Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis
Summary (3 min read)
Introduction
- Angiogenesis microtubules chemotaxis protein kinase C Because tumors can grow only to a size of 1–2 mm3 without being supplied with oxygen and nutrients by blood vessels, the inhibition of angiogenesis has gained clinical relevance in cancer therapy (1), also known as Key Words.
- Thus, the application of cell-cycle-inhibiting compounds such as tubulin antagonists is promising in antiangiogenic therapy.
- Thus, the determination of the functions of interphase microtubules in endothelial cells, especially those involved in angiogenic processes, is essential to conceive the mechanisms of microtubule inhibitors in angiogenesis.
Compounds
- Spongistatin 1 and CA4P were provided by George R. Pettit (Cancer Research Institute, Tempe, AZ, USA).
- Paclitaxel, propidium iodide, and phorbol 12-myristate 13-acetate were purchased from Sigma (Taufkirchen, Germany), vinblastine from Hexal (Holzkirchen, Germany), nocodazole from AppliChem GmbH (Darmstadt, Germany), and staurosporine from Cayman Chemical (Ann Arbor, MI, USA).
Cell culture and media
- Primary human umbilical vein endothelial cells were isolated by collagenase treatment of umbilical cords (17) and used at third passage.
- Cells were cultivated on 0.001% collagen G in endothelial growth medium (Provitro, Berlin, Germany), containing 10% inactivated fetal calf serum (FCS) and growth factors (basic fibroblast growth factor 1.0 ng/ml, Heparin 0.004 ml/ml, and epidermal growth factor 0.1 ng/ml).
- M199 medium (PAN Biotech, Aidenbach, Germany) was used as starvation medium.
Live-cell imaging
- HUVECs were transfected with 7 g pEGFP-C1-tubulin (kindly supplied by Stefan Linder, Institute for Cardiovascular Diseases, University of Munich, Munich, Germany), using the Nucleofector II and the HUVEC Nucleofector Kit (Amaxa, Cologne, Germany).
- At days 2 and 3 after transfection, cells were used for live-cell imaging (customized cell observation chamber; Emblem, Heidelberg, Germany; and confocal microscope (LSM 510 Meta; Zeiss, Jena, Germany).
Tubulin fractionation
- The supernatants were collected (fraction of soluble tubulin).
- After centrifugation (10,000 g for 10 min at 4°C), the supernatant contained the fraction of PIPES-insoluble tubulin.
- The anti-tubulin antibody (D-10) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the anti- -actin antibody (MAB1501R) from Chemicon International (Hofheim, Germany).
Cell cycle analysis
- After stimulation, cell culture supernatants containing apoptotic cells were collected.
- After another wash with PBS, cells were resuspended in hypotonic fluorochrome solution (HFS) (0.1% Na3-Citrat, 0.1% Triton X-100, and 50 g/ml propidium iodide).
- Cell cycle phases were analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany).
- Cell viability in HUVECs was measured by determination of the metabolical reduction of resazurin to resorufin, using the CellTiter-BlueTM assay (Promega, Madison, WI, USA).
Proliferation assay
- The proliferation assay was performed according to the NCI protocols for angiogenesis.
- Briefly, 1500 HUVECs/well were seeded into 96-well plates.
- Then HUVECs were treated with increasing concentrations of spongistatin 1, vinblastine, CA4P, or paclitaxel for 72 h.
- After stimulation, cells were stained with crystal violet solution (0.5% crystal violet in 20% methanol) for 10 min.
- Next, crystal violet, which mainly binds to DNA, was eluted from cells with 0.1 M sodium citrate in 50% ethanol.
Migration scratch assay
- HUVECs were seeded into 24-well plates and grown to confluency.
- The wells were washed with PBS to remove detached cells and incubated for 16 h at 37°C in either starvation medium (no migration), culture medium (100% migration), or culture medium containing increasing concentrations of spongistatin 1, vinblastine, CA4P, or paclitaxel.
- One image per well (center position) was taken on an inverted light microscope (Axiovert 200; Zeiss, Jena, Germany) using the Imago-QE camera system and the appending software (Till Photonics, Graefelfing, Germany).
- For quantification, these images were analyzed with the S.CORE imaging analysis tool (S.CO LifeScience, Munich, Germany).
- The cell-free area correlates to the ability of the HUVECs to migrate into the scratch.
