Changes in gene expression of CXCR4, CCR7 and BCL2 after treatment of breast cancer cells with saponin extract from Tribulus terrestris.

UNLABELLED: Saponins are natural substances produced by a large number of plants, one of which is Tribulus terrestris L. (TT). They have been reported to possess an antitumor activity exerted by regulating various signaling pathways in the cell. Although the mechanisms of action of saponin extracts from various plants have been widely studied, limited data are available about TT. The present study aimed to analyze the impact of saponin extract from TT on cell processes in breast carcinoma cell lines. The variations in expression of a group of 32 selected genes were examined by real-time PCR after saponin treatment of MCF7 and MCF10A cell lines. Only three genes - CXCR4, CCR7 and BCL2, showed changes in their mRNA levels after the application of the herb extract. While CXCR4 expression was reduced in both cell lines, CCR7 and BCL2 levels decreased only in tumorigenic MCF7 cells, implying cell-specificity of the saponin action. Our results suggested that TT extract containing saponins was likely to affect the processes of apoptosis and metastasizing of cancer cells. Further in vivo studies will show its applicability as an anticancer therapeutic agent. KEYWORDS: saponins, Tribulus terrestris, breast cancer, CXCR4, CCR7, BCL2.

Macrophageal infiltration and microvessel density in laryngeal carcinoma: study of 52 cases.

Angiogenesis is one of the six originally constituted hallmarks of cancer that has been extensively studied in the last two decades. The aim of our study is to assess the microvessel and macrophageal density in laryngeal carcinoma and its clinicopathological correlations. We immunohistochemically assessed microvessel density (CD34) and macrophage count (CD68) using microarray techniques and then looked for clinicopathological correlations. The mean micro-vessel density in the study group was 14.27 ± 12.92 vessels in a ×200 field with a mean macrophageal infiltration density of 5.19 ± 4.32. Median microvessel density was significantly higher in patients with metastasis than in patients without metastasis. Additionally, linear regression established that macrophageal infiltration density could predict microvessel density in laryngeal carcinoma. We found no association between either factor and recurrence rate or other clinical characteristics. Our study adds additional data to a problem that has been widely studied during the last two decades, even if controversies in this area still remain.

APC promoter 1B deletion in familial polyposis--implications for mutation-negative families.

Over 1500 adenomatous polyposis coli (APC) gene mutations have already been identified as causative of familial adenomatous polyposis (FAP). However, routine genetic testing fails to detect mutations in about 10% of classic FAP cases. Recently, it has been shown that a proportion of mutation-negative FAP cases bear molecular changes in deep intronic and regulatory sequences. In this study, we used direct sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) of genomic DNA from family members, affected by classic FAP. We first reported the family as mutation negative. With the launch of a new version of MLPA kit, we retested the family and a novel full deletion of promoter 1B was detected. The exact breakpoints of the deletion were determined by array comparative genomic hybridization (CGH) and long range polymerase chain reaction (PCR), followed by direct sequencing. The total APC expression levels were investigated by quantitative polymerase chain reaction (qPCR) assay and allele-specific expression (ASE) analysis. The APC gene expression was highly reduced, which indicates causative relationship. We suggest that there is a significant possibility that APC promoter 1B mutations could be found in mutation-negative FAP patients. In the light of our findings it seems reasonable to consider targeted genetic re-analysis of APC promoter 1B region in a larger cohort of unsolved cases.

The basal ganglia and cerebellum interact in the expression of dystonic movement.

Dystonia is a neurological disorder characterized by excessive involuntary muscle contractions that lead to twisting movements or abnormal posturing. Traditional views place responsibility for dystonia with dysfunction of basal ganglia circuits, yet recent evidence has pointed towards cerebellar circuits as well. In the current studies we used two strategies to explore the hypothesis that the expression of dystonic movements depends on influences from a motor network that includes both the basal ganglia and cerebellum. The first strategy was to evaluate the consequences of subthreshold lesions of the striatum in two different animal models where dystonic movements are thought to originate from abnormal cerebellar function. The second strategy employed microdialysis to search for changes in striatal dopamine release in these two animal models where the cerebellum has been already implicated. One of the animal models involved tottering mice, which exhibit paroxysmal dystonia due to an inherited defect affecting calcium channels. In keeping with prior results implicating the cerebellum in this model, surgical removal of the cerebellum eliminated their dystonic attacks. In contrast, subclinical lesions of the striatum with either 6-hydroxydopamine (6OHDA) or quinolinic acid (QA) exaggerated their dystonic attacks. Microdialysis of the striatum revealed dystonic attacks in tottering mice to be associated with a significant reduction in extracellular striatal dopamine. The other animal model involved the induction of dystonia via pharmacological excitation of the cerebellar cortex by local application of kainic acid in normal mice. In this model the site of stimulation determines the origin of dystonia in the cerebellum. However, subclinical striatal lesions with either 6OHDA or QA again exaggerated their generalized dystonia. When dystonic movements were triggered by pharmacological stimulation of the cerebellum, microdialysis revealed significant reductions in striatal dopamine release. These results demonstrate important functional relationships between cerebellar and basal ganglia circuits in two different animal models of dystonia. They suggest that expression of dystonic movements depends on influences from both basal ganglia and cerebellum in both models. These results support the hypothesis that dystonia may result from disruption of a motor network involving both the basal ganglia and cerebellum, rather than isolated dysfunction of only one motor system.

