Cancer Research Annual Meeting 2010 Protein Translation and Cancer
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Cancer Research 68, 8678, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-0294
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Bryostatin-5 Blocks Stromal Cell–Derived Factor-1 Induced Chemotaxis via Desensitization and Down-regulation of Cell Surface CXCR4 Receptors

Xing He1, Liyan Fang1, Jue Wang1, Yanghua Yi2, Shuyu Zhang2 and Xin Xie1

1 National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Graduate University of the Chinese Academy of Sciences; 2 Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai, China

Requests for reprints: Xin Xie, National Center for Drug Screening, 189 Guo Shou Jing Road, Shanghai, China. Phone: 86-21-5080-1313, ext. 156; Fax: 86-21-5080-0721; E-mail: xxie{at}mail.shcnc.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The chemokine receptor CXCR4 and its ligand, stromal cell–derived factor-1 (SDF-1), play important roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, including organogenesis, vascularization, and embryogenesis. The receptor is also involved in HIV infection and tumor growth and metastasis. Antagonists of CXCR4 have been widely evaluated for drugs against HIV and tumors. In an effort to identify novel CXCR4 antagonists, we screened a small library of compounds derived from marine organisms and found bryostatin-5, which potently inhibits chemotaxis induced by SDF-1 in Jurkat cells. Bryostatin-5 is a member of the macrolactones, and its analogue bryostatin-1 is currently being evaluated in clinical trials for its chemotherapeutic potential. The involvement of bryostatins in the SDF-1/CXCR4 signaling process has never been reported. In this study, we found that bryostatin-5 potently inhibits SDF-1–induced chemotaxis but does not affect serum-induced chemotaxis. Further studies indicate that this inhibitory effect is not due to receptor antagonism but rather to bryostatin-5–induced receptor desensitization and down-regulation of cell surface CXCR4. We also show that these effects are mediated by the activation of conventional protein kinase C. [Cancer Res 2008;68(21):8678–86]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Chemokines are a family of structurally related glycoproteins with potent leukocyte activation and/or chemotactic activity. They are divided into four subfamilies (C, CC, CXC, and CX3C) based on the arrangement of their first two conserved cysteine residues at the amino terminus (1). Stromal cell–derived factor-1 (SDF-1; CXCL12) is a member of the CXC chemokine subfamily, and it seems to be the only ligand for chemokine receptor CXCR4. CXCR4 belongs to the superfamily of G protein–coupled receptors (GPCR). CXCR4 is constitutively expressed in a wide variety of tissues and cell types, including various subtypes of leukocytes, hematopoietic progenitor cells, and nonhematopoietic cells, such as endothelial and epithelial cells (2). The SDF-1/CXCR4 axis plays important and unique roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, such as organogenesis, vascularization, and embryogenesis (3–5). CXCR4 is also one of the principal coreceptors for CD4-mediated HIV infection of T cells (6).

CXCR4 is also involved in the growth and metastasis of various types of cancers. At least 23 different types of human cancers, including hematopoietic and solid tumors, overexpress CXCR4 (7), and cancer cells with higher levels of CXCR4 show greater incidence of metastasis (8–10). The CXCR4 antagonist AMD 3100 has been reported to inhibit intracranial growth of primary brain tumors (11), whereas the antagonist T140 effectively inhibited pulmonary metastasis of human breast cancer cells in SCID mice (12). Other studies have indicated roles for SDF-1/CXCR4 in the metastasis of neuroblastoma, melanoma, and prostate cancer cells (13–15). These findings implicate the SDF-1/CXCR4 axis in a variety of cancers, which may make this pathway an interesting target for the development of drugs against cancer and HIV.

To identify antagonists of this signaling pathway, we screened a small library of compounds derived from marine organisms (Supplementary Fig. S1A) and found a class of macrolactones, bryostatins, with potent inhibitory activity against SDF-1–induced chemotaxis in a human acute T-cell leukemia cell line (Jurkat). Jurkat cells have high endogenous levels of CXCR4. Bryostatins were first isolated from the marine organism Bugula neritina. Currently, >20 natural bryostatins are known. Bryostatin-1 is the most well studied, as it possesses a unique pharmacologic profile as a cancer chemotherapeutic agent and inhibits tumor growth, metastasis, and angiogenesis in vitro and in vivo (16–18). Bryostatin-1 is currently being evaluated alone and in combination with other chemotherapeutic agents in >40 clinical trials for numerous cancer types, including melanoma, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, AIDS-related lymphoma, breast cancer, and non–small cell lung cancers (19, 20). The primary mechanism by which bryostatins suppress tumor growth is believed to involve modulation of conventional and novel protein kinase C (PKC) activities. These activities induce tumor necrosis factor-{alpha} (TNF-{alpha}) expression and activation of extrinsic apoptotic cascades, such as increasing the release of cytochrome c mediated by 1-β-D-arabinofuranosyl cytosine and altering the phosphorylation status of Bcl-2 (21, 22). However, the exact mechanisms of bryostatins in inhibiting tumor metastasis remain to be elucidated.

