Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism
<p>Recombinant type 1 repeat domain (rTSR1) induces caspase-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). (<b>A</b>) SDS-PAGE and immunoblot analysis of purified rTSR1 showed a band at ~10 kDa; (<b>B</b>) rTSR1 dose-dependently induced apoptosis in HUVECs in the presence of VEGF. Data shown are apoptosis observed at 24 h post-1000 nM rTSR1 treatment; (<b>C</b>) Pan-caspase inhibitor (Z-VAD-FMK) significantly reduced the 1000 nM rTSR1-induced apoptosis at 24 h post treatment. Data represent the mean of triplicates ± SD. Statistical analysis performed by one-way ANOVA. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 2
<p>Systemically delivered rTSR1 suppressed B16F10 tumor growth in mice. (<b>A</b>) Tumor growth curve from day 9 to 15 post tumor cell injection (<span class="html-italic">n</span> = 5). Data represents mean ± SD. The rTSR1 was systemically delivered through intra-peritoneal (IP) injection. Statistical analysis performed by Student’s <span class="html-italic">t</span> test. * <span class="html-italic">p</span> < 0.05; (<b>B</b>) Tumor weight at the end of experiment, control and rTSR1 treated group, <span class="html-italic">n</span> = 5 for each group. The graph represents the mean ± SD of the tumor weight. Statistical analysis performed by Student’s <span class="html-italic">t</span> test. * <span class="html-italic">p</span> < 0.05; (<b>C</b>) Dissected tumor nodules from control and rTSR1 treated groups—scale bar: 1 cm; (<b>D</b>) rTSR1-treated mice showed a reduced peri-tumor vascular network compared to tumors of similar sizes in the control group—scale bar: 0.5 cm. Arrows indicate the blood vessels surrounding the tumor.</p> "> Figure 3
<p>The rTSR1 suppressed angiogenesis and cell proliferation but induced apoptosis in tumors. Tumor paraffin sections were probed for microvessel density, tumor cell proliferation, and apoptosis through immunofluorescence staining, using (<b>A</b>) anti-endomucin, (<b>B</b>) TUNEL staining, and (<b>C</b>) anti-PCNA (Proliferating cell nuclear antigen) respectively. The corresponding quantification of the staining is presented in the bar graph on the right. The percentage of proliferating cells/field is the ratio of the nuclear PCNA-positive cells to the total number of nuclei in the field. Scale bar: 200 μm. Corresponding quantifications are presented in the bar graph on the right. Data represent the mean ± SD of four fields per section, four sections per tumor, and two tumors per group. Statistical analysis was performed by Student’s <span class="html-italic">t</span> test. ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 4
<p>The rTSR1 suppressed tumor endothelial cell proliferation. Representative microscopic images show decreased proliferating endothelial cells in an rTSR1-treated tumor compared to the control. Green indicates the endothelial cells (endomucin-positive), and red indicates the nuclear PCNA-positive proliferating cells. DAPI (4′,6-diamidino-2-phenylindole) was used as the nuclear counter stain. The arrow heads indicate the nuclear PCNA-positive tumor endothelial cells. Scale bar: 30 μm.</p> "> Figure 5
<p>The rTSR1 induced apoptosis in tumor endothelial cells. Representative microscopic images show the increased apoptotic endothelial cells in an rTSR1-treated tumor compared to the control. Red indicates the endothelial cells (endomucin-positive) and green indicates the cleaved caspase-3-positive cells. DAPI was used as the nuclear counter stain. The arrow heads indicates the cleaved caspase-3-positive endothelial cells. Scale bar: 20 μm.</p> "> Figure 6
<p>IP-injected rTSR1 reached the tumor. Immunoblot of serum and tumor lysate displayed band at ~10 kDa, corresponding to the molecular weight of rTSR1. The band is observed both in serum (<b>A</b>) and with the whole tumor lysate (<b>B</b>) from mice that received rTSR1 only. The rTSR1 input is used as the positive control. M1 to M3 refer to mouse 1 to mouse 3.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Recombinant Type 1 Repeat Domain Induces Caspase-Dependent Apoptosis in Human Umbilical Vein Endothelial Cells
2.2. Recombinant Type 1 Repeat Domain Suppressed the B16F10 Melanoma Growth in Mice
2.3. Recombinant Type 1 Repeat Domain Suppresses B16 Melaonma Growth by Inhibiting Tumor Angiogenesis and Tumor Cell Proliferation While Inducing Tumor Cell Apoptosis
3. Discussion
4. Materials and Methods
4.1. Recombinant Type 1 Repeat Domain Production and Purification
4.2. Cell Culture
4.3. Apoptosis Determination by IncuCyte ZOOM Live-Cell Analysis System
4.4. Western Blot
4.5. Immunohistochemistry
4.6. Xenograft Mouse Tumorigenesis Assay
