Nonmalignant Tissue Specimens Research Articles

Selenium levels in colorectal cancer: A systematic review and meta-analysis of serum, plasma, and colorectal specimens

BackgroundColorectal cancer (CRC) is a growing public health problem. Several clinical studies have shown a potentially protective effect of selenium (Se), but the reports are inconsistent. The objective of the study was to examine the evidence for relation between serum/tissue Se status and CRC. Method and materialsIn this Systematic Review and Meta-Analysis, we searched Cochrane Library, EBSCOhost, EMBASE, ProQuest, PubMed/MEDLINE, Scopus, and Web of Science for studies reporting serum/plasma/whole blood/tissue Se concentrations in CRC patients and controls for articles published till August 2023. Meta-analysis was performed, and study quality, heterogeneity, and small study effects were assessed. Based on a random effects model, summary mean differences in serum levels of Se between CRC patients and healthy controls, and Se levels between malignant and matched non-malignant tissue specimens were assessed. ResultsAfter initial screening, a total of 24 studies (18 serum and 6 tissue studies) with a pooled total of 2640 participants were included in the meta-analysis. CRC patients had significantly lower serum Se levels than healthy controls, being the difference between the two equal to 3.73 µg/dl (95% CI: 6.85–0.61). However, the heterogeneity was very high, I2= 99% (p < 0.01). Our meta-analysis showed higher Se levels in CRC cancerous specimens than in matched healthy colon tissue: the increase was equal to 0.07 µg/g wet tissue weight (95% CI: 0.06–0.09; p= 0.02). ConclusionsCRC patients have lower serum and higher colon cancerous tissue Se levels. Some factors, such as Se levels in different tumor grades of CRC need to be further considered for a more conclusive association between Se levels and risk of CRC.

Tumor microbiome variation in young versus average onset colorectal cancer.

144 Background: The incidence of young onset colorectal cancer (yoCRC) is rising at alarming rates. The gut microbiome may be a factor accounting for the increase. We analyzed differences in the intratumoral microbiome of yoCRC vs average onset CRC (aoCRC) and its clinical impact. Methods: We identified 314 histologically confirmed cases of stage I-IV CRC that underwent surgical resection at our institution from 2000-2020, diagnosed &lt;50 years of age for yoCRC and &gt;60 years for aoCRC, who consented to a prospective biorepository. 36 cases were excluded due to nonmalignant, non-adenocarcinoma or metastatic site specimens. Fresh frozen tissue from the primary tumor with paired adjacent nonmalignant tissue specimens were analyzed. 16S rDNA was isolated and sequence reads were assigned to genus level amplicon sequence variants in DADA2 and analyzed for alpha and beta diversity using Phyloseq. Statistical tests included analysis of variance (ANOVA), permutational multivariate analysis (PERMANOVA), linear regression, and Wilcoxon test. Differential abundance and correlation analysis were adjusted for sex and ethnicity as confounding factors. Correlation analysis was adjusted with Benjamini Hochberg correction. Clinical differences were analyzed using Fisher's exact test. Results: Of the cohort of 278 patients, 137 had yoCRC (median age 43 years, range 16-49) and 141 had aoCRC (median age 73 years, range 61-95). yoCRC patients were more likely to have stage III or IV disease at presentation (29% vs 14%, p =0.002; 29% vs 18%, p =0.024 respectively), left sided tumors (74% vs 58%, p =0.003) and receive neoadjuvant therapy (29% vs 15%, p =0.004). yoCRC had significantly higher tumor microbial alpha diversity than aoCRC ( p &lt;2.22e−16, Wilcoxon rank-sum test). Beta diversity analysis demonstrated significantly different diversity of genera between the groups (R2=0.12, p =0.001, PERMANOVA). The prevalent taxa identified in both groups were Lactobacillus, Bacillus and Listeria. Differential abundance analysis (ANOVA, p &lt;0.05) revealed a significant variation of intratumoral microbiome (Table). Correlation analysis revealed an association of longer overall survival (OS) with the presence of Akkermansia in yoCRC (R2 =0.36, p &lt;0.001), but not in aoCRC. Conclusions: We found significant differences between the intratumoral microbiome of yoCRC and aoCRC. In particular, Akkermansia, considered a healthy gut microbe, was found in greater relative abundance in yoCRC and correlated with improved OS. Further studies are warranted to understand the nature of association of these microbes with the development of and outcomes in yoCRC. [Table: see text]