Bryostatin-5 has a very similar structure to bryostatin-1 (Supplementary Fig. S2A). It inhibits the growth of murine melanoma K1735-M2 similar to bryostatin-1 but with fewer side effects in animal experiments (17). Thus far, no study has addressed the involvement of bryostatins in SDF-1/CXCR4 signaling processes. Understanding this involvement may help explain the inhibitory effects of bryostatins on cancer cell metastasis. In this study, we report that bryostatin-5 can potently inhibit SDF-1–induced chemotaxis in Jurkat cells, as well as in Chinese hamster ovary (CHO) cells, stably expressing human CXCR4, but no effect is observed on serum-induced chemotaxis. Further studies indicate that this inhibitory effect is not due to receptor antagonism but rather to desensitization and down-regulation of cell surface CXCR4 induced by bryostatin-5. These effects are reversible, and they are mediated by activation of conventional PKCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Reagents. A mammalian expression vector encoding G{alpha}16 was purchased from the UMR cDNA Resource Center. A plasmid encoding N-terminal myc-tagged CXCR4 was kindly provided by Dr. Gang Pei (Shanghai Institutes of Biological Sciences). Human SDF-1 and T140 were synthesized by GL Biochem. Hoechst 33342 and sulfinpyrazone were purchased from Sigma-Aldrich. The PKC inhibitors GF109203X and rottlerin were obtained from Calbiochem. PKC{zeta} antibody (C20) was obtained from Santa Cruz Biotechnology, and all other antibodies against PKC isoforms, phosphorylated PKC isoforms, β-actin, myc-tag (9B11), and horseradish peroxidase (HRP)–conjugated antirabbit IgG were purchased from Cell Signaling Technology. Donkey anti-mouse antibody conjugated to Alexa Fluor 488, donkey anti-rabbit antibody conjugated to Alexa Fluor 555, and Fluo-4 AM were purchased from Invitrogen. FlashBlue GPCR scintillation beads and [35S]GTP{gamma}S were obtained from PerkinElmer.

Cells culture and transfection. Jurkat and CHO-K1 cells obtained from American Type Culture Collection were maintained in RPMI 1640 and F12 medium (Life Technologies), respectively, supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 mg/L penicillin, and 100 mg/L streptomycin at 37°C in a humidified atmosphere of 5% CO2. CHO-K1 cells were cotransfected with plasmids encoding CXCR4 and G{alpha}16 by electroporation. To generate stable cell lines, transfected cells were seeded into 10-cm dishes and 1 mg/mL G418 and 40 µg/mL blasticidin were added to the culture medium 24 h later. The selection medium was changed every 3 d until colonies formed. A single colony was isolated, expanded, and tested with calcium mobilization assay to confirm the expression and proper function of the transfected genes.

Chemotaxis assay. Jurkat cells were collected by centrifugation and resuspended to a density of 1.0 x 106/mL in RPMI 1640 supplemented with 2.5% FBS. The cells were preincubated with various compounds for 15 min, and then 100 µL of the cells were added to the upper chamber of a 24-well transwell plate (6.5-mm diameter, 5-µm pore size; Corning) whereas the lower chamber received the same medium supplemented with 30 nmol/L SDF-1. The wells were incubated for 5 h at 37°C in 5% CO2. Cells that had traversed the membrane to the lower chamber were collected and stained with the nuclear dye Hoechst 33342 and then counted with an ArrayScan 4.0 HCS reader (Cellomics).

CHO cells stably transfected with CXCR4 were collected by centrifugation and resuspended to a density of 2.0 x 106/mL in F12 medium containing 0.1% bovine serum albumin. Cells were preincubated with various compounds for 15 min, and then 50 µL of the cells were added to the upper chamber of a 48-well chemotaxis chamber (AP48, Neuro Probe) containing 5-µm pore polycarbonate membranes. SDF-1 (100 nmol/L, 25 µL) was added to the lower chamber, and the wells were incubated for 5 h at 37°C in 5% CO2. Nonmigrated cells on the upper side of the membrane were wiped off with a blade, and the membrane with migrated cells on the other side were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. The membranes were scanned, and chemotaxis index was calculated as the staining density of the cells that migrated toward medium containing SDF-1 divided by the staining density of cells that migrated toward medium without SDF-1.

