Ling-Ling Hsieh

Arsenic methylation capacity, body retention, and null genotypes of glutathione S-transferase M1 and T1 among current arsenic-exposed residents in Taiwan

In order to elucidate the relationships among arsenic methylation capacity, body retention, and genetic polymorphisms of glutathione S-transferase (GST) M1 and T1, a total of 115 study subjects were recruited from Lanyang Basin located on the northeast coast of Taiwan. Specimens of drinking water, blood, urine, hair and toenail were collected from each study subject. Urinary inorganic and methylated arsenic were speciated by high performance liquid chromatography combined with hydride-generation atomic absorption spectrometry. Arsenic concentration in hair and toenail were quantitated by atomic absorption spectrophotometry. The polymerase chain reaction was used to determine genetic polymorphisms of GST M1 and T1. Arsenic concentrations in urine, hair, and toenail of study subjects were positively correlated with arsenic levels in their drinking water. Percentages of various arsenic species in urine (mean +/- standard error (SE) were 11.8 +/- 1.0, 26.9 +/- 1.2 and 61.3 +/- 1.4, respectively, for inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Men and women had similar arsenic methylation capability. No associations were observed between arsenic methylation capability and arsenic content in either drinking water or urine. Ratios of arsenic contents in hair and toenail to urinary arsenic content (mean +/- standard error) were 6.2 +/- 0.7 and 16.5 +/- 1.7, respectively. Genetic polymorphisms of GST M1 and T1 were significantly associated with arsenic methylation. Subjects having the null genotype of GST M1 had an increased percentage of inorganic arsenic in urine, while those with null genotype of GST T1 had an elevated percentage of DMA in urine. Arsenic contents in hair and toenail were significantly correlated with the increase in arsenic concentrations of drinking water and urine, while no significant associations were observed between arsenic contents in hair and toenail and polymorphisms of GST M1 and T1. The relationship between arsenic methylation capability and body retention was modified by genetic polymorphisms of GST M1 and T1. Arsenic contents in hair and toenail were negatively associated with MMA percentage and positively associated with DMA percentage among subjects having null genotypes of GST M1 and T1, but not among those with non-null genotypes.

Identification of collapsin response mediator protein‐2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS‐PAGE combined with MALDI‐TOF MS. Among the 325 proteins identified, collapsin response mediator protein‐2 (CRMP‐2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP‐2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP‐2‐positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP‐2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 ± 34.6 vs. 40.2 ± 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP‐2 may be a novel CRC biomarker.

Physical activity, water intake and risk of colorectal cancer in Taiwan: a hospital-based case-control study.

The age‐adjusted mortality rates of colorectal cancer have been rising in Taiwan over the past 2 decades, and colorectal cancer is now the third leading cause of cancer mortality in the country. We conducted a hospital‐based case‐control study to clarify the nature of the association between physical activity, water intake and colorectal‐cancer risk in Taiwan. A total of 163 subjects (aged 33–80 years) with histologically confirmed primary colorectal cancer and 163 hospital controls were enrolled during 1992. Dietary intake, physical activity and other lifestyle activities were assessed using a comprehensive food‐frequency and lifestyle‐activity questionnaire. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated using conditional logistic‐regression analysis. A strong inverse dose‐response relation between increased water intake and rectal cancer was found among men after adjustment for other risk factors (p for trend = 0.0005). The OR for rectal cancer among men in the highest tertile of water intake was 0.08 (95% CI, 0.02–0.35) compared with that among men in the lowest tertile (OR = 1). Similar but not significant trends were seen among women (p = 0.29). The OR for colon cancer among men with active leisure‐time physical activity was 0.19 (95% CI, 0.05–0.77) times that among sedentary men (p for trend = 0.03). However, physical activity was not associated with colon‐cancer risk among women (p = 0.48). No differences in the amount of water intake were found related to level of physical activity. These findings add to the evidence that leisure‐time activity may reduce colon‐cancer risk, not only in high‐risk but also in low‐risk populations, and support the potential beneficial effect of increased water intake in reducing colorectal‐cancer risk. Int. J. Cancer 82:484–489, 1999.