Whole-exome sequencing identifies biosignatures that predict adverse survival outcomes in surgically treated patients with oral cavity squamous cell carcinoma

ObjectivesThe postoperative outcomes of patients with oral cavity squamous cell carcinoma (OCSCC) vary greatly. To improve risk stratification, we sought to identify genetic biosignatures by whole-exome sequencing (WES). Materials and MethodsWe retrieved patients with OCSCC patients with paired freshly frozen malignant and non-malignant tissue specimens and performed WES by Illumina HiSeq4000 platform. We further applied a tree-based method to analyze copy number variations and obtain signature classification and driver-gene identification. We further confirmed the prognostic impact of the WES biosignature in an external independent validation set. ResultsWe examined 168 paired samples from patients with surgically treated OCSCC. Similar to the literature, the most commonly mutated genes were TP53 (66%), FAT1 (32%), and NOTCH1 (24%). The signatures 13 (APOBEC Cytidine deaminase [C > G]), 1 (spontaneous deamination of 5-methylcytosine), and 7 (UV exposure) showed the highest concordance rates. Using the MutSigCV, MuSiC, 20/20+, OncodriveFML, e-Driver, OncodriveCLUST, and tree-based methods, we identified a nine-gene OCSCC panel (RYR1, HLA-B, TSHZ2, PCDH17, DNAH17, GRID1, SBNO2, KSR2, and GCN1L1) predicting survival outcomes in our sample. We used the TCGA database to validate the prognostic value of the panel independently. Furthermore, gene-gene covariance analysis confirmed the coexistence of several gene alterations. ConclusionWe identified and independently validated a WES biosignature that predicts outcomes in surgically treated OCSCC in Taiwan, a betel-quid-chewing-prevent area. We proposed that the panel might help clinical trial designation for adjuvant therapy based on the risk stratification from the novel gene panel and identify targets for liquid biopsy monitoring during surveillance.

Abstract 39: Expression of uPA-system members correlate with the clinico-pathological parameters of PCa patients

Abstract Background: Members of the urokinase-type plasminogen activator (uPA) system play an important role in tumor progression and angiogenesis in different human malignancies including breast, kidney, colon, stomach and prostate cancer (PCa). The objective of this study was to determine the mRNA expression and protein levels of uPA system components in tissue specimens and serum samples, respectively, from patients with PCa, and to assess their potential association with clinic-pathological parameters and overall survival (OS). Material and methods: mRNA levels of uPA, its receptor (uPAR) and its inhibitor type 1 (PAI-1) were analyzed in corresponding malignant and adjacent non-malignant prostate tissue specimens from 132 PCa patients. Relative mRNA expression levels were determined by quantitative PCR and normalized to the reference gene TATA-box-binding protein. Pre-operative serum samples from 81 of the 132 PCa patients and 36 patients with benign prostatic hyperplasia (BPH) were analyzed for antigen levels of uPA system members by ELISA. Results: mRNA levels of the uPA system components displayed significant correlations with each other in the tumor tissues (uPA vs. uPAR: rs = 0.550; uPA vs. PAI-1: rs = 0.552; uPAR vs. PAI-1: rs = 0.643; all P &amp;lt; 0.001). A significantly decreased uPA mRNA expression in PCa tissue compared to the corresponding non-malignant tissue specimens was detected (data not shown). High mRNA expression of uPA and PAI-1 was significantly associated with a high Gleason score (P = 0.001 and P = 0.007, respectively; Fig. 2). Antigen levels of uPA and soluble uPAR (suPAR) in serum correlated weakly, but significantly, with each other (rs = 0.382, P &amp;lt; 0.001). A significant association was found between elevated suPAR concentrations in serum and poor OS of PCa patients (P = 0.022, log rank test). In multivariate Cox's regression analysis (adj. for age, lymph node status, tumor stage and Gleason sum) a high suPAR level was associated with a 7.12-fold increased risk of death (P=0.027). Conclusion: The observed association of high suPAR levels with poor survival of PCa patients is consistent with published data on patients afflicted with other types of solid cancer and strongly suggests a general prognostic impact of uPAR antigen levels in serum of cancer patients. Citation Format: Omar Al-Janabi, Helge Taubert, Andrea Lohse-Fischer, Michael Fröhner, Sven Wach, Robert Stöhr, Bastian Keck, Max Burger, Wolf Wieland, Kati Erdmann, Manfred P. Wirth, Bernd Wullich, Gustavo Baretton, Viktor Magdolen, Matthias Kotzsch, Susanne Füssel. Expression of uPA-system members correlate with the clinico-pathological parameters of PCa patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2014-39

