Soluble guanylate cyclase isoenzymes: The expression of α1, α2, ß1, and ß2 subunits in the benign and malignant breast tumors.

Soluble guanylate cyclase (sGC) encompasses α and ß subunits. This study examined the expression of α1, α2, ß1, and ß2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and ß2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and ß2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.

Expression level of VLDL receptor and VLDL-c levels in the malignant and benign breast tumors: The correlation with miRNA-4465 and miRNA-1297.

Breast cancer as one of the most prevalent cancers has high morbidity and mortality. Very low-density lipoprotein receptor (VLDLR) is a multifunctional receptor which plays a principal role in the tumor development through affecting cell metastasis and proliferation. The VLDLR as a target for miRNA-4465 and miRNA-1297 was predicted using bioinformatics analysis. Tissue specimens of malignant (n = 50), benign (n = 35) and corresponding normal breast (n = 20) were considered to evaluate the expression of VLDLR using RT-qPCR and western blotting. The VLDL cholesterol (VLDL-C) levels were quantified using a colorimetric assay. The relative VLDLR expression was found in the malignant tumors, which was significantly lower than that in the normal tissues (P<0.05). The expression levels of VLDLR had no significant difference between malignant and benign tissues (P>0.05). Correlation analysis revealed that the VLDLR expression level had a direct correlation with miRNA-1297 (R=0.566, P<0.05), but a reverse one with miRNA-4465 (R = -0.663, P<0.0001). The VLDL-C level in the malignant and normal tissues was lower than that in the benign tumors, which was not significant (P>0.05). The expression levels of VLDLR in E+P-H- (ER+,PR-,HER2-) tumors were higher than those in other subtypes (P<0.05). Furthermore, a negative correlation was found between the VLDLR expression level and the Ki 67% score. These data revealed that the lower expression of VLDLR mediated by the high expression levels of miRNA-4465 may be involved in the development of breast cancer. These results might provide some evidence for the effect of VLDLR on the breast cancer.

Differential expression of alternative transcripts of soluble guanylyl cyclase, GYCY1a3 and GUCY1b3 genes, in the malignant and benign breast tumors.

Extensive alterations in splicing is one of the molecular indicator for human cancers. Soluble guanylyl cyclase (sGC), an obligatory heterodimer, is composed of α1 and ß1 subunits. Each subunit is encoded by a separate gene, GUCY1a3 and GUCY1b3, correspondingly. sGC activity has been regulated by an alternative splicing and it has an important effect on the breast cancer. sGC alternative splicing has been evaluated in the 55 malignant, 25 benign and 30 normal breast tissues using qRT-PCR and RT-PCR. The differences between groups were analyzed by Mann-Whitney U. The expression of six different splice forms have been detected, three for α1 and three for ß1 sGC. Expressions of Tr1, Tr2 ß1 sGC and Tr7, Tr6 α1 sGC mRNA in the malignant breast tumors were significantly lower than those of benign and normal breast tissues. However, the expression of Tr3 α1 sGC mRNA was significantly higher than that of benign and normal tissues. Present data have provided some evidences for an alteration in the expression of α1 and ß1 sGC alternative splicing forms which may contribute to the loss of sGC functions in the breast cancer. The observed information might be discussed by the cGMP status.

Evaluation of RIP1K and RIP3K expressions in the malignant and benign breast tumors.

