Marta Celegato

Toward the selective delivery of chemotherapeutics into tumor cells by targeting peptide transporters: tailored gold-based anticancer peptidomimetics.

Complexes [Au(III)X(2)(dtc-Sar-AA-O(t-Bu))] (AA = Gly, X = Br (1)/Cl (2); AA = Aib, X = Br (3)/Cl (4); AA = l-Phe, X = Br (5)/Cl (6)) were designed on purpose in order to obtain gold(III)-based anticancer peptidomimetics that might specifically target two peptide transporters (namely, PEPT1 and PEPT2) upregulated in several tumor cells. All the compounds were characterized by means of FT-IR and mono- and multidimensional NMR spectroscopy, and the crystal structure of [Au(III)Br(2)(dtc-Sar-Aib-O(t-Bu))] (3) was solved and refined. According to in vitro cytotoxicity studies, the Aib-containing complexes 3 and 4 turned out to be the most effective toward all the human tumor cell lines evaluated (PC3, DU145, 2008, C13, and L540), reporting IC(50) values much lower than that of cisplatin. Remarkably, they showed no cross-resistance with cisplatin itself and were proved to inhibit tumor cell proliferation by inducing either apoptosis or late apoptosis/necrosis depending on the cell lines. Biological results are here reported and discussed in terms of the structure-activity relationship.

IRF4 silencing inhibits Hodgkin lymphoma cell proliferation, survival and CCL5 secretion

Interferon regulatory factor 4 (IRF4) expression is detected in many lymphoid and myeloid malignancies, and may be a promising therapeutic target. IRF4 is strongly expressed in classical Hodgkin lymphoma (cHL) and its expression is up‐regulated by CD40L and down‐regulated by both anti‐proliferative and pro‐apoptotic stimuli. This study analysed the effects of IRF4 silencing in a panel of HL‐derived cell lines. We demonstrated that IRF4 down‐modulation determined a remarkable decrease of both cell number and clonogenic growth in L‐1236, L‐428, KM‐H2 and HDLM‐2 cells, but not in IRF4‐negative L‐540 cells. IRF4 silencing induced apoptosis, as evaluated by caspase‐3 activation and Annexin‐V staining and up‐regulation of the pro‐apoptotic molecule Bax. CD40 engagement by both soluble and membrane bound‐CD40L almost totally reduced IRF4 down‐modulation and growth inhibition by IRF4 silencing in both L‐1236 and L‐428 cells. Finally, IRF4 silencing decreased CCL5 secretion in all HL cell lines tested and CCL17 in KM‐H2 cells. Taken together, our results demonstrated that IRF4 down‐modulation by IRF4 silencing was reversed by CD40 engagement, inhibited HL cells proliferation, induced apoptosis and decreased CCL5 secretion, thus suggesting that IRF4 may be involved in HL pathobiology.

Gefitinib inhibits the cross‐talk between mesenchymal stem cells and prostate cancer cells leading to tumor cell proliferation and inhibition of docetaxel activity

Increasing evidence suggests that bone marrow derived mesenchymal stem cells (BM‐MSCs) are recruited into the stroma of developing tumors where they contribute to progression by enhancing tumor growth and metastasis, or by inducing anticancer‐drug resistance. Prostate cancer cells secrete ligands of epidermal growth factor receptor (EGFR) and EGFR signaling could play an important role in the cross‐talk between mesenchymal stem cells and prostate cancer cells. In this study, we showed that treatment of human primary MSCs with conditioned medium (CM) derived from the bone metastatic PC3 carcinoma cells (PC3‐CM) resulted in: a significant activation of EGFR; increased proliferation; increased osteoblastic but decreased adipocitic differentiation; inhibition of senescence induced by serum starvation; increased CCL5 secretion. These activities were significantly inhibited in the presence of the EGFR tyrosine kinase inhibitor gefitinib. PC3‐CM directly inhibited osteoclastogenesis as well as the ability of osteoblasts to induce osteoclast differentiation. The increased MSCs migration by PC3‐CM and PC3 cells was partially mediated by CCL5. MSC‐CM increased the formation of colonies by PC3 cells and inhibited the anti‐proliferative activity of Docetaxel. Activation of EGFR expressed on MSCs by PC3‐CM enhanced their capability to increase PC3 cells proliferation and to inhibit Docetaxel activity. These findings, by showing that the tumor‐promoting interactions between PC3 cells and MSCs are mediated, at least in part, by EGFR, suggest a novel application of the EGFR‐tyrosine kinase inhibitors in the treatment of prostate cancer. J. Cell. Biochem. 114: 1135–1144, 2013.

