Molecular Mechanisms Associated with the Inhibitory Role of Long Chain n-3 PUFA in Colorectal Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the world. Multiple evidence suggests that there is an association between excess fat consumption and the risk of CRC. The long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for human health, and both in vitro and in vivo studies have shown that these fatty acids can prevent CRC development through various molecular mechanisms. These include the modulation of arachidonic acid (AA) derived prostaglandin synthesis, alteration of growth signaling pathways, arrest of the cell cycle, induction of cell apoptosis, suppression of angiogenesis and modulation of inflammatory response. Human clinical studies found that LC n-3 PUFA combined with chemotherapeutic agents can improve the efficacy of treatment and reduce the dosage of chemotherapy and associated side effects. In this review, we discuss comprehensively the anti-cancer effects of LC n-3 PUFA on CRC, with a main focus on the underlying molecular mechanisms.

Krill Oil and Its Bioactive Components as a Potential Therapy for Inflammatory Bowel Disease: Insights from In Vivo and In Vitro Studies.

Krill oil is extracted from krill, a small crustacean in the Antarctic Ocean. It has received growing attention because of krill oil's unique properties and diverse health benefits. Recent experimental and clinical studies suggest that it has potential therapeutic benefits in preventing the development of a range of chronic conditions, including inflammatory bowel disease (IBD). Krill oil is enriched with long-chain n-3 polyunsaturated fatty acids, especially eicosapentaenoic and docosahexaenoic acids, and the potent antioxidant astaxanthin, contributing to its therapeutic properties. The possible underlying mechanisms of krill oil's health benefits include anti-inflammatory and antioxidant actions, maintaining intestinal barrier functions, and modulating gut microbiota. This review aims to provide an overview of the beneficial effects of krill oil and its bioactive components on intestinal inflammation and to discuss the findings on the molecular mechanisms associated with the role of krill oil in IBD prevention and treatment.

Bone marrow-derived mesenchymal stem cells mitigate chronic colitis and enteric neuropathy via anti-inflammatory and anti-oxidative mechanisms.

Current treatments for inflammatory bowel disease (IBD) are often inadequate due to limited efficacy and toxicity, leading to surgical resection in refractory cases. IBD's broad and complex pathogenesis involving the immune system, enteric nervous system, microbiome, and oxidative stress requires more effective therapeutic strategies. In this study, we investigated the therapeutic potential of bone marrow-derived mesenchymal stem cell (BM-MSC) treatments in spontaneous chronic colitis using the Winnie mouse model which closely replicates the presentation and inflammatory profile of ulcerative colitis. The 14-day BM-MSC treatment regimen reduced the severity of colitis, leading to the attenuation of diarrheal symptoms and recovery in body mass. Morphological and histological abnormalities in the colon were also alleviated. Transcriptomic analysis demonstrated that BM-MSC treatment led to alterations in gene expression profiles primarily downregulating genes related to inflammation, including pro-inflammatory cytokines, chemokines and other biomarkers of inflammation. Further evaluation of immune cell populations using immunohistochemistry revealed a reduction in leukocyte infiltration upon BM-MSC treatment. Notably, enteric neuronal gene signatures were the most impacted by BM-MSC treatment, which correlated with the restoration of neuronal density in the myenteric ganglia. Moreover, BM-MSCs exhibited neuroprotective effects against oxidative stress-induced neuronal loss through antioxidant mechanisms, including the reduction of mitochondrial-derived superoxide and attenuation of oxidative stress-induced HMGB1 translocation, potentially relying on MSC-derived SOD1. These findings suggest that BM-MSCs hold promise as a therapeutic intervention to mitigate chronic colitis by exerting anti-inflammatory effects and protecting the enteric nervous system from oxidative stress-induced damage.

Role of ICAM-1 in the Adhesion of T Cells to Enteric Glia: Perspectives in the Formation of Plexitis in Crohn's Disease.

BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.

Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines.

BACKGROUND: Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. METHODS: We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) cells. RESULTS: Increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK. CONCLUSIONS: The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.

Differential Gene Expression of Checkpoint Markers and Cancer Markers in Mouse Models of Spontaneous Chronic Colitis.