Receptor-interacting protein kinase 1 (RIP1K) and RIP3K belong to RIPK family, which regulate cell survival and cell death. In the present investigation, the expression levels of RIP1K and RIP3K were evaluated in the 30 malignant, 15 benign, and 20 normal breast tissues, and their correlation with clinicopathological characteristics was also studied. The expression levels of RIP1K and RIP3K were determined, by western blot analysis. The relative RIP1K expression was significantly higher in the malignant and benign tumors when compared to those of normal tissues (P < 0.0001 and P < 0.001, respectively). However, the expression level of RIP3K was significantly lower in the malignant tumors than those of normal and benign values (P < 0.001 and P < 0.01, respectively). Positive significant correlation was found for RIP1K expression with tumor size (P < 0.001), grades (P < 0.0001), and c-erbB2 (P < 0.001), but negative significant correlation was detected with patient's age (P < 0.001), estrogen receptor (ER) (P < 0.001), progesterone receptor (PR) (P < 0.01), and P53 (P<0.01) status. RIP3K expression was significantly lower in the pre-menopauses (P < 0.01), grade III (P < 0.05), ER-negative (P < 0.05), and c-erbB2-negative malignant tumors, but no correlation was detected with tumor size, PR, and P53 status. No significant correlation was observed for RIP1K and RIP3K expressions with Ki67 and Her2. Based on the present results, it is concluded that reduction of RIP3K expression in the malignant breast tumor might be an important evidence to support the antitumor activity of this enzyme in vivo. However, RIP1K expression was shown to be higher in the malignant breast tumors than those of normal and benign breast tissues, which probably designates as a poor prognostic factor.

Personalized evolutionary hypothesis in genomics and auxiliary lymph node through diverse subtelomeric signal profile.

Few available data on the genomic-somatic evolution in breast cancer create limitation to provide the appropriate clinical managements. As an example, human subtelomeres (ST) are diverse-prone and variable targets. STs, as hot spots, have positive and negative impacts on the status of health and malady. We showed higher subtelomere signal copy number (SCN) of specific chromosomes in genomics than in auxiliary lymph node (ALN). Dissimilarity of signal intensity (SI) is found for all chromosomes. Significantly higher SI in genomics than in ALN cells were specified as chromosomes 5, 6, 9-12, 16-19 for weak; 1, 5-9, 19, X for medium; and 2, 5, 9, 10, 16, 18 for strong SI. For lacking, and presence of one and two SCNs; p/q ratio reflected differences for all chromosomes; but, 2, 3, 5, 7, 8, 10, 16, 18, 20, and X chromosomes were involved for three SCN. Chromosomes 1, 4, 9, 12, 17-19 lacked three SCN in ALN and lymphocytes. Weak SI ratio was higher in p- than in q-arm in majority of chromosomes. Manner of evolution and diversity in p- and q-arms is expressive of a novel definition as two diverse domains with a personalized insight. These data have been accompanied by periodic charts as ST array profiles which provide specific and individualized pattern in breast neoplasm. Such profiling at genomics level could be considered as a prediction through the patients' life. Moreover, subtelomere territory by interacting with protein expression of Ki67, cyclin D1, and cyclin E; and molecular targets including telomere length at genomics and somatic level provides package of information to bridge cancer cell biology to the cancer clinic as "puzzling paradigm."

Plasma total homocysteine level in association with folate, pyridoxine, and cobalamin status among Iranian primary breast cancer patients.

Recently the elevated plasma total homocysteine (tHcy) concentration has been concerned as the secondary feature of tumoral proliferation and enhances the likelihood of thrombogenesis in cancer patients. The objective of this study was to determine the associations between folate, cobalamin, and pyridoxine with fasting plasma tHcy concentration in breast cancer (BC) patients. The intake levels of nutrients were assessed using a validated food frequency questionnaire in 141 newly diagnosed BC patients. The plasma tHcy and pyridoxal-5-phosphate were measured using high performance liquid chromatography with fluorescence detector. Plasma tHcy levels were observed to be significantly higher among BC participants with Stage III where the plasma concentrations of folate was also comparatively less (P < 0.05) than other stages. Dietary pyridoxine was even being consumed less at this stage (P < 0.05). The plasma, dietary, and residual variables of folate were inversely correlated with plasma tHcy concentration (P < 0.05). Dietary cobalamin was also associated negatively with tHcy (P < 0.05). The odds ratio of comparing the highest tertile of plasma cobalamin (>394 pmol/l) and folate (>11.4 ng/ml) vs. the lowest categories were associated with reduced odds of high tHcy occurrence with 0.20 (95% confidence interval: 0.04-0.98) and 0.14 (95% confidence interval: 0.03-0.64), respectively. In conclusion, nutrition-related methyl-group insufficiency could lead to imbalance in tHcy metabolism, as a possible cancer marker.

Essential oil variation in the populations of Artemisia spicigera from northwest of Iran: chemical composition and antibacterial activity.