Abstract 4683: Correlation of transcript levels of members of the urokinase-type plasminogen activator system with clinico-pathological parameters in prostate cancer .

Abstract Background: Members of the urokinase-type plasminogen activator (uPA) system play an important role in tumor progression and angiogenesis in different human malignancies including breast, kidney, colon, stomach and prostate cancer (PCa). Aim of the study was to characterize mRNA expression levels of members of the uPA system in tissue specimens from primary PCa and their potential association with clinico-pathological parameters. Methods: Transcript levels of members of the uPA system, i.e., uPA, uPA receptor (uPAR) and plasminogen activator inhibitor type 1 (PAI-1) were analyzed in a cohort of 132 corresponding pairs of malignant and adjacent non-malignant prostate tissue samples. Relative gene expression levels were determined by quantitative PCR (qPCR) and normalized to the reference gene TATA-box-binding protein (TBP). Potential correlations between the three markers themselves and the clinico-pathological parameters were assessed by Spearman's Rho test and by Fisher's exact test. Results: There were no significant differences in expression levels of uPA system genes between the corresponding malignant and non-malignant tissue specimens. Transcript levels of all members of the uPA system displayed a significant correlation with each other in the tumor tissues. At stratifying uPA, uPAR and PAI-1 expression levels by the median a significant correlation between an elevated expression of uPA and PAI-1 was correlated with higher Gleason score (P = 0.001 and P = 0.007). In addition, a significant positive correlation between uPAR transcript levels and tumor stage was detected (P = 0.025). Conclusion: Our results demonstrate that mRNA expression levels of members of the uPA system correlated with each other and with clinico-pathological parameters. We suggest that transcript levels of uPA system members can be helpful markers for the study of tumor behavior of prostate cancer. Citation Format: Omar Al-Janabi, Helge Taubert, Sven Wach, Robert Stöhr, Maximilian Burger, Wolf Ferdinand Wieland, Andrea Lohse-Fischer, Michael Fröhner, Manfred P. Wirth, Bernd Wullich, Viktor Magdolen, Matthias Kotzsch, Susanne Fuessel. Correlation of transcript levels of members of the urokinase-type plasminogen activator system with clinico-pathological parameters in prostate cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4683. doi:10.1158/1538-7445.AM2013-4683

Cytogenetic Analysis of False-Positive Mucosa by Photodynamic Diagnosis Using 5-Aminolevulinic Acid – Possible Existence of Premalignant Genomic Alterations Examined by in vitro Experiment

