Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.

Our data suggest that concurrent or sequential administration of BPs with conventional chemotherapeutic drugs may have significant therapeutic potential for CLL patients.

Inhibitors of apoptosis proteins (IAPs) are a group of structurally related proteins that block apoptosis either by binding and inhibiting caspases or through caspases-independent mechanisms. Overexpression of IAPs has been detected in several types of cancers, including hematological malignancies, and it is correlated with chemotherapeutic resistance. B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature lymphocytes due to defective apoptosis. During disease progression, many patients acquire resistance to chemotherapeutic drugs, perhaps attributable, in large part, to the defects in programmed cell death. To verify whether IAPs expression could be responsible for chemotherapeutic resistance or disease progression we analyzed, by immunocitochemistry, the expression of c-IAP1, c-IAP2 and XIAP in 30 peripheral blood samples from B-CLL patients (16 samples from untreated patients) and compared it to well-known prognosis factors such as clinical stage (Rai and Binet), lymphocyte doubling time, gender, age and previous chemotherapeutic treatment or not. High levels of c-IAP1 and XIAP expression were not associated with any prognosis factor. On the other hand, interestingly, high levels of c-IAP2 expression were correlated with advanced clinical stage (p = 0,021 and p= 0,031 for Rai and Binet respectively) and previous chemotherapeutic treatment (p = 0,043). Our results suggest that the analysis of c-IAP2 expression in B-CLL cells might be considered a powerful tool as a marker for poor prognostic and disease progression.

Recent studies demonstrate that bisphosphonates - anti-resorptive drugs - have direct anti-tumour effect in many tumour cell lines, including hematopoietic ones. The aim of this study was to evaluate the apoptotic effect of Zoledronate in cells from B chronic lymphocytic leukemia (B-CLL) and low-grade lymphoma (LGL) patients. Samples of 19 B-CLL or LGL patients were incubated with Zoledronate 100 μM in RPMI medium supplemented with fetal bovine serum 10% at 370C for 12h. Apoptosis using Annexin V assay, by flow cytometry (FC), was observed in 8 out of 19 (42,1%) patients despite previous treatment. Multidrug resistance (MDR) phenotype was performed using rhodamine-123 efflux assay by FC. Our results demonstrate that Zoledronate 100 m M can induce apoptosis in B malignant lymphocytes despite previous treatment and MDR phenotype. So, patients previously treated with distinct therapeutic agents or not can potentially benefit from the anti-tumour effect of Zoledronate. This is the first work demonstrating the anti-tumour effect of Zoledronate in newly obtained cells from patients with B-CLL and LGL. These results in association with evidences from recent studies suggest that Zoledronate may have an important and complementary role in hematological malignant diseases.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature lymphocytes due to defective apoptosis. Inhibition of apoptosis also contributes to chemoresistance, mainly in patients in advanced clinical stages. Fludarabine is a potent chemotherapeutic drug used for patients with B-CLL. Despite good initial clinical responses, some patients acquire resistance to this drug. One of the mechanisms through which malignant cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins (IAPs). IAPs are a family of proteins able to block apoptosis either by binding and inhibiting caspases or through caspases-independent mechanisms. To date, overexpression of IAPs has been detected in many types of malignant diseases, including B-CLL. As fludarabine can induce apoptosis trough caspases activation and this proteases could be inhibited by IAPs, we evaluated in this current study, the expression of three members of IAP family (c-IAP1, c-IAP2 and XIAP) by immunocitochemistry in 30 peripheral blood B-CLL specimens and correlated it to fludarabine-induced apoptosis. In parallel, we also evaluated the expression of these IAPs before and after in vitro fludarabine treatment. B-CLL cells were incubated with fludarabine25 m M for 12 hours and the induction of apoptosis were measured by annexin-V assay. The apoptosis index by fludarabine was extremely significant (p < 0.001). Different levels of c-IAP1, c-IAP2 and XIAP expression were commonly found in circulating B-CLL cells. High levels of c-IAP1, c-IAP2 and XIAP expression were observed in 20%, 60,7% and 60% of samples, respectively. However, the overexpression of these IAPs was not correlated with an in vitro fludarabine apoptosis-resistance. Besides, fludarabine was not able to change the levels of IAPs expression as described for other drugs in literature. These results suggest that fludarabine might cause apoptosis by a caspase-independent manner.