Hong-Tai Chang

Molecular characterization of germline mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan

Abstract A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation.

Risk factors of mortality in perforated peptic ulcer

OBJECTIVE To assess the risk factors that influence mortality from perforated peptic ulcer. DESIGN Retrospective study. SETTING General hospital, Taiwan. SUBJECTS 179 patients who had their perforated peptic ulcers operated on and who had minimum follow-up of one year. MAIN OUTCOME MEASURES Mortality. RESULTS The overall mortality was 15% (26/179). Of the 26 patients who died, the cause of death was uncontrolled systemic infection in 21 (81%), hypovolaemic shock in 2, and fatal arrhythmia and heart failure in 1 each. 15 of the patients who died of sepsis did not have fulminant abdominal sepsis. Most deaths occurred early after operation, (range 1-96 days). Old age, preoperative shock, and type of operation seemed to be related to these deaths on univariate analysis, but multivariate analysis showed that coexisting medical illness, delayed treatment, and low albumin concentration were independent risk factors for mortality. CONCLUSIONS To improve the result of treatment of perforated peptic ulcer, the diagnosis and treatment should not be delayed, the associated medical illnesses should be treated, and nutritional support should be given.

Effect of Thymol on Ca2+ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

Aims: The effect of the natural product thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was examined. Methods: The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Results: Thymol at concentrations of 200–1,000 µmol/l induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited the thymol-induced [Ca²⁺]_i rise. Incubation with thymol also inhibited the thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished the thymol-induced [Ca²⁺]_i rise. At concentrations of 100–600 µmol/l, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/AM. Annexin V/propidium iodide staining data suggest that thymol (200 and 400 µmol/l) induced apoptosis in a concentration-dependent manner. Thymol (200 and 400 µmol/l) also increased levels of reactive oxygen species. Conclusions: In MG63 cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis via mitochondrial pathways.

APACHE II score: a useful tool for risk assessment and an aid to decision-making in emergency operation for bleeding gastric ulcer

BACKGROUND Operating for bleeding gastric ulcer remains controversial. Gastric resection bears a higher surgical risk while limited operation may result in more postoperative hemorrhage. There has been little discussion of effective risk assessment of patients. The aim of this study is to define surgical risk by using the APACHE II scoring system, and to determine optimal management. STUDY DESIGN Records from October 1990 to December 1996 were retrospectively reviewed for patients (n=101) with bleeding gastric ulcer who had undergone emergency operation after failed endoscopic therapy. Mortality rates were examined according to different APACHE II scores, and the surgical risk was defined. From January 1997 to December 1997, 35 consecutive patients were enrolled for prospective study. Partial gastric resection (PGR) was performed for patients with huge ulcers (>2 cm) and for low-risk patients with ulcers at the antrum or angularis, while limited operation (oversewing or excision of bleeding ulcer) was reserved for others. The results were compared with the retrospective study. RESULTS In the retrospective study, the mortality rates for the group with a score < 15 and > or = 15 were 5% (3 of 63) and 58% (22 of 38), respectively (p < 0.05). In the group with a score < 15, PGR was performed on 27 patients, and one died. For those patients with a score > or = 15, PGR carried a lower mortality than limited operation, although this was not statistically significant (47% vs 65%). Limited operation resulted in an overall rate of 22% postoperative hemorrhage and 12% reoperation rate, in which all patients with a score > or = 15 died. In the prospective study, the mortality rates in those scoring <15 and > or = 15 were 6% and 50%, respectively. This is not significantly different than the retrospective study. However, the rate of postoperative hemorrhage was diminished (5%). CONCLUSIONS APACHE II score is a useful tool for assessing risk in patients with bleeding gastric ulcer. The mortality is minimal in those with a score <15, and PGR can be performed with low risk. Although high-risk patients have dreadful outcomes, limited operation cannot improve them if postoperative hemorrhage occurs. Decision making in emergency operation for such patients should be based on the ulcer conditions and the patients hemodynamic status.

EFFECTS OF ECONAZOLE ON Ca2+ LEVELS IN AND THE GROWTH OF HUMAN PROSTATE CANCER PC3 CELLS

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura‐2 and tetrazolium as fluorescent dyes.