[35S]GTP{gamma}S binding assay. Cell membranes were isolated as previously described (23). Briefly, cells were pelleted by centrifugation and resuspended in lysis buffer [5 mmol/L Tris-HCl, 5 mmol/L EDTA, and 5 mmol/L EGTA (pH 7.5)]. Next, cells were homogenized, and crude membranes were pelleted by centrifugation at 12,000 x g for 15 min at 4°C. The membranes were resuspended in reaction buffer [20 mmol/L HEPES, 100 mmol/L NaCl, 5 mmol/L MgCl2 (pH 7.4)], and the protein concentration was determined. The exchange of [35S]GTP{gamma}S was measured using a scintillation proximity assay, as described previously (24). The reaction was carried out at 30°C for 3 h in 100 µL reaction buffer containing 5 µg membrane, 100 µg FlashBlue GPCR beads, 10 µmol/L GDP, 10 µg/mL saponin, 0.2 nmol/L [35S]GTP{gamma}S, 10 nmol/L SDF-1, and the indicated concentration of compounds. To measure nonspecific binding, 2 µmol/L GTP{gamma}S were added. Membrane-bound [35S]GTP{gamma}S was detected with a Microbeta scintillation counter (PerkinElmer).

Calcium mobilization assay. CHO cells stably expressing CXCR4 and G{alpha}16 were loaded with 2 µmol/L Fluo-4 AM in HBSS at 37°C for 45 min. After thorough washing with assay buffer, 50 µL of HBSS containing either antagonists or 1% DMSO (negative control) were added. After incubation at room temperature for 10 min, 25 µL of SDF-1 were dispensed into the well using a FlexStation II microplate reader (Molecular Devices), and intracellular calcium change was recorded at an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

Western blot. CHO cells stably expressing CXCR4 and G{alpha}16 were serum-starved for 24 h and then treated with various concentrations of bryostatin-5 or SDF-1 for 15 min at 37°C. Cells were lysed, sonicated, and boiled at 95 to 100°C for 5 min in sample buffer [62.5 mmol/L Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mmol/L DTT, 0.01% bromophenyl blue (pH 6.8)]. Aliquots (20 µg) of proteins were fractionated by SDS-PAGE on 12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were first incubated with blocking buffer (TBS with 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature and then incubated overnight at 4°C in buffer containing anti–β-actin (1:1,500) and anti-PKC antibodies (1:1,500). The membranes were washed thrice and incubated with goat anti-rabbit IgG HRP (1:5,000) for 1 h. After washing, immunostaining was visualized using Amersham ECL Plus Western blotting detection reagents (GE Healthcare).

Immunofluorescence microscopy. CHO cells stably expressing N-terminal myc-tagged CXCR4 and G{alpha}16 were seeded onto 96-well plates at a density of 3 x 104 per well. After overnight incubation, cells were treated with GF109203X at 37°C for 15 min and then stimulated with 200 nmol/L SDF-1, bryostatin-5, or 1% DMSO (negative control) at 37°C for the indicated time. After fixation with 4% formaldehyde in PBS and permeabilization with 0.5% Triton X-100, cells were incubated overnight with antibodies against the myc epitope or PKC{alpha} (1:200) at 4°C. To stain cell surface receptors, the Triton X-100 permeabilization step was omitted. On the next day, cells were washed and incubated with the appropriate secondary antibodies conjugated to Alexa Fluor 488 or 555 for 1.5 h at room temperature. Finally, cell nuclei were stained with Hoechst 33342 for 10 min at room temperature. Fluorescent images were obtained with an Olympus IX51 inverted fluorescent microscope. The fluorescent intensity of the nonpermeabilized cells was measured with a Cellomics ArrayScan 4.0 HCS Reader, which can automatically identify and outline each cell. Experiments were run in triplicate, and ~1,000 cells from each well were analyzed.

Data analysis. Data were analyzed with GraphPad Prism software (GraphPad). Nonlinear regression analyses were performed to generate dose-response curves and calculate EC50 or IC50 values. Mean ± SE was calculated automatically using this software. Two-tailed Student's t tests were performed to determine statistically significant differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Bryostatin-5 inhibits SDF-1–induced chemotaxis of Jurkat cells. To determine the specific roles of bryostatin-5 in cancer cell metastasis mediated by SDF-1/CXCR4, we investigated its effects on chemotaxis of Jurkat cells, a human T leukemia cell line that is reported to express abundant CXCR4 receptor and shows chemotaxis when exposed to SDF-1 (25, 26). As shown in Fig. 1A , pretreating the cells with bryostatin-5 blocked the chemotaxis of Jurkat cells toward SDF-1 in a dose-dependent manner (IC50 = 0.6 nmol/L), whereas bryostatin-5 alone did not induce Jurkat cell chemotaxis (Supplementary Fig. S1B). T140, a specific antagonist of CXCR4 (27), also blocked SDF-1–induced chemotaxis in Jurkat cells (Fig. 1A). We also found that bryostatin-1 blocks SDF-1–induced chemotaxis in Jurkat cells in a dose-dependent manner, with an IC50 value of 0.9 nmol/L (Supplementary Fig. S2B). Because we had only limited amounts of bryostatin-1, further studies were performed with bryostatin-5.


