Uncovering the clinical impact of kallikrein-related peptidase 5 (KLK5) mRNA expression in the colorectal adenoma-carcinoma sequence.

Background Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases located on chromosome 19q13.3. Most KLKs have been extensively studied as potential biomarkers for several carcinomas and other diseases. KLK5 was originally identified from a keratinocyte library, and its enzyme was purified from the stratum corneum of human skin. KLK5 was shown to be differentially expressed in a variety of endocrine tumors, although it is not as yet examined widely in colorectal cancer (CRC). Methods In this study, we quantitatively assessed the mRNA expression status of KLK5 in 197 colorectal tissues from 133 patients (70 cancerous and their paired normal colonic mucosa for 64 of them, as well as 63 colorectal adenomas) by quantitative real-time PCR (qPCR) using TaqMan probes. Statistical analysis evaluated the results. Results It was shown that KLK5 expression is reduced following the histologically non-cancerous-adenoma sequence (p<0.001), whereas it is increased during the sequence adenoma-carcinoma (p<0.001). Furthermore, KLK5 positive expression is associated with positive nodal status (p=0.022), advanced tumor stage (p=0.038) and high histological grade (p=0.033). Cox univariate analysis revealed that KLK5 positive expression is associated with disease-free survival (DFS) (p=0.028) and overall survival (OS) of patients (p=0.048). Kaplan-Meyer survival models showed that patients with positive KLK5 expression have lower DFS (p=0.009) and OS (p=0.019). Receiver operating characteristic (ROC) analysis demonstrated for first time that KLK5 expression had significant discriminatory values between cancer and adenoma tissues (area under the curve [AUC] 0.77; 95% confidence interval [CI]=0.69-0.85, p=0.03). Conclusions KLK5 mRNA expression may be useful for the differentiation of CRC from colorectal adenoma and represents a potential unfavorable prognostic biomarker for CRC.

Kallikrein-related peptidase 6 (KLK6) expression in the progression of colon adenoma to carcinoma.

KLK6 is a secreted trypsin-like serine protease. KLK6 mRNA expression and its association with colon cancer (CC) progression was studied using quantitative real-time PCR. We examined the expression of KLK6 in 232 colon tissues (cancerous, non-cancerous, and adenomatous). We proved that KLK6 expression in CC behaves as a continuous variable, as its expression correlates significantly with increasing tumor stage (p=0.004) and histological grade (p=0.007). Interestingly, the expression of KLK6 in adenomas was significantly higher than that in the cancerous or non-cancerous tissues examined (p<0.001). Cox proportional hazard regression model using univariate analysis revealed that positive KLK6 expression is a significant factor for disease-free survival (DFS) (p=0.017) and overall survival (OS) (p=0.002) of patients. Kaplan-Meier survival curves demonstrated that KLK6-negative expression is significantly associated with longer DFS (p=0.009) and OS (p=0.001). ROC analysis showed that KLK6 expression has significant discriminatory power in distinguishing cancerous from non-cancerous colon tissues (p<0.001), or cancerous from adenoma tissues (p=0.001), or adenoma from non-cancerous colon tissues (p<0.001). Additionally, strong KLK6 immunostaining was seen in the cancer cells of selected CC sections, as well as in glandular cells and inflammatory cells of adenomas. In conclusion, KLK6 may represent a potential unfavorable prognostic biomarker for CC.

Significant alterations in the expression pattern of kallikrein-related peptidase genes KLK4, KLK5 and KLK14 after treatment of breast cancer cells with the chemotherapeutic agents epirubicin, docetaxel and methotrexate.

Given that 1.3 million new cases of breast cancer are universally registered among women and approximately 36 % of the patients die annually, the revelation of new predictive markers for treatment efficiency is of vital importance. Recently, our group has depicted that KLK4, KLK5, and KLK14 are differentially expressed in breast carcinoma. The objective of this study was to determine and investigate the expression pattern of the KLK4, KLK5, and KLK14 genes in breast cancer cells after treatment with established chemotherapeutic agents. We evaluated these genes' expression after treatment of the BT-20 cells with epirubicin, docetaxel and methotrexate, determining their cytotoxic effect by MTT and trypan blue assays. The relative quantification of genes' mRNA levels was performed by using the SYBR Green® chemistry, and the HPRT1 served as an endogenous control gene. The drugs triggered apoptosis in treated cells and induced deregulations in the expression of the above KLKs. The most significant alterations were a 12-fold and tenfold increase of KLK5 in docetaxel and methotrexate-treated cells, respectively, while the KLK4 levels decreased by ten-fold in epirubicin, five-fold in docetaxel and twenty-fold in methotrexate treated-cells, compared to the untreated ones. In the case of KLK14 levels, a twofold increase in epirubicin and threefold decrease in methotrexate-treated cells were observed. Present significant alterations in the expression pattern of KLK4, KLK5, and KLK14 could comprise an initial stage for predicting chemotherapy response in breast cancer and should be further investigated as predictive markers in the future.