CONTEXT: Artemisia spicigera C. Koch (Asteraceae) is a perennial shrubby herb and is generally distributed in Armenia, Iran, and Middle Anatolia. This species traditionally has been used in medicines. OBJECTIVE: The aim of this research is to study the chemical composition and antibacterial activity of essential oils from Artemisia spicigera populations in northwest of Iran. MATERIALS AND METHODS: The essential oil of A. spicigera was obtained by hydrodistillation from eight populations collected from different regions of East Azerbaijan and West Azerbaijan provinces (Iran) and analyzed by gas chromatography-mass spectrometry (GC-MS). The antibacterial activity of the oils was investigated against four Gram-positive and four Gram-negative bacteria using MIC determinations and the agar-gel diffusion method. RESULTS: Fourteen compounds were identified as the main components of the essential oils and the most abundant constituents are 1,8-cineole, camphor, α-thujone, camphene, ß-thujone and p-cymene. Essential oil of population No. 1 showed the highest activity against Escherichia coli, Enterobacter aerogenes, Serratia marcescens and Staphylococcus aureus but the highest activity against St. saprophyticus, Bacillus megaterium, and B. cereus was found with population No. 6 and for Citrobacter amalonaficus with population No. 5. MIC values of essential oils ranged from 6 µg/mL against Bacillus megaterium to 12 µg/mL against Citrobacter amalonaficus. DISCUSSION: This study demonstrates the occurrence of 1,8-cineole/camphor/camphene chemotype of A. spicigera but there is also significant chemical variation between the studied populations. The findings showed the studied oils have good antibacterial activity, and thus potential to be used as natural health products.

Expression of cGMP-dependent protein kinase, PKGIα, PKGIß, and PKGII in malignant and benign breast tumors.

Cyclic GMP-dependent protein kinases (PKG) constitute a small family of enzymes that are encoded by two genes. Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated as Iα and Iß. There are increasing evidences that PKG can play an important role in the inhibition of cell proliferation and induction of apoptosis. In our previous studies, the inhibitory effects of cGMP/PKG on the cell growth were indicated using breast cancer cell lines. Accordingly, the present study was designed to compare the expression levels of three PKG isoforms in normal, benign, and malignant breast tissues. The expression level of PKG isoforms was assayed using quantitative real-time RT-PCR. The correlation between relative expression of PKG isoforms and clinicopathological characteristics were also analyzed. Downregulation of PKG isoforms was observed in the malignant and benign tumors when compared to those of respective normal tissues. No significant correlation was found between PKGIα, PKGIß, and PKGII expression and clinicopathological features. The present study is the first to evaluate the expression level of PKG isoforms PKGIα, PKGIß, and PKGII in the malignant and benign breast tumors. Reduction in the PKG expression is an important evidence to support the antitumor activity of this enzyme in vivo.

Evolutionary hypothesis of telomere length in primary breast cancer and brain tumour patients: a tracer for genomic-tumour heterogeneity and instability.

It was previously reported that tumour samples had shorter telomeres than the surrounding normal tissue. Hereby, the initial sign of correlation between malignant tissue and telomere behaviour could be noticed. Bridging knowledge between germ and somatic cells could facilitate understanding cellular evolution. The aim of our investigation was to provide evidence for the evolutionary hypothesis of TL (telomere length) in primary BC (breast cancer) and BTs (brain tumours), which might be applied as a prognostic and/or predictive marker. DNA extraction from the frozen tissues was performed using high pure PCR template preparation kit. Standard protocol of Telo TTAGGG Telomere Length Assay kit, a non-radioactive chemiluminescent assay, was used. The protein expression in extracted cells was analysed by immunofluorescence. We also detected telomerase activity. The G/T (genomic/tumour ratio) for TL in two groups of patients affected with primary BC and primary BT revealed significant differences in both BC patients (P = 0.025) and in BTs (P = 0.001). The pattern of telomere signals by Q-FISH (quantitative fluorescent in situ hybridization) show that in all samples, except one, SI (signal intensity) has been significantly decreased in tissue related to blood, either in BC patients or in patients with BTs (0.041≥P≥0.001). However, the data achieved by Q-FISH support the results of Southern blot. These data reflect a significant diversity either in BC or in BT patients, providing evidence for the evolutionary hypothesis of TL in cancer development and progression.