Objective: Although photodynamic diagnosis is a powerful tool for the detection of flat urothelial tumors, false-positive fluorescent mucosa still requires further elucidation. Thus, we aimed to study the significance of nonmalignant fluorescent mucosa by a cytogenetic approach. Methods: Sixty specimens of bladder mucosa were collected from 20 patients who were suspected of having carcinoma in situ by fluorescence cystoscopy with 5-aminolevulinic acid. To detect the copy number aberrations, the multi-color fluorescence in situ hybridization technique was performed, and the variant fractions (total fractions other than those of the modal copy number) of chromosomes 7, 9 and 17 and the chromosomal instability were determined. To delineate the relevant gene to the fluorescent mucosa, a comparative genomic hybridization technique was applied for 8 established bladder cancer cell lines, and these results were compared with the in vitro fluorescent expression experiment. Results: Fluorescent mucosa was detected in 33 of the 34 malignant tissue specimens (16 carcinoma in situ, 18 other transitional cell carcinomas) and in 11 of the 26 nonmalignant tissue specimens (6 dysplasia, 20 normal mucosa), with a false-positive rate of 42.3%. The variant fraction of chromosome 9 was significantly higher in fluorescent than in non-fluorescent mucosa, not only for all tissues (33 vs. 17%; p = 0.0069), but also for nonmalignant tissues (28 vs. 15%; p = 0.0225). There was no alteration in chromosome 9 in 1 cell line without fluorescent mucosa, while 5 of the 7 cell lines with fluorescent mucosa had a common deleted region on 9q24.1. Conclusion: These data suggest that a substantial portion of nonmalignant fluorescent mucosa harbors alterations in chromosome 9.

Kallikrein 7 enhances pancreatic cancer cell invasion by shedding E‐cadherin

Pancreatic cancer (PaC) is characterized by local invasion and early metastasis. Serine proteases have been associated with invasion and metastasis of many cancers due to their ability to degrade extracellular matrix (ECM) proteins and to activate other proteases; thus, the serine proteases expressed in PaC were investigated. An expression profile of serine proteases was generated from both normal and malignant pancreatic tissues using a polymerase chain reaction (PCR)-based screen and differential expression of kallikrein 7 was examined by reverse-transcriptase PCR (RT-PCR) and immunohistochemical analyses. The ability of human kallikrein 7 (hK7) to cleave the epithelial cell adhesion molecule E-cadherin was tested in vitro using both recombinant E-cadherin and BxPC-3 cells and the effects of hK7 proteolytic activity on pancreatic cell invasion and aggregation were examined. Expression profiling revealed that kallikrein 7 (KLK7) was overexpressed in pancreatic adenocarcinomas and its differential expression was confirmed by RT-PCR analysis. hK7 was observed in neoplastic cells of all tumors examined with moderate-to-intense staining in 70% of tumors examined (16/23). In contrast, only 15% of nonmalignant tissue specimens (2/13) displayed moderate hK7 staining, whereas the remaining specimens yielded weak, if any, immunoreactivity. Using in vitro assays, hK7 was shown to cleave E-cadherin and the soluble E-cadherin fragment produced significantly enhanced Panc-1 cell invasion through ECM proteins with a corresponding reduction in Panc-1 cell aggregation. These results suggest that aberrant expression of KLK7 plays an important role in PaC and provides novel insight into the effects of elevated hK7 proteinase activity in this, and perhaps other, adenocarcinomas.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

BackgroundHousekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.ResultsThe expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel.ConclusionOur study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

The flavonoid apigenin inhibits the proliferation of prostatic stromal cells via the MAPK-pathway and cell-cycle arrest in G1/S

Introduction Phytoestrogen apigenin, a member of flavonoids, has been described for its ability to induce growth arrest in carcinomas of colon, breast and prostate. In order to identify its influence on the prostatic stroma, which plays a crucial role in the pathogenesis of benign prostatic hyperplasia (BPH), we investigated the effect of apigenin on cell proliferation and cell-cycle progression. Methods Prostatic stromal cells were isolated from nonmalignant tissue specimens obtained from patients undergoing radical prostatectomy. After incubation with increasing concentrations of apigenin for 24, 48 and 72 h the cell proliferation was determined by MTT assay. Cell-cycle regulation was examined by FACS analysis and the expression level of proteins involved in the G1/S transition was determined by immunoblot analysis. Results Apigenin treatment resulted in a significant inhibition of proliferation starting at a concentration of 30 μM of the flavonoid. FACS analysis showed cell-cycle arrest at a G1/S transition point. Furthermore, apigenin modified the expression levels of cell-cycle regulatory proteins leading to a dose-dependent decrease of cyclin D1 and an increase of p21/WAF1 expression, as determined by immunoblot analysis. As apigenin lead to a decrease of the phosphorylation status of ERK1 and 2, we postulated that the mechanism of apigenin action is mediated through mitogen activated kinases (MAPK)-pathway. Conclusion Taken together, in response to apigenin treatment prostatic stromal cells showed a dose-dependent inhibition of cell proliferation, which may be due to a cell-cycle growth arrest and seems to occur via MAPK-pathway. These results suggest possible beneficial effects of apigenin in the prevention and/or treatment of benign prostatic hyperplasia.