Kallikrein-related peptidase 6 (KLK6)gene expression in intracranial tumors.

Kallikrein-related peptidases (KLKs) are emerging novel new biomarkers for prognosis, diagnosis and therapeutic intervention of cancer. Kallikrein-related peptidase 6 (KLK6) has the highest expression in normal brain among other tissues. Although its expression has been extensively studied in many types of cancer and in neurodegenerative diseases, very little is known for its expression in intracranial tumors. In the present study, 73 intracranial tumor samples were examined for KLK6 messenger ribunucleic acid (mRNA) gene expression using quantitative real-time polymerase chain reaction. Statistical analysis revealed the significant association of KLK6 expression with clinical and pathological parameters. Follow-up information was available for a median time of 20 months (range 1-59 months). KLK6 is expressed more frequently in tumors of high malignancy like the glioblastomas (70.6 %) and less in tumors of low malignancy like the meningiomas (12.5 %). KLK6 positive expression is associated with tumor grade (p < 0.001), malignancy status (p < 0.001), and tumor histologic type (p = 0.001). Cox proportional hazard regression model using univariate analysis revealed for the first time that positive KLK6 expression is a significant factor for disease-free survival (DFS; p = 0.041) of patients suffering from intracranial tumors. Kaplan-Meier survival curves demonstrated that negative KLK6 expression is significantly associated with longer DFS (p = 0.032). KLK6 gene expression may have clinical utility as a marker of unfavorable prognosis for intracranial tumors, and consequently, it could be used as target for therapeutic intervention.

Human kallikrein-related peptidase 12 (KLK12) splice variants expression in breast cancer and their clinical impact.

Kallikrein-related peptidases (KLKs) are a group of 15 serine proteases, hormonally regulated, and localized on chromosome 19q13.4. Alternative splicing is a process that plays significant role in the development, physiology, and different diseases, like cancer. Kallikrein family numbers more than 82 alternative transcripts. Understanding the role that those gene transcripts play in various cancer types, could lead to the discovery of diagnostic markers or drug targets. The present study was designed to analyze the expression profile of the splice variants of kallikrein-related peptidase 12 (KLK12) in breast cancer patients and to evaluate their clinical significance. KLK12 splice variants (KLK12sv3 and KLK12sv1/KLK12sv2) were examined in 69 tissue samples of breast cancer using quantitative real-time PCR as well as semi-quantitative PCR. Relative quantitative expression of KLK12 was statistically associated to clinicopathological parameters. From the splice variants examined, statistical associations with clinicopathological parameters were obtained only from KLK12sv3 variant. KLK12sv3 is more frequently expressed in tumors of lower grade (p = 0.040), early patient TNM stage (p = 0.024), and smaller tumor size (p = 0.023). Positive KLK12sv3 expression is associated with longer patient disease-free survival (DFS) (p = 0.042) and higher progesterone receptor concentration (p = 0.008). KLK12sv1/KLK12sv2 expression is statistically associated with KLK12sv3 expression (p = 0.001). KLK12sv3 can be regarded as a marker of good prognosis in breast cancer.

Clinical evaluation of PRMT1 gene expression in breast cancer.