Microenvironmental interactions in classical Hodgkin lymphoma and their role in promoting tumor growth, immune escape and drug resistance

Classical Hodgkin lymphoma (cHL) is characterized by few tumor cells surrounded by immune cells, fibroblasts, specialized mesenchymal stromal cells and endothelial cells, representing together with their products an active part of the disease. Hodgkin and Reed-Sternberg (HRS) cells can secrete cytokines/chemokines and angiogenic factors capable of recruiting and/or inducing the proliferation of the surrounding cells and can also interact with distant sites of the microenvironment by secreting exosomes. To escape from a useful anti-tumor response due to the recognition by T and NK cells, HRS cells down-regulate HLA molecules, produce immune suppressive cytokines that inhibit cytotoxic responses, and induce an immunosuppressive phenotype on T lymphocytes and Monocytes. HRS cells survive, proliferate and are protected from the cytotoxic effects of chemotherapy agents by soluble factors or by the direct contact with inflammatory and stromal cells of the tumor microenvironment (TME). A summary of the current knowledge about classical Hodgkin Lymphoma focusing on the cross-talk between tumor cells and the microenvironment leading to immune-escape, angiogenesis tumor growth/survival and drug resistance will be reviewed here.

Preclinical evaluation of a new liposomal formulation of cisplatin, lipoplatin, to treat cisplatin-resistant cervical cancer

OBJECTIVE Cisplatin-based chemotherapy has been shown to improve survival in cervical cancer; however, treatment is associated with tumor resistance and significant toxicity. Lipoplatin is a new liposomal formulation of cisplatin, developed to reduce cisplatin toxicity, to improve drug accumulation at tumor sites and to overcome drug resistance. The aim of this study is to analyze the antitumoral activity of lipoplatin against cisplatin-resistant cervical cancer cells and to investigate its mechanism of action. METHODS The activity and mechanism of action of lipoplatin were studied in the ME-180 cervical cancer cell line and its cisplatin-resistant clone R-ME-180 and HeLa cells using cell proliferation assays, flow cytometry, ELISA assay, cell migration, spheroids and tumor xenograft. RESULTS We demonstrated that lipoplatin exhibited a potent antitumoral activity on HeLa, ME-180 cells and its cisplatin-resistant clone R-ME-180. Lipoplatin inhibited cell proliferation in a dose-dependent manner and was more active than the reference drug cisplatin in R-ME-180 cells and induced apoptosis, as evaluated by Annexin-V staining and DNA fragmentation, caspases 9 and 3 activation, Bcl-2, and Bcl-xL down-regulation, but Bax up-regulation inhibited thioredoxin reductase (TrxR) enzymatic activity and increased reactive oxygen species (ROS) accumulation; reduced EGFR expression and inhibited both migration and invasion. R-ME-180, but not ME-180 cells, generated three-dimensional (3D)-multicellular spheroids expressing the cancer stem cell marker ALDH. The ability of R-ME-180 cells to form spheroids in vitro and tumors in nude mice was also remarkably decreased by lipoplatin. CONCLUSIONS Overall, our results suggest that lipoplatin has potential for the treatment of cisplatin-resistant cervical cancer.

New bipyridine gold(III) dithiocarbamate-containing complexes exerted a potent anticancer activity against cisplatin-resistant cancer cells independent of p53 status

We synthesized, characterized and tested in a panel of cancer cell lines, nine new bipyridine gold(III) dithiocarbamate-containing complexes. In vitro studies demonstrated that compounds 1, 2, 4, 5, 7 and 8 were the most cytotoxic in prostate, breast, ovarian cancer cell lines and in Hodgkin lymphoma cells with IC50 values lower than the reference drug cisplatin. The most active compound 1 was more active than cisplatin in ovarian (A2780cis and 2780CP-16) and breast cancer cisplatin-resistant cells. Compound 1 determined an alteration of the cellular redox homeostasis leading to increased ROS levels, a decrease in the mitochondrial membrane potential, cytochrome-c release from the mitochondria and activation of caspases 9 and 3. The ROS scavenger NAC suppressed ROS generation and rescued cells from damage. Compound 1 resulted more active in tumor cells than in normal human Mesenchymal stromal cells. Gold compounds were active independent of p53 status: exerted cytotoxic effects on a panel of non-small cell lung cancer cell lines with different p53 status and in the ovarian A2780 model where the p53 was knocked out. In conclusion, these promising results strongly indicate the need for further preclinical evaluation to test the clinical potential of these new gold(III) complexes.

