The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells

The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Table 1. Characteristics of the patients with chronic lymphocytic leukemia.Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients' cells with mutated p53 or ATM.PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status.These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.

section_content: C hronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature malignant CD5 + B lymphocytes. 1 Although many drugs have been used in the therapy of CLL, at present there is no curative therapy, and the search for new candidate drugs for future treatment of CLL is an active area of research. 4] 8 Hence, new therapies that overcome these defects by acting independently of p53 are of great interest. 9 3] [24] PK11195 was ini-tially described as a ligand for peripheral benzodiazepine receptor (PBR), 25 whose new proposed name is translocator protein (18 KDa) (TSPO). 26 TSPO is a transmembrane protein that is located mainly in the outer mitochondrial membrane, but is also expressed in other subcellular compartments. TSPO is associated with the regulation of cholesterol transport, the synthesis of steroid hormones, porphyrin transport, heme synthesis, apoptosis and cell proliferation. 26 Other chemically unrelated TSPO ligands induce apoptosis in different cell types, 15, 17, [19] [20] [21] indicating a TSPOdependent mechanism; however, TSPO-independent mechanisms have been proposed to inhibit cell proliferation or sensitize cells to apoptosis. 24, 27, 28 It has been reported that PK11195 induces apoptosis by altering the mitochondrial permeability transition. 10, 11 Interestingly, in a variety of systems, PK11195 can reduce or abrogate the antiapoptotic effect of BCL-2-like proteins, including BCL-2 and BCL-X L. 22, 29 Since primary CLL cells overexpress TSPO 30 and BCL-2, 31, 32 we decided to examine the effects of PK11195 on CLL cells.
section_name: ABSTRACT

section_name: Design and Methods

section_content: Samples from patients with CLL (Table 1 ) or healthy donors were studied. Most of the patients had not been treated previously (patients number 1, 13, 17, 23, 24, 27, 28 and 33 were, however, treated). The patient number in Table 1 corresponds to the patient number in the figures. CLL was diagnosed according to standard clinical and laboratory criteria. Blood samples were obtained from the Hospital de Bellvitge, Barcelona, Spain. Written informed consent was obtained from all patients in accordance with the Hospital de Bellvitge Ethical Committee. Mononuclear cells from peripheral blood samples were isolated by centrifugation on a Ficoll-Hypaque (Seromed, Berlin, Germany) gradient and cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide (DMSO).
section_name: Samples, patients with CLL, healthy donors and cell isolation

section_content: DNA was isolated and analyzed by MLPA using SALSA MLPA kits P037 and P038 from MRC-Holland (Amsterdam, The Netherlands). These kits were used to determine the loss of p53 (17p13; 8 probes), the RB1/DLEU/MIR15-16 region on 13q14 (12 probes) and the ATM gene on 11q23 (7 probes) in DNA samples obtained from CLL cells. RNA was isolated from cultured cells by the RNeasy Micro kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. RNA was analyzed by RT-MLPA using the SALSA MLPA kit R011 Apoptosis mRNA from MRC-Holland (Amsterdam, The Netherlands) for the simultaneous detection of 38 mRNA molecules. 7, 33
section_name: Multiplex ligation-dependent probe amplification (MLPA) for genomic alterations and reverse transcriptase (RT)-MLPA

section_content: Apoptosis was assessed by exposure of phosphatidylserine and membrane integrity. This was determined by annexin V-fluorescein isothiocyanate (FITC), propidium iodide (PI) double staining, and flow cytometric analysis using FACSCalibur and CellQuest software (Becton Dickinson, Mountain View, CA, USA), as described previously. 34 Cell viability was measured as the percentage of annexin V and PI double-negative cells. To
section_name: Analysis of apoptosis by flow cytometry

section_content: Release of cytochrome c from mitochondria into the cytosol was measured by western blot as previously described 35 with some modifications. Cells (25×10 6 ) were harvested, washed once with ice-cold PBS and gently lysed for 30 seconds in 80 µL ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA, 0. 05% digitonin, 25 mM Tris, pH 6. 8, 1 mM dithiothreitol, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 µg/mL aprotinin, 1 mM benzamidine, and 0. 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged at 12,000×g at 4°C for 3 minutes to obtain the supernatants (cytosolic extracts free of mitochondria) and the pellets (the fraction containing the mitochondria). Supernatants (50 µg) were electrophoresed on a 15% polyacrylamide gel and then analyzed by western blot using anti-cytochrome c antibody (7H8. 2C12, from Pharmingen, San Diego, CA, USA) and an electrochemiluminescence system, as described in online supplementary data.
section_name: Cytochrome c release measurements