The presence of checkpoint markers in cancer cells aids in immune escape. The identification of checkpoint markers and early cancer markers is of utmost importance to gain clarity regarding the relationship between colitis and progressive inflammation leading to cancer. Herein, the gene expression levels of checkpoint makers, cancer-related pathways, and cancer genes in colon tissues of mouse models of chronic colitis (Winnie and Winnie-Prolapse mice) using next-generation sequencing are determined. Winnie mice are a result of a Muc2 missense mutation. The identification of such genes and their subsequent expression and role at the protein level would enable novel markers for the early diagnosis of cancer in IBD patients. The differentially expressed genes in the colonic transcriptome were analysed based on the Kyoto Encyclopedia of Genes and Genomes pathway. The expression of several oncogenes is associated with the severity of IBD, with Winnie-Prolapse mice expressing a large number of key genes associated with development of cancer. This research presents a number of new targets to evaluate for the development of biomarkers and therapeutics.

Effect of the Cannabinoid Agonist WIN 55,212-2 on Neuropathic and Visceral Pain Induced by a Non-Diarrheagenic Dose of the Antitumoral Drug 5-Fluorouracil in the Rat.

5-fluorouracil (5-FU) is an antineoplastic drug used to treat colorectal cancer, but it causes, among other adverse effects, diarrhea and mucositis, as well as enteric neuropathy, as shown in experimental animals. It might also cause neuropathic pain and alterations in visceral sensitivity, but this has not been studied in either patients or experimental animals. Cannabinoids have antimotility and analgesic effects and may alleviate 5-FU-induced adverse effects. Our aim was to evaluate the effects of the cannabinoid agonist WIN 55,212-2 on neuropathic and visceral pain induced by a non-diarrheagenic dose of 5-FU. Male Wistar rats received a dose of 5-FU (150 mg/kg, ip) and gastrointestinal motility, colonic sensitivity, gut wall structure and tactile sensitivity were evaluated. WIN 55,212-2 (WIN) was administered to evaluate its effect on somatic (50-100 µg ipl; 1 mg/kg, ip) and visceral (1 mg/kg, ip) sensitivity. The cannabinoid tetrad was used to assess the central effects of WIN (1 mg/kg, ip). 5-FU decreased food intake and body weight gain, produced mucositis and thermal hyperalgesia, but these effects were reduced afterwards, and were not accompanied by diarrhea. Tactile mechanical allodynia was also evident and persisted for 15 days. Interestingly, it was alleviated by WIN. 5-FU tended to increase colonic sensitivity whereas WIN reduced the abdominal contractions induced by increasing intracolonic pressure in both control and 5-FU-treated animals. Importantly, the alleviating effects of WIN against those induced by 5-FU were not accompanied by any effect in the cannabinoid tetrad. The activation of the peripheral cannabinoid system may be useful to alleviate neuropathic and visceral pain associated with antitumoral treatment.

Oxidative Stress and Neural Dysfunction in Gastrointestinal Diseases: Can Stem Cells Offer a Solution?

Oxidative stress is involved in many gastrointestinal (GI) disorders as either the primary pathogenesis (radiation, chemotherapy, toxicity, ischemia-reperfusion) or a secondary driving force of disease progression (inflammation and diabetes). The GI tract is innervated intrinsically by the enteric nervous system (ENS) with a diverse role in maintaining gut homeostasis and GI motility. Complications in the physiological functioning of the ENS results in GI dysfunction that can result in debilitating sequelae from dysmotility greatly impacting quality of life and leading to potentially fatal complications. Therapeutics to remedy either oxidative stress or enteric neuronal dysfunction are severely limited, resulting in a critical gap in clinical care for GI disease and neurointestinal complications. Stem cell therapies have shown great promise in the treatment of several gut disorders via mechanisms including cell regeneration, anti-inflammatory activity, providing trophic support, and emerging evidence of antioxidant and neuroprotective functions. The potential of mesenchymal stem cell (MSC) therapies and recent evidence of their antioxidant and neuroprotective activity in several GI conditions are discussed. Finally, future therapeutic aspects of stem cell-based tools for combatting oxidative stress and enteric neuropathies in GI disease are considered.

A Green Synthesis Route to Derive Carbon Quantum Dots for Bioimaging Cancer Cells.

Carbon quantum dots (CQDs) are known for their biocompatibility and versatile applications in the biomedical sector. These CQDs retain high solubility, robust chemical inertness, facile modification, and good resistance to photobleaching, which makes them ideal for cell bioimaging. Many fabrication processes produce CQDs, but most require expensive equipment, toxic chemicals, and a long processing time. This study developed a facile and rapid toasting method to prepare CQDs using various slices of bread as precursors without any additional chemicals. This fast and cost-effective toasting method could produce CQDs within 2 h, compared with the 10 h process in the commonly used hydrothermal method. The CQDs derived from the toasting method could be used to bioimage two types of colon cancer cells, namely, CT-26 and HT-29, derived from mice and humans, respectively. Significantly, these CQDs from the rapid toasting method produced equally bright images as CQDs derived from the hydrothermal method.

