α2‐Adrenergic effect on human breast cancer MCF‐7 cells

(-)Epinephrine (Epi) and (-)Norepinephrine (NEpi) significantly stimulated tritiated Thymidine incorporation in MCF-7 cells at concentrations 10-30pM to 10nM, with an EC50 of 10pM for Epi and 14.2pM for NEpi. To characterize this action, cells were incubated in the presence of NEpi or Epi and different antagonists. The beta-adrenergic antagonist Propanolol showed no effect on the agonist's stimulation, whereas the alpha-adrenergic antagonist Phentolamine, reverted it completely at high concentrations (100 microM). The alpha1-adrenergic antagonist Prazosin (Pra) acted only at high concentrations, while the alpha2-adrenergic antagonist Yohimbine (Yo) reverted the stimulation at an EC50 of 0.11 microM. Likewise, when the cells were incubated in the presence of the specific alpha2-adrenergic agonist Clonidine (Clo), Thymidine incorporation was significantly stimulated at an EC50 of 0.298 pM. Again, the incubation of the cells in the presence of the alpha1-adrenergic antagonist Pra exerted its action at high concentrations, whereas the alpha2-adrenergic antagonist Yo showed a clear reversal of the agonist's enhancement at an EC50 of 0.136 microM. Moreover, Clo caused a clear and significant inhibition of stimulated cAMP levels both in the intracellular and the extracellular fractions. Yo showed a complete reversion of cAMP levels to control values in the presence of Clo, while Pra had the opposite effect. These data suggest that the stimulation provoked in Thymidine incorporation by the agonists Epi, NEpi, and Clo is, at least in part, due to an alpha2-adrenergic mechanism directly on tumoral cells, and that the effect is coupled with inhibition of cAMP levels, as described for this kind of receptors.     

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL2 expression by VP16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF7, MDAMB435S, and MDAMBA231 following in vitro treatment with 50–100M VP16. Elevation of BAX protein expression in the presence of VP16 alone did not correlate with reduced viability or induction of apoptosis in MCF7, MDAMB435S, or MDAMBA231. VP16 did effectively block the breast cancer cell lines evaluated (MCF7 and MDAMB435S) at G₂/M phase of the cell cycle, confirming activity of the drug in vitro. MCF7 and MDAMB435S cells that were pretreated with VP16 and subsequently exposed to 1.0–12.0g/m1 4hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.     

Pharmacokinetics in mice and metabolism in murine and human liver fractions of the putative cancer chemopreventive agents 3��,4��,5��,5,7-pentamethoxyflavone and tricin (4��,5,7-trihydroxy-3��,5��-dimethoxyflavone)

Purpose

The flavones tricin (4??,5,7-trihydroxy-3??,5??-dimethoxyflavone) and 3??,4??,5??,5,7-pentamethoxyflavone (PMF) are under development as potential colorectal cancer chemopreventive agents as they reduced adenoma development in the Apc ^Min mouse model of intestinal carcinogenesis. Here, the pharmacokinetic properties and metabolism of these flavones after oral administration were compared in mice.

Methods

C57BL/6?J mice received an oral bolus of PMF or tricin (807???mol/kg). Parent flavone and metabolites were analyzed by HPLC/UV in plasma, liver and gastrointestinal tissues. Flavones were incubated with mouse or human hepatic microsomes or 9000xg supernatant (S9), both fortified with a NADPH-generating system and either uridine 5??-diphosphoglucuronic acid (UDPGA, microsomes) or 3??-phosphoadenosine-5??-phosphosulfate (PAPS, S9). Disappearance of substrate was assessed by HPLC/UV, metabolites were characterized by HPLC/MS/MS.

Results

Plasma concentrations and area under the plasma concentration versus time curve for PMF were higher than those for tricin. A mono-O-desmethyl PMF and several isomeric mono-O-desmethyl PMF glucuronides and sulfonates were major PMF metabolites in murine plasma, liver and intestinal tissue. In murine and human liver fractions, in vitro metabolic removal of tricin was faster than that of PMF. On kinetic analysis of metabolite generation in these incubations, apparent maximal velocity (V _max) values for the generation of tricin O-glucuronide or O-sulfonate were consistently several fold higher than those characterizing the production of mono-O-desmethyl PMF glucuronides or sulfonates via the intermediacy of O-desmethyl PMF.

Conclusions

The results suggest that inclusion of methoxy moieties confers metabolic stability onto the flavone scaffold.     

Inhibitory effect of soluble platelet-derived growth factor receptor β on intraosseous growth of breast cancer cells in nude mice

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor β (PDGFRβ) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRβ signaling using soluble PDGFRβ (sPDGFRβ) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ. A stable transfectant (TNBCT/Bo-sPDGFRβ) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRβ cells. Intratibial injection of TNBCT/Bo-sPDGFRβ cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRβ is useful for the treatment of breast cancer bone metastasis.     

