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Keywords:

  • D-tyrosinol;
  • chronic lymphocytic leukaemia;
  • p38 mitogen-activated protein kinase (p38 MAPK);
  • apoptosis;
  • BIRC5

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.

Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the western world, with an incidence of around 3·5 cases per 100 000 people per year (Dighiero & Hamblin, 2008). While the outcome of patients with CLL vary by many factors, such as age, disease status, associated genetic abnormalities and other co-morbid illnesses, the available therapies including alkylating agents, purine analogues, bendamustine, alemtuzumab, rituximab and, more recently, combination therapy with chemoimmunotherapy have not been curative (Montserrat & Moreno, 2008). Similar to chemotherapy combinations in other malignancies, drug resistance often ensues when treatment has been initiated. In particular, patients relapsing after chemoimmunotherapy have a poor outcome with a survival <2 years, warranting alternative therapies in CLL.

One alternative to circumvent drug resistance is to utilize agents that act through mechanisms that are different from that of the currently used therapies. The p38 mitogen-activated protein kinase (MAPK) pathway, initially identified for its role in stress and inflammatory response, was found to have a tumour suppressor function (Nebreda & Porras, 2000; Ono & Han, 2000; Dolado et al, 2007; Han & Sun, 2007; Hui et al, 2007a,b; Kennedy et al, 2007). The p38 MAPK pathway has been implicated in negative control of tumourigenesis through cell cycle control (Ambrosino & Nebreda, 2001), cell differentiation (Puri et al, 2000), cell proliferation, oncogene-induced senescence, replicative senescence, contact inhibition, DNA-damage response and induction of apoptosis (Nebreda & Porras, 2000; Bulavin & Fornace, 2004; Wada & Penninger, 2004; Han & Sun, 2007). Some chemotherapeutic drugs have been reported to induce cell apoptosis via p38 MAPK activation, including all-trans retinoic acid in medulloblastoma cells, vinka alkaloids in HeLa cells, taxol and cisplatin in several non-haematological cell lines (Deacon et al, 2003; Hallahan et al, 2003; Losa et al, 2003; Olson & Hallahan, 2004). Although the mechanism(s) by which p38 MAPK induces cell apoptosis is not fully understood, studies in myeloma and rat pheochromocytoma cells suggest a link between p38 MAPK and the Bcl-2 family protein and mitochondrial pathway (Seo et al, 2007; Cai & Xia, 2008). Together, these data suggest a potential use for p38 MAPK targeted therapeutic agents in cancer treatment.

In an attempt to develop a new class of agents targeting p38 MAPK activation, we used the immunosuppressive agent FTY720 as a lead compound to conduct structural optimization, which has been reported to mediate apoptosis in human Jurkat T lymphocytes, in part, via a p38-dependent mechanism (Matsuda et al, 1999). Among various derivatives examined, OSU-DY-7 [(R)-2-amino-3-(4-heptyloxy-phenyl)-propan-1-ol] represented the optimal agent, which exhibits unique ability to activate p38 MAPK in a variety of lymphoid malignancies including primary CLL cells. Here, we demonstrate that OSU-DY7 mediates cytotoxicity in lymphocytic cell lines and primary B-CLL cells, in part, via p38 MAPK-dependent down-regulation of BIRC5 expression.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

Cells and culture conditions

Blood from patients with CLL was obtained under a protocol approved by The Ohio State University hospital internal review board. All patients had understood and signed the informed consent in accordance with the Declaration of Helsinki. B-CLL cells were isolated from freshly collected whole blood using Rosette-Sep kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Human B-lymphocyte cell lines MEC-1, Raji, Ramos, 697 and isolated primary B-CLL cells were incubated using procedures previously described (Liu et al, 2008). MEC-1 cell line was obtained from the German cell line bank (Braunschweig, Germany) and Raji, 697 and Ramos cell lines were from American Type Culture Collection (ATCC, Manassas, VA, USA).

