Introduction

Prodigiosins represent a family of naturally occurring red pigments produced by various microorganisms.¹ Some members of this family are potent apoptosis inducers. Thus, prodigiosin induces apoptosis in various human hematopoietic cancer cell lines, including acute T-cell leukemia, promyelocytic leukemia, myeloma, and Burkitt lymphoma cells.² Prodigiosin also induces apoptosis in cells derived from other human cancers, including gastric³ and colon.⁴ Interestingly, prodigiosin has no marked toxicity in nonmalignant cell lines.² However, the effect of prodigiosin in primary human tumor cells or normal human lymphocytes has not been reported.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal CD5⁺ B lymphocytes.^5,6 Most circulating cells appear to be nondividing and the clonal excess of B cells results from decreased apoptosis rather than increased proliferation.⁷ Importantly, chemotherapy agents induce apoptosis in B-CLL cells.⁸ Purified mononuclear cells from B-CLL samples contain a low percentage of normal T CD3⁺ lymphocytes, thus analysis of drug-induced apoptosis in these two cell populations by flow cytometry is a very useful method to study the cell-type selectivity of anticancer drugs or combinations.^9,10,11 The aim of this study was to analyze whether prodigiosin induces apoptosis in B-CLL cells and in normal T lymphocytes from B-CLL patients.

Materials and methods

Patients

A total of 32 patients with B-CLL, who had not received treatment for the previous 6 months, were studied. B-CLL was diagnosed according to standard clinical and laboratory criteria. Written informed consent was obtained from all patients.

Isolation of B-CLL cells and cell culture

Peripheral blood lymphocytes from B-CLL patients were obtained from the Hematopathology Unit at Hospital Clínic, Barcelona, Spain. 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). Cells were cultured immediately after thawing at a concentration of 2–5 10⁶ cells/ml in RPMI 1640 culture medium (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO BRL, Paisley, Scotland), 2 mmol/l glutamine, 100 U penicillin, and 100 ng/ml streptomycin at 37°C in a humidified atmosphere containing 5% carbon dioxide. Factors were added at the beginning of the culture and cells were incubated for the indicated periods of time.

Purification of prodigiosin

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) was obtained from supernatants from Serratia marcescens and purified as described previously.²

Cell viability assay

Cell viability was determined by the MTT assay.¹² Cells (5 10⁵ cells/well) were incubated in 96-well plates in the absence or in the presence of prodigiosin or fludarabine, in a final volume of 100 l. After 48 h, 10 l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Chemicals Co, St Louis, MO, USA) (5 mg/ml in phosphate-buffered saline (PBS)) was added to each well for an additional 6 h. The blue MTT formazan precipitate was dissolved in 100 l of isopropanol:1 M HCl (24:1) and the absorbance values at 550 nm were measured on a multiwell plate reader. Fludarabine was obtained from Sigma and dissolved in DMSO at 10 mg/ml.

Analysis of apoptosis by flow cytometry

Apoptosis was quantified by a reduction in cell forward scatter (FSC) and an increase in cell side scatter (SSC).¹³ After incubation, cells were washed in PBS and resuspended in 200 l of PBS before flow cytometry analysis. To analyze B and T lymphocytes, cells were incubated in 50 l of PBS with allophycocyanin (APC)-conjugated anti-CD3 (Becton Dickinson) for 10 min in the dark and diluted with PBS to a final volume of 500 l before flow cytometric analysis. To analyze a sufficient number of cells, a live-gate in FSC vs CD3 was drawn and at least 5000 CD3⁺ cells were acquired. Cells were analyzed using a Becton Dickinson FACS Calibur flow cytometer (Mountain View, CA, USA). Samples were acquired and data were analyzed using Cell Quest software.

Western blot analysis

Cells were lysed with Laemmli sample buffer and Western blot analysis was performed as previously described¹⁴ using polyclonal antibodies against poly (ADP-ribose) polymerase (PARP) (Boehringer Mannheim, Mannheim, Germany) and cleaved caspase-9 (New England Biolabs, Beverly, MA, USA), and monoclonal anti-p53 Ab-5 (Neomarkers, Fremont, CA, USA). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and the ECL detection system (Amersham, Bucks, UK).

