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. 2019 Aug 17:16:53.
doi: 10.1186/s12986-019-0382-3. eCollection 2019.

Krill oil extract suppresses the proliferation of colorectal cancer cells through activation of caspase 3/9

Abilasha Gayani Jayathilake  1 , Elif Kadife  1 , Rodney Brain Luwor  2 , Kulmira Nurgali  1   3   4 , Xiao Qun Su  1
Affiliations

Krill oil extract suppresses the proliferation of colorectal cancer cells through activation of caspase 3/9

Abilasha Gayani Jayathilake et al. Nutr Metab (Lond). .

Abstract

Background: Currently available treatments for colorectal cancer (CRC) associate with numerous side-effects that reduce patients' quality of life. The effective nutraceuticals with high anti-proliferative efficacy and low side-effects are desirable. Our previous study has reported that free fatty acids extract (FFAE) of krill oil induced apoptosis of CRC cells, possibly associated with changes in mitochondrial membrane potential (MMP). The aims of this study were to compare the anti-proliferative efficacy of FFAE from krill oil on CRC cells with commonly used chemotherapeutic drug, Oxaliplatin, and to investigate the molecular mechanisms underlying the anti-proliferative effects of krill oil with a focus on intrinsic mitochondrial death pathway.

Methods: Three human CRC cell lines, including DLD-1, HT-29 and LIM-2405, and one mouse CRC cell line, CT-26, were treated with FFAE of KO and the bioactive components of krill oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 24 h and 48 h. Similarly, these cell lines were treated with Oxaliplatin, a commonly used drug for CRC treatment, for 24 h. The effects of FFAE of KO, EPA, DHA and Oxaliplatin on cell proliferation, mitochondrial membrane potential and reactive oxygen species (ROS) were determined via WST-1, JC-10, and ROS assays respectively. The expression of caspase-3, caspase-9 and DNA damage following treatments of FFAE of KO was investigated via western blotting and immunohistochemistry.

Results: The FFAE of KO, EPA and DHA significantly inhibited cell proliferation and increased formation of ROS in all four cell lines (P < 0.01). A small dose of FFAE from KO ranged from 0.06 μL/100 μL to 0.12 μL/100 μL containing low concentrations of EPA (0.13-0.52 μM) and DHA (0.06-0.26 μM) achieved similar anti-proliferative effect as Oxaliplatin (P > 0.05). Treatments with the FFAE of KO, EPA and DHA (2:1 ratio) resulted in a significant increase in the mitochondrial membrane potential (P < 0.001). Furthermore, the expression of active forms of caspase-3 and caspase-9 was significantly increased following the treatment of FFAE of KO.

Conclusions: The present study has demonstrated that the anti-proliferative effects of krill oil on CRC cells are comparable with that of Oxaliplatin, and its anti-proliferative property is associated with the activation of caspase 3/9 in the CRC cells.