section_content: Results are shown as the mean ± standard deviation (SD) of values obtained in independent experiments. The paired Student's t test was used to compare differences between paired samples. Data were analyzed using the SPSS 11. 5 software package (Chicago, IL, USA). Additive and synergistic effects were analyzed using the fractional product method. 36
section_name: Statistical analysis

section_name: Results

section_content: As FasL does not induce apoptosis in CLL cells 37 we first examined whether PK11195 reverses this resistance in CLL cells, but PK11195 was not able to do so (Figure 1A ). However, when combined with the chemotherapeutic drugs, dexamethasone, doxorubicin, chlorambucil, fludarabine and mafosfamide, PK11195 had an additive effect in inducing apoptosis in CLL cells (Figures 1B, 1C and 1D). It is interesting to note that 50 µM PK11195 alone induced apoptosis in most of the samples analyzed in Figure 1. Thus, we studied the effect of several doses of PK11195 on the viability of CLL cells. Although there was heterogeneity in the sensitivity of CLL cells, PK11195 induced apoptosis in all the samples analyzed in a dose-dependent manner (Figure 2A ) and the IC 50 was 60±21 µM (n=29) at 24 hours. The viability after incubation for 24 hours with 50 µM PK11195 decreased from 66±18% to 38±21% (n=33), and with 100 µM PK11195 it decreased from 71±16% to 16±13% (n=19). Fresh or frozen-thawed CLL cells showed the same sensitivity to PK11195 (Table 1 ). The average of the effect of 50 µM PK11195 was 54±21% for fresh cells and 53±15% for thawed cells. Furthermore, PK11195 had the same effect on viability of fresh or frozen-thawed CLL cells from the same sample (patients 2 and 24, Table 1 ). PK11195 induced apoptosis independently of sex, ZAP70 or CD38 status (Table 1 ). Next, we analyzed whether other compounds described as TSPO ligands could induce apoptosis in CLL cells. Thus, CLL cells were incubated for 24 hours with PK11195 and the TSPO ligands RO 5-4864 and FGIN-1-27 26 in a range of concentrations (12. 5-100 µM). Similarly to PK11195, both RO 5-4864 and FGIN-1-27 induced apoptosis in a dose-dependent manner and their IC50 were 83±18 µM and 88±31 µM, respectively (n=9) (Figure 2B ). Moreover, PK11195 had a synergistic effect with regards to induction of apoptosis in CLL cells when combined with the other TSPO ligands, RO 5-4864 and FGIN-1-27 (Figure 2C ).
section_name: PK11195 and other TSPO ligands induce apoptosis in CLL cells

section_content: The effect of PK11195 on the loss of mitochondrial membrane potential (∆ψm) and cytochrome c release was analyzed in CLL cells. First, we analyzed the effect of 50 µM PK11195 on ∆ψm using the JC-1 dye. PK11195 induced apoptosis (Figure 3A ), and decreased ∆ψm (Figure 3B ) at 6 hours. The caspase inhibitor Z-VAD. fmk did not inhibit loss of ∆ψm but did inhibit PK11195-induced apoptosis, indicating that early loss of ∆ψm is caspase-independent. Furthermore, to analyze the involvement of cytochrome c release in PK11195-induced apoptosis, cytosolic fractions were obtained and the presence of cytochrome c was analyzed by western blotting. Treatment with 50 µM PK11195 for 6 hours produced an increase in cytochrome c in the cytosolic fraction of CLL cells from two different representative patients (Figure 3C ), demonstrating that PK11195 induced cytochrome c release. Z-VAD. fmk did not inhibit PK11195-induced cytochrome c accumulation in the cytosol. Furthermore, PK11195 induced cleavage and activation of caspase-9 at 9 hours (Figure 3D ). These results demonstrate that the mitochondrial effects of PK11195 are caspase-independent and precede the activation of caspases. 

To study the mechanism involved in PK11195-induced cytochrome c release, we used RT-MLPA to analyze changes in the expression of genes involved in the control of apoptosis. 7, 33 PK11195 treatment for 3 and 6 hours did not modify the expression of any of the genes of the BCL-2 family analyzed (data not shown). Furthermore, treatment with PK11195 for 3 or 6 hours neither increased the levels of the pro-apoptotic proteins BIM, PUMA, and BMF, nor decreased the levels of the anti-apoptotic proteins MCL-1, BCL-X L and BCL-2 (Figure 4A ). Furthermore, treatment with PK11195 did not change the levels of the pro-apop- totic protein BAX and induced an increase in the levels of MCL-1 protein in most of the samples analyzed (Figure 4A and 4B). Moreover, inhibition of cAMP-dependent protein kinase (PKA) or JNK with the specific inhibitors (H89 or SP600125, respectively) did not affect PK11195-induced apoptosis. Interestingly, DIDS, a voltage-dependent anion channel (VDAC) inhibitor, 38 reduced the pro-apoptotic activity of PK11195 in CLL cells (Figure 4C ). As a control, DIDS did not inhibit apoptosis induced by fludarabine or doxorubicin (data not shown).
section_name: Characterization of PK11195-induced apoptosis in CLL cells: loss of mitochondrial membrane potential and cytochrome c release precede caspase activation