Reagents

OSU-DY7 was prepared from D-tyrosine, of which the synthetic procedure will be published elsewhere. The identity and purity were confirmed by nuclear magnetic resonance and mass spectrometry. The chemical structure of OSU-DY7 is shown in Fig 1A. The pharmacological agents were purchased from the respective vendors: D-tyrosine (Sigma-Aldrich; Saint Louis, MO, USA), p38 MAPK inhibitor SB202190 and MAP kinase kinase (MEK) inhibitor PD98059 (Calbiochem, Gibbstown, NJ, USA); Z-VAD-FMK (BIOMOL, Plymouth, PA, USA); caspase-3 substrate (Ac-DMQD)2-Rh110 (AnaSpec, San Jose, CA, USA); TRIzol reagent (Invitrogen, Carlsbad, CA, USA); MG132 (Cayman Chemical, Ann Arbor, MI, USA).

image

Figure 1.  Chemical structure of OSU-DY7 and cytotoxicity study in B-lymphocytic cell lines and primary B CLL cells. (A) Chemical structure of OSU-DY7 with a molecular weight of 265·39. (B) MEC-1, 697, Raji, and Ramos cells (0·25 × 106 cells/ml) were incubated with indicated concentrations of OSU-DY7 or DMSO vehicle for 24 h (□) or 48 h (inline image). The cells were analysed with the MTS assay, as described in the “Materials and methods” section (n = 3). (C) Purified B-CLL cells (1 × 106 cells/ml) were incubated with OSU-DY7 or DMSO for 24 h (□) or 48 h (inline image). The cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide for accessing cell viability (n = 16).

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Cell viability and apoptosis assay

Cell viability was assessed by dual staining with annexin V conjugated to flourescein isothiocyanate (FITC) and propidium iodide (PI). Cells (1 × 106) were stained by annextin V-FITC (BD Pharmingen, San Diego, CA, USA) and PI (BD Pharmingen) using a procedure previously published (Liu et al, 2008). Cells were analysed by a Beckman-Coulter EPICS XL cytometer (Beckman-Coulter, Miami, FL, USA). Annexin V-FITC and/or PI positive cells were identified as apoptotic cells. Viable cells were those with both annexin V-FITC negative and PI negative staining. The viable cells in each sample were expressed as % by normalizing annexin V/PI cells to untreated control.

Analysis of caspase-3 activity

Caspase-3 activity was determined by using (Ac-DMQD)2-Rh110 (AnaSpec) as the fluorogenic substrate for caspase-3 using a procedure previously described (Hung et al, 2008).

MTS assay

Measurement of cell growth was performed using CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay kit purchased from Promega (Madison, WI, USA). Cells (0·25 × 106/ml for cell lines and 1 × 106/ml for primary B-CLL) were placed in 200 μl volume in 96-well microtitre plates with indicated reagent and incubated in 37°C. MTS solution [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)- 2H-tetrazolium] and PMS (phenazine methoxulfate) solution were mixed in 20:1 in volume. The colourimetric measurements were performed 4 h later at 490-nm wavelength by a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cell viability was expressed as a percentage of absorbance value in treated sample compared to that observed in control vehicle-treated sample.

Western blotting

Cell lysates were prepared using radioimmunoprecipitation assay RIPA buffer (150 mmol/l NaCl, 50 mmol/l Tris PH 8·0, 1% Nonidet P40, 0·5% sodium deoxycholate and 0·1% sodium dodecyl sulphate) supplemented with protease inhibitor (Sigma) and phosphatase inhibitor cocktail (Calbiochem). Antibodies against various proteins were obtained from the following sources: poly-ADP-ribose polymerase (PARP), Akt, p-Akt (Ser473), ERK1/2, p-ERK1/2 (pThr202Tyr204), JNK, p-JNK (Thr183Tyr185), p38 MAPK, p–p38 MAPK (Thr180Tyr182), MAPKAPK2, p-MAPKAPK2 (Thr334) (Cell Signaling, Danvers, MA, USA); BIRC5 (R&D, Minneapolis, MN), tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); actin (MP Biomedicals, Solon, OH, USA). The goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and goat anti-mouse IgG-HRP conjugates were purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA).