Chemotaxis assay
- Chemotaxis of spongistatin 1-treated HUVECs was investigated by -slide chemotaxis (IBIDI, Martinsried, Germany).
- The chemotaxis assay was performed as described in the manual, “ -Slide Chemotaxis” (http://www.ibidi.de/applications/ap_chemotaxis.html).
- Two opposing medium reservoirs were connected to the seeding chamber by a thin slit.
- One of these reservoirs was filled with starvation medium without FCS; the other was filled with HUVEC culture medium containing 10% FCS, generating by diffusion a linear and stable ( 48 h) FCS gradient from 0 to 10% FCS in the seeding chamber of the -slide.
- Cell movement was observed every 10 min over 24 h by live cell imaging in a cell observation chamber mounted to a confocal microscope (LSM 510 Meta; Zeiss).
Tube formation assay
- MatrigelTM (BD Discovery Labware, Bedford, MA, USA) was placed into the lower chambers of -slide angiogenesis wells and hardened for 30 min at 37°C.
- The images were analyzed with the tube formation module of S.CORE (S.CO LifeScience).
- Aortic rings were incubated for 2 days in HUVEC culture medium before stimulation with spongistatin 1.
- The controls received vehicle treatment only (n 5).
- The length of new vessels in the cornea was measured from the inside margin of vessels around the limbus to the tip of the longest neovascular sprout.
Kinome chip analysis (PepChip)
- PepChip performance and analysis of the results were done as described previously (20, 21) by Pepscan Presto BV (Lelystad, The Netherlands; for detailed protocol see http://www. pepscanpresto.com/files/PepChip%20Kinase%20Lysate% 20Protocol_v5.pdf).
- On PepChip, 1152 different peptides with specific phosphorylation motifs for upstream kinases were spotted in triplicates.
- Afterward, chips 1129THE TUBULIN ANTAGONIST SPONGISTATIN INHIBITS PKC were rinsed 3 times with distilled water and then air-dried.
- The phosphorylation status of the chips with untreated or treated cells (spongistatin 1, vinblastine, or CA4P) was compared spot by spot.
PKC activity: (ser)-substrate phosphorylation
- The supernatants were prepared in 5 SDS sample buffer for immunoblotting.
- Phospho-(Ser) PKC substrate antibody was purchased from Cell Signaling Technology (CST, Frankfurt, Germany).
- PKC in vitro activity assay Potential direct inhibition of PKC isozymes in vitro by spongistatin 1 was assessed using a micellar-based assay.
- The samples were incubated for 10 min at 30°C and subsequently loaded onto phosphocellulose filter disks (Whatman, Dassel, Germany).
- Then 2 ml of scintillation fluid (Ultima Gold; PerkinElmer) was added to each filter disk, and radioactivity was counted with a liquid scintillation counter.
PKC translocation
- The supernatant was discarded, and the pellet was solubilized with lysing buffer containing 20 mM 3-[(3-cholamidopropyl) diethylammonio]-1 propane sulfonate at 4°C for 30 min.
- Both fractions were used for Western blotting.
- Antibodies against PKC , PKC , and PKCε were purchased from Santa-Cruz, the anti-VE-Cadherin antibody from Cell Signaling Technology.
Statistical analysis
- Data were expressed as means se, and analyzed using 1-way ANOVA, Student’s t test, or rank-sum test.
- Values of P 0.05 were considered statistically significant.
RESULTS
- To test whether spongistatin 1 causes microtubule disassembly in endothelial cells, 2 different approaches were pursued.
- In migrating HUVECs (culture medium), F-actin is arranged in stress fibers within the cell body and lamellopodia at the leading edge.
- Spongistatin 1 suppresses neovascularization of the mouse cornea Antiangiogenic properties of spongistatin 1 in vivo were determined in the mouse corneal micropocket assay.
- To validate the PepChip data of reduced PKC activity, Western blot analysis of phosphorylated serine residues of PKC substrates was performed.
- Spongistatin 1 inhibits the translocation of PKC to the membrane Because most of the PKC isoforms have to be translocated from the cytoplasm to membranes to become activated and translocation processes can depend on microtubules, the tubulin antagonist spongistatin 1 may influence the activity of specific PKC isoforms via inhibition of their transport to membranes.
DISCUSSION
- In addition to classical cytostatic chemotherapy, antiangiogenic therapy has become a very important approach in cancer treatment.
