Fish oil suppresses cell growth and metastatic potential by regulating PTEN and NF-κB signaling in colorectal cancer

Abstract

Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF) play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP), NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1)&FO∶CO(2.5∶1) respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates apoptosis, diminishes DNA damage and inhibits inflammatory signalling in a dose and time dependent manner so as to inhibit progression of colon cancer.

Figure 1. Effect of different ratios of fish oil and corn oil on % of apoptotic cells in the initiation phase of experimentally induced colon cancer.

The isolated colonocytes were incubated with Hoechst 342 (H342) dye and PI. Photomicrographs were acquired using fluorescent microscope (Nikon Eclipse 80i) A- control group, B- DMH treated, C) FO+CO(1∶1)+DMH, D) FO+CO(2.5∶1)+DMH. Dark blue colour represents the normal live cells, faint blue or pink represents apoptotic cells (Magnification 400×). E) Graphical representation of % of live and apoptotic cells in different groups. The results are expressed as Mean±S.D. for n = 4 (^**p<0.01 wrt control, ^###p<0.001 wrt DMH).

Figure 2. Alterations in the % of apoptotic cells on administration of both the ratios of fish oil and corn oil in post-initiation phase.

The isolated colonocytes were incubated with Hoechst 342 (H342) dye and PI. Photomicrographs were acquired using fluorescent microscope (Nikon Eclipse 80i) A- control group, B- DMH treated, C) FO+CO(1∶1)+DMH, D) FO+CO(2.5∶1)+DMH. Dark blue colour represents the normal live cells, faint blue or pink represents apoptotic cells (Magnification 400×). E) Graphical representation of % of live and apoptotic cells in different groups. The results are expressed as Mean±S.D. for n = 4 (^###p<0.001 wrt DMH).

Figure 3. Inhibition of PARP activity on receiving different ratios of fish oil and corn oil in experimentally induced colon cancer.

Immunohistochemical staining of PARP in formalin fixed paraffin embedded sections of colon tissue. A–D depicts the representative photomicrographs of initiation phase and E–H represents the post-initiation phase (“arrow” depicts the expression of PARP). (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).

Figure 4. Effect of supplementation of fish oil on nuclear localization of NF-κBp65 in initiation and post-initiation phase.

The isolated fixed colonocytes were permeabilized and incubated with monoclonal antibody against NF-κB p65 and corresponding FITC conjugated secondary antibody The sections were counterstained with PI to visualise nuclear localisation. Red fluorescence represents the nuclear staining by PI with no expression of NF-κB (depicted by “dotted arrow”) and green fluorescence indicates the expression of NF-κB p65. Yellow colour indicates the localisation of NF-κB p65 from cytoplasm to nucleus (represented by “arrow”). A–D depicts the initiation phase and E–H represents the post-initiation phase. (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).

Figure 5. Inhibition of nuclear localization of NF-κBp50 on receiving the different ratios of fish oil in both the phases of experimentally induced colon cancer.

The isolated fixed colonocytes were permeabilized and incubated with monoclonal antibody against NF-κB p65 and corresponding FITC conjugated secondary antibody The sections were counterstained with PI to visualise nuclear localisation. Red fluorescence represents the nuclear staining by PI with no expression of NF-κB (depicted by “dotted arrow”) and green fluorescence indicates the expression of NF-κB p65. Yellow colour indicates the localisation of NF-κB p65 from cytoplasm to nucleus (represented by “arrow”). A–D depicts the initiation phase and E–H represents the post-initiation phase. (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).

Figure 6. Alterations in VEGF expression on administration of both the ratios of fish oil and corn oil.

The expression of VEGF was analyzed using immunohistochemistry in formalin fixed paraffin embedded sections of colon tissue. A–D depicts the representative photomicrographs of initiation phase and E–H represents the post-initiation phase (→ depicts the expression of VEGF). (A and E) Control, (B and F) DMH treated, (C and G) FO+CO(1∶1)+DMH, (D and H) FO+CO(2.5∶1)+DMH (Magnification 400X).