Therapeutic potential of cladribine in combination with STAT3 inhibitor against multiple myeloma

Cladribine or 2-chlorodeoxyadenosine (2-CDA) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). STAT3 inhibitor VI (S3I-201) was obtained from EMD Chemicals, Inc. (Gibbstown, NJ). Antibodies for western blot analysis were from following sources: caspase-8 mouse mAb (1C12), caspase-9 polyclonal antibody, caspase-3 rabbit mAb (8G10), Poly (ADP-ribose) polymerase (PARP) rabbit mAb, phospho-STAT3 rabbit mAb and STAT3 (Cell Signaling Technology, Inc., Beverly, MA); b-actin mouse mAb (clone AC-75) (Sigma). All other reagents were purchased from Sigma unless otherwise specified.

Human MM cell line U266 was kindly provided by Dr. Lisheng Wang (Institute of Radiology, Academy of Military Medical Sciences, Beijing, China). Human MM cell line RPMI8226 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Human MM cell line MM1.S was kindly provided by Dr. Steven Rosen (Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL). All cell lines were maintained in RPMI1640 cell culture medium supplemented with 10% fetal bovine serum (FBS) at a 37°C humidified atmosphere containing 95% air and 5% CO2 and were split twice a week.

The CellTiter96™ AQ non-radioactive cell proliferation kit (Promega Corp., Madison, WI) was used to determine cell viability as we previously described [28]. In brief, cells were plated onto 96-well plates with either 0.1 ml complete medium (5% FBS) as control, or 0.1 ml of the same medium containing a series of doses of cladribine, and incubated for 72 hrs. After reading all wells at 490 nm with a micro-plate reader, the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS (3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt).

Flow cytometric analyses were performed as described previously [28] to define the cell cycle distribution and apoptosis for treated and untreated cells. For cell cycle analysis, cells grown in 100-mm culture dishes were harvested and fixed with 70% ethanol. Cells were then stained for total DNA content with a solution containing 50 μg/ml propidium iodide and 100 μg/ml RNase A in PBS for 30 min at 37°C. Cell cycle distribution was analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA). For apoptosis analysis, harvested cells were stained with Annexin V-FITC and propidium iodide according to the manufacturer's instruction and then subjected to the same analyzer.

An apoptosis ELISA kit (Roche Diagnostics) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as previously reported [28].

Protein expression levels were determined by western blot analysis as previously described [28]. Briefly, cells were lysed in a buffer containing 50 mM Tris, pH 7.4, 50 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, and 25 μg/ml aprotinin. The protein concentrations of the total cell lysates were determination by the Coomassie Plus protein assay reagent (Pierce Chemical Co., Rockford, IL). Equal amounts of cell lysates were boiled in Laemmli SDS-sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), and probed with specific antibodies as described in the figure legends. After the blots were incubated with horseradish peroxidase-labeled secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), the signals were detected using the enhanced chemiluminescence reagents (Amersham Life Science, Piscataway, NJ).

To explore whether cladribine might be a potential therapeutic agent against MM, we investigated its anti-proliferative/anti-survival effects on three MM cell lines: U266, RPMI8226 with mutant p53; and MM1.S which retains and expresses WT p53 [23]. Although the three MM cell lines exhibited different sensitivities, cladribine was able to inhibit proliferation/survival of all cells in a dose-dependent manner (Figure 1). While U266 was the least sensitive cell line, MM1.S was the most sensitive one to cladribine. The IC50s of cladribine for U266, RPMI8226, MM1.S cells were approximately 2.43, 0.75, and 0.18 μmol/L, respectively. To determine the molecular mechanisms by which cladribine inhibited proliferation/survival of MM cells, we first investigated the effects of cladribine on cell cycle progression. Both U266 and RPMI8226 cells with mutant p53 were treated with cladribine at the same concentration (2 μmol/L). U266 cells were collected at different time points (24, 48, or 72 hrs), and then analyzed with flow cytometry. Treatment with cladribine gradually increased the percentage of cells in the G1 phase of the cell cycle and reduced the percentage of cells in S phase (Figure 2A). Similar results were obtained in RPMI8226 cells with the treatment of cladribine for 24 hrs (Figure 2B). Cladribine appeared to increase G2-M phase in U266 cells upon 24 hr-treatment, it had no significant effect on G2-M phase either in U266 cells with 48 or 72 hr-treatment or in RPMI8226 cells (Figure 2A & 2B). It remains unclear why cladribine affected G2-M phase in U266 cells only by 24 hr-treatment. MM1.S cells were treated with cladribine at a much lower concentration (0.5 μmol/L) for 24 hrs. Cladribine induced a minor increase in G1 phase, decreased the percentage cells in S phase, and had no effect on G2-M phase in MM1.S cells (Figure 2C). Although our cell proliferation assays indicated that the IC50 of cladribine was much lower for MM1.S cells than the IC50s for U266 and RPMI8226 cells (Figure 1), it appeared G1 arrest-induced by cladribine in MM1.S cells was not as profound as that we observed in the other two cell lines (Figure 2). It is likely that the potent anti-proliferative/anti-survival effects of cladribine on MM1.S cells may be mainly due to its strong capability to induce apoptosis as we discovered in the following studies (Figures 3 & 4). Collectively, our data suggest that induction of cell cycle G1 arrest contributes to cladribine-mediated growth inhibition in MM cells.