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced, partly, by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quenching of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca2+ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C–dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca2+ channels.

Recurrence after skin-sparing mastectomy and immediate transverse rectus abdominis musculocutaneous flap reconstruction for invasive breast cancer

BackgroundThe aim of this study was to evaluate the recurrence pattern after skin-sparing mastectomy (SSM) and immediate breast reconstruction (IBR) using transverse rectus abdominis musculocutaneous (TRAM) flap in patients with invasive breast cancer.MethodsFrom 1995 to 2010, patients with invasive breast cancer who underwent SSM followed by IBR using TRAM flap were retrospectively reviewed. The pattern of the first recurrence event was recorded.ResultsWe identified 249 consecutive patients with invasive breast cancer, two-thirds of whom (67.1%) were diagnosed with stage II or stage III disease. During a median follow-up period of 53 months, three (1.2%) local, 13 (5.2%) regional, 34 (13.7%) distant, and five (2.0%) concurrent locoregional and distant recurrences were observed. The median time to recurrences was 26 months (range, 2 to 70 months) for all recurrences, 23 months (range, 2 to 64 months) for locoregional recurrences, and 26 months (range, 8 to 70 months) for distant recurrences. All local recurrent lesions were detectable by careful physical examination, and detection of local recurrence suggested the presence of distant metastasis (60.0%). In contrast to distant metastasis, the risk of locoregional recurrence did not increase significantly with an increase in disease stage. The 5-year overall, locoregional relapse-free, and distant relapse-free survival rates were 89.7%, 90.8%, and 81.6%, respectively.ConclusionsSSM followed by immediate reconstruction using TRAM flap is an oncologically safe procedure even in patients with advanced-stage disease. Detection of local recurrence is crucial and can be aided by a thorough physical examination.

Effect of MK-886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK-886 on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability in human PC3 prostate cancer cells. [Ca(2+)](i) in suspended cells was measured by using fura-2. MK-886 at concentrations of 1 microM and above increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 20 microM. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). MK-886 evoked Mn(2+) quenching of fura-2 fluorescence, implicating Ca(2+) entry. MK-886-induced Ca(2+) influx was inhibited by store-operated Ca(2+) entry inhibitors nifedipine, econazole and SKF96365. In Ca(2+)-free medium, after pre-treatment with 10 microM MK-886, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were abolished; and conversely, thapsigargin pre-treatment abolished MK-886-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with U73122 did not alter MK-886-induced [Ca(2+)](i) rises. MK-886 at concentrations of 1-100 microM concentration-dependently decreased cell viability with an IC(50) value of 60 microM. The cytotoxic effect of MK-886 was not inhibited by pre-chelating cytosolic Ca(2+) with BAPTA/AM. Together, in PC3 cells, MK-886 induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum; and Ca(2+) influx via store-operated Ca(2+) channels. Independently, MK-886 was cytotoxic to cells in a Ca(2+)-independent manner.

Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells.

AbstractEsculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca2+ homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca2+ signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca2+ concentration ([Ca2+]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca2+ signal response was reduced by removing extracellular Ca2+ and was inhibited by the store-operated Ca2+ channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca2+-free medium, pre-treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca2+]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca2+]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca2+]i rises by releasing Ca2+ from the ER and causing Ca2+ influx through 2-APB-sensitive store-operated Ca2+ entry. Furthermore, esculetin activated Ca2+-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstractThe summary of esculetin-evoked [Ca2+]i rises and -activated Ca2+-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca2+ influx via 2-APB-sensitive store-operated Ca2+ entry and induced Ca2+ release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca2+-associated manner that involved G2/M arrest.

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca2+ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca2+ concentrations ([Ca2+]i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Celecoxib-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A2 inhibitor, aristolochic acid. In Ca2+-free medium, 30 μM of celecoxib failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca2+ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca2+ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca2+]i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca2+ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca2+]i rises by causing phospholipase C–independent Ca2+ release from the ER and Ca2+ influx via non-L-type, phospholipase A2-regulated Ca2+ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.