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients

OBJECTIVES Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Identification of differentially expressed genes in oral squamous cell carcinoma (OSCC) : Overexpression of NPM, CDK1 and NDRG1 and underexpression of CHES1

To identify cellular genes that could potentially serve as predictive molecular markers for human oral cancer, we employed differential display analysis to compare the gene expression profiles between oral squamous cell carcinoma (OSCC) and histopathologically normal epithelium tissues. Comparative real‐time RT‐PCR was used to confirm the gene expression in 52 OSCC patients, and a 2‐fold difference was defined as over‐ or underexpression. A total of 7 genes were identified: NPM, CDK1, NDRG1, HMGCR, EF1A, NAC and CHES1. In the cancer tissues, NPM, CDK1 and NDRG1 were significantly overexpressed (an average of 7.6‐, 17.2‐ and 12.9‐fold, respectively), and CHES1 was underexpressed (15‐fold). The frequencies of the differential expression were 40, 56, 67 and 46%, respectively in NPM, CDK1, NDRG1 and CHES1. In Western blot analysis, the protein expressions of NPM, CDK1 and NDRG1 were also increased in the cancer tissues, consistent with the mRNA expression results. To further evaluate clinicopathological associations in these genes, Pearson chi‐square analysis was employed. Levels of CDK1 and NDRG1 were associated with poorly differentiated tumors (p = 0.043 and 0.023), suggesting that these genes participate in the mechanism of tumor transformation. Expressions of CDK1 and NDRG1, and CDK1 and CHES1 were mutually statistically correlated (p = 0.001 and 0.014), indicating that these genes share a very close regulatory relationship or interact synergistically in oncogenesis. In conclusion, we identified 7 genes that are differentially expressed in OSCC, and we provide the first evidence that NPM, CDK1 and NDRG1 are overexpressed and CHES1 is underexpressed in oral cancer. These results serve as a fundamental base for employing these genes in future clinical applications.

RISK FACTORS FOR COLORECTAL CANCER IN TAIWAN: A HOSPITAL-BASED CASE-CONTROL STUDY

BACKGROUND AND PURPOSE There have been few studies of the risk factors associated with colorectal cancer in Taiwan, a country of low incidence of the disease. This study investigated whether dietary and lifestyle factors correlate with colorectal cancer risks in Taiwan. METHODS A total of 352 patients with colon cancer and 375 patients with rectal cancer histologically confirmed between 1995 to 1999 at a medical center in northern Taiwan were included in the study. They were age- and gender-matched with 736 healthy controls who were recruited from the health examination clinic at the same hospital. Dietary intake and lifestyle variables were ascertained using a standardized questionnaire. Unconditional multiple logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS The risk of colon cancer and of rectal cancer was inversely associated with vegetable/fruit consumption in both men and women. The adjusted ORs based on the highest versus the lowest tertile consumption were 0.36 (95% CI, 0.21 to 0.61) and 0.44 (95% CI, 0.27 to 0.72) for men, respectively. The corresponding ORs for women were 0.32 (95% CI, 0.19 to 0.56) and 0.39 (95% CI, 0.23 to 0.69), respectively. However, the highest versus the lowest tertile meat consumption was associated with significantly elevated risk in both men and women for both colon cancer (ORs, 1.85 and 2.29, respectively) and rectal cancer (ORs, 2.32 and 2.42, respectively). Risk also increased with less exercise, low or moderate coffee consumption, cigarette smoking and alcohol intake, and decreased with the frequency of fish/shrimp consumption among men. CONCLUSIONS Consistent with the findings of previous studies in Western populations, this study found that vegetable and fruit consumption, less meat consumption, and exercise were associated with a reduced incidence of colorectal cancer in Taiwanese.

Humoral response to p53 in human colorectal tumors: A prospective study of 1,209 patients

p53 Antibodies (p53‐Abs) have been detected in the serum of a proportion of colorectal cancer (CRC) patients. It is not yet known at which stage during colorectal tumor progression p53‐Abs appear in the serum. The utility of these antibodies as markers for CRC prognosis remains to be clarified. Using a quantitative enzyme‐linked immunosorbent assay, we analyzed serum samples from 998 CRC patients and from 211 patients with polyp. Levels of p53‐Abs were defined as negative (<10 U/μL), low (10–76 U/μL) and high (>76 U/μL). Overall, 13.0% of CRC patients and less than 1% of polyp patients had increased serum p53‐Ab levels. High p53‐Ab levels were only seen in patients with invasive carcinomas. The parameters that were significantly and independently associated with a greater frequency of high p53‐Ab levels were the left colon (odds ratio [OR] = 3.4; 95% CI = 1.1–10.5), the rectum (OR = 2.9; 95% CI, 1.0–8.8) and advanced lymph node metastasis (OR = 4.6; 95% CI, 2.2–9.6). In univariate analysis, patients with high p53‐Ab levels had a shorter survival times than did those without (p = 0.007). However, the significant effect disappeared in a Cox regression model adjusting for sex, age, tumor location, carcinoembryonic antigen levels, gross findings, histologic grade, mucin production and TNM stage. Thus, autoantibodies against p53 occur with tumor progression in multistep colorectal carcinogenesis and increase with advanced node metastasis. Furthermore, the seemingly adverse effect of high p53‐Ab levels on the survival of CRC patients may be explained by other prognostic factors.

