A novel retinoic/butyric hyaluronan ester for the treatment of acute promyelocytic leukemia: preliminary preclinical results.

All-trans retinoic acid (ATRA) represents the therapy of choice for patients with acute promyelocytic leukemia (APL). However, patients often relapse due to ATRA-resistance. The molecular basis of APL alterations indicates that addition of a histone deacetylase inhibitor to ATRA may restore the sensitivity to retinoids. We explored the in vitro and in vivo effects of a novel retinoic/butyric hyaluronan ester (HBR) on a retinoic acid (RA)-sensitive human myeloid cell line, NB4, and on its RA-resistant subclone, NB4.007/6. In vitro, HBR induced growth arrest and terminal differentiation in RA-sensitive NB4 cells (as confirmed by an increased expression of CD11 family members and nitroblue tetrazolium assay), whereas it inhibited the growth of RA-resistant cells by apoptosis, paralleled by an increase in the levels of caspase 3 and 7. In vivo, HBR treatment of NB4-inoculated severe combined immunodeficient mice resulted in a statistically significant increase in survival time (P<0.0001), comparable to that induced by a maximum tolerated dose of RA alone. Also on P388-inoculated mice, HBR was active in contrast to RA that was completely ineffective. Present findings suggest that, owing to the simultaneous presence of RA and an histone deacetylases inhibitor, HBR might be useful in controlling the proliferation of RA-resistant cells and the differentiation of RA-sensitive cells.

Hypoxia and estrogen receptor profile influence the responsiveness of human breast cancer cells to estradiol and antiestrogens.

Angiogenesis activation mediated by vascular endothelial growth factor (VEGF) is one of the factors that can cause antiestrogen treatment failure in estrogen receptor (ER)?positive breast cancer patients. Since VEGF synthesis is modulated not only by hypoxia but also by steroid hormones, we investigated the relationship between hypoxic and estrogenic/antiestrogenic stimuli in two human breast cancer cell lines expressing both ER6alpha and ERbeta (MCF7) or only ERbeta (MDA-MB231). In both cell lines, the VEGF level was significantly influenced by hypoxic conditions and in antiestrogen-responsive MCF7 cells, this effect was not counteracted by tamoxifen or ICI 182780, thus providing an experimental explanation for the resistance to endocrine treatment observed in patients with ER-positive tumors. In MDA-MB231 cells, estradiol significantly reduced the VEGF level, suggesting that through the ERbeta isoform it may function as a negative modulator of VEGF synthesis under hypoxia, and providing evidence for a complex interplay of the estrogen-dependent and hypoxia-dependent pathways.

Vascular endothelial growth factor in node-positive breast cancer patients treated with adjuvant tamoxifen.

In 212 postmenopausal women with node-positive oestrogen receptor-positive (ER(LBA)) breast cancer subjected to radical surgery and adjuvant tamoxifen, the risk of 6-year relapse increased with increasing values of intratumoral vascular endothelial growth factor (VEGF) in patients whose tumours had a low/intermediate ER(LBA) content compared to patients with high-ER(LBA) tumours. These findings indicate that tumour progression, activated or sustained by high VEGF levels, may be counteracted in high-ER(LBA) cancers by tamoxifen, which in contrast fails to contrast the metastatic potential in low-ER(LBA) tumours.

Infiltrating ductal and lobular breast carcinomas are characterised by different interrelationships among markers related to angiogenesis and hormone dependence.

To obtain a more integrated understanding of the different breast cancer phenotypes and to investigate whether bio-molecular profiles can distinguish between specific histotypes, we explored the interrelations among several biologic variables indicative of, or related to, hormone dependence, proliferation and apoptosis control, and angiogenesis in ductal and lobular carcinomas, the most common histotypes. Oestrogen and progesterone receptors, tumour proliferative activity, the expression of cyclin A, p16(ink4A), p27(kip1), p21(waf1), p53, bcl-2, and levels of vascular endothelial growth factor and hypoxia-inducible factor-1alpha (HIF-1alpha) were evaluated in 190 in ductal and 67 lobular carcinomas. Our findings support the hypothesis that in ductal and lobular carcinomas are two distinct, partially phenotypically unrelated entities, the latter being characterised by the presence of features indicative of differentiation such as oestrogen receptors, low proliferation and lack of p53 expression and associated with low vascular endothelial growth factor content compared to angiogenesis in ductal carcinomas. Conversely, no significant difference was found between lobular carcinomas and in ductal carcinomas considering the frequency distribution of PgR-positive cases, cyclin-dependent kinase inhibitors acting at the G1/S boundary, bcl-2 and HIF-1alpha protein expression. Although both generally defined as hormone responsive, in ductal and lobular carcinomas are also characterised by biologic patterns in which proteins related to hormone responsiveness, cell-cycle control, apoptosis and angiogenesis were differently associated. This finding suggests the need to refine breast cancer characterisation in order to provide detailed information about individual tumours, or subsets of tumours, that will help in defining optimal treatment approaches.