Preclinical Activity of the Liposomal Cisplatin Lipoplatin in Ovarian Cancer

Purpose: Cisplatin and its platinum derivatives are first-line chemotherapeutic agents in the treatment of ovarian cancer; however, treatment is associated with tumor resistance and significant toxicity. Here we investigated the antitumoral activity of lipoplatin, one of the most promising liposomal platinum drug formulations under clinical investigation. Experimental Design: In vitro effects of lipoplatin were tested on a panel of ovarian cancer cell lines, sensitive and resistant to cisplatin, using both two-dimensional (2D) and 3D cell models. We evaluated in vivo the lipoplatin anticancer activity using tumor xenografts. Results: Lipoplatin exhibited a potent antitumoral activity in all ovarian cancer cell lines tested, induced apoptosis, and activated caspase-9, -8, and -3, downregulating Bcl-2 and upregulating Bax. Lipoplatin inhibited thioredoxin reductase enzymatic activity and increased reactive oxygen species accumulation and reduced EGF receptor (EGFR) expression and inhibited cell invasion. Lipoplatin demonstrated a synergistic effect when used in combination with doxorubicin, widely used in relapsed ovarian cancer treatment, and with the albumin-bound paclitaxel, Abraxane. Lipoplatin decreased both ALDH and CD133 expression, markers of ovarian cancer stem cells. Multicellular aggregates/spheroids are present in ascites of patients and most contribute to the spreading to secondary sites. Lipoplatin decreased spheroids growth, vitality, and cell migration out of preformed spheroids. Finally, lipoplatin inhibited more than 90% tumor xenograft growth with minimal systemic toxicity, and after the treatment suspension, no tumor progression was observed. Conclusion: These preclinical data suggest that lipoplatin has potential for clinical assessment in aggressive cisplatin-resistant patients with ovarian cancer. Clin Cancer Res; 20(21); 5496–506. ©2014 AACR.

Preclinical activity of the repurposed drug auranofin in classical Hodgkin lymphoma

To the editor: The treatment of classical Hodgkin lymphoma (cHL) patients with refractory/relapsed disease remains a clinical challenge.[1][1] To find a new therapeutic option for cHL, we investigated the preclinical activity of the repurposed drug auranofin (AF).[2][2] AF is an anti-inflammatory

The NF-κB inhibitor DHMEQ decreases survival factors, overcomes the protective activity of microenvironment and synergizes with chemotherapy agents in classical Hodgkin lymphoma

The NF-κB inhibitor DHMEQ has shown preclinical activity in classical Hodgkin Lymphoma (cHL). Here we evaluated if DHMEQ could affect microenvironmental interactions and formation and improve the activity of drugs used in relapsed/refractory cHL. We demonstrated that DHMEQ down-regulated the NF-κB target genes IRF4 and CD40, the secretion of IL-6, CCL5, CCL17 and generated ROS. Cytotoxicity, CD30 down-modulation and CD30 shedding by DHMEQ were prevented by ROS scavenger NAC. DHMEQ overcame stimuli from CD40 engagement and fibroblasts and enhanced doxorubicin, cisplatin and gemcitabine activity. Our results suggest that DHMEQ may be a promising agent for future therapeutic strategies in cHL.

Bortezomib down-modulates the survival factor interferon regulatory factor 4 in Hodgkin lymphoma cell lines and decreases the protective activity of Hodgkin lymphoma-associated fibroblasts.

Abstract Bortezomib is a proteasome inhibitor active in classical Hodgkin lymphoma (cHL) cell lines, but poorly active in the clinic when used as a single agent, suggesting that the microenvironment could protect from drug efficacy. Therefore, we investigated the effects of bortezomib activity in the presence of HL-associated fibroblasts (HL-AFs) and sCD40L. We found that co-cultivation with human HL-AFs or the addition of sCD40L during bortezomib treatment protected cHL cells from apoptosis and cytotoxicity and rescued the down-regulation of the survival factor interferon regulatory factor 4 (IRF4). In contrast, bortezomib treatment before co-cultivation with HL-AFs inhibited in a dose-dependent manner cHL cell adhesion to HL-AFs and completely overcame HL-AF protection against drug activity. Consistently, we found that bortezomib treatment down-regulated the surface expression of CD49d and CD44, which mediate the adhesion of cHL cells to HL-AFs, and of CD54 and CD40, which mediate the adhesion to CD40L+ rosetting T-cells. These preclinical findings suggest that the low in vivo activity of bortezomib as a single agent may be due to a protective influence of the microenvironment. However, inclusion of bortezomib in the cHL drug regimen, by reducing IRF4 expression and interactions with the microenvironment, could increase the efficacy of current chemotherapeutic treatment of relapsed/refractory cHL.