Ilaria Rocco

Proteasome inhibitor-induced apoptosis in human monocyte-derived dendritic cells.

Proteasome inhibitors possess potent antitumor activity against a broad spectrum of human malignancies. However, the effects of these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effects of proteasome inhibitors on dendritic cells (DC), antigen‐presenting cells playing a key role in the initiation of immune responses. Exposure to the proteasome inhibitors bortezomib, MG132 or epoxomicin was found to promote apoptosis of human monocyte‐derived DC and to reduce the yield of viable DC when given to monocytes early during differentiation to DC. DC apoptosis via proteasome inhibition was accompanied by mitochondria disruption and subsequent activation of the caspase cascade. Up‐regulation and intracellular redistribution of Bcl‐2‐associated X protein (Bax), a pro‐apoptotic Bcl‐2 family protein, were observed in DC treated with these compounds and represent a suitable mechanism leading to activation of the intrinsic apoptotic pathway. Finally, active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by proteasome inhibitors, since the translation inhibitor cycloheximide blocked all of the steps of the observed apoptotic response. In conclusion, induction of apoptosis in DC may represent a novel mechanism by which proteasome inhibitors affect the immune response at the antigen‐presenting cell level.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cooperates with Anticancer Drugs to Overcome Chemoresistance in Antiapoptotic Bcl-2 Family Members Expressing Jurkat Cells

Purpose: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-xL overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model Experimental Design: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-xL overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC6 and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. Results: Bcl-2 and Bcl-xL overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-xL Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation Conclusions: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.

Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-α, IL-1β) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as α-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Patient-Tailored Treatments with Anti-EGFR Monoclonal Antibodies in Advanced Colorectal Cancer: KRAS and Beyond

Personalized medicine emphasizes the practice of considering individual patient characteristics as opposed to that centered on standards derived from epidemiological studies which, by definition, do not take into account the variability of individuals within a given population. When applied to oncology, personalized medicine is an even more complex concept because it extends the variability beyond the individual patient to the individual tumor. Indeed, the great genotypic and phenotypic variability (both in primary and metastatic sites of cancer) the development of targeted therapies, and the growing availability of biological assays complicate the scenario of personalized medicine in the oncological field. In this paper we review the results of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) therapy in metastatic colorectal cancer (mCRC) in the context of tumor biology, delineating the future prospects of patient-tailored medicine in this area. In particular, we deal with EGFR inhibition by Cetuximab, a chimeric mouse human IgG1 mAb, and panitumumab, a fully human IgG2 mAb. We discuss the clinical impact of anti-EGFR mAbs on wild-type (WT) KRAS mCRC, also taking into account the feasibility of novel multi-marker approaches to treatment decision-making, aimed at increasing the predictive power of pre-therapy biomarkers. Experimental topics and fields of ongoing research, such as targeting microRNAs (miRNAs) with novel anticancer drugs and epigenetics in CRC are also addressed.

Reverse-transcriptase polymerase chain reaction of the maspin gene in the detection of bone marrow breast carcinoma cell contamination

Maspin is a molecular marker used for the detection of contaminating breast carcinoma (BC) cells in peripheral blood and lymph nodes. However, its specificity has been questioned recently. The objective of this study was to verify the specificity of this marker and to determine the incidence of positive bone marrow results in patients with BC who are eligible for high‐dose chemotherapy (HDT) both in early and advanced disease stages and before and after treatment.

APO866 activity in hematologic malignancies: a preclinical in vitro study

To the editor: Nahimana and coworkers have recently reported that the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor APO866 elicited massive cell death in primary leukemia cells and in numerous leukemia/lymphoma cell lines.[1][1] In particular, in 32 primary leukemias (including 12 B-cell

Ras-Induced Resistance to Lapatinib is Overcome by MEK Inhibition

Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor is highly active in HER2+ breast cancer. However, its efficacy is limited by either primary or acquired resistance. Although mutations in ras genes are rarely found in breast cancer, H-ras overexpression is frequently observed. Moreover, genetic alterations that do not directly involve ras such as Brk amplification, ultimately result in increased ras signaling. Using SKBR3 cells, a HER2+ breast cancer cell line that is naturally devoid of mutations in PI3KCA, PTEN, BRAF, and ras we show that both H-ras overexpression and expression of an oncogenic ras allele (ras V12) reduce susceptibility to lapatinib in analogy to what observed with activating PI3KCA mutations and with a constitutively active form of Akt. Importantly, we found that resistance to lapatinib due to ras overexpression or to ras V12 is overcome by MEK inhibition with U0126, suggesting a key role for the MEK-Erk pathway in ras-induced resistance. Similar results were obtained in BT474 cells, another HER+ breast cancer cell line. Therefore, our data indicate that overexpressed/mutated ras may act as a biological modifier of the response to lapatinib. Combining MEK inhibitors with lapatinib may help overcome this form of resistance and increase the efficacy of lapatinib in these tumors.

Kinase domain mutations of BCR-ABL identified at diagnosis before imatinib-based therapy are associated with progression in patients with high Sokal risk chronic phase chronic myeloid leukemia

Acquired resistance to imatinib in the advanced phase of chronic myeloid leukemia (CML) has been associated with mutations in the kinase domain (KD) of BCR-ABL. On the contrary, the prognostic implication of KD mutations in early chronic phase (CP) patients at diagnosis before imatinib-based therapy has not yet been established. We have reviewed the status of mutations in 43 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequencing (DS) with BidDye Terminator v 1.1. cycle sequencing kit and analyzed with a 3130 ABI capillary electrophoresis system. Eight out 13 (61.5%) high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V. Three patients progressed during imatinib and second-line inhibitors and died of blastic phase CML at 23, 33, and 69 months. Another patient with intermediate Sokal risk showed D363G mutation at diagnosis, progressed under imatinib, was allografted and he is now alive in major molecular remission (MMR). No low-risk patient carried KD mutation at diagnosis. In conclusion, KD mutations conferring high-level imatinib resistance are present in patients with de novo CML and in some of them lead to disease progression.

Peripheral blood vs. bone marrow for molecular monitoring of BCR-ABL1 levels in chronic myelogenous leukemia, a retrospective analysis in allogeneic bone marrow recipients.

Molecular monitoring of the BCR‐ABL1 transcript in chronic myelogenous leukemia (CML) using quantitative real‐time PCR (RQ‐PCR) can be performed using either bone marrow (BM) or peripheral blood (PB). However, a recent report by Stock et al. [International Journal of Oncology 28 (2006) 1099] questioned the reliability of PB samples for BCR‐ABL1 detection as performed by RQ‐PCR. We report a study on 114 CML patients who received allogeneic stem cell transplantation (ASCT), and who were monitored by RQ‐PCR using paired samples of BM and PB: the total number of determinations was 428, with a median follow‐up after transplant of 8 years. BCR‐ABL1 transcript was undetectable or <0.1%, in 106 (49.57%) and 62 (29%) paired determinations, respectively. BCR‐ABL1 was >0.1% in 36 (16.8%) paired determinations and was discordant in 10 (4.7%). Agreement between PB and BM results was quantified by the kappa test (k = 0.85; 95% CI 0.76–0.94). This study shows that BCR‐ABL1 RQ‐PCR monitoring of CML patients after ASCT with PB is concordant with BM in 95.3% of cases, and thus may be used to monitor the disease. This may be relevant when discussing both quality of life issues and the need for post‐transplant monitoring with the patient.