Danila Coradini

Isolation and In vitro Propagation of Tumorigenic Breast Cancer Cells with Stem/Progenitor Cell Properties

Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24(-/low) cells, which exclusively retain tumorigenic activity and display stem cell-like properties. However, at present, direct evidence that breast cancer-initiating cells can be propagated in vitro is still lacking. We report here the isolation and in vitro propagation of breast cancer-initiating cells from three breast cancer lesions and from an established breast carcinoma cell line. Our breast carcinoma-derived cultures encompassed undifferentiated cells capable of self-renewal, extensive proliferation as clonal nonadherent spherical clusters, and differentiation along different mammary epithelial lineages (ductal and myoepithelial). Interestingly, cultured cells were CD44+/CD24- and Cx43-, overexpressed neoangiogenic and cytoprotective factors, expressed the putative stem cell marker Oct-4, and gave rise to new tumors when as few as 10(3) cells were injected into the mammary fat pad of SCID mice. Long-term cultures of breast tumorigenic cells with stem/progenitor cell properties represent a suitable in vitro model to study breast cancer-initiating cells and to develop therapeutic strategies aimed at eradicating the tumorigenic subpopulation within breast cancer.

Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line

In vivo and in vitro studies have shown an antitumor activity of Lactobacilli in colon cancer, and some epidemiologic studies have indicated a reduced risk of breast cancer in women who consume fermented milk products. We studied the direct effect of milk fermented by five bacteria strains (Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus paracasei) on the growth of the MCF7 breast cancer cell line. Our results showed a growth inhibition induced by all fermented milks, even though B. infantis and L. acidophilus were the most effective (85% inhibition after 9 days). The antiproliferative effect was not related to the presence of bacteria in fermented milk, and neither whole milk (crude or ultrahigh temperature sterlizied) nor its main fractions (lactalbumin or beta-lactoglobulin fraction) affected cell growth. Our findings suggest the presence of an ex novo soluble compound produced by lactic acid bacteria during milk fermentation or the microbial transformation of some milk components in a biologically active form. Although the mechanism of the antitumor activity is not clear, the present study suggests the potentiality offered by fermented milk as producers of compounds with antiproliferative activity useful in the prevention and therapy of solid tumors like breast cancer.

Hyaluronic acid as drug delivery for sodium butyrate: Improvement of the anti‐proliferative activity on a breast‐cancer cell line

The potential clinical utility of sodium butyrate, a natural compound known to inhibit tumor‐cell growth, is hampered by the difficulty of achieving effective in‐vivo concentrations. The short half‐life (about 5 minutes) of sodium butyrate results in rapid metabolism and excretion. To increase the availability of sodium butyrate over a longer period of time, we co‐valently linked it to hyaluronic acid (a component of the extracellular matrix). Its major advantages as a drug carrier consist in its high biocompatibility and its ability to bind CD44, a specific membrane receptor frequently over‐expressed on the tumor‐cell surface. The degree of substitution of hyaluronic acid with butyrate residues ranged from d.s. = 0.10 to d.s. = 2.24 (1.8–28.4% w/w). The biological activity of hyaluronic‐acid‐butyric‐ester derivatives was evaluated in terms of the inhibition of the growth of the MCF7 cell line and compared with that of sodium butyrate. After 6 days of treatment, we observed a progressive improvement of the anti‐proliferative activity up to d.s. = 0.20; thereafter, the anti‐proliferative effect of the ester derivatives decreased. Fluorescence microscopy showed that after 2 hr of treatment fluorescein‐labelled compounds appeared to be almost completely internalized into MCF7 cells, expressing CD44 standard and variant isoforms. These findings indicate that hyaluronic acid could offer an important advantage in drug delivery, in addition to its biocompatibility: the ability to bind to CD44, which are known to be frequently over‐expressed on the tumor‐cell surface. Int. J. Cancer 81:411–416, 1999.