Microsatellite instability in Bulgarian patients with endometrial cancer.

PURPOSE: Microsatellite instability (MSI) is a frequent event in different types of cancer. In several studies MSI was shown to have both clinical and prognostic value. The aim of our study was to determine the frequency of MSI in Bulgarian patients with endometrial cancer (EC) and the possible relation of this phenomenon to their clinicopathological characteristics. PATIENTS AND METHODS: A total of 33 histologically confirmed EC patients were analyzed for tumor MSI using a panel of 6 microsatellite markers. RESULTS: We identified MSI in 30% of endometrial cancer cases. Six of them had high degree of MSI (MSI-H), and 4 displayed low degree of MSI (MSI-L). CONCLUSION: The frequency of MSI in Bulgarian EC patients does not differ significantly from that reported in other European studies.

Saponins from Tribulus terrestris L are less toxic for normal human fibroblasts than for many cancer lines: influence on apoptosis and proliferation.

The objective of the study was to explore the influence of saponins derived from Tribulus terrestris L. (TT) on normal human skin fibroblasts and to compare it with their anticancer properties. In this study, [3H]thymidine incorporation and MTT to assess cell proliferation and viability, respectively, and immunoblotting and HPLC analysis to explore intracellular signal transduction pathways have been used. We found that TT caused a dose-dependent decrease in [3H]thymidine incorporation into the DNA of treated fibroblast compared to the untreated controls. Viability of treated cells remained within the control levels with treatment of up to 5 micro g TT/ml medium. It was significantly depressed with incubation in > or =6 micro g TT/ml medium with an IC50 of 12.6 micro g TT/ml of cultivating media. ERK1/2 was significantly dephosphorylated at 5 mins of incubation with TT until the 48th hour, when phosphorylation slightly recovered, but was still below the control levels. In contrast, p38 and JNK phosphorylation was positively influenced, with peaks at 1 hr and 24 hrs of incubation respectively. Phosphorylation/dephosphorylation events of SAPK/MAPK clearly correlated with Mkp-1 induction. Procaspase 3 was activated after 5 mins of incubation and coincided with a rapid actin cleavage. There was a significant decrease of putrescine concentration and a concomitant increase of spermidine and spermine at 2 mins of treatment. According to our results, TT is less toxic for normal human skin fibroblasts in comparison to many cancer lines investigated in previous studies. The molecular mechanism of this cytotoxicity involves up- and downregulation of polyamines' homeostasis, suppression of proliferation, and induction of apoptosis. Further research in this field using animal models would help to explore and interpret the potential properties of TT as an anticancer supplement.

The aphrodisiac herb Tribulus terrestris does not influence the androgen production in young men.

OBJECTIVE: The aim of the current study is to investigate the influence of Tribulus terrestris extract on androgen metabolism in young males. DESIGN AND METHODS: Twenty-one healthy young 20-36 years old men with body weight ranging from 60 to 125 kg were randomly separated into three groups-two experimental (each n=7) and a control (placebo) one (n=7). The experimental groups were named TT1 and TT2 and the subjects were assigned to consume 20 and 10 mg/kg body weight per day of Tribulus terrestris extract, respectively, separated into three daily intakes for 4 weeks. Testosterone, androstenedione and luteinizing hormone levels in the serum were measured 24 h before supplementation (clear probe), and at 24, 72, 240, 408 and 576 h from the beginning of the supplementation. RESULTS: There was no significant difference between Tribulus terrestris supplemented groups and controls in the serum testosterone (TT1 (mean+/-S.D.: 15.75+/-1.75 nmol/l); TT2 (mean+/-S.D.: 16.32+/-1.57 nmol/l); controls (mean+/-S.D.: 17.74+/-1.09 nmol/l) (p>0.05)), androstenedione (TT1 (mean+/-S.D.: 1.927+/-0.126 ng/ml); TT2 (mean+/-S.D.: 2.026+/-0.256 ng/ml); controls (mean+/-S.D.: 1.952+/-0.236 ng/ml) (p>0.05)) or luteinizing hormone (TT1 (mean+/-S.D.: 4.662+/-0.274U/l); TT2 (mean+/-S.D.: 4.103+/-0.869U/l); controls (mean+/-S.D.: 4.170+/-0.406U/l) (p>0.05)) levels. All results were within the normal range. The findings in the current study anticipate that Tribulus terrestris steroid saponins possess neither direct nor indirect androgen-increasing properties. The study will be extended in the clarifying the probable mode of action of Tribulus terrestris steroid saponins.

Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland.

A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.

The difference between cytosol and membrane growth-related protein kinase activities in lactating mouse mammary gland.

1. A protein kinase activity which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary gland cells was isolated and partially purified from cytosol. 2. Another protein kinase activity was demonstrated in crude membranes of lactating mouse mammary gland. 3. By the use of several different synthetic peptides as a substrate, it was demonstrated that the cytosol enzyme was a serine kinase, while the membrane protein kinase activity was mainly due to tyrosine kinase.

Insulin action in vivo on a growth-related protein kinase in lactating mouse mammary gland.

1. A cytosol serine-protein kinase was isolated and partially purified from lactating mouse mammary gland which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary cells. 2. After supra-physiological doses of insulin (0.4 IU daily) given in vivo the activity of this kinase rises 2.4 times, but at 5 times higher doses of insulin the activity was completely inhibited. 3. The biphasic effect of insulin suggests that this protein kinase might be connected to the growth effect of the hormone.