Figure 1
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  		Fig. 1. Bryostatin-5 inhibits SDF-1–induced chemotaxis but not [35S]GTP{gamma}S binding. A, Jurkat cells were pretreated with the indicated concentrations of bryostatin-5 or T140 for 15 min. Chemotaxis was then induced using 30 nmol/L SDF-1. Both bryostatin-5 and T140 inhibit SDF-1–induced chemotaxis in a dose-dependent manner. B, bryostatin-5 completely inhibits SDF-1–induced chemotaxis in CHO cells stably expressing CXCR4 at concentrations as low as 2 nmol/L. C, bryostatin-5 does not inhibit 10% FBS–induced chemotaxis in CHO cells stably expressing CXCR4. Columns, mean (n = 3); bars, SE. *, P < 0.001 versus SDF-1–induced control. D, T140 inhibits SDF-1–induced binding of [35S]GTP{gamma}S in a dose-dependent manner. Bryostatin-5 has no effect on SDF-1–induced binding of [35S]GTP{gamma}S at concentrations up to 1 µmol/L. Representative of two independent experiments, each carried out in triplicate.

 
At concentrations as low as 2 nmol/L, bryostatin-5 completely inhibited SDF-1–induced chemotaxis in CHO cells stably expressing CXCR4 (Fig. 1B) but did not inhibit chemotaxis of CHO cells induced by 10% FBS even at a high concentration (10 nmol/L; Fig. 1C). No significant change in cell viability was detected after exposure to 10 nmol/L bryostatin-5 for 24 hours (data not shown). These results indicate that bryostatin-5 specifically inhibits SDF-1–induced chemotaxis.

Bryostatin-5 inhibits SDF-1–induced calcium response but not [35S]GTP{gamma}S binding. To explore the direct effects of bryostatin-5 on SDF-1–induced G-protein activation, we used a cell-free system, namely the [35S]GTP{gamma}S binding scintillation proximity assay. The EC50 of SDF-1 obtained from this assay was 1.77 nmol/L (Supplementary Fig. S1C), which agrees well with previously reported values (28). The CXCR4 antagonist T140 potently inhibited G-protein activation induced by 10 nmol/L of SDF-1 (IC50 = 3.2 nmol/L), whereas bryostatin-5 had no effect at any of the concentrations tested from 0.0001 to 1,000 nmol/L (Fig. 1D).

Next, we tested the effect of bryostatin-5 on the SDF-1–induced calcium response in CHO cells stably expressing CXCR4 and G{alpha}16. G{alpha}16 is a G protein that greatly enhances the coupling of GPCRs to the PLC pathway, thus augmenting calcium response (29, 30). Cells were first stimulated with either 30 nmol/L SDF-1 or bryostatin-5, whereas exposure to 1% DMSO served as the negative control (Fig. 2A, first arrow ). SDF-1 induced a strong calcium response, whereas bryostatin-5 failed to cause any change in the intracellular calcium level. Cells were then washed and restimulated after 20 min with 30 nmol/L SDF-1 (Fig. 2A, second arrow). Prestimulation with both SDF-1 and bryostatin-5 led to receptor desensitization, i.e., reduced calcium response at the second stimulation with SDF-1. In a dose-response experiment, both bryostatin-5 (Fig. 2B) and bryostatin-1 (Supplementary Fig. S2C) potently inhibited calcium signaling induced by 30 nmol/L SDF-1, with IC50 values of 17.9 and 13.7 nmol/L, respectively. However, bryostatin-5 only partially desensitized the {delta}-opioid receptor and CCR5 chemokine receptor even at micromolar concentrations (data not shown). A kinetic analysis (Fig. 2C) revealed that this inhibition was time-dependent (t1/2, ~50 seconds), indicating that a short period of pretreatment was necessary for desensitization. These data indicate that bryostatin-5 does not antagonize CXCR4 but rather induces CXCR4 desensitization in a time- and dose-dependent manner.