Reverse transcription and polymerase chain reaction (PCR)

Cells (5 × 106) were washed with phosphate-buffered saline twice and mRNA was extracted by TRIzol reagent (Invitrogen). Reverse transcription to cDNA was performed using BioRad iScript cDNA Synthesis Kit (BIO-RAD Laboratories, Hercules, CA, USA). Briefly, 1 μg of mRNA, 4 μl of 5× iScript-Reaction Mix and 1 μl of iScript Reverse Transcriptase were mixed in nuclease-free water with a final volume of 20 μl. The conditions for reverse transcription were: 25°C for 5 min, 42°C for 30 min and 85°C for 5 min. The real-time PCR for BIRC5 was performed using Taqman Gene Expression Assay (Applied Biosystems, Foster City, CA) using RN18S1 as an internal control. Briefly, 1 μl of cDNA, 12·5 μl of Taqman Universal PCR Master Mix (Applied Biosystems) and 1·25 μl BIRC5 probe (Hs 00977611_g1; Applied Biosystems) or RN18S1 control (Hs 03003631_g1; Applied Biosystems) were mixed to DNAse-free water to a final volume of 25 μl. The real-time PCR reaction was carried on 7900HT Real-Time PCR System (Applied Biosystems).

Statistical analysis

All statistical analyses were performed by biostatisticians in the Center for Biostatistics at the Ohio State University. Nonlinear mixed models were used to obtain the half maximal inhibitory concentration (IC50). For comparisons, linear mixed models were used for modelling treatment effect and patient random effect. Holm’s method was applied to adjust for multiplicity and control the overall Type I error rate at α = 0·05. SAS software (version 9·1; SAS Institute, Inc., Cary, NC, USA) was used for all statistical analyses.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

OSU-DY7 mediates cytotoxicity in B-lymphocytic cell lines and primary B cells from CLL patients

Lymphoid cell lines representative of CLL (MEC-1), ALL (697), Burkitt lymphoma (Raji and Ramos) were cultured with 0, 1, 2, 4, 6, 8, 10, 12, and 15 μmol/l concentrations of OSU-DY7, which induced dose- and time-dependent decrease in cell survival (Fig 1B). The IC50 value for each of the cell lines is shown in Table I.

Table I.   IC50 values*.
Time (h)MEC-1 (μmol/l)697 (μmol/l)Raji (μmol/l)Ramos (μmol/l)Primary B-CLL (μmol/l)
  1. *The IC50 values were obtained from three different experiments. The values in parenthesis represent 95% confidence intervals.

242·40 (1·23–1·58)10·39 (8·47–12·31)6·04 (5·75–6·33)3·75 (3·40–4·10)3·58 (2·60–4·57)
481·02 (1·00–1·54)8·04 (6·43–9·65)5·21 (4·54–5·88)2·94 (2·94–3·50)3·26 (2·20–4·32)

In order to determine the cytotoxic efficacy of OSY-DY7 in B-CLL cells, freshly isolated CD19+ B-CLL cells were treated with OSU-DY7 (ranging from 0, 1, 2, 4, 6, 8, and 10 μmol/l) and the cell viability was evaluated by annexin-V/PI staining analysis at 24 or 48 h. The IC50 for 16 patients was 3·58 μmol/l (95% confidence interval [CI]: 2·60–4·57) and 3·26 μmol/l (95% CI: 2·20–4·32) at 24 h and 48 h, respectively (Fig 1C, Table I) demonstrating maximal apoptosis was observed at 24 h with no advantage for extended exposure beyond this time period.

OSU-DY7 induces caspase-3 activation and PARP cleavage in B-lymphocytic cell lines and primary B-CLL cells

To investigate the relationship of OSU-DY7-mediated cytotoxicity and activation of caspase-3, cells were treated with OSU-DY7 0, 4 and 8 μmol/l for 24 h. There was a significant linear increase in caspase-3 activity as the concentration of OSU-DY7 increased (Fig 2A, *P < 0·0001 in Raji cells and *P = 0·0048 in primary B-CLL cells).