Methylation of RASSF1A, RASSF2A, and HIN-1 Is Associated with Poor Outcome after Radiotherapy, but not Surgery, in Oral Squamous Cell Carcinoma

Purpose: Radiotherapy is the standard adjuvant treatment for oral squamous cell carcinoma (OSCC). The Ras/PI3K/AKT pathway is the major mechanism associated with radioresistance. To evaluate the potential significance on the outcome of radiotherapy in OSCC of the Ras/PI3K/AKT pathway with respect to methylation of negative regulators, we examined the methylation status of genes known to be involved in Ras/PI3K/AKT pathway and aberrantly methylated in human cancers together with the mutation status of K-ras/H-ras. Experimental Design: PCR–denaturing high-performance liquid chromatography was used to examine the methylation status of the RASSF1A, RASSF2A, PTEN, and HIN-1 genes, and PCR-RFLP was used to determine the mutation status of K-ras/H-ras in 482 OSCCs. Associations between mutation, methylation, clinicopathologic parameters, and outcome were evaluated. Results: The frequencies of K-ras/H-ras mutation and promoter methylation of the RASSF1A, RASSF2A, PTEN, and HIN-1 genes were 6.6%, 22.4%, 27.8%, 1.2%, and 7.3%, respectively. A combination of RASSF1A and RASSF2A methylation was found to be significantly associated with poor disease-free survival (DFS). Furthermore, a gene dosage effect of the activated Ras/PI3K/AKT signal on DFS was observed in patients treated with radiotherapy after surgery but not in patients treated with surgery alone. The Ras/PI3K/AKT pathway was activated in 140 primary OSCCs among 286 patients treated with radiotherapy after surgery and methylation of RASSF1A/RASSF2A (75.7%) was the most common mechanism. Conclusion: Our study indicates that epigenetic silencing of tumor suppressor genes involved in the Ras/PI3K/AKT pathway plays an important role in OSCC radioresistance and this provides a rationale for exploring novel treatment strategies.

MCP-1 Promoter Polymorphism at −2518 Is Associated with Metastasis of Nasopharyngeal Carcinoma after Treatment

Purpose: We herein examined whether the single nucleotide polymorphism (SNP) at −2518 of the MCP-1 gene promoter region influences clinical outcomes among nasopharyngeal carcinoma (NPC) patients. Experimental Design: The study population consisted of 411 NPC patients without metastasis at diagnosis. All patients were treated at the Chang Gung Memorial Hospital from March 1994 to November 2004. The MCP-1 SNP−2518 genotype of each patient was determined by TaqMan genotyping kit. Statistical analyses were conducted to compare disease-specific survival (DSS), progression-free survival (PFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) of patients according to genotype. MCP-1 expression in tumor biopsies was examined by immunohistochemistry. Results: Among 411 NPC patients, carriers of AA and AG genotypes were prone to distant metastasis than that of GG genotype (hazard ratio, 2.21; P = 0.017, and hazard ratio, 2.23; P = 0.005, for AA and AG genotype, respectively) after initial radiotherapy. No genotype-specific significant difference was found in DSS, PFS, and LRFS. Furthermore, immunohistochemistry revealed that MCP-1 expression level was higher in NPC tumor cells from GG carriers compared with those from AA and AG carriers. Conclusions:MCP-1 SNP−2518 may be a valuable genetic marker for assessing the risk of developing distant metastasis after the radiotherapy in NPC patients. Carriers of A allele may require more aggressive chemotherapy implicating a potential marker for personalized medicine. We speculate that a regulatory SNP may be associated with the distant metastasis of NPC. Validation studies are warranted.

Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility.

Oral squamous cell carcinoma (OSCC) is the 11th most common cancer worldwide, and is associated with a high death rate. At present, there are no suitable markers for detecting and/or monitoring OSCC in body fluids/tissues. Here, we used 1D SDS-PAGE and MALDI-TOF MS to systematically analyze the secretomes of two OSCC cell lines (OEC-M1 and SCC4). The putative OSCC-related proteins identified in this analysis included the Mac-2 binding protein (Mac-2 BP), which was further found to be overexpressed in OSCC specimens and significantly elevated in the sera of OSCC patients compared to healthy controls. Finally, RNA interference-based knock-down of Mac-2 BP expression in OSCC cells revealed for the first time that Mac-2 BP is involved in regulating growth and motility of OSCC cells.