Methylation of arginine residues has been implicated in many cellular activities like mRNA splicing, transcription regulation, signal transduction and protein-protein interactions. Protein arginine methyltransferases are the enzymes responsible for this modification in living cells. The most commonly used methyltransferase in man is protein arginine methyltransferase 1 (PRMT1). Since methylation processes appear to interfere in the emergence of several diseases, including cancer, we investigated the localisation of the protein in cancer tissue and, for the first time, the relation that possibly exists between the expression of PRMT1 gene and breast cancer progression. We used tumour specimens from 62 breast cancer patients and semi-quantitative RT-PCR to determine the expression of PRMT1 gene and was found to be associated with patient's age (p = 0.002), menopausal status (p = 0.006), tumour grade (p = 0.03), and progesterone receptor status (p = 0.001). Survival curves revealed that PRMT1-v1 status-low expression relates to longer disease-free survival (DFS; p = 0.036). To the contrary, PRMT1-v2 status is not associated neither with the clinical or pathological parameters nor with DFS (p = 0.31). PRMT1-v3 was not statistically significantly expressed in breast cancer tissue. Selected cancer and normal breast samples were stained for PRMT1. In both normal and cancerous breast tissues, staining was in the cytoplasm and only in rare cases the cell nucleus appeared stained. Present results show a potential use for this gene as a marker of unfavourable prognosis for breast cancer patients.

Expression analysis and clinical evaluation of kallikrein-related peptidase 10 (KLK10) in colorectal cancer.

Kallikrein-related peptidases (KLKs) represent a serine protease family having 15 members. KLK10 is a secreted protease with a trypsin-like activity. The function of KLK10 is poorly understood, although it has been suggested that KLK10 may function as a tumor suppressor gene. In human cancer, KLK10 gene shows organ-specific up- or down-regulation. Since KLKs are promising tumor biomarkers, the examination of KLK10 mRNA expression and its association with colorectal cancer (CRC) progression was studied using semi-quantitative PCR. One hundred and nineteen primary CRC specimens were examined for which follow-up information was available for a median period of 29 months (range, 1-104 months). KLK10 expression was found to be significantly associated with TNM stage (p=0.028). Cox proportional hazard regression model using univariate analysis revealed for the first time that high status KLK10 expression is a significant factor for disease-free survival (DFS; p=0.002) and overall survival (OS; p=0.026) of patients. Kaplan-Meier survival curves demonstrated that KLK10 expression of low status is significantly associated with longer DFS (p=0.001) as well as OS (p=0.021), suggesting that KLK10 gene expression may be used as a marker of unfavorable prognosis for CRC. As the epigenetics of cancer are unraveled, KLK10 may represent not only a novel biomarker, but also a promising future therapeutic target for the disease.

KLK5 gene expression is severely upregulated in androgen-independent prostate cancer cells after treatment with the chemotherapeutic agents docetaxel and mitoxantrone.

Kallikrein-related peptidases (KLKs), including KLK5, have been proposed as promising biomarkers for prostate cancer diagnosis and prognosis. In the present study, we report that distinct augmentations (up to 6.4-fold) of KLK5 mRNA expressional levels, calculated via quantitative real-time PCR, occur after treatment of DU145 cells with appropriate concentrations, determined by the MTT method, of docetaxel and mitoxantrone. Our data reveal the endogenous need of prostate cancer cells for modified KLK5 expression to cope with the administration of chemotherapeutic drugs. Furthermore, it is proposed that the expression profile of KLK5 could serve as a putative biomarker for monitoring the treatment response in hormone refractory prostate cancer patients.

Treatment of PC3 prostate cancer cells with mitoxantrone, etoposide, doxorubicin and carboplatin induces distinct alterations in the expression of kallikreins 5 and 11.

Several of the novel kallikrein-related peptidases (tissue kallikreins; KLKs) are emerging new serum and/or tissue biomarkers for prostate cancer (CaP) diagnosis, prognosis and monitoring. In the present research approach, our objective was to investigate the possible alterations in the mRNA expression levels of KLK5 and KLK11 genes in prostate cancer cells PC3 as a response to treatment with mitoxantrone, etoposide, doxorubicin and carboplatin. Viability was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after cell treatment with either mitoxantrone (2 microM), etoposide (20 microM), doxorubicin (1 microM), or carboplatin (15 microM), for 24, 48 and 72 hours. Additionally, trypan blue staining revealed that in PC3 cells all drugs displayed almost the same limited necrotic effects which appeared mainly at 72 hours of treatment. PC3 prostate cancer cells showed a concentration- and time-dependent increased cytotoxicity to the drugs under study which was mainly due to reduction of cell proliferation efficiency. Distinct modulations of KLK5 and KLK11 genes, at the mRNA level, were observed, supporting a drug-dependent cell response. Our experimental data demonstrate that the molecular profile mainly of KLK5 gene may serve as a new potential molecular biomarker predicting treatment response in CaP cells.

