Jenq Chang Lee

Overexpression of Pyruvate Dehydrogenase Kinase 3 Increases Drug Resistance and Early Recurrence in Colon Cancer

The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and is associated with tumor malignancy. Pyruvate dehydrogenase kinase-1 (PDK1) and PDK3 participate in the metabolic switch of cancer cells; however, the medical significance of PDK1 and PDK3 in cancer progression is not known. Here, we assessed the expression profiles of PDK1 and PDK3 in colorectal cancer. Western blot analysis (n = 74) demonstrated that PDK3 was markedly increased in colon cancer compared to that in adjacent normal tissues, whereas PDK1 was decreased in cancer cells. In addition, PDK3 expression was positively correlated with that of hypoxia inducible factor-1α (HIF-1α) in cancer cells. Further analysis using immunohistochemical staining revealed that PDK3 levels were positively associated with severity of cancer and negatively associated with disease-free survival. In vitro studies using several colon cancer cell lines showed that PDK3 expression was controlled by HIF-1α and contributed to hypoxia-induced increased drug resistance, perhaps explaining why patients with PDK3 overexpression have a greater incidence of treatment failure. Taken together, our findings suggest that PDK3 plays an important role in the metabolic switch and drug resistance of colon cancer and is potentially a novel target for cancer therapy.

EPS8 Facilitates Cellular Growth and Motility of Colon Cancer Cells by Increasing the Expression and Activity of Focal Adhesion Kinase

In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.

Suppression of dual-specificity phosphatase-2 by hypoxia increases chemoresistance and malignancy in human cancer cells.

Hypoxia inducible factor-1 (HIF-1) is the master transcriptional regulator of the cellular response to altered oxygen levels. HIF-1α protein is elevated in most solid tumors and contributes to poor disease outcome by promoting tumor progression, metastasis, and resistance to chemotherapy. To date, the relationship between HIF-1 and these processes, particularly chemoresistance, has remained largely unexplored. Here, we show that expression of the MAPK-specific phosphatase dual-specificity phosphatase-2 (DUSP2) is markedly reduced or completely absent in many human cancers and that its level of expression inversely correlates with that of HIF-1α and with cancer malignancy. Analysis of human cancer cell lines indicated that HIF-1α inhibited DUSP2 transcription, which resulted in prolonged phosphorylation of ERK and, hence, increased chemoresistance. Knockdown of DUSP2 increased drug resistance under normoxia, while forced expression of DUSP2 abolished hypoxia-induced chemoresistance. Further, reexpression of DUSP2 during cancer progression caused tumor regression and markedly increased drug sensitivity in mice xenografted with human tumor cell lines. Furthermore, a variety of genes involved in drug response, angiogenesis, cell survival, and apoptosis were found to be downregulated by DUSP2. Our results demonstrate that DUSP2 is a key downstream regulator of HIF-1-mediated tumor progression and chemoresistance. DUSP2 therefore may represent a novel drug target of particular relevance in tumors resistant to conventional chemotherapy.

Regulation of CD151 by Hypoxia Controls Cell Adhesion and Metastasis in Colorectal Cancer

Purpose: The first step of metastasis is the detachment of cancer cells from the surrounding matrix and neighboring cells; however, how cancer cells accomplish this process remains unclear. Thus, we aimed to investigate the underlying mechanism that controls the early event of metastasis. Experimental Design: One hundred and thirty-seven paired colorectal carcinoma and normal colon tissues were examined by immunohistochemical staining and Western blot for the expression of CD151, a member of the tetraspanin family that plays important roles in cell adhesion and motility. The effect of CD151 on cancer cell adhesion was investigated under normoxia and hypoxia conditions. Results: The level of CD151 was down-regulated in colon cancer compared with the paired normal counterparts. Expression of CD151 was negatively regulated by hypoxia inducible factor-1–dependent hypoxic stress. Suppression of CD151 by hypoxia caused the detachment of cancer cells from the surrounding matrix and neighboring cells whereas restoration of CD151 expression during reoxygenation facilitated the adhesion capacity. Clinical examination further showed that metastasized cancer cells expressed a greater level of CD151 compared with that of primary tumor. Conclusion: Regulation of CD151 by oxygen tension may play an important role in cancer metastasis by regulating the detachment from the primary site and homing in the secondary site.

