (cIAP2) and increased the caspase‐3 activity in HNSCC cells. In addition, elevated expression of BRF1 decreased the expression level of enhanced green fluorescent protein (EGFP) linked to a 3′ terminal AU‐rich element (ARE) of cIAP2 mRNA. These findings demonstrate that BRF1 expression enhanced cisplatin sensitivity in HNSCC cells by reducing the levels of cIAP2 mRNA.

Combination Chemotherapy with 5-Fluorouracil and Heptaplatin as First-line Treatment in Patients with Advanced Gastric Cancer

Heptaplatin is a recently developed platinum derivative. This agent has been reported to have a response rate of 17% as a single agent, and tolerable toxicity in the treatment of advanced gastric cancer. The aim of this study was to evaluate the efficacy and toxicity of a combination of 5-fluorouracil (5-FU) and heptaplatin in patients with advanced gastric cancer. Forty-seven chemotherapy-naive patients with advanced or recurred gastric cancer were recruited. 5-FU was administered over 120 hr by continuous intravenous infusion from day 1 to 5, at a daily dose of 1,000 mg/m2 and heptaplatin was administered over 1 hr by intravenous infusion on day 1 at 400 mg/m2, and this cycle was repeated every 4 weeks. The response rate was 21%, median progression-free survival was 1.9 months (95% CI, 1.6 to 2.2 months). Median overall survival was 6.2 months (95% CI, 4 to 8.4 months) and the 1-yr survival rate was 29% for all patients. The most frequent toxicity was proteinuria. Toxicities were generally mild and reversible. This study demonstrates that the combination of 5-FU/heptaplatin combination is less active but tolerated in patients with advance gastric cancer.

Analysis op alloimmuno T cell activation in 4-IBB KO mice

BB, a member of the TNF receptor superfamily, functions mainly as a costimulatory molecule in T cells. Since signaling through 4-1BB provides T cells with costimulation independently of CD28, it has been postulated that 4-1BB plays an important role in allograft rejection. In this study, we demon- strated the critical role of 4-1BB in heart allograft rejection using 4-1BB-deficient mice. When 4-1BB- deficient C57BL/6 mice received MHC-mis- matched cardiac transplant of Balb/c origin, there was a markedly-delayed graft rejection as com- pared with the wild type. The delayed graft rejec- tion in the mutant mice correlated with less severe lymphocytic infiltration and vasculitis in the donor hearts. The cardiac grafts were harvested on days 1,3, 5, and 7, and the transcription level of various T cell cytokines and antigens were analyzed by RT- PCR. Furthermore, T cells of 4-1BB-deficient mice showed lower proliferation and cytokine produc- tion when challenged with allostimulatory den- dritic cells. We found that the mRNA levels of FasL and T cell activation cytokines such as IL-2, IFN-y, iNOS were decreased in 4-1BB-/- mice compared to wild type mice. Whereas, transcription of B7-2 antigen, CD28 related costimulatory molecule, was increased in 4-1BB/- mice. These findings have a clinical implication that the blockade of 4-BB sig- naling may prolong the survival of solid organ transplants.