Identification and Validation of Suitable Endogenous Reference Genes for Gene Expression Studies of Human Bladder Cancer

Housekeeping genes as endogenous references are generally used for the relative quantification of target genes in gene profiling studies. To date that issue has not been sufficiently investigated in bladder cancer. From a panel of 9 potential candidates we selected the most stable housekeeping genes for gene normalization in bladder cancer tissue. Expression profiles of the 9 genes ACTB, ALAS1, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, SDHA and TBP were established in matched malignant and nonmalignant tissue specimens from 14 patients with bladder cancer. Quality assessment of isolated RNA was performed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California) and real-time reverse transcriptase-polymerase chain reaction was performed with LightCycler. The software geNorm and NormFinder (Aarhus University, Aarhus, Denmark) were used to identify the most suitable reference genes. RNA was isolated with high purity and integrity. Candidate reference genes showed a broad range of between 20 and 34 polymerase chain reaction cycles. Expression did not depend on patient sex or tumor stage. GAPD, G6PD and HMBS showed significant differences in expression between malignant and nonmalignant pairs (at least p <0.04). Expression of the remaining genes did not differ between the matched pairs. SDHA and TBP were the most stably expressed genes, covering higher and lower expression levels. For normalization purposes in gene profiling studies of bladder cancer the genes SDH and TBP are recommended as single reference genes depending on the expression level of the target gene or more favorably in combination.

Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?

Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and >or=7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.

Up-regulation of cathepsin X in prostate cancer and prostatic intraepithelial neoplasia.

Evidence is accumulating that several proteases are involved in prostate cancer progression. A locus which is often amplified in prostate cancer is the chromosomal region 20q13. Interestingly, one of the genes encoding the cysteine protease cathepsin X maps to this region. The aim of this study was to assess the expression pattern of cathepsin X in malignant and non-malignant prostatic tissue samples. Matched malignant and non-malignant tissue specimens were obtained from 56 men after radical prostatectomy. Cathepsin X was quantified at both protein and mRNA levels using several detection methods: Western blotting, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. Furthermore, genomic DNA was analyzed by PCR for possible gene amplification. Immunohistochemical analysis of formalin-fixed, paraffin-embedded sections of radical prostatectomy specimens was performed utilizing a polyclonal antibody against human procathepsin X and revealed staining of acinar basal cells in normal prostate glands. Prostatic intraepithelial neoplasias (PINs) and prostate carcinomas stained highly positive for cathepsin X, showing a significant difference to the staining of normal prostate glands. In contrast, relatively weak and heterogeneous staining was observed for cathepsins F, B, and L. Up-regulation of cathepsin X at the protein level was confirmed by Western blotting. No statistically significant difference was observed at the mRNA level. PCR of genomic DNA revealed that cathepsin X up-regulation most likely occurs in the absence of genomic amplification. The high expression levels of cathepsin X both in PIN and invasive adenocarcinomas of the prostate suggest that cathepsin X may play a role in the early tumorigenesis of prostate cancer. Further studies are needed to define the utility of this cysteine protease as a diagnostic marker for the early detection of prostate cancer.

Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR

The identification of new diagnostic markers and potential treatment targets for prostate carcinoma (PCa) necessitates the evaluation of expression patterns in both malignant and non-malignant tissue specimens. In this study, we compared the mRNA expression of recently identified prostate-associated genes, prostate stem cell antigen (PSCA) and transient receptor potential p8 (trp-p8), to the mRNA expression of the most commonly used markers for PCa, prostate-specific antigen (PSA) and human kallikrein 2 (hK2). For these four candidates we performed highly specific quantitative real-time LightCycler RT-PCR assays with cDNA originating from matched tissue specimens of 40 patients with primary PCa. The highest transcript amounts were found for PSA in malignant as well as in non-malignant tissue specimens followed by hK2, trp-p8 and PSCA with an mRNA expression remarkably lower. The relative transcript levels of PSA, hK2 and trp-p8 were elevated in malignant in comparison to non-malignant tissues, but only for trp-p8 this increased expression was statistically significant. Focussing on organ confined tumors, we found a significant difference of the mRNA expression of PSA and trp-p8 between malignant and non-malignant tissue specimens. The marker trp-p8 is also suited to differentiate between the tumor stages when quantifying its transcript levels within tumor tissue specimens. The evaluation of the mRNA expression patterns of these markers by quantitative real-time RT-PCR could provide new tools for differential diagnosis and molecular staging. According to our data, the novel marker trp-p8 seems to represent a highly prostate-specific and PCa-associated gene qualifying it as a potential target for specific therapies.

MRNA expression of the five membrane‐type matrix metalloproteinases MT1–MT5 in human prostatic cell lines and their down‐regulation in human malignant prostatic tissue

The aim of this study was to assess the expression of membrane-type matrix metalloproteinases (MT-MMPs) 1-5 in the human prostatic cell lines BPH-1, LNCaP, DU 145, PC-3, in malignant and non-malignant prostatic tissue samples, and in epithelial cells cultured from these tissue samples. Matched malignant and non-malignant tissue specimens were obtained from 12 men with untreated prostate carcinoma after radical prostatectomy. Expression of mRNA for the five MT-MMPs was quantified by real-time PCR technique and normalized to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of the five MT-MMPs was distinctly different not only between the prostate cell lines but also varied in the same cell line. There was a general higher expression of all MT-MMPs except for MT3-MMP in the androgen-insensitive cells DU 145 and PC-3 compared with that in the androgen-sensitive LNCaP cells. Their relatively high expression in the benign prostatic cell line BPH-1 and also in the primary cell cultures from malignant and non-malignant tissue samples argues against a simple association between MT-MMP expression and invasiveness. In malignant tissue samples and their corresponding cell cultures, the expression of most MT-MMPs was down-regulated in comparison to the normal counterparts. There was no correlation between tumor classification data and the MT-MMP expression results. In contrast to other carcinoma, the down-regulation of most MT-MMPs is typical for prostate carcinoma. It seems to occur mainly in epithelial cells and has to be examined as special characteristic of this tumor entity in further studies.

The expression of leptin in colorectal cancer

Introduction: Weight loss is a recognised feature of advanced stage colorectal cancer; the precise aetiology remains obscure. Leptin, a 16 kDa protein and encoded by the ob gene, is sectreted from adipocytes and has been shown to play an important role in the regulation of energy balance and therefore body weight in animal models. Recently, non-adipocyte sources of leptin have been reported. The purpose of our study was to determine whether leptin may playa part in the aetiology of weight loss in colorectal cancer. We therefore assessed leptin expression in colonic mucosa and compared mailgnant to non-malignant tissue specimens. Methods: Twenty-seven cancer and 17 corresponding normal mucosa specimens were collected from patients undergoing surgical resection of colorectal cancer. Samples were mechanically disaggregated and homogenised on ice in lysis buffer. Following centrifugation the supernatant was removed for leptin analysis. Leptin was measured by using a Human Leptin RIA Kit (Linco Research Inc.), which utilises radioaminoassay, and leptin levels were standardised against protein concentration . Results: Leptin was found to be present in both normal and malignant colonic mucosa. The expression of leptin was significantly higher in normal mucosa (mean 1.03 ng/f.Lg protein) when compared to cancer specimens (mean 0.68 ng/f.Lg, p=0.0l5). There was no significant relationship between histophathological stage of cancer and leptin expression. Conclusions: This is the first study demonstrating the presence of leptin in both normal and malignant colonic mucosa. Interestingly, the relatively low leptin levels in colorectal cancers do not appear to explain the weight loss seen in patients with advanced colorectal malignancy. However, whether there is a hypothalamic insensitivity to leptin, as reported in lung cancer patients, remains to be elucidated.