Contribution of vascular endothelial growth factor to the Nottingham prognostic index in node-negative breast cancer.

The prognostic contribution of intratumour VEGF, the most important factor in tumour-induced angiogenesis, to NPI was evaluated by using flexible modelling in a series of 226 N-primary breast cancer patients in which steroid receptors and cell proliferation were also accounted for. VEGF provided an additional prognostic contribution to NPI mainly within ER-poor tumours.

The effect of formulation and concentration of cholesteryl butyrate solid lipid nanospheres (SLN) on NIH-H460 cell proliferation.

Experimental factorial design was used to evaluate the influence of two factors involved in producing cholesteryl butyrate (chol-but) solid lipid nanospheres (SLN), microemulsion formulation and microemulsion/water ratio, on the effect of the SLN on the proliferation of NIH-H460, a non-small-cell lung carcinoma; six experimental settings were tested. The cells were treated with scalar concentrations of cholesteryl butyrate (from 0.008 to 1.000 mM) for each experimental condition; NIH-H460 cell growth was inhibited in all cases. The best experimental setting provided complete inhibition at 0.125 mM chol-but, while at the same concentration sodium butyrate provided only 38% inhibition.

Modulation of cell cycle-related protein expression by sodium butyrate in human non-small cell lung cancer cell lines.

To elucidate the mechanism of action of sodium butyrate (NaB), we examined its effect on the expression of some cell cycle-related proteins (cyclins D1 and E, p16(ink4), p21(waf1), p27(kip1)) in 2 human non-small cell lung cancer cell lines (NCI-460 and NCI-H23) characterized by wild- type and mutant TP53, respectively. The growth of both cell lines was inhibited in a dose-dependent manner and this process was accompanied by a modulation of cell cycle-related proteins. In NCI-H460, the p27(kip1) and p16(ink4) protein levels were markedly increased following NaB treatment, whereas p21(waf1) was only slightly elevated, with a peak at 2 mM NaB, and p53 was unaffected by any concentration. By contrast, in NCI-H23, a marked increase in p21(waf1) protein was paralleled by decreased p53 levels, whereas all the other investigated proteins remained stable. The results suggest that NaB blocks the growth of both cell lines by induction of cyclin-dependent kinase inhibitors (in particular, p21(waf1) in NCI-H23 and p27(kip1) and p16(ink4) in NCI-H460) through a p53-dependent or p53-independent mechanism, and open up interesting perspectives for the use of NaB as an alternative or additional strategy in the treatment of non-small cell lung carcinoma.

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.

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.

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.

Cholesteryl butyrate in solid lipid nanospheres as an alternative approach for butyric acid delivery.

BACKGROUND: Cholesteryl-butyrate chosen as lipid matrix of solid lipid nanospheres (SLNs) could be a suitable pro-drug to deliver butyric acid and overcome one of the most limiting disadvantages of the compound: the short half-life due to a rapid metabolism. METHODS: We evaluated the antiproliferative effect, with respect to that of sodium butyrate, of four SLNs (SLN1, SLN2, SLN3 and SLN4) characterized by a different concentration of cholesteryl-butyrate (range, 1.7-30 mM) on NIH-H460, a non-small cell lung carcinoma cell line. RESULTS: After 6 days of treatment, all SLN preparations induced a dose-dependent inhibition of NHI-H460 cell growth: the most effective SLN preparation (SLN1) was able to induce a complete growth inhibition already at 0.25 mM, a concentration at which sodium butyrate induced only a 55% inhibition. Fluorescence microscopy showed that 6-coumarin-tagged SLNs were almost completely internalized by cells after 5 min of treatment. CONCLUSIONS: The present results indicate that owing to their peculiar characteristics, SLNs could be an interesting alternative approach for butyric acid delivery into tumor cells.

Effect of liposome-encapsulated alpha- or beta-interferon on breast cancer cell lines.