Keywords: Caspase 3/9; Docosahexaenoic acid; Eicosapentaenoic acid; Human colorectal cancer cells; Krill oil extract.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
Effects of FFAE of krill oil on the proliferation of CRC cells compared to the anti-cancer drug Oxaliplatin. Cell viability of DLD-1 (a), HT-29 (b), LIM-2405 (c) and CT-26 (d) cells were determined using WST-1 assay following 24 h of treatment with FFAE of krill oil (KO) at the concentrations of 0.03 μL/100 μL (containing 0.13 μM EPA/0.065 μM DHA), 0.06 μL/100 μL (containing 0.26 μM EPA/0.13 μM DHA), and 0.12 μL/100 μL (containing 0.52 μM EPA/0.26 μM DHA) or chemotherapeutic drug, Oxaliplatin (OXAL). The experiment was repeated three times for each cell line. Data are expressed as mean ± SEM (n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001 indicate a significant difference between the treatment and Ethanol (vehicle) control
Fig. 2
Fig. 2
Proliferation of CRC cells following treatment with EPA and DHA. Cell viability of DLD-1 (a), HT-29 (b), LIM-2405 (c) and CT-26 (d) cells were determined using WST-1 assay following treatment with DHA and EPA for 24 and 48 h. The experiment was repeated three times for each cell line. Data are expressed as mean ± SEM (n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001 indicate a significant difference between the treatment and Ethanol (vehicle) control
Fig. 3
Fig. 3
ROS formation in mitochondria of CRC cells after 24 h of treatment with FFAE of krill oil, EPA and DHA. The mitochondrial superoxide level was measured using the MitoSox™ and was presented as a percentage comparison to the ROS level in Ethanol (vehicle) treated cells. Three replicates for each treatment and two individual experiments were performed. Data are expressed as mean ± SEM (n = 3). **p < 0.01 indicates a significant difference between the treatment and Ethanol (vehicle) control
Fig. 4
Fig. 4
Mitochondrial membrane potential (MMP) in CRC cells following treatment with FFAE of krill oil, EPA and DHA. (a) MMP of DLD-1, HT-29, LIM-2405 and CT-26 cells was measured using the JC-10 fluorescent MMP microplate assay following 24 h of treatment with FFAE of krill oil (0.12 μL/100 μL, containing 0.52 μM EPA/0.26 μM DHA), DHA (250 μM) or EPA (200 μM). (b) Effects of treatment with combined EPA and DHA at a 2:1 volume ratio. Three replicates for each treatment and two individual experiments were performed. Data are expressed as mean ± SEM (n = 3), **p < 0.01 and ***p < 0.001 indicate a significant difference compared to Ethanol (vehicle) control
Fig. 5
Fig. 5
Activation of caspase-9 in CRC cells after treatment with FFAE of krill oil. The expression of caspase-9 and cleaved caspase-9 was measured by western blotting in DLD-1 (A) and HT-29 (A’) cells following treatment with FFAE of krill oil at 0.03 μL/100 μL (containing 0.13 μM EPA/0.065 μM DHA) and 0.12 μL/100 μL (containing 0.52 μM EPA/0.26 μM DHA) for 4 h and 8 h. Fluorescent intensity of subcellular distribution of cleaved caspase-9 in DLD-1 (B-C) and HT-29 (B′-C′) cells was determined using a monoclonal antibody for cleaved caspase-9 following 8 h of treatment with FFAE of krill oil at 0.12 μL/100 μL(containing 0.52 μM EPA/0.26 μM DHA). Scale bar = 50 μM. Magnification = 60X. The results were verified through at least three individual experiments. Data are expressed as mean ± SEM. ***p < 0.001 compared to Ethanol control
Fig. 6
Fig. 6
Activation of caspase-3 in DLD-1 and HT-29 cells following treatment with FFAE of krill oil. The expression of caspase-3 and cleaved caspase-3 was measured by western blotting in DLD-1 (A) and HT-29 (A’) following treatment with FFAE of krill oil at 0.03 μL/100 μL (containing 0.13 μM EPA/0.065 μM DHA) and0.12 μL/100 μL (containing 0.52 μM EPA/0.26 μM DHA) for 4 h and 8 h. Fluorescent intensity of subcellular distribution of cleaved caspase-3 and DNA damage in DLD-1 (B-C) and HT-29 (B′-C′) cells was determined using monoclonal antibodies for cleaved caspase-3 and DNA/RNA damage (anti-8-OHdG) following 8 h of treatment with FFAE of krill oil at 0.12 μL/100 μL (containing 0.52 μM EPA/0.26 μM DHA) . Scale bar = 50 μM. Magnification = 60X. The results were verified through at least three individual experiments. Data are expressed as mean ± SEM. **p < 0.01 and ***p < 0.001 compared to Ethanol control
Fig. 7
Fig. 7
Schematic summary of the death signaling pathways initiated by the FFAE of krill oil in DLD-1 and HT-29 cells. The FFAE of krill oil and a combination of EPA/DHA exert their effects on cancer cells by changing the mitochondrial membrane potential (MMP). That results in the activation of caspase-9 and caspase-3 and lead to nuclear DNA damage hence possible apoptosis of cancer cells
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References

    1. Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683–691. - PubMed
    1. El-Shami K, Oeffinger KC, Erb NL, Willis A, Bretsch JK, Pratt-Chapman ML, et al. American Cancer Society colorectal Cancer survivorship care guidelines. CA Cancer J Clin. 2015;65(6):428–455. - PMC - PubMed
    1. Bardhan K, Liu K. Epigenetics and colorectal cancer pathogenesis. Cancers. 2013;5(2):676–713. - PMC - PubMed
    1. Armaghany T, Wilson JD, Chu Q, Mills G. Genetic alterations in colorectal cancer. Gastrointest Cancer Res. 2012;5(1):19–27. - PMC - PubMed
    1. Yang P, Cartwright C, Chan D, Ding J, Felix E, Pan Y, et al. Anticancer activity of fish oils against human lung cancer is associated with changes in formation of PGE2 and PGE3 and alteration of Akt phosphorylation. Mol Carcinog. 2014;53(7):566–577. - PMC - PubMed