Figure 2
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  		Fig. 2. Bryostatin-5 induces desensitization of CXCR4. A, CHO cells coexpressing CXCR4 and G{alpha}16 were loaded with Fluo-4 AM and then stimulated (first arrow) with 30 nmol/L SDF-1, 30 nmol/L bryostatin-5, or DMSO (negative control). Cells were then washed and restimulated (second arrow) with 30 nmol/L SDF-1 after 20 min. The initial stimulation of SDF-1 increases the intracellular calcium level, whereas bryostatin-5 has no such effect. Cells prestimulated with both SDF-1 and bryostatin-5 show reduced reactivity upon the second stimulation with SDF-1, indicating CXCR4 desensitization. Representative of two independent experiments, each carried out in triplicate. B, bryostatin-5 inhibits SDF-1–induced calcium elevation in a dose-dependent manner, with an IC50 value of 17.9 nmol/L. Points, mean (n = 3); bars, SE. C, bryostatin-5 inhibits SDF-1–induced calcium elevation in a time-dependent manner. Representative of two independent experiments, each carried out in triplicate.

 
Bryostatin-5 induces down-regulation of cell surface but not total CXCR4. Receptor internalization is a common phenomenon after GPCR stimulation, and it plays important roles in receptor desensitization and resensitization (31, 32). To explore the effects of bryostatin-5 on CXCR4 internalization, we investigated the levels of CXCR4 on the cell surface and inside the cell after stimulation of 200 nmol/L SDF-1 and bryostatin-5 using immunofluorescent staining in nonpermeabilized (Fig. 3A ) or permeabilized cells (Fig. 3B). In nonpermeabilized cells, antibody staining only revealed cell surface CXCR4. Treatment with SDF-1 and bryostatin-5 significantly reduced the amount of CXCR4 on the cell surface. Statistical analysis of fluorescent intensity revealed an ~30% reduction in CXCR4 levels on the cell surface after 30 to 60 minutes of stimulation with SDF-1 or bryostatin-5. Staining with permeabilized cells revealed internalized receptors after stimulation (Fig. 3B). Additional results from Western blot analysis (Fig. 3C and D) indicated different fates of internalized receptors. Prolonged stimulation by SDF-1 caused a reduction in total CXCR4 (Fig. 3C), indicating degradation of the internalized receptors. However, bryostatin-5 did not alter total CXCR4 levels even after 24-hour incubation (Fig. 3D). This indicates the involvement of a receptor recycling pathway.


Figure 3
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  		Fig. 3. Bryostatin-5 induces down-regulation of cell surface, but not total CXCR4. A, representative immunofluorescent images and statistical analysis of cell surface CXCR4 in nonpermeabilized cells. Reduction in fluorescent staining after 30 to 60 min of stimulation with SDF-1 or bryostatin-5 indicates the removal of CXCR4 from the cell surface. B, staining of total CXCR4 in permeabilized cells. Treatment with SDF-1 or bryostatin-5 causes internalization of CXCR4. C and D, Western blots and statistical analysis of total CXCR4 levels after stimulation with SDF-1 or bryostatin-5. Long-term SDF-1 treatment induces a down-regulation of total CXCR4, whereas bryostatin-5 has no effect even after 24 h of treatment. The CXCR4 level is normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01.

 
Bryostatin-5 activates conventional and novel PKC isoforms but not atypical PKCs. PKC is one of the major protein kinases responsible for heterologous desensitization of many GPCRs (33, 34), including CXCR4 (35). Because bryostatin-1 is reported to activate various isoforms of PKC, we examined the function of bryostatin-5 on PKC activation. Translocation and phosphorylation of PKC allow us to detect PKC activation after stimulation. Immunofluorescent staining revealed that PKC{alpha} can rapidly translocate to the cell membrane after only 5 minutes of exposure to bryostatin-5 (Fig. 4A ). Western blot analysis was applied to study bryostatin-5–induced PKC phosphorylation. CHO cells stably expressing CXCR4 were treated with DMSO, SDF-1 (1 µmol/L), or bryostatin-5 (0.01, 0.1, and 1 µmol/L) for 15 minutes. Cell lysates were resolved with SDS-PAGE, and the phosphorylation levels of the various PKC isoforms were determined using subtype-specific antibodies. We used an antibody specific to a phosphorylation site at the carboxy-terminal residue homologous to Ser660 of PKCβ, which has been shown to detect primarily the {alpha} and β isoforms of PKC. We found that bryostatin-5 induced a strong, dose-dependent phosphorylation of conventional PKC (Fig. 4B).


Figure 4
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  		Fig. 4. Bryostatin-5 induces phosphorylation of conventional and novel PKC isozymes but not atypical PKC. A, translocation of PKC{alpha} to the cell membrane in response to stimulation with 200 nmol/L bryostatin-5 for 5 min. B, C, and D, Western blot results of brystatin-5–induced PKC phosphorylation and the respective statistical analyses. CHO cells expressing CXCR4 were treated with 1 µmol/L SDF-1 or with the indicated concentrations of bryostatin-5. Equal amounts of cell lysates were resolved by SDS-PAGE, and Western blotting was performed with subtype-specific antibodies. Statistical analysis was performed after densitometric scanning of films. Both SDF-1 and bryostatin-5 strongly induce conventional PKC phosphorylation (B), although they only moderately induce phosphorylation of novel PKC (C). Neither SDF-1 nor bryostatin-5 induces phosphorylation of atypical PKC isoform PKC{zeta}/{lambda} (D). Insulin was used as a positive control to stimulate PKC{zeta}/{lambda} phosphorylation. *, P < 0.05; **, P < 0.01. Columns, mean (n = 3); bars, SE.