Inhibition of ��-secretase induces G2/M arrest and triggers apoptosis in breast cancer cells

γ-Secretase activity is vital for the transmembrane cleavage of Notch receptors and the subsequent migration of their intracellular domains to the nucleus. Notch overexpression has been associated with breast, colon, cervical and prostate cancers. We tested the effect of three different γ-secretase inhibitors (GSIs) in breast cancer cells. One inhibitor (GSI1) was lethal to breast cancer cell lines at concentrations of 2 μM and above but had a minimal effect on the non-malignant breast lines. GSI1 was also cytotoxic for a wide variety of cancer cell lines in the NCI60 cell screen. GSI1 treatment resulted in a marked decrease in γ-secretase activity and downregulation of the Notch signalling pathway with no effects on expression of the γ-secretase components or ligands. Flow cytometric and western blot analyses indicated that GSI1 induces a G2/M arrest leading to apoptosis, through downregulation of Bcl-2, Bax and Bcl-XL. GSI1 also inhibited proteasome activity. Thus, the γ-secretase inhibitor GSI1 has a complex mode of action to inhibit breast cancer cell survival and may represent a novel therapy in breast cancer.     

The effect of 14-3-3ζ expression on tamoxifen resistance and breast cancer recurrence: a Danish population-based study

Purpose

Overexpression of 14-3-3ζ has been linked to breast cancer recurrence in several studies, including studies assessing its effect on tamoxifen resistance. The study was performed to estimate the effect of 14-3-3ζ and differentiate potential prognostic or predictive utility.

Methods

A case–control study, nested in a population of 11,251 females residing on the Jutland Peninsula of Denmark, was performed. Participants were aged 35–69, diagnosed with stage I, II, or III breast cancer between 1985 and 2001, and registered with the Danish Breast Cancer Cooperative Group. We identified 541 recurrent breast cancer cases with estrogen receptor-positive disease treated with tamoxifen for at least 1 year (ER+/TAM+) and 300 cases with estrogen receptor-negative disease never treated with tamoxifen (ER?/TAM?). We matched cases to controls on ER/TAM status, date of surgery, menopausal status, stage, and county. 14-3-3ζ expression was assessed using immunohistochemistry on tissue microarrays. We computed the odds ratio (OR) associating 14-3-3ζ expression with breast cancer recurrence adjusting for confounding using logistic regression. A quantitative bias analysis was performed to account for bias due to expression assay methods.

Results

Associations for cytoplasmic and nuclear 14-3-3ζ staining above the 50th percentile were near null in both ER+/TAM+ and ER?/TAM? patients. When examining combined 14-3-3ζ staining, the association increased in the ER+/TAM+ group (adjusted OR 1.44, 95% confidence interval (CI) 1.05, 1.99). A nearly twofold increase in odds of recurrence was observed in above the 75th percentile staining of combined 14-3-3ζ, both for ER+/TAM+ patients (adjusted OR 1.93, 95% CI 1.15, 3.24) and ER?/TAM? patients (adjusted OR 1.93, 95% CI 1.03, 3.62), indicating potential prognostic utility.

Conclusion

Evidence is lacking to conclude that 14-3-3ζ is a useful marker of tamoxifen resistance; however, 14-3-3ζ expression is a potentially useful prognostic marker of breast cancer recurrence. Independent utility beyond established prognostic markers needs to be determined.

     

Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP‐470 on human breast cancer cells

The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDAMB231, T47D, MCF7, KPL1, and MKLF). In all five cell lines, EPA and TNP470 alone both showed tumor growth inhibition in a time and dosedependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bc1x_S, the apoptosisenhancing proteins, was more upregulated and that of Bcl2 and Bclx_L, the apoptosissuppressing proteins, was more downregulated compared to the use of EPA or TNP470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.     

Inhibitory effect of a TGF�� receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells

Background:

Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.

Methods:

Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.

Results:

TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.

Conclusion:

A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.     

BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β(3) expression

Background

The transforming growth factor (TGF)-?? superfamily comprises cytokines such as TGF-?? and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-?? are associated with poor outcome, whereas inhibition of TGF-??-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.

Methods

In this study, we investigated the effect of BMP-7 on TGF-??-induced invasion in a 3 dimensional invasion assay.

Results

BMP-7 inhibited TGF-??-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-??-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-??-induced expression of integrin ??_v??₃ in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin ??₃ negatively affected TGF-??-induced invasion. On the other hand, overexpression of integrin ??₃ counteracted the inhibitory effect of BMP7 on TGF-??-induced invasion.

Conclusion

Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-??-induced expression of integrin ??₃.     