Inhibition of Hepatocellular Carcinomas in vitro and Hepatic Metastases in vivo in Mice by the Histone Deacetylase Inhibitor HA-But

Purpose: The purpose is to evaluate the CD44-mediated cellular targeting of HA-But, a hyaluronic acid esterified with butyric acid (But) residues, to hepatocellular carcinoma cell lines in vitro and to hepatic tumor metastases in vivo. Experimental Design: In vitro, the CD44-dependent cytotoxicity in two human hepatocellular carcinoma cell lines (HepB3 and HepG2) with high and low CD44 expression was investigated; in vivo, the effect on liver metastases originating from intrasplenic implants of Lewis lung carcinoma (LL3) or B16-F10 melanoma in mice was compared with the pharmacokinetics of organ and tissue distribution using different routes of administration. Results: HepB3 and HepG2 cell lines showed different expression of CD44 (78 and 18%, respectively), which resulted in a CD44-dependent HA-But inhibitory effect as demonstrated also by the uptake analysis performed using radiolabeled HA-But (99mTc-HA-But). Pharmacokinetic studies showed different rates of 99mTc-HA-But distribution according to the route of administration (i.v., i.p., or s.c.): very fast (a few minutes) after i.v. treatment, with substantial accumulation in the liver and spleen; relatively slow after i.p. or s.c. treatment, with marked persistence of the drug at the site of injection. The effect of s.c. and i.p. treatment with HA-But on liver metastases originating from intrasplenic implants of LL3 carcinoma or B16-F10 melanoma (both CD44-positive: 68 and 87%, respectively), resulted in 87 and 100% metastases-free animals, respectively (regardless of the route of administration), and a significant prolongation of the life expectancy compared with control groups. Conclusions: HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions.

Loss of the Inactive X Chromosome and Replication of the Active X in BRCA1-Defective and Wild-type Breast Cancer Cells

In females, X chromosome inactivation (XCI) begins with the expression of the XIST gene from the X chromosome destined to be inactivated (Xi) and the coating of XIST RNA in cis. It has recently been reported that this process is supported by the product of the BRCA1 tumor suppressor gene and that BRCA1-/- cancers show Xi chromatin structure defects, thus suggesting a role of XCI perturbation in BRCA1-mediated tumorigenesis. Using a combined genetic and epigenetic approach, we verified the occurrence of XCI in BRCA1-/- and BRCA1wt breast cancer cell lines. It was ascertained that the Xi was lost in all cancer cell lines, irrespective of the BRCA1 status and that more than one active X (Xa) was present. In addition, no epigenetic silencing of genes normally subjected to XCI was observed. We also evaluated XIST expression and found that XIST may be occasionally transcribed also from Xa. Moreover, in one of the BRCA1wt cell line the restoring of XIST expression using a histone deacetylase inhibitor, did not lead to XCI. To verify these findings in primary tumors, chromosome X behavior was investigated in a few BRCA1-associated and BRCA1-not associated primary noncultured breast carcinomas and the results mirrored those obtained in cancer cell lines. Our findings indicate that the lack of XCI may be a frequent phenomenon in breast tumorigenesis, which occurs independently of BRCA1 status and XIST expression and is due to the loss of Xi and replication of Xa and not to the reactivation of the native Xi.

Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells

Sodium butyrate (NaB), a product of colonic fermentation of dietary fibre, has been shown to inhibit cell proliferation by blocking cells in the G0/G1 phase of the cell cycle through a mechanism of action still not completely understood. We investigated the effect of NaB on the level of some G1 phase‐related proteins in a colon carcinoma cell line (HT29). In particular, we addressed our attention to cyclin D1 (the key regulator of G1S progression), p21waf1/cip1 (the main inactivator of the cyclin D/cdk complex), and p53 (the most important regulator of p21waf1/cip1 gene transcription). At inhibitory concentrations (higher than 1 mM) NaB reduced cyclin D1 and p53 level in a dose‐dependent manner and sustained the synthesis of p21waf1/cip1, probably in a p53‐independent way, accounting for the G0/G1 block observed by flow cytometry. Present results provide further evidence on the molecular mechanism at the basis of the physiological role of NaB and support the hypothesis that an unbalanced diet, poor in carbohydrates and therefore in NaB, could result in functional alterations with clinical and carcinogenic implications.

Is axillary lymph node dissection necessary in elderly patients with breast carcinoma who have a clinically uninvolved axilla

Axillary dissection in elderly patients with early‐stage breast carcinoma who do not have palpable axillary lymph nodes is controversial because of the associated morbidity of the surgery, reduced life expectancy of the patients, and efficacy of hormone therapy in preventing recurrences and axillary events.

Biomolecular prognostic factors in breast cancer.