image

Figure 2.  OSU-DY7-mediated cytotoxicity is dependent on caspase activation and apoptosis. (A) Raji cells (0·25 × 106 cells/ml) and primary B-CLL cells (1 × 106 cells/ml) were incubated with OSU-DY7 or DMSO for 24 h. Cells (1 × 106) were analysed for caspase-3 activity as described in the Methods section. The numbers in each graph represent the percentage and the range of caspase-3 positive cells (n = 3). (B) OSU-DY7 induces PARP cleavage in Raji cell, MEC-1 cell and primary B-cell CLL in 24 h. Raji cells (0·25 × 106 cells/ml), MEC-1 cells (0·25 × 106 cells/ml) and primary B-CLL cells (1 × 106 cells/ml) were incubated with OSU-DY7 for 24 h. Total cell lysates (10 μg) were used for Western blot. The data represents results from two of six patient samples with similar observation. (C) OSU-DY7-induced cytotoxicity can be rescued in part by Z-VAD-FMK in MEC-1 and Raji cells. In MEC-1 study, cells (0·25 × 106 cells/ml) were pretreated with medium or 100 μmol/l Z-VAD-FMK, followed by incubation with DMSO or 2 μmol/l OSU-DY7 for 24 h. In Raji study, cells (0·25 × 106 cells/ml) were pretreated with medium or 50 μmol/l Z-VAD-FMK, followed by incubation with DMSO or 4 μmol/l OSU-DY7 for 24 h. The viability of cells was checked with the MTS assay (n = 4). (D) OSU-DY7-induced cytotoxicity can be rescued in part by Z-VAD-FMK in primary CLL cells. Primary B-CLL cells (1 × 106 cells/ml) were pretreated with medium or 100 μmol/l Z-VAD-FMK, followed by incubation with DMSO or 4 μmol/l OSU-DY7 for 24 hours. At 24 hours, the cells were stained with annexin V-FITC and PI for assessing cell viability (n = 5).

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In order to determine if caspase activation results in PARP cleavage, Raji cells were incubated with OSU-DY7 at 0, 2, 4, or 8 μmol/l for 24 h. MEC-1 cells were incubated with OSU-DY7 at 0, 0·5, 1, and 2 μmol/l for 24 h. Lower concentrations of OSU-DY7 were chosen for MEC-1 cells due to their relatively higher sensitivity compared to Raji cells (see Table I). The results showed that OSU-DY7 induced PARP cleavage in Raji and MEC-1 cell lines in a dose-dependent manner, as evidenced by appearance of the cleaved 89 kDa band (Fig 2B, left panels). Similar to the results obtained in cell lines, OSU-DY7 lead to PARP cleavage in primary B-CLL cells in 6 independent patient samples. Representative results from two patients are shown in Fig 2B.

In order to determine the relevance of caspase activation in OSU-DY7 mediated cytotoxicity, we tested the effect of OSU-DY7 on cells pretreated with pancaspase inhibitor Z-VAD-FMK. As shown in Fig 2C, OSU-DY7-mediated cytotoxicity was partially yet significantly rescued by Z-VAD-FMK, in both cell lines and primary B-CLL cells. In MEC-1 cells, 100 μmol/l Z-VAD-FMK pretreatment rescued the OSU-DY7-mediated cytotoxicity as evidenced by the significant increase in viable CLL cells from 21·3% to 44·5%. Thus, Z-VAD-FMK significantly decreased OSU-DY7-mediated MEC-1 cell killing by 52% (95% CI: 48·3–55·7%, n = 4, *P < 0·0001). Similar rescue effect of Z-VAD-FMK was also observed in Raji cells where OSU-DY7-mediated cytotoxicity was partially rescued with the increased viable cells from 34·1% to 44·5%. Thus, Z-VAD-FMK significantly decreased OSU-DY7-mediated Raji cell killing by 23% (95% CI: 21·0–25·6%, n = 4, *P < 0·0001; Fig 2C). Consistent with the cell line data, Z-VAD-FMK also rescued OSU-DY7-mediated cytotoxicity in primary B-CLL cells (Fig 2D). Without Z-VAD-FMK, the ratio in survival between OSU-DY7 and dimethyl sulfoxide (DMSO) was 62·1% (95% CI: 55·3–69·7%), and the ratio was 74·3% (95% CI: 60·6–91·0%) with Z-VAD-FMK. The Z-VAD-FMK significantly decreased the cell killing by OSU-DY7 by 16·5% (95% CI: 3·9–27·4%, n = 5, *P = 0·0298).