 
Antibodies against phosphorylated PKC{delta}/{theta} (Ser643/676) and phosphorylated PKC{zeta}/{lambda} (Thr410/403) were used to detect novel and atypical PKC phosphorylation. Novel PKC isoforms were found to be moderately, but significantly, phosphorylated in response to bryostatin-5 (Fig. 4C), although phosphorylation levels of atypical PKC isoforms were unaffected (Fig. 4D). SDF-1 treatment resulted in a PKC activation pattern similar to that in response to bryostatin-5 treatment. These results indicate that bryostatin-5 activates conventional and novel PKCs but not atypical PKCs.

Bryostatin-5–induced CXCR4 desensitization and internalization are mediated by conventional PKC isoforms. Several PKC inhibitors were studied to explore the link between bryostatin-5–induced activation of PKC, CXCR4 desensitization, and inhibition of chemotaxis mediated by SDF-1/CXCR4. GF109203X, an inhibitor of both conventional and novel PKCs, enhanced SDF-1–induced calcium elevation and chemotaxis in a dose-dependent manner (Fig. 5A ). This indicates that SDF-1–induced CXCR4 desensitization is partially due to the activation of conventional and novel PKCs. GF109203X also reversed, in a dose-dependent manner, the inhibitory effect of bryostatin-5 on SDF-1–induced calcium response and chemotaxis (Fig. 5A). In addition, at concentration of 1 µmol/L, GF109203X partially inhibited SDF-1–induced CXCR4 internalization. This same concentration of GF109203X almost completely inhibited CXCR4 internalization induced by bryostatin-5 (Fig. 5C). These results indicate that PKC activation is a critical step in bryostatin-5–induced CXCR4 desensitization and inhibition of chemotaxis mediated by SDF-1/CXCR4.


Figure 5
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  		Fig. 5. Effects of PKC inhibitors on bryostatin-5–induced CXCR4 desensitization and internalization. A, GF 109203X (GF), an inhibitor of both conventional and novel PKCs, enhances SDF-1–induced calcium elevation and chemotaxis in a dose-dependent manner. It also reverses the inhibitory effect of bryostatin-5 (Bryo 5) on SDF-1–induced calcium response and chemotaxis in a dose-dependent manner. B, rottlerin, a selective inhibitor for the novel PKC isoform PKC{delta}, has no effect on SDF-1–induced calcium response and chemotaxis. Similarly, it does not reverse the inhibitory effects of bryostatin-5 on SDF-1–induced calcium response and chemotaxis. C, GF109203X (1 µmol/L) partially inhibits SDF-1–induced CXCR4 internalization and significantly inhibits bryostatin-5–induced CXCR4 internalization. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01.

 
To further clarify which PKC isoforms are involved in bryostatin-5–induced CXCR4 desensitization, rottlerin, a selective inhibitor of the novel PKC isoform PKC{delta}, was used (36, 37). Rottlerin was found to have no effect on SDF-1–induced calcium response and chemotaxis at concentrations up to 20 µmol/L. Similarly, it had no effect on the inhibition by bryostatin-5 of SDF-1–induced calcium response and chemotaxis (Fig. 5B). These data reveal that bryostatin-5–induced CXCR4 desensitization and internalization is mediated primarily by conventional PKC isoforms.

Bryostatin-5–induced CXCR4 desensitization and internalization are reversible. Although both SDF-1 and bryostatin-5 induce receptor desensitization and internalization, Western blot analysis of whole cell lysates (Fig. 3C and D) revealed different fates for receptors internalized in response to different stimuli. Bryostatin-5, unlike SDF-1, did not cause a reduction in total CXCR4 levels even after a 24-hour incubation. These results imply that bryostatin-5–induced CXCR4 desensitization and internalization are reversible. Therefore, we performed a long-term desensitization assay. As shown in Fig. 6A , SDF-1–induced desensitization was irreversible. This was consistent with the Western blot results, which showed that long-term exposure to SDF-1 caused a reduction in the total levels of CXCR4 (Fig. 3C).