section_content: Next, we analyzed the sensitivity of normal B and T cells to apoptosis induced by PK11195. The number of apoptotic CD3 + T cells was measured in blood samples from four CLL patients and four healthy donors exposed to several doses of PK11195, ranging from 12. 5 µM to 100 µM, for 24 hours (Figure 5A ). Treatment with 50 µM PK11195 reduced the percentage of viable CLL CD19 + B cells to 46±7% and the IC50 was 47±2 µM. In contrast, under the same treatment the percentage of viable T cells from the same samples was 95±4% with an IC50 of 87±8 µM. Similar results were obtained with cells obtained from healthy donors such that 50 µM PK11195 reduced the viability of B cells to 42±3%, with an IC50 of 49±4 µM, while the viability of T cells was 98±6% with an IC50 of 82±8 µM. At doses of PK11195 higher than 50 µM, T cells from CLL patients and healthy donors showed sensitivity to apoptosis although to a lesser extent than B cells. These dose-response experiments show that B cells are more sensitive than T cells to PK11195-induced apoptosis. Similarly, T cells are more resistant than B cells from CLL samples to RO 5-4864-and FGIN-1-27-induced apoptosis (Figure 5B ).
section_name: Differential effect of PK11195 on B and T cells from patients with CLL and healthy donor

section_content: To study the role of p53 in PK11195-induced apoptosis we analyzed the effect of this compound on p53 protein levels (Figure 6A ). The patients with mutated p53 (patients 24 and 33) or altered expression were described previously. 7 Patient 24 has a frame-shift mutation in one allele (nucleotide deletion in codon 272) and a 17p deletion in the other allele in 86% of cells from peripheral blood leukocyte (PBL) samples, and patient 33 has the M246V muta- tion, which has been described to interfere with wild type p53. 39 When CLL cells were incubated with 50 µM PK11195 there was no change in p53 levels (Figure 6A ). As a control of p53 activation we used doxorubicin (0. 8 µM), which induced apoptosis and p53 protein accumulation in wild type p53 cells (from patients 16 and 34) but not in mutated p53 cells (from patients 24 and 33). In patient 24, doxorubicin induced a low accumulation of mutated p53. Interestingly, PK11195 induced apoptosis in CLL cells with mutated p53 (Figure 6A ). Furthermore, chemotherapeutic drugs that activate p53 did not increase the apoptotic effect of PK11195 in p53-mutated samples (data not shown). 

Next, we analyzed the effect of PK11195 on CLL cells with ATM alterations. ATM expression was analyzed by western blotting in several patients with and without 11q deletion (Figure 6B ). We found loss of ATM expression in three patients with an 11q deletion (patients 5, 8 and 39). Patients 5 and 8 have the 11q deletion in one allele in 90% and 86% of cells from PBL samples, respectively. The low expression of ATM likely corresponds to the percentage of contaminating normal cells. PK11195 induced apoptosis in CLL cells with 11q deletion irrespectively of the expression of ATM protein (Figure 6C ).
section_name: PK11195 induces apoptosis irrespective of p53 and ATM status in CLL cells

section_content: In this work we show that the potential anticancer agent PK11195 induces apoptosis in primary CLL cells. Several targets for the induction of apoptosis by PK11195 have been suggested. The best known target of PK11195 is TSPO, 25 which is overexpressed in CLL cells. 30 Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in primary CLL cells. However, the role of TSPO in PK11195-induced apoptosis is controversial. PK11195 binds to TSPO in vivo at nanomolar concentrations, 25 although PK11195 induces apoptosis at much higher concentrations (50-100 µM). Furthermore, siRNA experiments demonstrate that TSPO is not necessary to inhibit cell proliferation 27 or sensitize cells to apoptosis. 28 Multidrug resistance (MDR) has been proposed as a PK11195 target to sensitize cells to apoptosis and could explain the synergism between PK11195 and other chemotherapeutic drugs that are transported by MDR proteins. 24 PK11195 inhibits MDR in CLL cells (data not shown) and this could contribute to the additive effect of PK11195 and chemotherapeutic drugs. However, PK11195 was able to induce apoptosis alone in CLL, whereas it is not clear how inhibition of MDR could induce apoptosis in the absence of other drugs. Furthermore, cyclosporine A inhibits MDR without affecting the viability of CLL cells (data not shown). Thus, there is no correlation between inhibition of MDR and induction of apoptosis by PK11195 alone. Taken together, these results indicate that the apoptotic activity of PK11195 cannot be attributed to its inhibitory effect on MDR. 