4.7. Mice Serum Isolation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- Bergers, G.; Benjamin, L.E. Angiogenesis: Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 2003, 3, 401–410. [Google Scholar] [CrossRef] [PubMed]
- Folkman, J. Role of angiogenesis in tumor growth and metastasis. Semin. Oncol. 2002, 29, 15–18. [Google Scholar] [CrossRef] [PubMed]
- Hagedorn, M.; Zilberberg, L.; Wilting, J.; Canron, X.; Carrabba, G.; Giussani, C.; Pluderi, M.; Bello, L.; Bikfalvi, A. Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors. Cancer Res. 2002, 62, 6884–6890. [Google Scholar] [CrossRef] [PubMed]
- Sund, M.; Hamano, Y.; Sugimoto, H.; Sudhakar, A.; Soubasakos, M.; Yerramalla, U.; Benjamin, L.E.; Lawler, J.; Kieran, M.; Shah, A.; et al. Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors. Proc. Natl. Acad. Sci. USA 2005, 102, 2934–2939. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Glasson, S.S.; Askew, R.; Sheppard, B.; Carito, B.; Blanchet, T.; Ma, H.-L.; Flannery, C.R.; Peluso, D.; Kanki, K.; Yang, Z.; et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature 2005, 434, 644–648. [Google Scholar] [CrossRef] [PubMed]
- Larkin, J.; Lohr, T.A.; Elefante, L.; Shearin, J.; Matico, R.; Su, J.-L.; Xue, Y.; Liu, F.; Genell, C.; Miller, R.E.; et al. Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification. Osteoarthr. Cartil. 2015, 23, 1254–1266. [Google Scholar] [CrossRef] [PubMed]
- Cross, N.A.; Chandrasekharan, S.; Jokonya, N.; Fowles, A.; Hamdy, F.C.; Buttle, D.J.; Eaton, C.L. The expression and regulation of ADAMTS-1, -4, -5, -9, and -15, and TIMP-3 by TGFbeta1 in prostate cells: Relevance to the accumulation of versican. Prostate 2005, 63, 269–275. [Google Scholar] [CrossRef] [PubMed]
- Porter, S. Dysregulated expression of adamalysin-thrombospondin genes in human breast carcinoma. Clin. Cancer Res. 2004, 10, 2429–2440. [Google Scholar] [CrossRef] [PubMed]
- Kim, Y.-H.; Lee, H.C.; Kim, S.-Y.; Yeom, Y.I.; Ryu, K.J.; Min, B.-H.; Kim, D.-H.; Son, H.J.; Rhee, P.-L.; Kim, J.J.; et al. Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations. Ann. Surg. Oncol. 2011, 18, 2338–2347. [Google Scholar] [CrossRef] [PubMed]
- Li, C.; Xiong, Y.; Yang, X.; Wang, L.; Zhang, S.; Dai, N.; Li, M.; Ren, T.; Yang, Y.; Zhou, S.-F.; et al. Lost expression of ADAMTS5 protein associates with progression and poor prognosis of hepatocellular carcinoma. Drug Des. Dev. Ther. 2015, 9, 1773–1783. [Google Scholar] [CrossRef] [PubMed]
- Nakada, M.; Miyamori, H.; Kita, D.; Takahashi, T.; Yamashita, J.; Sato, H.; Miura, R.; Yamaguchi, Y.; Okada, Y. Human glioblastomas overexpress ADAMTS-5 that degrades brevican. Acta Neuropathol. 2005, 110, 239–246. [Google Scholar] [CrossRef] [PubMed]
- Haraguchi, N.; Ohara, N.; Koseki, J.; Takahashi, H.; Nishimura, J.; Hata, T.; Mizushima, T.; Yamamoto, H.; Ishii, H.; Doki, Y.; et al. High expression of ADAMTS5 is a potent marker for lymphatic invasion and lymph node metastasis in colorectal cancer. Mol. Clin. Oncol. 2017, 6, 130–134. [Google Scholar] [CrossRef] [PubMed]
- Kumar, S.; Sharghi-Namini, S.; Rao, N.; Ge, R. ADAMTS5 functions as an anti-angiogenic and anti-tumorigenic protein independent of its proteoglycanase activity. Am. J. Pathol. 2012, 181, 1056–1068. [Google Scholar] [CrossRef] [PubMed]
- Sharghi-Namini, S.; Fan, H.; Sulochana, K.N.; Potturi, P.; Xiang, W.; Chong, Y.S.; Wang, Z.; Yang, H.; Ge, R. The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor. Biochem. Biophys. Res. Commun. 2008, 371, 215–219. [Google Scholar] [CrossRef] [PubMed]
- Shojaei, F. Anti-angiogenesis therapy in cancer: Current challenges and future perspectives. Cancer Lett. 2012, 320, 130–137. [Google Scholar] [CrossRef] [PubMed]
- Rao, N.; Lee, Y.F.; Ge, R. Novel endogenous angiogenesis inhibitors and their therapeutic potential. Acta Pharmacol. Sin. 2015, 36, 1177–1190. [Google Scholar] [CrossRef] [PubMed]
- Miao, W.M.; Seng, W.L.; Duquette, M.; Lawler, P.; Laus, C.; Lawler, J. Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms. Cancer Res. 2001, 61, 7830–7839. [Google Scholar] [PubMed]
- Kumar, S.; Rao, N.; Ge, R. Emerging roles of ADAMTSS in angiogenesis and cancer. Cancers 2012, 4, 1252–1299. [Google Scholar] [CrossRef] [PubMed]
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Renganathan, B.; Durairaj, V.; Kirman, D.C.; Esubonteng, P.K.A.; Ang, S.K.; Ge, R. Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism. Cancers 2018, 10, 192. https://doi.org/10.3390/cancers10060192
Renganathan B, Durairaj V, Kirman DC, Esubonteng PKA, Ang SK, Ge R. Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism. Cancers. 2018; 10(6):192. https://doi.org/10.3390/cancers10060192
Chicago/Turabian StyleRenganathan, Bhuvanasundar, Vinoth Durairaj, Dogan Can Kirman, Paa Kow A. Esubonteng, Swee Kim Ang, and Ruowen Ge. 2018. "Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism" Cancers 10, no. 6: 192. https://doi.org/10.3390/cancers10060192