Oxaliplatin-Induced Damage to the Gastric Innervation: Role in Nausea and Vomiting.

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility.

Contractility of isolated colonic smooth muscle strips from rats treated with cancer chemotherapy: differential effects of cisplatin and vincristine.

Background: Certain antineoplastic drugs cause gastrointestinal disorders even after the end of treatment. Enteric neuropathy has been associated with some of these alterations. Our goal was to assess the impact of repeated treatment with cisplatin and vincristine on the contractility of circular and longitudinal muscle strips isolated from the rat colon. Methods: Two cohorts of male rats were used: in cohort 1, rats received one intraperitoneal (ip) injection of saline or cisplatin (2 mg kg-1 week-1) on the first day of weeks 1-5; in cohort 2, rats received two cycles of five daily ip injections (Monday to Friday, weeks 1-2) of saline or vincristine (0.1 mg kg-1 day-1). Body weight and food and water intake were monitored throughout the study. One week after treatment, responses of colonic smooth muscle strips to acetylcholine (10-9-10-5 M) and electrical field stimulation (EFS, 0.1-20 Hz), before and after atropine (10-6 M), were evaluated in an organ bath. Results: Both drugs decreased body weight gain. Compared to saline, cisplatin significantly decreased responses of both longitudinal and circular smooth muscle strips to EFS, whereas vincristine tended to increase them, although in a non-significant manner. No differences were observed in the muscle response to acetylcholine. Atropine abolished the contractile responses induced by acetylcholine, although those induced by EFS were only partially reduced in the presence of atropine. Conclusion: The findings suggest that although both drugs cause the development of enteric neuropathy, this seems to have a functional impact only in cisplatin-treated animals. Understanding the effects of chemotherapy on gastrointestinal motor function is vital for enhancing the quality of life of cancer patients.

Cancer Immunotherapy: The Checkpoint between Chronic Colitis and Colorectal Cancer.

Inflammatory Bowel Disease (IBD) is a group of diseases that cause intestinal inflammation and lesions because of an abnormal immune response to host gut microflora. Corticosteroids, anti-inflammatories, and antibiotics are often used to reduce non-specific inflammation and relapse rates; however, such treatments are ineffective over time. Patients with chronic colitis are more susceptible to developing colorectal cancer, especially those with a longer duration of colitis. There is often a limit in using chemotherapy due to side effects, leading to reduced efficacy, leaving an urgent need to improve treatments and identify new therapeutic targets. Cancer immunotherapy has made significant advances in recent years and is mainly categorized as cancer vaccines, adoptive cellular immunotherapy, or immune checkpoint blockade therapies. Checkpoint markers are expressed on cancer cells to evade the immune system, and as a result checkpoint inhibitors have transformed cancer treatment in the last 5-10 years. Immune checkpoint inhibitors have produced long-lasting clinical responses in both single and combination therapies. Winnie mice are a viable model of spontaneous chronic colitis with immune responses like human IBD. Determining the expression levels of checkpoint markers in tissues from these mice will provide insights into disease initiation, progression, and cancer. Such information will lead to identification of novel checkpoint markers and the development of treatments with or without immune checkpoint inhibitors or vaccines to slow or stop disease progression.

Methamphetamine Induces Systemic Inflammation and Anxiety: The Role of the Gut-Immune-Brain Axis.

Methamphetamine (METH) is a highly addictive drug abused by millions of users worldwide, thus becoming a global health concern with limited management options. The inefficiency of existing treatment methods has driven research into understanding the mechanisms underlying METH-induced disorders and finding effective treatments. This study aims to understand the complex interactions of the gastrointestinal-immune-nervous systems following an acute METH dose administration as one of the potential underlying molecular mechanisms concentrating on the impact of METH abuse on gut permeability. Findings showed a decreased expression of tight junction proteins ZO-1 and EpCAm in intestinal tissue and the presence of FABP-1 in sera of METH treated mice suggests intestinal wall disruption. The increased presence of CD45+ immune cells in the intestinal wall further confirms gut wall inflammation/disruption. In the brain, the expression of inflammatory markers Ccl2, Cxcl1, IL-1ß, TMEM119, and the presence of albumin were higher in METH mice compared to shams, suggesting METH-induced blood-brain barrier disruption. In the spleen, cellular and gene changes are also noted. In addition, mice treated with an acute dose of METH showed anxious behavior in dark and light, open field, and elevated maze tests compared to sham controls. The findings on METH-induced inflammation and anxiety may provide opportunities to develop effective treatments for METH addiction in the future.