Lipid-Soluble Antioxidants: β-Carotene and α-Tocopherol Levels in Breast and Gynecologic Cancers

Free radical-induced damage is etiologically implicated in many chronic diseases including cancer. Epidemiologic data suggest an association between increased dietary intake of nutrients that are high in antioxidant vitamins and protection against the incidence of some human cancers. The purpose of this study was (a) to determine whether specific tissue antioxidants (β-carotene and α-tocopherol) and any differences in their levels were measurable in randomly selected human breast and gynecologic malignant neoplasms and nonneoplastic tissue samples obtained from the same patient and (b) to establish normal ranges of these two antioxidant levels in human female reproductive tract tissues. Tissue samples were excised immediately from surgical specimens and released by staff pathologists from a spectrum of human female cancers. Neoplastic and adjacent nonneoplastic tissues samples were obtained from the same patient. Normal reproductive tract tissue samples were obtained from women undergoing hysterectomy for benign gynecologic conditions. Breast carcinoma and adjacent nonmalignant tissue specimens were obtained from women undergoing mastectomy. The concentrations of β-carotene and α-tocopherol were measured by high-pressure liquid chromatography. In the same patient, β-carotene levels were significantly lower in the cervical ( P < 0.01) and endometrial ( P < 0.005) carcinoma tissues than the levels detectable in adjacent nonneoplastic sites. In contrast, β-carotene levels were higher in the ovarian ( P < 0.05), breast ( P < 0.005), and vulva ( P < 0.05) carcinoma tissues. The α-tocopherol concentrations were significantly higher in the cancer tissues of cervix ( P < 0.01) and endometrium ( P < 0.001) than those in adjacent noninvolved tissue sites. The tissue concentrations of α-tocopherol in malignant and adjacent normal sites in breast, ovary, and vulva were comparable. For the first time, the ranges for β-carotene and α-tocopherol levels in the normal female reproductive tract tissues were also established. The present findings of contrasting tissue levels of the antioxidants (β-carotene and α-tocopherol) in breast, cervix, endometrium, ovary, and vulva cancers and in nonneoplastic tissues of the same patient suggest an organ-specific and heterogenous distribution. These antioxidants appear to be essential nutritional requirements of the human female reproductive tract and breast and are implicated in the pathophysiology and carcinogenesis of these human organs. The findings require further study of the role of these antioxidant nutrients in epithelial cell proliferation, maturation, and differentiation.

Comparison of the in vitro conversion of estradiol-17β to estrone of normal and neoplastic human breast tissue

Specific activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) was measured in 48 tissue specimens of human female breast cancer and, in addition, 48 nonmalignant tissue specimens obtained in each case from the same cancer-bearing breast. In all cases the nonmalignant tissue showed greater conversion of estradiol-17β into estrone than the neoplastic tissues. In normal human breast tissue of premenopausal women specific enzyme activity depended on the phase of the menstrual cycle: the highest values of 17β-HSD activity were found in the early secretory phase. To determine the intracellular distribution of the 17β-HSD, purified microsomes, mitochondria, peroxysomes, lysosomes, nuclei and cytosol fractions were prepared. The purity of each fraction was monitored by marker enzymes. It was found that the 17β-HSD was mainly located in mitochondria and microsomes. Furthermore it could be demonstrated that the microsomal enzyme was bound tightly to the membranes of the endoplasmic reticulum, while the mitochondrial 17β-HSD was mainly associated with the outer membranes of the organelle. Kinetic parameters ( K m -values, coenzyme requirements and maximal velocities) of a cytoplasmic, nuclear, mitochondrial and microsomal 17β-HSD of normal and neoplastic human mammary tissue were compared. Maximal velocity was highest in enzyme preparations of normal mammary tissue obtained from premenopausal women in the early secretory phase. K m values were nearly identical in normal and neoplastic mammary tissue preparations (approx. 1 × 10 −6 M). NAD was more efficient than NADP as a cofactor. For the conversion of estradiol to estrone the optimum temperature was approximately 40°C and the optimum pH 9.S. For the reduction of estrone the optimum pH was 6.5. Sulphydryl groups were shown to be essential for catalysis.