Figure 6
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  		Fig. 6. Bryostatin-5–induced CXCR4 desensitization and internalization are reversible. A, calcium response after long-term treatment with 200 nmol/L SDF-1 or bryostatin-5. SDF-1 causes persistent desensitization, whereas bryostatin-5–induced desensitization reverses after ~7 h. B, cell surface CXCR4 level after long-term treatment with 200 nmol/L SDF-1 or bryostatin-5. SDF-1 induces persistent loss of cell surface CXCR4, whereas bryostatin-5 induces reversible receptor internalization. C, bryostatin-5 induces down-regulation of conventional and novel PKC isoforms. Columns, mean (n = 3); bars, SE.

 
In contrast, byrostatin-5–induced desensitization reversed after ~7 hours despite the continued presence of the compound in the culture medium (Fig. 6A). Immunofluorescent staining also revealed that the loss of cell surface CXCR4 induced by SDF-1 was persistent, whereas the receptor internalization induced by bryostatin-5 reversed after a long-term treatment (Fig. 6B). These reversible effects are likely due to the down-regulation of conventional and novel PKC isoforms after a long-term treatment with bryostatin-5 (Fig. 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Bryostatins are macrolactones isolated from marine organisms. Of the >20 known bryostatins, bryostatin-1 is the best characterized. Bryostatin-1 potently inhibits tumor growth, metastasis, and angiogenesis in vitro and in vivo (16–18), and it is currently being evaluated in clinical studies for its therapeutic potential against various types of cancer (19, 20). A variety of studies have been performed to better understand the molecular mechanisms of tumor suppression induced by bryostatin-1 (21, 22, 38). These studies revealed that bryostatin-1 binds to the phorbol ester/diacylglycerol-responsive C1 domain of certain PKC isoforms and activates them. This activation of PKC leads to downstream changes in apoptosis-related proteins and pathways, including overexpression of TNF-{alpha} and altered Bcl-2 phosphorylation (21, 22), which culminate in tumor cell growth inhibition and death. However, the specific pathways involved in bryostatin-induced metastasis inhibition remain largely unknown. The present study is the first, to our knowledge, that identifies bryostatin-5 (Supplementary Fig. S2A), an analogue of bryostatin-1, as a potent inhibitor of chemotaxis mediated by SDF-1 and CXCR4. This may help to explain the inhibitory effect of bryostatins on tumor metastasis.

Metastasis continues to be the leading cause of mortality for cancer patients. It is a highly organized, nonrandom, and organ-selective process (39, 40). It involves several sequential steps, including detachment of tumor cells from the primary site, intravasation to vascular or lymphatic vessels, migration to remote sites, adhesion to microvessel walls, and extravasation into target tissue (41–43). A number of molecules have been implicated in cancer metastasis, including the chemokine SDF-1 and its receptor CXCR4. Chemokines are small, proinflammatory chemoattractant cytokines that bind to G protein–coupled seven-transmembrane receptors that regulate cellular trafficking (32). SDF-1 and CXCR4 were originally characterized as playing important roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, such as organogenesis, vascularization, and embryogenesis (3–5). However, recent studies indicate that CXCR4 is overexpressed in >20 different types of human cancers, including hematopoietic and solid tumors (7); in fact, higher levels of CXCR4 expression correlate with higher incidence of metastasis and poor prognosis (8–10). In animal studies, the CXCR4 antagonist T140 has been shown to inhibit pulmonary metastasis of human breast cancer cells (10). These findings indicate that compounds able to block the SDF-1/CXCR4 interaction or SDF-1–induced chemotaxis may inhibit metastasis of tumor cells and, therefore, be effective anticancer agents.

In a search for new CXCR4 antagonists derived from marine organisms, we identified bryostatin-5, which potently inhibited SDF-1–induced chemotaxis in Jurkat cells and CHO cells expressing human CXCR4. We believe that this inhibitory effect is related to CXCR4 rather than to downstream factors involved in cytoskeletal rearrangement, because bryostatin-5 did not inhibit FBS-induced chemotaxis in CHO cells (Fig. 1A–C). These promising results led us to speculate that bryostatin-5 might be the first representative of a new class of CXCR4 antagonists. However, we were surprised to find that bryostatin-5 did not block SDF-1–induced G-protein activation in the GTP{gamma}S binding experiment with membranes containing CXCR4 (Fig. 1D). This result suggests that bryostatin-5 failed to inhibit binding of SDF-1 to CXCR4 and the subsequent G-protein activation.

Receptor desensitization is another possibility that might cause the loss of function in GPCRs (44). We therefore tested whether bryostatin-5 can induce CXCR4 desensitization using a calcium flux assay. As predicted, bryostatin-5 caused dose-dependent and time-dependent desensitization of CXCR4 (Fig. 2). Previous work has shown that receptor internalization also plays important roles in receptor desensitization and resensitization (31, 32). Using immunocytochemical staining to detect surface and intracellular CXCR4, we discovered that bryostatin-5 induces CXCR4 internalization (Fig. 3A and B). These data indicate that bryostatin-5 inhibits SDF-1–mediated chemotaxis not by blocking the CXCR4 receptor but by causing its desensitization and internalization.