Pilot Study: Immune Checkpoints Polymorphisms in Greek Primary Breast Cancer Patients.

Background: Breast cancer is the most prevalent and second leading cause of cancer-related death in women worldwide. Despite early detection and better treatment therapies, 30% of early-stage breast cancer patients still develop recurrent disease. Breast cancer is a heterogeneous disease comprising several molecular subtypes, commonly classified into clinical subtypes based on the hormone receptor status. These subtypes included luminal A and luminal B, which have different prognoses. Breast cancer development and progression involve many factors. Polymorphisms of PD-1, PD-L1, and PD-L2 genes have been previously associated with high risk and prognosis of cancer. However, no studies have associated PD-1, PD-L1, and PD-L2 polymorphisms with primary breast cancer subtypes. Hence, this study evaluated functional single nucleotide polymorphisms of PD-1, PD-L1, and PD-L2 with primary breast cancer subtypes, luminal A, and luminal B. In addition, we evaluated the PD-L1 protein expression in relation to primary breast cancer subtypes and stages. Results: There were no significant differences in the allele frequencies of PD-1 polymorphisms (rs2227981 G>A, rs7421861 A>G, and rs11568821 C>T) and PD-L1 polymorphisms (rs10815225 C>T and rs2282055 T>G) when compared with the general European population. However, a significant difference was detected in one of the PD-L2 polymorphisms (rs1009759 A>G), with the G allele higher in breast cancer patients than in the general European population. A higher prevalence of the T allele of PD-L1 polymorphism rs2282055 T>G was observed in luminal B breast cancer patients compared with luminal A. No significant difference was detected in other polymorphisms. We also observed that the PD-L1 rs2282055 TT genotype was more prevalent in luminal B breast cancer patients compared with luminal A. Our results found no association of the selected SNPs in the PDCD1 gene with breast cancer risk. Similarly, the protein expression data showed that PD-L1 and PD-L2 are associated with an aggressive phenotype, Luminal B, and advanced breast cancer stage. Conclusion: These findings suggest that immune checkpoint polymorphisms are associated with the risk and subtypes of breast cancer.

Checkpoint Markers and Tumor Microenvironment: What Do We Know?

The cancer microenvironment, or tumor microenvironment (TME), describes the non-cancerous cells present in the tumor, such as fibroblasts, immune cells, and cells that comprise the blood vessels and proteins produced by all of the cells present in the tumor that support the growth of the cancer cells [...].

Mesenchymal stem cell treatment for enteric neuropathy in the Winnie mouse model of spontaneous chronic colitis.

Inflammatory bowel disease (IBD) is a chronic gut inflammation with periods of acute flares and remission. Beneficial effects of a single dose of mesenchymal stem cell (MSC)-based treatment have been demonstrated in acute models of colitis. No studies investigated therapeutic effects of MSCs for the attenuation of enteric neuropathy in a chronic model of colitis. The short and long-term effects of MSC treatment in modulating inflammation and damage to the enteric nervous system (ENS) were studied in the Winnie mouse model of spontaneous chronic colitis highly representative of human IBD. Winnie mice received a single dose of either 1 × 106 human bone marrow-derived MSCs or 100µL PBS by intracolonic enema. C57BL/6 mice received 100µL PBS. Colon tissues were collected at 3 and 60 days post MSC administration to evaluate the short-term and long-term effects of MSCs on inflammation and enteric neuropathy by histological and immunohistochemical analyses. In a separate set of experiments, multiple treatments with 4 × 106 and 2 × 106 MSCs were performed and tissue collected at 3 days post treatment. Chronic intestinal inflammation in Winnie mice was associated with persistent diarrhea, perianal bleeding, morphological changes, and immune cell infiltration in the colon. Significant changes to the ENS, including impairment of cholinergic, noradrenergic and sensory innervation, and myenteric neuronal loss were prominent in Winnie mice. Treatment with a single dose of bone marrow-derived MSCs was ineffective in attenuating chronic inflammation and enteric neuropathy in Winnie.

Mechanisms of Chemotherapy-Induced Neurotoxicity.