OSU-DY7 induces phosphorylation of p38 MAPK in lymphoid cell lines and primary CLL cells

To investigate the possible mechanisms involved in OSU-DY7-mediated cytotoxicity, pathways of Akt, ERK, p38 MAPK, JNK, NF-κB were examined. No differences in levels of p-JNK (Thr183Tyr185), JNK and NF-κB between vehicle and OSU-DY7 treated groups were noted (data not shown). However, OSU-DY7 induced significant increase of phosphorylation of p38 MAPK in a dose-dependent manner in Raji (n = 4, *P < 0·0001) and MEC-1 (n = 4, *P = 0·0325) when comparing OSU-DY7-treated group with DMSO (Fig 3A). In Raji cells, the ratio of p–p38 MAPK (Thr180Tyr182) versus p38 MAPK after OSU-DY7 at 2, 4 and 8 μmol/l concentrations increased 2·2-fold (1–3·8), 4·7-fold (1·2–11·8) and 11-fold (2·9–31·2) compared with control group. A similar increase in p–p38 MAPK was also observed in MEC-1 cells. The ratio after treatment with OSU-DY7 0·5, 1 and 2 μmol/l for 24 h increased 1·1-fold (1–1·3), 1·9-fold (1·2–2·8) and 2·9-fold (1·4–5·5), respectively, compared with the control group. The phosphorylated MAPKAPK2 (Thr334), a downstream target of p38 MAPK, was also increased after OSU-DY7 treatment.

image

Figure 3.  OSU-DY7 induces phosphorylation of p38 MAPK. (A) Raji cells and MEC-1 cells (0·25 × 106 cells/ml) were incubated with OSU-DY7 or DMSO for 24 h. Cell lysates of 15 μg were used for Western blot analysis. The ratio of p–p38 MAPK versus p38 MAPK in indicated concentrations of OSU-DY7 was compared with that in DMSO. inline image represents average of the values from four different experiments. (B) Primary B-CLL cells (1 × 106 cells/ml) were incubated with OSU-DY7 or DMSO for 24 h. Cell lysates of 15 μg at each point were used for Western blot analysis. The ratio of p–p38 MAPK versus p38 in indicated concentrations of OSU-DY7 was compared with that in DMSO. inline image represents average of the values from six patients. *P = 0·0004 when comparing OSU-DY7-treated group with DMSO control. (C) OSU-DY7 induces phosphorylation of p38 MAPK in Raji cells in a time-dependent manner. Cell lysates of 15 μg at each point were used for Western blot analysis. The data shown here is a representative of two independent experiments with similar results.

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Consistent with the findings in cell lines, OSU-DY7 induced phosphorylation of p38 MAPK and MAPKAPK2 in primary B-CLL cells (Fig 3B). The ratio of p–p38 MAPK and p38 MAPK increased 1·3-fold (1–1·8), 2·3-fold (1·2–3·4) and 90-fold (1–412) in cells from six CLL patients after treatment with OSU-DY7 2, 4 and 8 μmol/l for 24 h (n = 6, *P = 0·0004 when comparing OSU-DY7-treated group with DMSO vehicle control).

The time course analysis of OSU-DY7-induced phosphorylation of p38 MAPK in Raji cells revealed increased phosphorylation of p38 MAPK as early as 2 h post-treatment with the progressive increase leading to approximately 44-fold increased phosphorylation by 24 h. The difference in phosphorylation of p38 MAPK between the trends of OSU-DY7 and the negative control was significant (P = 0·0022). (Fig 3C).

OSU-DY7-mediated cytotoxicity is dependent on phosphorylation of p38 MAPK

To further examine the role of p38 MAPK pathway in OSU-DY7-mediated cytotoxicity, p38 MAPK-specific inhibitor SB202190 was used. OSU-DY7 induced increased phosphorylation of MAPKAPK2 and PARP cleavage in Raji cells. SB202190 reversed the phosphorylated MAPKAPK2 and reduced the degree of PARP cleavage (Fig 4A). Meanwhile, SB202190 rescued OSU-DY7-mediated cytotoxicity in Raji cells partially either at 24 or 48 h (Fig 4B). At 24 h, without SB202190, the ratio in survival between OSU-DY7 and DMSO was 34·1% (95% CI: 32·7–35·6%, n = 4); with SB202190, the ratio was 53·9% (95% CI: 51·7–56·2%, n = 4). The SB202190 significantly decreased OSU-DY7-mediated Raji cell killing by 36·7% (95% CI: 33·9–39·4%, *P < 0·0001 when SB202190 and OSU-DY7 interaction were compared). Similarly, at 48 h treatment, there was significant interaction between SB202190 and OSU-DY7 (Fig 4B). Without SB202190, the ratio in survival between OSU-DY7 and DMSO was 24·3% (95% CI: 22·7–26·1%, n = 4), with SB202190, the ratio was 48·2% (95% CI: 51·7–56·2%, n = 4). SB202190 significantly decreased OSU-DY7-mediated Raji cell killing by 49·5% (95% CI: 45·0–53·6%, *P < 0·0001 when comparing SB202190 and OSU-DY7 interaction).