Purpose of review To update clinicians on recent findings concerning the clinical usefulness of biomarkers in breast cancer, this review examines recently published papers dealing with promising prognostic/predictive biological factors. These factors can be classified according to their involvement in the main alterations characterizing tumor cells: self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Recent findings Despite relevant research efforts and the identification of many putative good prognosticators, few of these factors are proving clinically useful for identifying patients at minimal risk of relapse, patients with a worse prognosis, or patients likely to benefit from specific treatments. Most of them, such as HER-2/neu, epidermal growth factor receptor, cyclin E, p53, bcl-2, vascular endothelial growth factor, urokinase-type plasminogen activator-1 and the recently discovered anti-apoptosis protein survivin, are suggested for possible inclusion in the category of biomarkers with a high level of clinico-laboratory effectiveness. However, no single biomarker was able to identify those patients with the best (or worst) prognosis or those which would be responsive to a given therapy. Novel findings derived from gene-expression analysis indicate that the simultaneous consideration of molecular alterations contributing to the hallmarks of cancer might provide clinically useful prognostic, and perhaps therapeutic, information. Summary Rapid translation of laboratory findings to clinical practice was hampered by many difficulties, including technical and statistical concerns, a lack of assay standardization and comparability, and the modest design of translational studies. Many studies are performed on too small series of patients to provide reliable results; the studies are often heterogeneous in terms of treatment, patients and tumor characteristics, and data may be evaluated using different analytical approaches and are thus not easily comparable. Adequately planned prospective studies are required to assess the clinical utility of biomarker determinations.

Prognostic factors for metachronous contralateral breast cancer: A comparison of the linear Cox regression model and its artificial neural network extension

The purpose of the present study was toassess prognostic factors for metachronous contralateral recurrence ofbreast cancer (CBC). Two factors were of particularinterest, namely estrogen (ER) and progesterone (PgR) receptorsassayed with the biochemical method in primary tumortissue. Information was obtained from a prospective clinicaldatabase for 1763 axillary node-negative women who hadreceived curative surgery, mostly of the conservative type,and followed-up for a median of 82 months.The analysis was performed based on both astandard (linear) Cox model and an artificial neuralnetwork (ANN) extension of this model proposed byFaraggi and Simon [9]. Furthermore, to assess theprognostic importance of the factors considered, model predictiveability was computed.In agreement with already published studies, the resultsof our analysis confirmed the prognostic role ofage at surgery, histology, and primary tumor site,in that young patients (≤ 45 years) withtumors of lobular histology or located at inner/centralmammary quadrants were at greater risk of developingCBC. ER and PgR were also shown tohave a prognostic role. Their effect, however, wasnot simple in relation to the presence ofinteractions between ER and age, and between PgRand histology. In fact, ER appeared to playa protective role in young patients, whereas theopposite was true in older women. Higher levelsof PgR implied a greater hazard of CBCoccurrence in infiltrating duct carcinoma or tumors withan associated extensive intraductal component, and a lowerhazard in infiltrating lobular carcinoma or other histotypes.In spite of the above findings, the predictivevalue of both the standard and ANN Coxmodels was relatively low, thus suggesting an intrinsiclimitation of the prognostic variables considered, rather thantheir suboptimal modeling. Research for better prognostic variablesshould therefore continue.

Time-Dependent Relevance of Steroid Receptors in Breast Cancer

PURPOSE To analyze the time-dependent prognostic role of the investigated variables, considered, when appropriate, on a continuous scale, for the purpose of evaluating and describing the interrelationships between clinically relevant patient and tumor characteristics (age, size and histology, and estrogen receptor [ER] and progesterone receptor content) and the risk of new disease manifestation. PATIENTS AND METHODS We applied a flexible statistical model to a case series of 1,793 patients with axillary lymph node-negative breast cancer with a minimal potential follow-up of 10 years. To avoid a potential confounding effect of adjuvant treatment, only patients given local-regional therapy until relapse were considered. RESULTS ER content and tumor size (adjusted for all the other covariates) showed a time-dependent relationship with the risk of new disease manifestations. In particular, ER content failed to show a prognostic effect within the first years of follow-up; thereafter, a positive association with risk of relapse was observed. For tumor size, within the first years of follow-up, the risk of relapse was directly related to size for only tumors up to 2.5 cm in diameter; thereafter, the impact on prognosis progressively decreased. CONCLUSION The availability of a long follow-up on a large breast cancer series, as well as the use of innovative statistical approaches, allowed us to explore the functional relation between steroid receptors and clinical outcome and to generate a hypothesis on the involvement of ER in favoring long-term metastasis development.