Since the first clinical trials conducted after World War II, chemotherapeutic drugs have been extensively used in the clinic as the main cancer treatment either alone or as an adjuvant therapy before and after surgery. Although the use of chemotherapeutic drugs improved the survival of cancer patients, these drugs are notorious for causing many severe side effects that significantly reduce the efficacy of anti-cancer treatment and patients' quality of life. Many widely used chemotherapy drugs including platinum-based agents, taxanes, vinca alkaloids, proteasome inhibitors, and thalidomide analogs may cause direct and indirect neurotoxicity. In this review we discuss the main effects of chemotherapy on the peripheral and central nervous systems, including neuropathic pain, chemobrain, enteric neuropathy, as well as nausea and emesis. Understanding mechanisms involved in chemotherapy-induced neurotoxicity is crucial for the development of drugs that can protect the nervous system, reduce symptoms experienced by millions of patients, and improve the outcome of the treatment and patients' quality of life.

The comparative anti-cancer effects of krill oil and oxaliplatin in an orthotopic mouse model of colorectal cancer.

BACKGROUND: Our in vitro studies demonstrated that krill oil (KO) has anti-cancer potential. This study aimed to compare the anti-cancer effects of KO with a commonly used chemotherapeutic drug, oxaliplatin and to identify the molecular mechanisms associated with KO supplementation in a mouse model of colorectal cancer (CRC). METHODS: Thirty-six male Balb/c mice were randomly divided into six groups. Five groups received standard chow diet supplemented with KO (150 g/kg)), corn oil (150 g/kg), KO combined with ½ dose of oxaliplatin (1.5 mg/kg body weight/3 times per week), corn oil combined with ½ dose of oxaliplatin (1.5 mg/kg body weight/3 times per week), or a full dose of oxaliplatin (3 mg/kg body weight/3 times per week). The control (sham) group received a standard chow diet. Treatments started three weeks before and continued for three weeks after orthotopic CRC induction. The number of metastases, tumour weight and volume were quantified ex-vivo. The expression of cytochrome c, cleaved caspase-9 and -3, DNA damage, PD-L1, PD-L2 and HSP-70 were determined. RESULTS: A significant reductions in the weight and volume of tumours were observed in mice treated with KO and KO plus a ½ dose of oxaliplatin compared to the sham group, similar to oxaliplatin-treated mice. KO, and KO plus ½ dose of oxaliplatin significantly increased the expression of cytochrome c, cleaved caspase-9 and -3, and DNA damage and decreased expression of PD-L1, PD-L2 and HSP-70 in tumour tissues compared to the sham group. CONCLUSIONS: The in vivo anti-cancer effects of KO are comparable with oxaliplatin. Thus, dietary KO supplementation has a great potential as a therapeutic/adjunctive agent for CRC treatment.

Krill oil supplementation reduces the growth of CT-26 orthotopic tumours in Balb/c mice.

BACKGROUND: We have previously reported that the free fatty acid extract (FFAE) of krill oil (KO) significantly inhibits the proliferation and migration, and induces apoptosis of colorectal cancer (CRC) cells. This study aimed to investigate the in vivo efficacy of various doses of KO supplementation on the inhibition of CRC tumour growth, molecular markers of proliferation, angiogenesis, apoptosis, the epidermal growth factor receptor (EGFR) and its downstream molecular signalling. METHODS: Male Balb/c mice were randomly divided into four groups with five in each group. The control (untreated) group received standard chow diet; and other three groups received KO supplementation at 5%, 10%, and 15% of their daily dietary intake respectively for three weeks before and after the orthotopic implantation of CT-26 CRC cells in their caecum. The expression of cell proliferation marker Ki-67 and angiogenesis marker CD-31 were assessed by immunohistochemistry. The expression of EGFR, phosphorylated EGFR (pEGFR), protein kinase B (AKT), pAKT, extracellular signal-regulated kinase (ERK1/2), pERK1/2, cleaved caspase-7, cleaved poly (ADP-ribose) polymerase (PARP), and DNA/RNA damage were determined by western blot. RESULTS: KO supplementation reduced the CRC tumour growth in a dose-dependent manner; with 15% of KO being the most effective in reduction of tumour weight and volume (68.5% and 68.3% respectively, P < 0.001), inhibition of cell proliferation by 69.9% (P < 0.001) and microvessel density by 72.7% (P < 0.001). The suppressive effects of KO on EGFR and its downstream signalling, ERK1/2 and AKT, were consistent with our previous in vitro observations. Furthermore, KO exhibited pro-apoptotic effects on tumour cells as indicated by an increase in the expression of cleaved PARP by 3.9-fold and caspase-7 by 8.9-fold. CONCLUSIONS: This study has demonstrated that KO supplementation reduces CRC tumour growth by inhibiting cancer cell proliferation and blood vessel formation and inducing apoptosis of tumour cells. These anti-cancer effects are associated with the downregulation of the EGFR signalling pathway and activation of caspase-7, PARP cleavage, and DNA/RNA damage.