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Oxidized-LDL promotes colorectal cancer progression and growth of human colonoides
Background
Expression levels of the oxidized-LDL receptor LOX-1 have been related to cancer progression in prostatic, gastric, pancreatic, and colorectal cancer (CRC); however, the relationship among pro-inflammatory markers, ox-LDL/LOX-1 and CRC are not completely elucidated. We tested the hypothesis that stimulation of CRC cell lines with TNF-α and ox-LDL promotes tumor progression. We also explore the effect of ox-LDL on human colonoids growth.
Methods
The colorectal cancer (CRC) cell lines HCT116 and COLO320 were treated with TNF-α and ox-LDL and tested for LOX-1 expression. The effect on cell proliferation of this proinflammatory signals were assayed by MTT and cell count with the exclusion dye trypan blue. LOX-1-negative cell lines were obtained by transduction with lentiviral vectors encoding a shRNA specific for LOX-1. The expression of genes involved in tumor progression were analyzed by qRT-PCR and Western blot in wild type and LOX-1 negative cells treated with TNF-α and ox-LDL. Cell migration and invasion was assayed in ox-LDL and TNF-α treated cells. Human colonoids were stimulated with ox-LDL in the presence of the NADPH oxidase inhibitors ML171, VAS2870 and ROS scavenger NAC. Surface area and survival were assessed by contrast microscopy and MTT. Proliferation was assessed by click-chemistry.
Results
LOX-1 receptor is expressed in the CRC cell lines HCT116 and COLO320. Additionally, we found in microarrays data stored in public databases that LOX-1 mRNA is over-expressed in colorectal carcinomas and adenocarcinomas compared with normal colon tissue. Treatment of CRC cell lines with TNF-α and ox-LDL augmented LOX-1 expression and LOX-1 silencing abrogated this effect. Moreover, both cell lines treated with ox-LDL proliferated in a dose-dependent manner. Importantly, ox-LDL stimulated human organoids proliferation and viability also in a dose-dependent manner, an effect partly mediated by ROS. The activation of LOX-1 by ox-LDL induced the expression of angiogenic markers such as MMP2, MMP7, MMP9 and VEGF. LOX-1 activation by oxLDL also promotes epithelial to mesenchymal transition of human colorectal cancer cells, characterized by reduced expression of the epithelial marker E-cadherin and increased expression of mesenchymal markers such as Vimentin, N-Cadherin, TWIST, ZEB1 and ZEB2. In addition, we determined that ox-LDL promotes invasion and migration of colorectal cancer cells. Nevertheless, co-treatment of colorectal cancer cells with TNF-α and ox-LDL did not show an additive effect.
Conclusions
LOX-1 activation by ox-LDL induces a transcription reprogramming of genes involved in angiogenesis, invasion, migration, and cell proliferation. In the context of inflammation and obesity, we speculate that overexpression of LOX-1 provides additional stimulation accelerating tumor progression.
Legal entity responsible for the study
The author.
Funding
Agencia Nacional de Innovación y Desarrollo (ANID) - Chile. Fondecyt Regular 1201217.
Disclosures
All authors have declared no conflicts of interest.