Results

Prodigiosin induces apoptosis of B-CLL cells

First, the effect of prodigiosin on the viability of B-CLL lymphocytes was studied. Cells from two B-CLL patients were incubated for 48 h with increasing doses of prodigiosin, ranging from 40 to 190 nM, and cell viability was determined by the MTT assay. A dose-dependent decrease in cell viability was observed in both cases (Figure 1). Next, we examined whether prodigiosin induced phosphatidylserine exposure using annexin V conjugated with phycoerythrin, APC or fluorescein-isothiocyanate. Prodigiosin is a fluorescent molecule and it interfered with these three fluorochromes, so we could not determine apoptosis using this assay. Thus, we examined whether this cytotoxic effect was because of apoptosis by changes in the FSC and SSC. Incubation of B-CLL cells with prodigiosin produced a decrease in FSC and an increase in cell SSC as determined by flow cytometry indicative of apoptosis induction (Figure 2a). This effect was observed in all 32 cases studied (Figure 2b). Prodigiosin induced apoptosis in B-CLL cells in a dose-dependent manner with a mean IC₅₀ (concentration of drug required to reduce the cell viability to 50%) of 11625 nM (n=12 patients). Nine representative dose–responses are shown in Figure 2c. To confirm that prodigiosin induces apoptosis in B-CLL cells, we assessed whether it induced caspase activation. Thus, we analyzed PARP proteolysis, which is a marker of caspase-3 activation.¹⁵ As shown in Figure 3, prodigiosin induced PARP cleavage. Furthermore, prodigiosin induced processing of caspase-9 in B-CLL cells, as shown by the appearance of its intermediate cleavage product of 37 kDa. These results demonstrate that prodigiosin induces apoptosis of B-CLL cells through caspase activation.

Figure 1.

Prodigiosin induces cytotoxicity in B-CLL cells. Cells from two patients were incubated for 48 h with different doses of prodigiosin. Cell viability was analyzed by the MTT assay and viability is expressed as a percentage with respect to control cells at the beginning of the culture. Experiments were performed in triplicate.

Full figure and legend (20K)

Figure 2.

Prodigiosin induces apoptosis of B-CLL cells. (a) Changes in FSC and SSC were used to differentiate normal cells from dying cells in two different populations, as represented. B-CLL cells were treated with or without 190 nM prodigiosin for 24 h and we determined these changes by flow cytometry. The apoptotic populations are marked and the percentages of apoptotic cells are indicated. (b) B-CLL cells from 32 patients were incubated for 24 h with or without 190 nM prodigiosin. Prodigiosin induced apoptosis in B-CLL cells from all the 32 patients studied. Apoptosis was determined by flow cytometry as described in Materials and methods. (c) Cells were incubated for 24 h with various concentrations of prodigiosin ranging from 40 to 190 nM. Apoptosis was assessed by flow cytometry assay as described in Materials and methods.

Full figure and legend (253K)

Figure 3.

Prodigiosin induces PARP cleavage and caspase-9 activation. Cells from four patients were cultured for 24 h with (P) or without (C) 190 nM of prodigiosin. PARP and caspase-9 cleavage were assessed by Western Blot, as described in Materials and methods. The position of the native PARP (116 kDa) and the cleaved fragment (85 kDa) and the active form of caspase-9 (37 kDa) are indicated in the figure.

Full figure and legend (37K)

Prodigiosin did not induce p53 accumulation

The p53 tumor suppressor protein plays a major role in the cellular response to a wide range of chemotherapeutic drugs.¹⁶ To study the role of p53 in prodigiosin-induced apoptosis, we analyzed the effect of this compound on p53 protein levels. Prodigiosin had no effect on p53 levels in the five B-CLL patients analyzed. The results corresponding to two representative cases are shown (Figure 4). As a control we used doxorrubicin, which induced p53 protein accumulation.

Figure 4.

Prodigiosin does not induce p53 accumulation. Cells from B-CLL patients were untreated (C) or treated with 190 nM prodigiosin (P) or 0.8 M doxorrubicin (D) for 24 h. Cells were lysed and the levels of p53 protein were analyzed by Western blot as described in Materials and methods.

Full figure and legend (29K)

Prodigiosin induces apoptosis on B and T cells from B-CLL patients

To analyze the induction of apoptosis by prodigiosin in T lymphocytes from B-CLL patients, we quantified the FSC/SSC changes in CD3⁺ lymphocytes (T cells) and CD3^- lymphocytes (mostly B cells) from B-CLL samples. B and T cells from B-CLL samples showed the same sensitivity to prodigiosin-induced apoptosis (Figure 5). In samples in which fludarabine was more cytotoxic for T cells than for B-CLL cells, prodigiosin showed the same toxicity for both cell types (Table 1). Furthermore, prodigiosin induced apoptosis in B-CLL cells that are resistant to fludarabine (patients 22 and 25 in Table 1).

Figure 5.

Prodigiosin induces apoptosis in B-CLL cells and T cells from B-CLL samples. Mononuclear cells were incubated for 24 h with several doses of prodigiosin. Apoptosis of B-cell and T-cell populations was assessed by flow cytometry as described in Materials and methods. The percentage of nonapoptotic T cells () and B-CLL cells () is represented.