image

Figure 4.  OSU-DY7-induced cytotoxicity is dependent on p38 MAPK activation. (A) OSU-DY7 induces upregulation of phosphorylation of MAPKAPK2, which is reversed by p38 MAPK inhibitor SB202190 in Raji cells. Cells (0·25 × 106 cells/ml) were pretreated with medium or 10 μmol/l SB202190 for 2 h, followed by incubation with DMSO or 4 μmol/l OSU-DY7 for 24 h. Cell lysates of 15 μg at each point were used for Western blot analysis. (B) OSU-DY7-induced cytotoxicity is partially rescued by SB202190 in Raji cells. Cells (0·25 × 106 cells/ml) were pretreated with medium or 10 μmol/l SB202190 for 2 h, followed by incubation with DMSO or 4 μmol/l OSU-DY7. The viability of cells was checked with the MTS assay (n = 4). (C) OSU-DY7-induced cytotoxicity is partially rescued by SB202190 in primary CLL cells. B-CLL cells (1 × 106 cells/ml) were pretreated with medium or 10 μmol/l SB202190 for 2 h, followed by incubation with DMSO or 4 μmol/l OSU-DY7 for 24 h. The cells were stained with annexin V- fluorescein isothiocyanate and propidium iodide to assess cell viability (n = 4).

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We then checked the salvage effect of SB202190 for OSU-DY7 in primary CLL cells (Fig 4C). Briefly, without SB202190, the ratio in survival between OSU-DY7 and DMSO was 58·4% (95% CI: 52·2–65·4%, n = 4). With SB202190, the ratio was 77·2% (95% CI: 68·5–81·7%, n = 4). The SB202190 significantly decreased OSU-DY7-mediated cell killing by 24·3% (95% CI: 14·1–33·3%, *P = 0·002 when comparing SB202190 and OSU-DY7 interaction).

OSU-DY7 down regulates BIRC5 via p38 MAPK activation

The underlying molecular mechanism by which p38 MAPK causes cell apoptosis is not completely understood. We investigated pro-apoptotic and anti-apoptotic proteins in response to OSU-DY7 treatment in Raji cells. Compared with the DMSO group, there was no significant change in expression of Bcl-2, Bcl-xl and cIAP2 proteins as detected by immunoblot analysis following OSU-DY7 treatment (data not shown). However, significant reduction in expression of BIRC5 protein was observed in OSU-DY7-treated group. The down-regulation of BIRC5 level could be partially reversed by SB202190 (Fig 5A, left panel). Without P38 MAPK inhibitor SB202190, OSU-DY7 significantly decreased BIRC5 expression (24% of DMSO, 95%CI: 21·5–25·7%, P < 0·0001, n = 3). The pretreatment with 10 μmol/l of SB202190 partially rescued BIRC5 expression level by 1·9-fold (1·5–2·1, *P < 0·0001, n = 3) (Fig 5A, right panel). To further delineate the cause of down-regulation of BIRC5 protein, RT-PCR analysis of BIRC5 transcripts was analysed. OSU-DY7 significantly decreased BIRC5 mRNA expression (P = 0·0002, n = 3, Fig 5B). Pretreatment with 10 μmol/l of SB202190 significantly prevented OSU-DY7-induced BIRC5 mRNA expression by 2·4-fold (Fig 5B, 95% CI: 1·56- to 3·84-fold, *P = 0·0026 when comparing SB202190 and OSU-DY7 interaction, n = 3). This implied that OSU-DY7 activated p38 MAPK, which suppressed BIRC5 expression by transcriptional inhibition. To investigate if the down-regulation of BIRC5 protein level was related to increased proteasome activity, Raji cells were treated with DMSO or 4 μmol/l OSU-DY7 for 12 h, followed by DMSO or 10 μmol/l of MG132, a proteasome inhibitor, for 12 h. In contrast to OSU-DY7 induced Mcl-1 protein, which was partially rescued, MG132 failed to modulate the BIRC5 protein level (Fig 5C). Collectively, these results suggest that OSU-DY7 induces activation of p38 MAPK, which leads to down regulation of BIRC5 protein expression through a transcriptional mechanism.