Androgen receptor gene CAG repeat polymorphism and breast cancer risk in Iranian women: a case-control study.

We investigated the association between polymorphic expansion of trinucleotide CAG repeats in androgen receptor (AR) gene and breast cancer risk among Iranian women in a matched case-control study. There was a strong overall association between per CAG repeat increments in average repeat length and the risk of the malignancy [OR=3.56; 95% CI, 2.80-5.29]. Women carrying one or two alleles with [CAG]n repeat ≥22 units were at increased risk of breast cancer [OR=2.03; 95% CI, 1.56-2.6]. The risk was significantly increased in homozygous longer repeats, versus homozygous alleles <22. We observed reduced risk of developing the tumor in positive familial breast cancer subjects carrying repeats ≥22 and 23. Homozygosity for the longer [CAG]n repeats may be linked to the increased breast cancer risk. In contrast to previous reports, longer AR [CAG]n repeat alleles may decline the risk among women with a familial breast cancer.

The effect of modifiable potentials on hypermethylation status of retinoic acid receptor-beta2 and estrogen receptor-alpha genes in primary breast cancer.

Epigenetic silencing of retinoic acid receptor-beta2 (RARbeta2) and estrogen receptor-alpha (ERalpha) expressions have been revealed to be important in the development of approaches for diagnosis and therapy of breast cancer. We aimed to explore the correlation of some potential factors with the hypermethylation status of RARbeta2 and ERalpha genes among Iranian breast cancer patients. The hypermethylation status was investigated in 137 dissected tissues from primary breast cancer patients through methylation-specific PCR. Overall, the methylation frequencies of RARbeta2 and ERalpha genes were observed in 36.5 and 51.1% of participants, respectively. The hypermethylated RARbeta2 was associated with younger age at diagnosis and negative family history of breast cancer. The hypermethylation of ERalpha was correlated positively with smoking, duration of estradiol exposure, ER-negativity in tumors and body mass index (at 5 years ago). The plasma levels of folate and vitamin B(12) were inversely related to the hypermethylation status of ERalpha, after controlling for covariates. The risk of ERalpha hypermethylation was increased with high plasma level of total homocysteine. In conclusion, our data provide new insights into the possible effect of some lifestyle-related factors on the aberrant methylation drift of ERalpha and RARbeta2 genes in breast cancer.

Intraspecific variability of the essential oil of Ziziphora clinopodioides ssp. rigida from Iran.

Hydrodistillated essential oils of Ziziphora clinopodioides ssp. rigida from nine populations of the Lashgardar protected region (Hamedan Province, Iran) were analyzed by using GC and GC/MS techniques to determine the intraspecific chemical variability. Altogether, 39 compounds were identified in the oils, and a relatively high variation in their contents was found. The main constituents of the essential oils were pulegone (0.7-44.5%), 1,8-cineole (2.1-26.0%), neomenthol (2.5-22.5%), 4-terpineol (0.0-9.9%), 1-terpineol (0.0-13.2%), neomenthyl acetate (0.0-7.1%), and piperitenone (0.0-5.4%). For the determination of the chemotypes and the intraspecific chemical variability, the essential oil components were subjected to cluster analysis (CA). The five different chemotypes characterized were Chemotype I (pulegone/neomenthol), Chemotype II (pulegone), Chemotype III (pulegone/1,8-cineole), Chemotype IV (neomenthol), and Chemotype V (1,8-cineole/4-terpineol).

Differential expression of miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant and benign breast tumors.