Full figure and legend (39K)

Table 1 - Effect of prodigiosin and fludarabine on the viability of B and T cells from B-CLL samples.

Full table

Discussion

These results demonstrate that prodigiosin induces apoptosis of B and T cells from B-CLL samples. This is the first report showing that prodigiosin induces apoptosis in human primary cancer cells.

We have previously demonstrated that prodigiosin induces apoptosis of human hematopoietic cancer cell lines derived from acute T-cell leukemia, promyelocytic leukemia, myeloma, and Burkitt lymphoma.² Prodigiosin also induces apoptosis in cells derived from other human tumors.^3,4 Cycloprodigiosin, another member of the prodigiosin family, induces apoptosis in various cancer cell lines including acute human T-cell leukemia,¹⁷ promyelocytic leukemia,¹⁸ human and rat hepatocellular cancer,¹⁹ human breast cancer,²⁰ and TNF-stimulated human cervix carcinoma (HeLa).²¹ The National Cancer Institute (Bethesda) has also shown that prodigiosin has an average IC₅₀ of 2.1 M against a panel of 57 different human cancer cell lines (this information is available on the Internet at www.dtp.nci.nih.gov the NSC number for prodigiosin is 47147-F). Interestingly, we demonstrate in this paper that prodigiosin induces apoptosis in B-CLL cells with an IC₅₀ of 11625 nM.

The effect of prodigiosins on normal lymphocytes is more controversial. It has been reported that prodigiosin inhibits the proliferation of murine splenic T lymphocytes at nontoxic concentrations with no effect on B-cell proliferation.^22,23 In contrast, cycloprodigiosin induces apoptosis of activated murine splenic T cells.²⁴ Although the toxicity of prodigiosin for normal human B cells has not been tested, the results presented here show that B and T cells from B-CLL samples have the same sensitivity to prodigiosin-induced apoptosis. Current B-CLL therapies do not demonstrate specificity, thus fludarabine is cytotoxic for T lymphocytes.^10,11 Interestingly, in a subset of samples, prodigiosin is less toxic than fludarabine for normal T cells, while having similar effect in B-CLL cells. Furthermore, prodigiosin induces apoptosis in B-CLL cells that are resistant to treatment with fludarabine.

The mechanism by which prodigiosin induces apoptosis is unknown. Other prodigiosins lack apoptotic activity, thus undecylprodigiosin and its derivative PNU15804 inhibit the proliferation of human B and T lymphocytes with no effects on cell death,^25,26 indicating that they may act through a different mechanism. PNU156804 blocks IL-2-dependent NF-B and AP-1 activation²⁶ and it is a selective inhibitor of Janus tyrosine kinase 3 (Jak3).²⁷ Cycloprodigiosin also inhibits NF-B activation,²¹ although prodigiosin does not.²³ Taken together, these results suggest that inhibition of these signaling pathways cannot explain the apoptotic activity of prodigiosins in B and T lymphocytes.

In vitro, prodigiosin binds to DNA, facilitating oxidative double-strand DNA cleavage that correlates with cytotoxicity.²⁸ DNA damage induces the accumulation of p53 tumor suppressor protein.¹⁶ However, p53 was not induced by prodigiosin in B-CLL cells. Furthermore, prodigiosin induces apoptosis in Jurkat and HL-60 cells that are p53-deficient.² These results suggest that prodigiosin induces apoptosis independently of p53 and DNA damage.

Prodigiosins promote H⁺/Cl^- symport activity leading to acidification of the cytosol. This activity has been implicated in cycloprodigiosin-induced apoptosis because imidazole inhibits both acidification and apoptosis in different human cancer cell lines.^18,19,20 The role of cytoplasmic acidification in the apoptosis of B-CLL cells will be analyzed in the future.

In conclusion, this is the first report showing that prodigiosin induces apoptosis in human primary cancer cells. Understanding the mechanism of prodigiosin-induced apoptosis and identification of its molecular target would help to design more potent agents to induce apoptosis of B-CLL cells.

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Acknowledgements

We thank Dr Ernest Giralt, Dr Marta Vilaseca, and Marc Martinell (Organic Chemistry Department and Mass Spectrometry Unit, University of Barcelona) for the purification of prodigiosin. We also thank Dr Esther Castaño, Dr José Manuel López-Blanco, Daniel Iglesias, Antonio Fernández and Llorenç Coll for helpful discussions and suggestions, and R Rycroft for language assistance. C Campàs and M Barragán are recipients of a research fellowship from the Fundación Ramón Areces and the Ministerio de Ciencia y Tecnología, respectively.