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Figure 5.  OSU-DY7-induced down regulation of BIRC5 protein and BIRC5 mRNA transcription in p38 MAPK activation-dependent manner. (A) Raji cells (0·25 × 106 cells/ml) were pretreated with medium or 10 μmol/l SB202190 for 2 hours, followed by DMSO or 4 μmol/l OSU-DY7 for 24 h. Cell lysates of 15 μg at each point were used for Western blot (left panel). The ratio of BIRC5 versus actin protein was compared between OSU-DY7 alone and OSU-DY7 plus SB202190 (right panel, n = 3). (B) Raji cells (0·25 × 106 cells/ml) were pretreated with DMSO or 10 μmol/l SB202190 for 2 h, followed by DMSO or 4 μmol/l OSU-DY7 for 24 h. mRNA was reverse transcribed to cDNA, which was further compared for BIRC5 expression using real-time PCR. The data shown here represents relative BIRC5 mRNA level compared with DMSO-treated cells. (C) Raji cells (0·25 × 106 cells/ml) were pretreated with DMSO or 4 μmol/l OSU-DY7 for 12 h, followed by DMSO or 10 μmol/l MG132 for 12 h. Cell lysates of 15 μg at each point were used for Western blot.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

We have described here development of a novel D-tyrosinol derivative, OSU-DY7, which mediates cytotoxicity in primary CLL B cells and B cell lines representing CLL (MEC-1), ALL (697), and Burkitt lymphoma (Raji and Ramos) cell lines. The cytotoxic effect of OSU-DY7 is dependent on the activation of caspase and downstream PARP cleavage. Z-VAD-FMK, at concentrations that inhibited the activation of caspases, prevented OSU-DY7-mediated apoptosis. The partial inhibitory effect of Z-VAD-FMK suggested a potential additional mechanism in OSY-DY7-mediated cytotoxicity. Consistent with this hypothesis, OSU-DY7 induced activation of p38 MAPK in B-CLL cells and B cells lines.

Three lines of evidence suggested a potential role for p38 MAPK phosphorylation in OSU-DY7-mediated apoptosis. First, concentrations of OSU-DY7 that induced apoptosis resulted in time-dependent phosphorylation of p38 MAPK on Thr180 and Tyr182 residues, which has been shown to promote apoptosis (Wada & Penninger, 2004; Seo et al, 2007). Second, SB202190, which resulted in inhibition of p38 MAPK activity, also resulted in inhibition of OSU-DY7-induced apoptosis. Third, the OSU-DY7 resulted in phosphorylation of MAPKAPK2, a downstream target of p38 MAPK that is implicated in apoptosis (Ono & Han, 2000; Dolado et al, 2007). Concentrations of SB202190 that inhibited MAPKAP2 phosphorylation prevented OSU-DY7-mediated apoptosis in B cell lines and B cells from CLL patients.

