Abstract
Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy inhibited almost totally the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and induction of a pro-apoptotic state in the tumor microenvironment (TME). These effects were favoured by the partial re-education of TAMs towards a M1 phenotype and maintained via suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the tumor microenvironment, by suppressing its most important malignant biological capabilities.
Highlights
Novel combined liposomal delivery of SIM and DMXAA inhibits melanoma tumor growth
LCL-SIM augments the anti-angiogenic effects of LCL-DMXAA
Combined liposomal therapy inhibits HIF-1α/VEGF axis, induces apoptosis and TAM re-education in tumors
This novel therapy suppresses the most important malignant capabilities of melanoma
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* Contact information: Alina Sesarman, PhD, Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babes-Bolyai University, 5-7, Clinicilor Street, 400006, Cluj-Napoca, Romania, Tel: +40 0264 431691; Fax: +40 0264 431858;, Email: alina.sesarman{at}ubbcluj.ro; sesarman{at}gmail.com
Abbreviations
- ANOVA
- Analysis of variance
- ARG-1
- arginase-1
- AUTCs
- Areas under the tumor growth curves
- Bax
- Bcl-2-associated X protein
- Bcl-xL
- B-cell lymphoma-extra-large
- bFGF
- Basic fibroblast growth factor
- CD31
- cluster of differentiation 31
- CHL
- cholesterol
- DMEM
- Dulbecco’s Modified Eagle’s Medium
- DMXAA
- 5,6-dimethylxanthenone-4-acetic acid
- DPPC
- 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; ECM-extracellular matrix
- PEG-2000-DSPE
- (N-(Carbonyl-methoxypolyethylene-glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt)
- EPR
- enhanced permeability and retention
- FasL
- Fas ligand
- G-CSF
- Granulocyte-colony stimulating factor
- GM-CSF
- Granulocyte-macrophage-colony stimulating factor
- HE
- Hematoxylin and Eosin
- HIF-1α
- hypoxia-inducible factor 1α
- HPLC
- High-Performance Liquid Chromatography
- HRP
- horseradish peroxidase
- iNOS
- Inducible nitric oxide synthase
- IFN-γ
- Interferon γ
- IGF-II
- Insulin-like growth factor 2
- IL-12p40
- Interleukin 12 p40
- IL-12p70
- Interleukin 12 p70
- IL-13
- Interleukin 13
- IL-1α
- Interleukin 1α
- IL-1β
- Interleukin 1β
- IL-6
- Interleukin 6
- IL-9
- Interleukin 9
- i.v.
- Intravenous
- LCL
- Long circulating liposomes
- MCP-1
- Monocyte chemoattractant protein-1
- M-CSF
- Monocyte-colony stimulating factor
- MDA
- Malondialdehyde
- MIG
- Monokine induced by IFN-γ
- MMPs
- Matrix metalloproteinases
- MMP-2
- matrix metalloprotease-2
- MMP-9
- matrix metalloprotease-9
- pAP-1-c-Jun
- Phosphorylated form of c-Jun subunit of AP-1: PBS, Phosphate buffered saline
- PF-4
- Platelet factor 4
- PDI
- polydispersity index
- ROS
- reactive oxygen species
- s.c.
- Subcutaneous
- SD
- Standard Deviation
- SDS
- Sodium dodecyl sulfate
- SIM
- Simvastatin
- TAC
- Total antioxidant capacity
- TAMs
- Tumor-associated macrophages
- TBS-T
- Tris Buffered Saline with Tween
- TIMP-1
- Tissue inhibitor of metalloproteinase 1
- TIMP-2
- Tissue inhibitor of metalloproteinase 2
- TME
- tumor microenvironment
- TNF-α
- Tumor necrosis factor α
- VDA
- vascular disrupting agent
- VEGF
- Vascular endothelial growth factor