Clinical significance of kallikrein-related peptidase 7 (KLK7) in colorectal cancer.

Human tissue kallikrein-related peptidases are a family of 15 secreted serine proteases, located at chromosome 19q13.4. Most of them have been reported to be potential biomarkers for several carcinomas and other diseases. Human tissue kallikrein-related peptidase 7 (KLK7) has been purified from human stratum corneum and resembles a chymotryptic endopeptidase originally called stratum corneum chymotryptic enzyme (SCCE). In this study, we examined for the first time, the prognostic value of KLK7 mRNA expression, using a semi-quantitative RT-PCR method, in 105 colorectal cancer tissues for 54 of which, paired normal colonic mucosa were available. Furthermore, we analysed the expression of KLK7 in 10 adenomas, in 18 biopsies of inflamed colon mucosa, as well as in 22 human cancer cell lines of various origin, four of them being of colon. A defined number of colon cancer samples were also examined by immunohistochemistry. KLK7 expression was higher in cancerous than in normal tissues. Less differentiated tumors of more advanced stage showed higher KLK7 expression. Follow-up analysis revealed that KLK7 was significantly associated with shorter overall survival (OS) and disease-free survival (DFS). In addition, selected colon cancer samples highly expressing KLK7 gene, showed intense immunohistochemical staining for KLK7, enhancing RT-PCR results. Present data suggest that KLK7 gene is up-regulated in colon cancer and its expression predicts poor prognosis for colon cancer patients.

Cathepsin B protein levels in endometrial cancer: Potential value as a tumour biomarker.

OBJECTIVE: Lysosomal cysteine protease Cathepsin-B has been implicated in the progression of various human tumours. We examined Cathepsin-B protein levels in endometrial carcinoma patients-mainly post-menopausal-and investigated their possible association with clinical and pathological parameters in order to assess Cathepsin-B's significance as a potential tumour biomarker. METHODS: The indirect immunoperoxidase method was used for Cathepsin-B immunohistochemical staining of 64 paraffin-embedded endometrial tumour tissues, having follow-up period of 18-240 months. Steroid hormone receptors were measured as well. Tissue samples were staged following the FIGO criteria. RESULTS: Positive Cathepsin-B immunostaining was observed in 27 patients (42.2%) and was significantly associated with the FIGO stage of the disease (p=0.006), as well as cervical and stromal invasion (p=0.001 and p=0.037, respectively) and progesterone receptor status (p=0.027). Positive Cathepsin-B expression was also inversely related to Disease-free Survival (p=0.034) and Overall Survival (p=0.035) in univariate analysis, as well as in multivariate analysis (p=0.022 and p=0.035, respectively). CONCLUSION: Increased Cathepsin-B expression was found to be predictive of more aggressive tumour behaviour over time and can be regarded as an unfavourable and independent tumour marker for endometrial cancer patients with a long follow-up.

Colon cancer and protein arginine methyltransferase 1 gene expression.

BACKGROUND: In this study, the possible relation of the expression pattern of arginine methyltransferase 1 and colon cancer progression is investigated. MATERIALS AND METHODS: Colon cancer samples as well as normal colon samples were used to define the arginine methyltransferase 1 expression by RT-PCR. The results were associated with clinical and histological parameters of the tissues. RESULTS: In colon cancer tissue, only PRMT1 variants v1 and v2 were often expressed. Statistical significance for the clinicopathological parameters examined was found only for PRMT1 variant v2. PRMT1 v2 expression was associated with nodal status and tumour grade. PRMT1 v2 expression analysis in 25 pairs of cancerous/non-cancerous colon tissue showed higher or equal expression in cancer versus normal tissue. In 18 inflamed colon tissues examined for PRMT1 expression and compared with the expression of 90 colon cancer tissue samples, statistical significance was found only for variants v1 and v2. A higher percentage of PRMT1 v2 expression was observed in older patients. CONCLUSION: From the present preliminary results, it can be said that PRMT1 variant v2 can probably be regarded as a marker of unfavourable prognosis in colon cancer patients.

Human kallikrein-related peptidase 12 (KLK12) splice variants discriminate benign from cancerous breast tumors.