We next studied whether cladribine might also induce apoptosis in these MM cells, using two different methods. U266 cells were double-stained with Annexin V and propidium iodide, and then analyzed by a FACScan flow cytometer. These studies showed that cladribine induced apoptosis in U266 cells in a dose-dependent manner. The percentages of apoptotic cells evidenced by Annexin V-positive staining were 5%, 15%, 21%, and 33% when U266 cells were untreated or treated with 2, 5, 10 μmol/L of cladribine, respectively (Figure 3A). When an ELISA methodology was used to quantify apoptosis in RPMI8226 and MM1.S treated with cladribine, a dose-dependent increase in apoptosis was seen in both RPMI8226 and MM1.S cells (Figure 3B & 3C). Consistent with the cell proliferation data (Figure 1), MM1.S was more sensitive to cladribine than RPMI8226 cells. To explore whether cladribine induced apoptosis through caspase-dependent mechanism, we carried out western blot assays to examine activation of caspases and PARP cleavage. In U266 cells, we were able to observe caspase-3 and caspase-8 activation and PARP cleavage only with cladribine at a higher concentration (10 μmol/L), however, it had no significant effect on caspase-9 activation (Figure 4A). Similar results were obtained in RPMI8226 cells treated with 1 μmol/L of cladribine for 48 hrs (Figure 4B). In contrast, treatment with cladribine at 0.2 μmol/L dramatically induced activation of caspase-3, -8, and -9 and PARP cleavage in a time-dependent manner in MM1.S (Figure 4C). Consistent with previous data derived from the apoptotic-ELISA (Figure 3B & 3C), the lowest concentration of cladribine induced strongest activation of caspases and PARP cleavage in MM1.S cells (Figure 4). Taken together, our studies indicate that caspase-dependent apoptosis contributes to cladribine-mediated anti-proliferation/anti-survival effects on MM cells. Among the three MM cell lines tested, MM1.S is the most sensitive one to cladribine-induced apoptosis.

It has been reported that constitutive activation of STAT3 is common in many human and murine cancer cells, and leads to cellular transformation [29, 30]. Since aberrant activation of STAT3 plays a critical role in the development of human cancers, including MM [27], numerous studies have tried to identify novel anticancer strategies/agents targeting STAT3 [27, 31]. To test whether cladribine's inhibitory activity against MM cells is due to STAT3 inactivation, we performed western blot analysis to determine the phosphorylation status of STAT3 in cladrabine-treated MM cells. In all three MM cell lines, cladribine significantly decreased the phospho-STAT3 (P-STAT3) levels in a dose-dependent manner, but had no effect on the total STAT3 protein levels (Figure 5). As with our cell proliferation (Figure 1) and apoptosis data (Figures 3 & 4), treatment with low doses of cladribine (0.2 μmol/L) was as effective in reducing P-STAT3 in MM1.S cells (Figure 5C) as high doses were when applied to U266 (2 μmol/L) and RPMA8226 (1 μmol/L) cells (Figure 5A & 5B). These data suggest that cladribine-induced growth inhibition and apoptosis in MM cells may be associated with its inactivation of STAT3.

Since STAT3 activation is important in the development of human cancers, including MM [27], and cladribine was able to inhibit STAT3 in MM cells (Figure 5), we hypothesized that the combinations of cladribine and a specific STAT3 inhibitor might exhibit super activity in inducing apoptosis in MM cells. S3I-201, which selectively inhibits STAT3 DNA-binding activity [32], was chosen to test this hypothesis. It has been shown that treatment with 30 μmol/L of S3I-201 for 48 hrs induces significant apoptosis in human breast cancer cell line MDA-MB-435, which harbors constitutive active STAT3 [32]. S3I-201 with 5 μmol/L was used in the following assays, as this concentration alone did not induce apoptosis in all the three MM cell lines (Figure 6). In contrast, different concentrations of cladribine were used in the combinational studies: 2 μmol/L for U266 cells, 1 μmol/L for RPMI8226 cells, and 0.2 μmol/L for MM1.S cells, because treatment with cladribine at this concentration for 24 hrs did decrease P-STAT3 levels (Figure 5), but had no significant induction of caspase activation and PARP cleavage for each of the three MM cell lines (Figure 4). As expected, the combinations of cladribine and S3I-201 induced strong activation of caspase-3 and -8, and PARP cleavage in all three MM cell lines (Figure 6A). Furthermore, apoptotic-ELISA demonstrated that their combinations, as compared to either agent alone, significantly promoted MM cells undergoing apoptosis (Figure 6B, P < 0.002, P < 0.0007, P < 0.002 for U266, RPMI8226, MM1.S, respectively).