Moreover, PK11195 was described as a phosphodiesterase (PDE) inhibitor in vitro. 40 PDE inhibitors induce cyclic AMP (cAMP) levels and have been described as apoptotic inducers in CLL cells. 41 However, H89, a cAMPdependent protein kinase inhibitor did not inhibit PK11195-induced apoptosis, indicating that PK11195 induces apoptosis in CLL cells through a different mechanism. JNK has also been involved in the induction of apoptosis by the combination of PK11195 and bortezomib, 23 but in CLL cells inhibition of JNK did not protect from PK11195-induced apoptosis. 

We have shown that PK11195 induces caspase-dependent apoptosis in CLL cells. Furthermore, PK11195 has effects in mitochondria upstream of caspase activation, as a caspase inhibitor was not able to inhibit the mitochondrial effects induced by PK11195, such as depolarization and cytochrome c release. However, the levels of the pro-apoptotic proteins BIM, PUMA, BMF and BAX did not increase, and the levels of the anti-apoptotic proteins MCL-1, BCL-XL and BCL-2 did not decrease. It should be pointed out that DIDS, a VDAC inhibitor, inhibited PK11195-induced apoptosis, suggesting an involvement of VDAC. Significantly, VDAC has been proposed as one of the components of the permeability transition pore complex (PTPC) that mediates cytochrome c release. 40 3] [44] BAX and BAK may co-operate with the PTPC to form a channel, but whether cytochrome c release is mainly mediated by the PTPC and/or the pore-forming function of BAX/BAK is still open to debate. 42, 43 Thus, the mechanism by which PK11195 induces cytochrome c release requires further investigation using cells lines in which the candidate proteins could be down-regulated efficiently. 

Our experiments indicate that CLL cells and normal B cells are more sensitive to PK11195-, RO 5-4864-, and FGIN-1-27-induced apoptosis than are T cells from the same samples. Chemotherapeutic drugs including fludarabine, chlorambucil, and doxorubicin induce apoptosis equally in both B and T cells 5, 45 leading to immunosuppression. 46 Thus, the differential effect of PK11195 in B and T lymphocytes is of interest. Most drugs currently used in the therapy of CLL act, at least partially, through activation of the p53 pathway. 2,5-7 [4] 8 Furthermore, ATM is inactivated in 10-20% of CLL cases thus providing an alternative way for disabling p53 function. 47, 48 Tumors with alterations upstream of p53 would not respond adequately to genotoxic chemotherapeutics that act through the p53 pathway, for example, alkylating agents (chlorambucil, cyclophosphamide), purine nucleosides (fludarabine, cladribine) or topoisomerase inhibitors (doxorubicin, mitoxantrone). Genetic alterations in P53 and ATM are among the worst prognostic factors in CLL patients, 3, 4, [46] [47] [48] [49] and p53 alterations confer resistance to conventional chemotherapy. 3, 4, 7 Thus, new approaches to induce apoptosis in cells with mutated p53 or ATM are needed. Here, we demonstrate that PK11195 induces apoptosis in CLL cells irrespectively of their p53 or ATM status. 

From a therapeutic perspective, it is interesting to note that the doses of PK11195 that induce apoptosis in CLL samples in vitro are achievable in vivo, as previously demonstrated by their potent antitumor effect in mouse models of human cancer. 11, 21, 50 PK11195 has been administered safely to patients. 51, 52 Furthermore, PK11195 has additive effects with chemotherapeutic drugs in CLL. This suggests that it could be possible to lower the doses of chemotherapeutic drugs used in the treatment of CLL and thus reduce cytotoxicity to normal T cells. In conclusion, the results presented here suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.
section_name: Discussion

section_content: Acknowledgments: the authors thank Dr. Montse Barragán and Dr. Esther Castaño for helpful discussions and suggestions; and Michael Maudsley for language assistance. We also thank the Unitat de Biologia and the Unitat de Genòmica from the Serveis Cientificotècnics at the Universitat de Barcelona for their

section_name: Authors' Contributions

section_content: AFS performed the research, analyzed the data and wrote the paper. AMC, LC-M, DI-S, MdF, DMG-G performed research; CC contributed with analytical tools; AD contributed with patients samples and data; GP designed the research and analyzed the data; JG designed and supervised the research, analyzed the data and wrote the paper. All authors revised the manuscript critically and approved the final version to be published.
section_name: Authors' Contributions

section_content: The authors reported no potential conflicts of interest.
section_name: Conflict of Interest