OBJECTIVES: As kallikrein-related peptidase 12 (KLK12) has been implicated in the cancer progression and alternative splicing plays significant role in this disease, the aim of this study was to examine the expression profile and the clinical impact of the KLK12 splice variants in breast cancer. DESIGN AND METHODS: Total RNA was isolated and reverse transcripted from 141 tissues. Afterwards, quantitative real-time PCR were conducted, followed by the performance of the comparative CT (2-ΔΔCT) method for relative quantification, whilst their correlation with the clinicopathological features of breast malignancies were assessed by statistical analysis. RESULTS: Both KLK12sv1/2 and KLK12sv3 showed higher expression in non-cancerous than in cancerous samples. KLKsv1/2 (P = 0.001) upregulated and KLK12sv3 (P < 0.001) downregulated in the malignant compared to the benign tumors and their discriminative ability was verified by ROC curve analysis. Moreover, KLK12sv3 was associated with grade (P = 0.012) and hormonal receptor status (P = 0.001). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with positive KLK12sv1/2 and KLK12sv3 levels presented a significantly longer disease-free survival (P = 0.014 and P = 0.013, respectively) and overall survival (P = 0.062 and P = 0.004, respectively). CONCLUSIONS: Our results demonstrate the discriminative value of KLK12sv1/2 and KLK12sv3 between benign and malignant breast tumors as well as their potential favorable prognostic significance in breast adenocarcinoma.

Prognostic value of the apoptosis related genes BCL2 and BCL2L12 in breast cancer.

Many members of BCL2 (Bcl-2) apoptosis-related genes were found to be differentially expressed in various malignancies and were proposed as prognostic cancer biomarkers. Recently, a new member of the BCL2 gene family, BCL2L12, was cloned and was found to be expressed in mammary gland. In the present study, 55 specimens from patients with, histologically confirmed, epithelial breast carcinoma were analyzed for BCL2 and BCL2L12 gene expression by RT-PCR. Increased expression of BCL2 gene was found in patients belonging to the age groups <45 or >55 years, as well as in estrogen receptors (ER)-positive patients and in BCL2L12-positive tumors. In addition, BCL2 or BCL2L12-positive patients were found to be almost four times less likely to relapse or die in comparison to BCL2 or BCL2L12-negative patients, respectively. Multivariate analysis revealed that BCL2 and BCL2L12 might be used as independent prognostic biomarkers in breast cancer.

Alterations in mRNA expression of apoptosis-related genes BCL2, BAX, FAS, caspase-3, and the novel member BCL2L12 after treatment of human leukemic cell line HL60 with the antineoplastic agent etoposide.

Apoptotic cell death is a highly regulated process, which plays a crucial role in many biological events. Etoposide is an antineoplastic drug, which targets the DNA unwinding enzyme, topoisomerase II. The aim of the present research approach to investigate the expression of the apoptosis-related genes BCL2 (Bcl-2), FAS, Caspase-3, BAX and the new member BCL2L12, cloned by our group, along with treatment of HL-60 leukemia cells with etoposide. The kinetics of apoptosis induction and cell toxicity was evaluated by DNA laddering and MTT method, respectively. The mRNA expression levels of the genes were analyzed by RT-PCR using gene-specific primers. Beta-actin was used as a control gene. An important downregulation of BCL2L12 was observed at 4 h of drug treatment, whereas BAX was upregulated at the same time point. No alteration in the expression pattern of the other apoptosis-related genes was detected. Since, the main anticarcinogenic effect of etoposide is due to the induction of apoptosis, these changes observed in the mRNA expression levels of the genes may be an underlying mechanism.

EGFR alterations in pancreatic ductal adenocarcinoma: a chromogenic in situ hybridization analysis based on tissue microarrays.

BACKGROUND/AIMS: To evaluate epidermal growth factor receptor (EGFR) gene status in pancreatic ductal adenocarcinoma correlating the results to protein expression and clinicopathological features METHODOLOGY: Using tissue microarray technology (TMArrayer 100), fifty (n = 50) paraffin-embedded tissue samples of histologically-confirmed primary tumors were cored twice at a diameter of 1 mm and re-embedded into the final recipient block. Immunohistochemistry was performed by the use of anti-EGFR monoclonal antibody (31G7). Also, a chromogenic in situ hybridization protocol was applied based on the use of EGFR gene and chromosome 7 centromeric probes, respectively. RESULTS: EGFR protein overexpression was observed in 29/50 (58%) cases and correlated to stage (p = 0.001) but not to grade (p = 0.206). EGFR gene analysis identified numerical alterations in 6/50 (12%), including 2 cases characterized by low-level gene amplification and 4 by absence of one allele. Gene status was associated to tumor grade (p = 0.023) and stage (p = 0.02). Chromosome 7 analysis detected aneuploidy in 14 (28%) cases. CONCLUSIONS: A subset of pancreatic ductal adenocarcinomas (PDACs) is characterized by EGFR gene numerical alterations including sporadic cases of amplification or absence of one allele (maybe due to gene deletion or intragenic point mutation and allelic silence). Those alternative mechanisms maybe influence the efficacy of novel targeted therapeutic strategies based on monoclonal antibodies or intracellular tyrosine-kinase inhibitors in PDACs.

Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA.

OBJECTIVES: To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation. DESIGN AND METHODS: The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells. RESULTS: The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose. CONCLUSIONS: The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples.

Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma.

Kallikreins are a subgroup of serine proteases that are involved in the post-translational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. Human kallikrein gene 7 (KLK7; HSCCE) is a new member of the human kallikrein gene family. KLK7 is expressed in normal breast tissue and is up-regulated in breast cancer cells by estrogens and glucocorticoids. In the present study, expression of the KLK7 gene in 92 breast cancer tissues was analyzed by reverse transcription-PCR (RT-PCR) and direct sequencing of several samples. The results were correlated with other clinicopathological variables and patient outcome. KLK7 gene expression was significantly lower in breast cancer patients of low stage (I/II) (p = 0.011) and patients with positive progesterone receptors (p = 0.022). Survival analysis showed that breast cancer patients with KLK7 positive tumors have relatively shorter disease-free survival (DFS) and overall survival (OS) than patients with KLK7 negative tumors. These data suggest that KLK7 gene expression may be used as a marker of unfavorable prognosis for breast cancer patients.

Expression of BCL2L12, a new member of apoptosis-related genes, in breast tumors.

Apoptosis, a normal physiological form of cell death, is critically involved in the regulation of cellular homeostasis. If the delicate balance between cell death and cell proliferation is altered by a defect in the normal regulation of apoptosis signaling, a cell population is able to survive and accumulate, thereby favoring the acquisition of further genetic alterations and promoting tumorigenesis. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in the responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-XL has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Recently, a new member of the Bcl-2 family, BCL2L12, was cloned. The BCL2L12 gene is constitutively expressed in many tissues, suggesting that the encoded protein serves an important function in different cell types. In the present study, the expression of BCL2L12 gene was analyzed by reverse transcription-PCR (PT-PCR) in 70 breast cancer tissues. Our results indicate that BCL2L12 positive breast tumors are mainly of lower stage (I/II) or grade (I/II) (p=0.02 or p=0.04 respectively). Cox regression analysis revealed that BCL2L12 expression is positively related to disease-free (DFS) and overall survival (OS) at both univariate and multivariate analysis (p=0.021, p=0.029, p=0.032, p=0.044 respectively). Kaplan-Meier survival curves also demonstrated that patients with BCL2L12-positive tumors have significantly longer DFS and OS (p=0.002 and p<0.001 respectively). BCL2L12 expression may be regarded as a new independent favorable prognostic marker for breast cancer.

Cathepsin B and cathepsin D expression in the progression of colorectal adenoma to carcinoma.

Lysosomal proteinases, cathepsin B (CB) and cathepsin D (CD) have been implicated in the progression of several human tumors. In the present study, the antigen levels of CB and CD, and their immunohistochemical staining were compared in paired colorectal tumors (n =64) and background colon tissue of the same patients with clinicopathological staging. The antigen levels, were found to be significantly higher in cancer tissue (mean 35.79 ng/mg protein for CB and 3.97 ng/mg protein for CD) than in corresponding normal mucosa (24.62 ng/mg protein for CB and 2.69 ng/mg protein for CD). CB antigen levels were positively correlated with differentiation grade and Duke's stage (P < 0.001 and P = 0.041, respectively), but not correlated with nodal status. CD antigen levels were not correlated with the previous parameters. Staining intensity for both antigens increased from adenoma to adenocarcinoma. The degree of staining for CB and CD was associated with differentiation grade (P = 0.004 and 0.001, respectively), Dukes' stage (P = 0.002 and 0.001, respectively) and lymph node involvement (P = 0.002 and P < 0.001, respectively).