OBJECTIVES: MicroRNAs (miRs) are a class of small non-coding RNAs which are associated with tumor growth and progression. In the present study, we assessed the expression of selected miRs in malignant, benign, and adjacent normal breast tissues. MATERIALS AND METHODS: The expression of miR-1297, miR-3191-5P, miR-4435, and miR-4465 were evaluated in malignant (n=50), benign (n=35), and adjacent normal breast tissues (n=20) using qRT-PCR. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated for evaluating the diagnostic values of miRs. To evaluate diagnostic efficacy, miRs-based score was obtained using the logistic regression model. RESULTS: Among malignant tumors, the expression of miR-1297, miR-3191-5p, and miR-4435 was significantly lower (P=0.024, P<0.001 and P=0.031), respectively. The expression of miR-4465 was higher (P=0.023) than that of normal tissue. The expression of these miRs was lower than those of benign tumors (P<0.01, P<0.001, P<0.0001, and P<0.01, respectively). We observed a positive correlation between miR-4465 expression levels and tumor stage (P=0.042) and a negative correlation with grade and Ki-67 score (P<0.05). The AUCs for miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant tumors versus normal tissues were 0.784, 0.700, 0.976, and 0.865 and versus benign tumors they were 0.938, 0.857, 0.981, and 0.785, respectively. The optimal logit(P) value of 0.262 distinguished malignant from normal subjects with a sensitivity of 0.91, specificity of 0.85, and an overall accuracy of 0.89. CONCLUSION: The panel of these miRs are suggested as possible onco-miRs(miR-4465) or tumor suppressor-miRs (miR-3191-5P, miR-1297, miR-4435). Overall, our results indicated that these miRs could be introduced as diagnostic biomarkers in breast cancer patients.

Evaluation of LDL receptor and Scavenger Receptor, Class B, Type 1 in the malignant and benign breast tumors: The correlation with the expression of miR-199a-5p, miR-199b-5p and miR-455-5p.

AIMS: The purpose of present study was to examine the correlations of LDL (LDLR) and HDL (SR-B1) receptors with lipoproteins, miR-199a-5p, miR-199b-5p, miR-455-5p in the malignant and benign breast tumors. METHODS: Total cholesterol-rich-lipoproteins and the receptors were determined using enzymatic-homogeneous and ELISA methods. The expression levels of miRNAs were detected by qRT-PCR. RESULTS: Receptor expressions and lipoproteins concentration were significantly higher in the malignant tumors (p < 0.05). Positive correlation was found for LDLR with Ki67% and Her2+. HDL-C content of TNBC tumors was higher than those of Non-TNBC (p < 0.05). The expression level of miR-199a-5p was found to be downregulated significantly in the malignant tumors of <2 cm, TNBC, HER2- or stage3. The expression of miR-199b-5p was downregulated in the malignant tumors and was negatively associated with TNBC, stage and Her2+. The expression of miR-455-5p was significantly correlated with Her2- (p < 0.05). A positive correlation was observed for SR-B1 or LDLR with HDL-C or LDL-C and also for SR-B1 with LDLR, although a reverse association was detected for the expression of miR-199b-5p with LDLR in the malignant tumors (p < 0.05). No significant correlations were found for miR-199a-5p or miR-455-5p with LDLR or SR-B1 expressions and also for LDL-C and SR-B1 with clinicopathological features (p ≥ 0.05). CONCLUSIONS: Mechanisms potentially involved in the present findings may be due to the lipid internalization and lipoprotein consumption through LDLR and SR-B1 over expression. It is noteworthy that the expression of miR-199b-5p is negatively correlated with LDLR which may suggest it as a suppressor for LDLR expression in the breast cancer.

Autologous dendritic cells loaded with apoptotic tumor cells induce T cell-mediated immune responses against breast cancer in vitro.