Experimental evidence and clinical studies have indicated that interferons (IFN) inhibit proliferation in a wide panel of neoplasms, including breast cancer. However, the antitumor activity of IFN requires the continuous presence of high concentrations of the drug and is associated with side effects. To explore the potential of liposomes as an IFN delivery system, we compared the effect of free or liposome-encapsulated alpha-IFN and beta-IFN on the growth of two breast cancer cell lines (MCF7 and MDA-MB231). Cells were cultured in the presence of IFN at different concentrations (500, 1000, 2000 IU/ml) or in the presence of multilamellar liposomes (phosphatidylcholine-phosphatidylserine at a molar ratio of 7:3) containing saline buffer, alpha-IFN or beta-IFN. Additional control groups consisted of cells cultured with alpha-IFN or beta-IFN plus empty liposomes. Empty liposomes were not cytotoxic and did not interfere with IFN activity. In both cell lines liposomes encapsulating alpha-IFN (at the highest lipid:drug ratio) inhibited cell growth in a manner similar to that of free alpha-IFN, whereas liposomes encapsulating beta-IFN showed slightly, lower inhibition than free beta-IFN, this was more evident in MCF7 cells. The present results indicate that liposomes encapsulating alpha-IFN or beta-IFN were effective on the growth of both breast cancer cell lines, which are characterized by a different estrogen responsiveness, and that they might be a useful carrier system for the delivery of high doses of IFN.

Simultaneous but not sequential treatment with sodium butyrate improves the antiproliferative effect of alpha- or beta-interferon on a breast cancer cell line.

Clinical evidence indicates that alpha- and beta-interferon (alpha-IFN, beta-IFN) are only partially effective in human breast cancer. To improve their effectiveness, it has been proposed that differentiation inducers, such as sodium butyrate (NaB), be used to increase the IFN sensitivity of tumors. Therefore, we assessed concomitant or sequential combinations of low/intermediate concentrations of alpha-IFN or beta-IFN (10, 50 and 100 IU/ml) with a low concentration (0.1 mM) of NaB on a human breast cancer cell line (MDA-MB231), which exhibited a moderate sensitivity to IFN. Moreover, to verify the capability of NaB to potentiate IFN effectiveness by increasing IFN receptor (IFN-R) concentration, we investigated the effect of NaB on the synthesis of IFN-R. The concomitant presence of NaB and alpha-IFN or beta-IFN significantly improved the antiproliferative effect of the corresponding IFN alone. Conversely, the sequential treatment NaB-IFN did not enhance the inhibitory activity of the cytokine, although NaB was able to induce the expression of IFN-R. More likely, NaB induced the expression of some component of the IFN system, such as Stat1, Stat2 or p48, whose higher availability in the cytoplasmic compartment promotes formation of the multimeric transcription factor ISGF3, which induces the transcription of IFN-stimulated genes.

Effect of sodium butyrate on human breast cancer cell lines.

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 mM. The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G2M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.

Combined effect of tamoxifen or interferon-beta and 4-hydroxyphenylretinamide on the growth of breast cancer cell lines.

To improve the effectiveness of 4-hydroxyphenylretinamide (4-HPR), an analogue of retinoic acid used in chemoprevention and treatment of breast cancer, we investigated the effect of concomitant administration of 4-HPR (0.1, 1 microM) and tamoxifen (TAM, 0.1, 1 microM), or 4-HPR and interferon-beta (IFN-beta, 10, 100, 500 IU/ml) on the growth of four cell lines (MCF7, T47D, MDA-MB231 and BT20) characterized by a different steroid receptor profile. A high concentration of 4-HPR caused a significant inhibitory effect not only on the estrogen receptor-positive cell lines (MCF7 and T47D), but also on one (BT20) of the two estrogen receptor-negative cell lines. IFN-beta displayed a dose-dependent inhibitory effect in all cell lines, but it was most evident in MCF7 cells. In all cell lines, the combination of 4-HPR (0.1 microM) and TAM (1 microM) or IFN-beta (500 IU/ml) generally caused additive or synergistic effects. In particular, the finding that in estrogen receptor-negative MDA-MB231 cells 4-HPR (which at 1 microM was singly ineffective) in combination with TAM at 1 microM or any concentration of IFN-beta produced a synergistic effect suggests that the compound could act through a pathway independent of specific receptors for retinoids. Our results indicate that intrinsic characteristics of cells can influence responsiveness to 4-HPR, TAM and IFN-beta, singly or in association, ever within cell lines with similar steroid receptor profiles. Thus, more attention should be payed to the biological characteristics of the single tumor in order to help choose the best combination of drugs to be applied.