Our finding, indicating that OSU-DY7 induced activation of p38 MAPK leading to cytotoxicity of CLL cells, is consistent with the potential role for p38 MAPK in a tumour suppressive effect (Nebreda & Porras, 2000; Ono & Han, 2000; Dolado et al, 2007; Han & Sun, 2007; Hui et al, 2007a,b; Kennedy et al, 2007). Interestingly, a negative role for p38 MAPK in cell survival has been documented, suggesting a complex role for p38 MAPK activation in cell growth and apoptosis (Juretic et al, 2001; Park et al, 2002; Wada & Penninger, 2004). Multiple key cell cycle controls are known to be targets of p38 MAPK. Lavoie et al (1996) demonstrated that p38 MAPK could inhibit cyclin D1 expression and was reversed by p38 MAPK inhibitor. Bulavin et al (2002) found that, in vivo, inactivated p38 MAPK expedited tumour formation by suppressing p53 activation. The same group further found p38 MAPK activation could suppress tumour appearance by modulating the CDKN2A tumour-suppressor gene (Bulavin et al, 2004). Importantly, a downstream signal of the p38 MAPK pathway, MAPKAPK-2, has been found to be a cell cycle checkpoint kinase, exhibiting its activity via phophorylation of CDC25 protein phosphatase (Manke et al, 2005). Several evidences suggest a correlation between p38 MAPK activation and apoptosis induction. For example, withdrawal of nerve growth factor from Rat PC-12 pheochromocytoma cell was found to cause sustained activation of p38 MAPK and JNK, as well as induction of apoptosis (Xia et al, 1995). Knockout studies further demonstrated decreased cell survival in cells lacking MKK6, p38αMAPK and MAPKAPK2 (Nebreda & Porras, 2000).

Despite the extensive description, the precise molecular mechanisms by which p38 MAPK causes cell apoptosis are not completely understood. A potential role for the regulation of Bcl-2 family and mitochondrial pathway has been implicated (Kennedy et al, 2007; Seo et al, 2007; Cai & Xia, 2008). Down-regulation of cIAP-1/2, XIAP and BIRC5, as well as accumulation of p53, Bax and Bak in mitochondria were noted in sulindac-induced p38 MAPK activation and cell apoptosis (Seo et al, 2007). OSU-DY7-induced downregulation of BIRC5 is consistent with the observations in human lung carcinoma cells where down-regulation of BIRC5 protein expression was caused by activation of the p38 MAPK pathway (Chao et al, 2004). Similar down modulation of BIRC5 by activation of p38 MAPK and JNK pathways was also observed in response to arsenic trioxide in lung adenocarcinoma cells (Cheng et al, 2006), and vitamin D3-mediated cell growth inhibition and apoptosis (Li et al, 2005). Together, these data suggest that p38 MAPK activation plays a role in tumour suppression. Although untreated primary CLL cells have low expression of BIRC5 compared with ALL or follicular lymphoma cells (de Graaf et al, 2005; Hui et al, 2006), up-regulation of BIRC5 is found in post-chemotherapy CLL cells, which may be one of the mechanisms by which CLL becomes chemoresistant (Hui et al, 2006).

The pro-apoptotic effect of p38 MAPK pathway has also been revealed in CLL. Pedersen et al (2002) demonstrated that rituximab, besides its activity to induce antibody-dependent cellular toxicity, could induce apoptosis in B-CLL through a p38 MAPK activation-dependent pathway, and inhibition of p38 MAPK reduced the degree of anti-CD20-induce apoptosis. In another study, a vitamin D3 analogue, EB1089, was reported to induce apoptosis in B-CLL cells via a mechanism involving p38 MAPK activation and ERK suppression (Pepper et al, 2003a). Interestingly OSU-DY7 also induced activation of p38 MAPK with concomitant down regulation of ERK1/2 phosphorylation (data not shown), suggesting a potential reciprocal regulation of these two signalling pathways. Pepper et al (2003b) also showed that flavopiridol-induced apoptosis in B-CLL was associated with activation of p38 MAPK and suppression of ERK activity. Altering the balance between these two pathways could provide a rationale for the p53-independent nature of flavopiridol-induced apoptosis.

In conclusion, these studies describe a newly synthesized D-tyrosinol derivative, OSU-DY7, which is active for B lymphocytic cell lines representing ALL, Burkitt lymphoma and CLL and primary B cells from CLL patients. p38 MAPK activation-dependent mechanisms involving down modulation of BIRC5 protein and BIRC5 mRNA and caspase-dependent apoptosis reveal OSU-DY7 as an attractive alternative therapy targeting the p38 MAPK pathway for CLL and other lymphocytic malignancies. Further studies are warranted to validate OSU-DY7 for clinical development for CLL and other B cell malignancies.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

This work was supported by the D Warren Brown Foundation, Specialized Center of Research from the Leukemia and Lymphoma Society and Experimental Therapeutics of Leukemia- SPORE grant (1 P50 CA 140158-01) from NCI.

Conflict of interest

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References

No potential conflicts of interest were disclosed.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
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