Dendritic cell (DCs) based immunotherapy has received increased interest in the treatment of specific malignancies including breast cancer. In this in vitro study, T cell responses, which are induced by monocyte-derived DCs pulsed with apoptotic breast tumor cells (ApTC), were analyzed in terms of proliferation, specific cytotoxicity, and cytokine release. Nylon wool-enriched T lymphocytes from five patients with breast cancer stimulated with monocyte-derived DCs pulsed with apoptotic tumor cells in vitro and their proliferation response were analyzed by [(3)H] thymidine uptake and specific cytotoxic activity of tumor antigen-primed T cells after three rounds of weekly stimulation by flow cytometry. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) cytokine release assay was carried out 24h after the last stimulation. The supernatant from primed T cells was collected and analyzed using commercially available ELISA kits. T cell proliferation assays revealed that DCs pulsed with apoptotic tumor cell could stimulate an autologous T cell proliferation response with stimulation indices of 5-21. The T cell-mediated cytotoxicity assay demonstrated that tumor antigen-primed T cells could kill significantly more autologous tumor cells than normal cells (P<0.05). These cells had variable amounts of cytotoxic activity against K562 cells. Primed T cells released both IFN-gamma and IL-4 in response to re-stimulation by antigen-pulsed DCs, but were dominated by IFN-gamma production in two out of five patients and IL-4 production in three out of five patients. In conclusion, our results suggested that DCs pulsed with apoptotic breast tumor cells could elicit effective specific antitumor T cell responses in vitro. Therefore, vaccination with DCs pulsed with apoptotic tumor cells may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.

Prognostic implication of CDC25A and cyclin E expression on primary breast cancer patients.

Defect in cell cycle control is a hallmark character of cancer. We have investigated the association of Ki67 labeling index, cyclin E and CDC25A expressions with clinical follow-up data in breast carcinomas. Flow cytometry was used to detect gene amplification of cyclins in 44 tumor tissue with invasive breast carcinomas. Multivariate Cox proportional hazard ratio test was used to show the correlations. Cyclin E or CDC25A were upregulated in 34% of the tumors. Among the whole total material, expression of cyclin E and of CDC25A were found upregulated in 31.9% and 39.4% of cells, respectively. Both CDC25A and cyclin E protein expression levels were correlated with Ki67 expression level (p<0.001). In addition, the expression of CDC25A was associated significantly with poor survival (P=0.028), whereas no correlation was found with cyclin E. These findings suggest a possible prognostic value for CDC25A as a cell cycle marker and may imply in characteristic of high risk breast cancer patients.

Hypermethylation pattern of ESR and PgR genes and lacking estrogen and progesterone receptors in human breast cancer tumors: ER/PR subtypes.

BACKGROUND: The option of endocrine therapy in breast cancer remains conventionally promising. OBJECTIVE: We aimed to investigate how accurately the pattern of hypermethylation at estrogen receptor (ESR) and progesterone receptor (PgR) genes may associate with relative expression and protein status of ER, PR and the combinative phenotype of ER/PR. METHODS: In this consecutive case-series, we enrolled 139 primary diagnosed breast cancer. Methylation specific PCR was used to assess the methylation status (individual test). Tumor mRNA expression levels were evaluated using real-time RT-PCR. Immunohistochemistry data was used to present hormonal receptor status of a tumor (as test reference). RESULTS: Methylation at ESR1 was comparably frequent in ER-breast tumors (83.0%, P< 0.001; sensitivity = 83.0%, specificity = 65.2% and diagnostic odds ratio, DOR = 12.0) and strongly correlated with ER-/PR- conditions (Cramer's V= 0.44, P< 0.001). Methylated PgRb promoter frequently was observed in tumors recognised as ER- or negative ER/PR (77.1%, P< 0.01). Assessment of DNA methylation of ESR1 harbouring methylation at PgRb was a case significantly suggested to be able to detect the lack of ER/PR expressions (55.6%, P< 0.01; sensitivity = 80.6%, specificity = 68.7% and DOR = 8.7). However, methylated PgRb was quite acceptable determinant to contribute with methylated ESR1 to rank tumors as ER-/PR- (64.4%, P< 0.01; sensitivity = 78.0%, specificity = 62.5% and DOR = 6.0). CONCLUSIONS: Despite the methylation status of ESR1 showed preponderant contribution to tumoral phenotypes of ER- and ER-/PR-, the hypermethylation of PgRb seem another epigenetic signalling variable actively associate with methylated ESR1 to show lack of ER+/PR+ tumors in breast cancer.

Prognostic values of proliferative markers ki-67 and repp86 in breast cancer.

