Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

sTRAIL coupled to liposomes improves its pharmacokinetic profile and overcomes neuroblastoma tumour resistance in combination with Bortezomib.

Neuroblastoma (NB), the most common and deadly extracranial solid tumour of childhood, represents a challenging in paediatric oncology. Soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting remarkable anti-tumour activities against paediatric malignancies both in vitro and in preclinical settings. However, due to its too fast elimination and to the undesired related side effects, the improvement of sTRAIL in vivo bioavailability and the specific delivery to the tumour is mandatory for increasing its therapeutic efficacy. In this manuscript, we developed an innovative pegylated liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) with the intent to improve its serum half-life and increase its efficacy in vivo, while reducing side effects. Furthermore, the possibility to combine sTRAIL-SL with the proteasome inhibitor Bortezomib (BTZ) was investigated, being BTZ able to sensitize tumour cells toward TRAIL-induced apoptosis. We demonstrated that sTRAIL preserved and improved its anti-tumour activity when coupled to nanocarriers. Moreover, sTRAIL-SL ameliorated its PK profile in blood allowing sTRAIL to exert a more potent anti-tumour activity, which led, upon BTZ priming, to a statistically significant enhanced life spans in two models of sTRAIL-resistant NB-bearing mice. Finally, mechanistic studies indicated that the combination of sTRAIL with BTZ sensitized sTRAIL-resistant NB tumour cells to sTRAIL-induced cell death, both in vitro and in vivo, through the Akt/GSK3/ß-catenin axis-dependent mechanism. In conclusion, our results suggest that sTRAIL-SL might be an efficient vehicle for sTRAIL delivery and that its use in clinic, in combination with BTZ, might represent an adjuvant strategy for the treatment of stage IV, sTRAIL-resistant, NB patients.

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b(+)/Gr-1(+) cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1(+) population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-ß1 (TGF-ß1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-ß1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

4-OH tamoxifen does not interfere with bicalutamide inhibitory effects on human prostatic cancer cells in vitro.

BACKGROUND: The present study was aimed at investigating the effects of the co-administration of 4-OH Tamoxifen (4HT) and bicalutamide (BIC) on the human prostate cancer cell line LNCaP in vitro. MATERIALS AND METHODS: The LNCaP FGC prostate cancer cell line was grown in vitro within a three-dimensional matrix formed by collagen gel under dihydrotestosterone (DHT) (0.1 nM) stimulation. Cells were incubated in the presence either of BIC at escalating concentrations (1 nM; 100 nM; 10 microM) or 4HT Tamoxifen (10 nM; 100 nM) or both compounds at the different concentrations studied. The cells were incubated for 144 hours, and growth was evaluated in non trypsinised cells by crystal violet vital dye staining assay. RESULTS: BIC appeared to exert a dose-dependent inhibitory action, with the maximum inhibitory effect achieved by the 10 microM concentration. A comparable inhibitory effect was also observed after exposure to 4HT at both doses tested. No statistically significant interference was observed when BIC was combined with 4HT. CONCLUSION: 4HT, even at the higher concentration employed here, showed no major interference with the inhibitory effects of BIC.

Chemoprevention of tumors: the role of RAR-beta.

Chemoprevention can be defined as the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. The knowledge of carcinogenic mechanisms provides the scientific rationale for chemoprevention. Epithelial carcinogenesis proceeds through multiple discernible stages of molecular and cellular alterations. Understanding of the multistep nature of carcinogenesis has evolved through highly controlled animal carcinogenesis studies, and these studies have identified three distinct phases: initiation, promotion and progression. Animal model studies have provided evidence that the development of cancer involves many different factors, including alterations in the structures and functions of different genes. Transitions between successive stages can be enhanced or inhibited in the laboratory by different types of agents, such activities providing the fundamental basis for chemoprevention.

Retinoid receptors: pathways of proliferation inhibition and apoptosis induction in breast cancer cell lines.

In this study we investigated the effects of several selective agonist retinoids (specific for RAR alpha, RAR beta, RAR gamma, and RXR alpha, respectively) on the proliferation and apoptosis of human breast cancer cell lines. All these retinoids inhibit proliferation through apoptosis induction, but with some differences among the tested molecules and the three cell lines. In particular, estrogen receptor positive (ER+) cells display a higher sensitivity to RARs selective compounds, the RAR alpha selective compound being the most effective agent, while estrogen receptor negative (ER-) cells show a greater responsiveness to the RXR alpha selective retinoid. In all tested cell lines a potent antiproliferative and apoptotic effect was also displayed by a high dose of the RAR gamma selective compound. The apoptosis induction is associated with bcl-2 down-regulation, while p53 expression is not modified by any retinoid. Only in one cell line (ZR-75.1), after RAR alpha selective retinoid treatment is there an induction of RAR beta: therefore not only RAR beta induction but also other mechanisms may contribute to the growth inhibitory effect of retinoids in tested breast cancer cell lines.

Different effects of the treatment with AGN 193836 and 9-cis-retinoic acid in breast cancer cells.

Retinoid effects have been well studied in different cellular models, and their in vitro action on breast cancer is well known. Much less is known about the function of the different retinoid receptors in mediating retinoid activity in this and other cellular models. In order to better understand these biological mechanisms, several synthetic compounds have been produced, that have specific binding affinity for selected nuclear receptors, and their effect has been evaluated and confronted with that of classic compounds able to bind to different receptors. The aim of this study was the evaluation of the biological activities in breast cancer cell lines of one of these new compounds, AGN 193836, with a very selective binding affinity (selective agonist retinoid) for one single retinoic acid receptor (RAR alpha), in respect to a classic retinoid able to bind to a broad spectrum of retinoic acid receptors (pan-agonist retinoid), 9cRA. Our results clearly indicate that the selective retinoid retains most of the biological activities of the pan-agonist compound, but its effect is probably aggravated by fewer side-effects in vivo: This evidences indicate that selective-agonist retinoids are an interesting research field for the future, not only because of their speculative interest, but also in view of future clinical applications.