The Prognostic Significance of RON and MET Receptor Coexpression in Patients with Colorectal Cancer

PurposeAlthough Recepteur d’Origine Nantais (RON), a member of the MET receptor tyrosine kinase family, is overexpressed and constitutively active in some primary tumors and tumor cell lines, its expression pattern and clinical significance in colorectal cancer are not well documented.MethodsBy using immunohistochemical staining, we examined RON and MET expression in 135 colorectal cancer specimens and investigated the association of the immunoreactivity of both receptors with colorectal cancer clinical parameters and prognosis.ResultsWe found moderate to strong expression in 99 cases (73 percent) for RON and 97 cases (72 percent) for MET. Univariate analysis showed that increased immunoreactivity of RON or MET was associated with shorter patient survival and that moderate to strong coexpression of both receptors was associated with a significantly worse prognosis. Multivariate Cox analysis showed that the risk of tumor recurrence for patients with high-RON/high-MET expression was approximately 11 times greater than for patients with low-RON/low-MET expression (P = 0.001). In addition, RON and MET expression levels were positively correlated (P ≤ 0.001; τ = 0.306).ConclusionsThe crosstalk between RON and MET in colorectal cancer seems important. Evaluating the expression patterns of RON and MET was predictive of clinical outcome for patients with colorectal cancer.

Morusin induces apoptosis and suppresses NF-κB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G(1) phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-alpha, IKK-beta and IkappaB-alpha, increased expression of IkappaB-alpha, and suppressed nuclear translocation of NF-kappaB and its DNA binding activity. Dephosphorylation of NF-kappaB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-kappaB.

Differences in survival between colon and rectal cancer from SEER data

Background Little is known about colorectal cancer or colon and rectal cancer. Are they the same disease or different diseases? Objectives The aim of this epidemiology study was to compare the features of colon and rectal cancer by using recent national cancer surveillance data. Design and setting Data included colorectal cancer (1995–2008) from the Surveillance, Epidemiology, and End Results Program (SEER) database. Only adenocarcinoma was included for analysis. Patients A total of 372,130 patients with a median follow-up of 32 months were analyzed. Main outcome measures Mean survival of patients with the same stage of colon and rectal cancer was evaluated. Results Around 35% of patients had stage information. Among them, colon cancer patients had better survival than those with rectal cancer, by a margin of 4 months in stage IIB. In stage IIIC and stage IV, rectal cancer patients had better survival than colon cancer patients, by about 3 months. Stage IIB colorectal cancer patients had a poorer prognosis than those with stage IIIA and IIIB colorectal cancer. After adjustment of age, sex and race, colon cancer patients had better survival than rectal cancer of stage IIB, but in stage IIIC and IV, rectal cancer patients had better survival than colon cancer. Limitations The study is limited by its retrospective nature. Conclusion This was a population-based study. The prognosis of rectal cancer was not worse than that of colon cancer. Local advanced colorectal cancer had a poorer prognosis than local regional lymph node metastasis. Stage IIB might require more aggressive chemotherapy, and no less than that for stage III.

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase‐8 to increase tBid, disrupted mitochondrial membrane potential (Δψ m), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl‐xL and increased Bax and Bak in mitochondria. Caspase‐8 inhibitor (Z‐IETD) attenuated the justicidin A‐induced disruption of Δψ m. Growth of Hep 3B implanted in NOD‐SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A‐induced apoptosis in these cells proceeds via caspase‐8 and is followed by mitochondrial disruption. Supplementary materials are available at http://myweb.ncku.edu.tw/~a725/.

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.

We report the culture conditions for successful amino‐acid‐type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide‐bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [α‐15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope‐labeled Rho. The labeling of [α‐15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [α‐15N]Leu, ‐Lys, and ‐Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [α‐15N]Asp and ‐Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide‐bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study. Proteins 2006.

Early presumptive therapy with fluconazole for occult Candida infection after gastrointestinal surgery.

The objective of this retrospective comparative study was to improve the outcome of patients with suspected occult Candida infection after gastrointestinal surgery by early presumptive therapy. It was conducted in the National Cheng Kung University Hospital in Taiwan. A total of 36 patients with prolonged ileus with fever after gastrointestinal tract surgery between January 1995 and December 2002 were examined for two time periods: those treated before and those treated after January 1999. One set of patients did not receive early presumptive therapy (EPT) until Candida infection was confirmed, and they were designated EPT(−). Another group of patients with suspected occult Candida infection received EPT and were designated EPT(+). Fluconazole, 400 mg/day, was given as EPT. Urine, wound, intraperitoneal drainage, and blood specimens were obtained from patients for fungus culture before starting treatment and weekly until symptoms subsided. The primary endpoints were the frequency of candidiasis and the persistence of candidemia; the secondary endpoint was the efficiency of EPT in the clinical outcome. There was no difference in Candida peritonitis, wound colonization, or urine colonization in the two treatment groups. Candida albicans accounted for 87.5%% of the isolated Candida species: 84.6%% in the EPT(+) group and 89.5%% in the EPT(−) group. In the EPT(+) group, the positive blood culture rate was 66.7%%. The fever subsided rapidly in 17 patients (94%%), the hospital stay and intensive care unit stay were shorter, and the mortality decreased significantly: 11%% vs. 78%%, P < 0.001. Persistent gastrointestinal ileus was the main cause of breakthrough candidemia. We concluded that EPT with fluconazole improves the prognosis of patients with occult Candida infection after gastrointestinal surgery. Surgical intervention was required in patients with breakthrough candidemia.