BACKGROUND: Breast cancer is the leading cause of carcinoma death in women. Proper treatment depends on the consideration of molecular biology status of tumor cells, which may determine the patient's treatment and prognosis. To determine the prognostic models for this disease, we evaluated the role of cell proliferation-related antigens including ki-67 (a nuclear antigen, expressed in G1, G2, and M phases of cell cycle) and repp86 (an 86-kDa nuclear protein expressed in S, G2, and M phases of cell cycle) for detection of biologic behavior of breast cancer. METHODS: We studied 60 women with grade I and II lymph node-negative and 27 with grade III lymph node-positive breast cancers. The mean follow-up periods for these two groups were 60 and 72 months, respectively. Tumor cell proliferation was measured by immunohistochemical methods with monoclonal antibodies directed against the nuclear antigens ki-67 and repp86. RESULTS: The ki-67, repp86 labeling indices (percentage of antibody-stained tumor cell nuclei) were not statistically different between the cases and controls of lymph node-negative patients (ki-67 with P = 0.33; repp86 with P = 0.40). The odds ratio (the mean chance of ki-67 labeling index > 10%, repp86 labeling index >10%) in patients with recurrence was 4 (CI = 0.2 - 76.5) for ki-67 and 3.6 (CI = 0.4 - 32.5) for repp86. Both indices were statistically different in lymph node-positive cases and controls (P < 0.0001). The odds ratio in patients with recurrence was 87 (CI = 4 - 18.71) for ki-67 and 71.5 (CI = 5.7 - 899.2) for repp86. CONCLUSION: The present study confirms the importance of cell proliferation as a determinant of biologic behavior of breast cancer. Measurement of ki-67 and repp86 labeling indices may be very helpful for physicians to detect high-risk patients and to adopt appropriate procedure such as adjuvant therapy.

The association between telomerase activity and expression of its RNA component (hTR) in breast cancer patients: the importance of DNase treatment.

Telomerase is a ribonucleoprotein enzyme that compensates for the telomere length shortening which occurs during the cell cycle. Telomerase activity has been detected in most tumours but not in somatic cells. However, hTR; the RNA component of telomerase; has been reported to be universally expressed in both cancerous and non-cancerous tissues. Tumour samples from 50 patients with primary invasive breast cancer were collected. The TRAP assay was used to detect telomerase activity. RT-PCR on cDNA and DNased cDNA samples and control groups was used to detect the expression of hTR, GAPDH and PGM1 genes. Seventy-two percent of samples showed telomerase activity. DNA contamination was detected in 36 (72%) of RNA samples. Without performing DNase treatment, 49 (98%) of all samples showed hTR expression, but with the application of this strategy, hTR expression decreased from 98% to 64%. A significant association (p < 0.001) between hTR expression and telomerase activity was observed. Among the 32 hTR positive samples, 30 had telomerase activity and among the 18 hTR negative samples, telomerase activity was observed in 6 cases. Thus the application of this strategy could provide an applicable tool to use instead of the TRAP assay thus facilitating telomerase research in cancer genetic investigations.

The expression of hTR and hTERT in human breast cancer: correlation with clinico-pathological parameters.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesizes telomeres after cell division and maintains chromosomal stability leading to cellular immortalization. Telomerase has been associated with negative prognostic indicators in some studies. The present study aims to detect any association between telomerase sub-units: hTERT and hTR and the prognostic indicators including tumour's size and grade, nodal status and patient's age. METHODS: Tumour samples from 46 patients with primary invasive breast cancer and 3 patients with benign tumours were collected. RT-PCR analysis was used for the detection of hTR, hTERT, and PGM1 (as a housekeeping) genes expression. RESULTS: The expression of hTR and hTERT was found in 31(67.4%) and 38 (82.6%) samples respectively. We observed a significant association between hTR gene expression and younger age at diagnosis (p = 0.019) when comparing patients < or = 40 years with those who are older than 40 years. None of the benign tumours expressed hTR gene. However, the expression of hTERT gene was revealed in 2 samples. No significant association between hTR and hTERT expression and tumour's grade, stage and nodal status was seen. CONCLUSION: The expression of hTR and hTERT seems to be independent of tumour's stage. hTR expression probably plays a greater role in mammary tumourogenesis in younger women (< or = 40 years) and this may have therapeutic implications in the context of hTR targeting strategies.