Differential effect of hormone therapy on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro

Breast tissue possesses the enzymes for local estrogen biosynthesis. We measured the effect of Estradiol (E2), Tibolone (OrgOD14) and its metabolite Org4094 on estrone sulfate (E1S)-sulfatase (STS) using breast cancer (MCF-7) and non-malignant breast cells (HBL-100). Cells were cultured in 5% steroid depleted fetal calf serum for 3 days and subsequently incubated with each steroid for either 24 h or directly in cell extracts. STS mRNA and protein expression, and its subcellular localization were determined by semi-quantitative RT-PCR, immunoblotting, and confocal immunofluorescence microscopy. STS activity was evaluated by incubating homogenized breast cells with [³H]-E1S. The products E1 and E2 were separated by thin layer chromatography. STS was co-localized with the Golgi marker protein GM130 and the endoplasmic reticulum marker protein calnexin. Treatment did not significantly alter STS mRNA expression. STS protein expression was increased by each steroid in HBL-100 cells but by E2 only in MCF-7 cells. 24 h incubation with OrgOD14 and Org4094 did not alter STS activity in both cell lines. However, STS activity was significantly diminished in HBL-100 but slightly increased in MCF-7 cells by 24 h treatment with E2. “Direct” incubation of cell extracts, eliminating cellular regulation of metabolism, reduced estrogen biosynthesis regardless of cell line and treatment. In conclusion, the immediate reduction of estrogen biosynthesis by OrgOD14 is counteracted by an increased STS protein expression. On the contrary, E2 exerts a differential effect on STS in HBL-100 and MCF-7 cells. The transition from normal to malignant breast cells may be accompanied by an abolished autoregulation of local estrogen formation.     

Pro-apoptotic effect of Δ2-TGZ in “claudin-1-low” triple-negative breast cancer cells: involvement of claudin-1

Background

Although some studies suggest that breast-conserving therapy (BCT) shows better psychosocial outcomes than mastectomy in patients with primary breast cancer, little is known about the outcomes of these surgical options in recurrent breast cancer. We investigated differences in overall survival and re-recurrence rates as well as psychosocial outcomes among patients who underwent BCT or mastectomy after the diagnosis of recurrent breast cancer in a single-center setting.

Methods

124 of 186 eligible patients who underwent surgical treatment for breast cancer recurrence completed the questionnaires on quality of life (EORTC QLQ-C30 and -BR23), fear of progression (PA-F-KF), anxiety and depression (HADS), and body image (BIS).

Results

Women after breast-conserving surgery (n = 46) showed significantly better outcomes than women after mastectomy (n = 61) with respect to body image (P < 0.001 in BIS and p < 0.001 in BR23), social functioning (p = 0.016), emotional functioning (p = 0.028), and role functioning (p = 0.043). There were no significant group differences regarding anxiety, depression, and fear of progression as well as re-recurrence and survival rates. Predictors of good quality of life were partnership (OR 2.46), higher monthly family income (OR 3.54), and higher professional qualification (OR 4.3) in our group of patients.

Discussion

Our results indicate that patients treated with breast-conserving therapy after recurrent breast cancer perceive lower impairments in body image and several aspects of quality of life than patients treated with mastectomy.

     

Tissue inhibitor of metalloprotease (TIMP)‐1 and proliferative behaviour of clonal breast cancer cells

In the present paper, we have examined whether human tissue inhibitor of metalloprotease1 (hTIMP1) is able to exert a growth factorlike effect on two clonal cell lines (BC3A and BC61), isolated from a parental line of human breast carcinoma cells (8701BC), and endowed with different growth and invasive behaviour in vitro and in nude mouse. The data obtained indicate that only the more tumorigenic clonal cell line (BC61) is responsive to hTIMP1 treatment by increasing its proliferative rate in a dosedependent manner. It was also found that BC61 cells selectively express a transmembrane protein of about 80kDa able to bind hTIMP1 in vitro and in vivo with high affinity (K_d of 0.07 ± 0.004 nM), and that treatment of BC61 cells with a proliferationpromoting concentration of hTIMP1 is able to stimulate tyrosinetargeted phosphorylation. The cumulative results obtained strongly support the hypothesis that hTIMP1, classically regarded as a collagenase inhibitor, may be a crucial element of the extracellular signalling network during breast cancer development by controlling cell growth phenotype in autocrine and paracrine manner, and that intratumoural heterogeneity for the biological response to TIMP1 may exist within the composite cell population of the primary tumour site.     

Oral (E)-5-(2-Bromovinyl)-2′-Deoxyuridine treatment of severe herpes zoster in cancer patients

BVDU [(E)-5-(2-bromovinyl)-2′-deoxyuridine] is a highly potent and selective anti-herpes drug. It is particularly active against Varicella zoster virus, as demonstrated in cell culture and animals (monkeys). BVDU has been administered orally, at a dose of 7.5 mg/kg/day for 5 days, to 20 patients with severe localised or disseminated Herpes zoster. All patients had a malignant disorder for which they had been given intensive chemo- or radiotherapy. Upon BVDU treatment a rapid cessation of the acute Herpes zoster episode was noted in all but one patient. In the majority of patients progression of the infection was arrested within 1 day of starting treatment. No toxic side-effects could be attributed to the drug at the dosage used.     

TRPM8 promotes aggressiveness of breast cancer cells by regulating EMT via activating AKT/GSK-3β pathway

Breast cancer already taken the first place of incidence in Chinese female cancer patients. TRPM8 is found to be over-expressed in breast cancer, but whether it promotes breast cancer aggressiveness remains unknown. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR-75-30, while it just could be detected in MCF-10A, the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore expression status of cancer-related proteins. The Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. All the results demonstrated that TRPM8 significantly up-expressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/GSK-3β pathway, indicating TRPM8 gets the prospects of to be developed as medication or diagnostic indicator to be applied in clinical work.