Desensitization of receptors, initiated by their phosphorylation, is an important regulatory step in GPCR-mediated signaling. There are two major types of GPCR desensitization. Firstly, heterologous desensitization is mediated by phosphorylation of the receptor by second messenger–dependent protein kinases, such as PKA and PKC. Secondly, homologous desensitization is mediated by phosphorylation of the receptor by G protein–coupled receptor kinases (GRK) and subsequent binding of β-arrestins (45–47). Because PKC is reported to be activated and causes desensitization in many GPCRs, including CXCR4, after ligand binding (33–35, 48), we tested whether bryostatin-5 induces CXCR4 desensitization and internalization by activating PKC isozymes. PKC translocation and phosphorylation are common methods for detecting PKC activation. Immunocytochemical staining revealed that PKC{alpha} translocated toward the plasma membrane after 5 minutes of treatment with bryostatin-5. Western blot analysis indicated that bryostatin-5 strongly stimulated the phosphorylation of conventional PKC isoforms in a dose-dependent manner. It also moderately activated novel PKC isoforms, although it had no effect on atypical PKC. SDF-1, the native ligand of CXCR4, showed an activation pattern of PKC isoforms very similar to that of bryostatin-5 (Fig. 4).

To further verify the involvement of PKC in bryostatin-5–induced CXCR4 desensitization and internalization, two PKC inhibitors were studied. GF109203X, an inhibitor of both conventional and novel PKCs, reversed bryostatin-5–induced CXCR4 internalization at a concentration of 1 µmol/L (Fig. 5C). It also reversed the inhibitory effect of bryostatin-5 on SDF-1–induced calcium response and chemotaxis in a dose-dependent manner (Fig. 5A). However, rottlerin, a selective inhibitor for the novel PKC isoform PKC{delta}, failed to reverse the inhibitory effect of bryostatin-5 on SDF-1–induced calcium response and chemotaxis (Fig. 5B). These results indicate that bryostatin-5–induced CXCR4 desensitization and internalization are mediated primarily by the activation of conventional PKC isoforms. In contrast, SDF-1–induced desensitization of CXCR4 involved both homologous and heterologous components. This explains why GF109203X enhanced SDF-1–induced calcium response and chemotaxis because it blocked the heterologous desensitization component, whereas it only partially reversed the receptor internalization induced by SDF-1 because it was unable to block the homologous desensitization mediated by GRK. Rottlerin did not affect SDF-1–induced calcium response and chemotaxis, indicating that the heterologous component of SDF-1–induced CXCR4 desensitization is due primarily to conventional PKC isoforms.

Interestingly, although both SDF-1 and bryostatin-5 induced receptor desensitization and internalization, Western blot analysis of whole cell lysates (Fig. 3C and D) indicated different fates for receptors internalized in response to the two stimuli. CXCR4 internalized after the stimulation of SDF-1 underwent a degradation pathway because the long-term treatment of SDF-1 caused down-regulation of total CXCR4. In contrast, bryostatin-5 failed to alter total CXCR4 levels even after a 24-hour incubation, indicating the involvement of a receptor recycling pathway. These results imply that bryostatin-5–induced receptor desensitization and internalization are reversible. Indeed, long-term treatment assays revealed that bryostatin-5, but not SDF-1, induced reversible CXCR4 desensitization and internalization (Fig. 6A and B). Long-term treatment of bryostatin-5 has previously been reported to down-regulate conventional and novel PKCs (49, 50), and our data confirm this finding (Fig. 6C). The down-regulation of conventional PKC isoform is likely involved in the reversible effects of bryostatin-5 because it is the major PKC isoform regulating CXCR4 desensitization.

In summary, we report, for the first time, that bryostatin-5 potently inhibits chemotaxis mediated by SDF-1 and CXCR4. Our results reveal that this inhibitory effect is not due to blockade of CXCR4 but rather to the desensitization and internalization of this receptor. Bryostatin-5 induces the desensitization and internalization of CXCR4 primarily by activating conventional PKC isoforms. Our results gave light to a new mechanism that might lead to a better understanding of the therapeutic effect of bryostatin-5 in cancer treatment.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Natural Sciences Foundation of China grants 90713047 and 30623008, Ministry of Science and Technology of China grant 2006AA020602, Chinese Academy of Sciences grant KSCX2-YW-R-18, and Shanghai Commission of Science and Technology grant 06DZ22907 (X. Xie).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jingbo Wei for her excellent technical support and Andria Leyden, Gusheng Wu, and Robert Ledeen for the critical reading and editing of the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/24/08. Revised 8/ 4/08. Accepted 8/20/08.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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