- Continuing investigations have shown that spongistatin 1 does not affect PKC activity by direct inhibition.
- This is the first time that microtubule-dependent PKC translocation is shown in endothelial cells.
- Further, it is known that VEGF-induced proliferation requires PKC (35), and it has been shown that PMA-induced angiogenesis depends on PKC activity (36).
- The kinase inhibition profile of the different tubulin antagonists exhibits common features as well as differences between the single compounds, indicating that effects of microtubule-inhibiting drugs could be compound specific.
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Frequently Asked Questions (15)
Q2. What is the effect of spongistatin 1 on endothelial cell?
the depolymerization of microtubules by spongistatin 1 might abrogate polarization in the endothelial cell and consequently reduce directed migration.
Q3. What is the role of spongistatin 1 in the apoptos?
Spongistatin 1 inhibits migration of endothelial cells and influences F-actin organizationMigration is a crucial step in angiogenesis.
Q4. What is the effect of spongistatin 1 on endothelial cells?
Spongistatin 1 is antiproliferative at a nontoxic concentrationAntiproliferative effects of spongistatin 1 on endothelial cells were investigated in a cell viability assay, DNA-fragmentation analysis, proliferation assay, and cell cycle analysis.
Q5. What is the role of spongistatin 1 in the neovascular?
Spongistatin 1 inhibits the translocation of PKC to the membraneBecause most of the PKC isoforms have to be translocated from the cytoplasm to membranes to become activated and translocation processes can depend on microtubules, the tubulin antagonist spongistatin 1 may influence the activity of specific PKC isoforms via inhibition of their transport to membranes.
Q6. At what concentration did spongistatin 1 cause apoptosis?
Acute cytotoxicity of spongistatin 1 on proliferating cells was observed at a concentration as low as 5.0 nM, as judged by the significant decrease in the metabolic activity of spongistatin 1-treated cells.
Q7. What is the effect of spongistatin 1 on PKC activity?
The well-established kinase inhibitor staurosporine, which is known to inhibit PKC activity by binding the ATP-site, caused complete inhibition of PKC serine-substrate phosphorylation, whereas spongistatin 1 reduced phosphorylation of not all but of distinct serine substrates (arrows in Fig. 7A).
Q8. What is the effect of spongistatin 1 on angiogenesis?
To identify microtubule-dependent signaling pathways involved in the initiation phase of angiogenesis and that might be affected by tubulin antagonism, a kinome array (PepChip) with spongistatin 1-, vinblastine-, or CA4P-treated HUVECs was performed.
Q9. What is the role of tubulin antagonists in angiogenesis?
inhibition of interphase microtubules by tubulin antagonists and subsequently reduced PKC translocation might provide a new mechanism by which tubulin antagonists act on angiogenesis.
Q10. How long did the spongistatin 1 treatment last?
Spongistatin 1 was administered intraperitoneally at a dose of 10 g/kg (dissolved in 2% dimethyl sulfoxide and isotonic saline) daily for 5 days, beginning from the first postoperative day (n 5).
Q11. What is the effect of spongistatin 1 on aortic ring?
Treatment of the aortic rings with 0.5 nM spongistatin 1 yielded reduced sprout formation, which was completely suppressed by treatment with 1.0 nM spongistatin 1 (Fig. 5C).
Q12. What is the effect of paclitaxel on endothelial cells?
As was to be expected, the microtubule-stabilizing compound paclitaxel caused an increase of tubulin in the fraction of polymerized microtubules.
Q13. What is the role of spongistatin 1 in the treatment of angiogenesis?
Meeting the increasing demand for new appropriate drugs for antiangiogenic therapy, this study presents the marine compound spongistatin 1 to be a strong antiangiogenic agent both in vitro and in vivo.
Q14. At what concentration did spongistatin 1 inhibit endothelial cells?
proliferation of HUVECs was already inhibited at a concentration as low as 100 pM spongistatin 1, whereas more than 25–500 higher concentrations of vinblastine (2.5 nM), C4AP (7.5 nM), or paclitaxel (50.0 nM), respectively, were needed to inhibit endothelial proliferation to a similar degree (Fig. 2C).
Q15. What was the protocol for -slide chemotaxis?
Cell tracking and analysis were done using the manual tracking plug-in (Fabrice Cordelieres, Orsay, France) and the chemotaxis and migration tool (IBIDI) for ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA), as described in the -slide chemotaxis protocol.