Category Archives: Colon Cancer

The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2

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Abstract

“BACKGROUND AND AIMS:

Cyclooxygenase 2 (COX-2) is upregulated in most colorectal cancers and is responsible for metabolism of the endogenous cannabinoid, anandamide, into prostaglandin-ethanolamides (PG-EAs). The aims of this study were to determine whether anandamide and PG-EAs induce cell death in colorectal carcinoma (CRC) cells, and whether high levels of COX-2 in CRC cells could be utilised for their specific targeting for cell death by anandamide.

METHODS:

We determined the effect of anandamide on human CRC cell growth by measuring cell growth and cell death, whether this was dependent on COX-2 protein expression or enzyme activity, and the potential involvement of PG-EAs in induction of cell death.

RESULTS:

Anandamide inhibited the growth of CRC cell lines HT29 and HCA7/C29 (moderate and high COX-2 expressors, respectively) but had little effect on the very low COX-2 expressing CRC cell line, SW480. Induction of cell death in HT29 and HCA7/C29 cell lines was partially rescued by the COX-2 selective inhibitor NS398. Cell death induced by anandamide was neither apoptosis nor necrosis. Furthermore, inhibition of fatty acid amide hydrolase potentiated the non-apoptotic cell death, indicating that anandamide induced cell death was mediated via metabolism of anandamide by COX-2, rather than its degradation into arachidonic acid and ethanolamine. Interestingly, both PGE2-EA and PGD2-EA induced classical apoptosis.

CONCLUSIONS:

These findings suggest anandamide may be a useful chemopreventive/therapeutic agent for colorectal cancer as it targets cells that are high expressors of COX-2, and may also be used in the eradication of tumour cells that have become resistant to apoptosis.”

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1774787/

The endogenous cannabinoid, anandamide, induces COX-2-dependent cell death in apoptosis-resistant colon cancer cells.

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Abstract

“Despite recent advances in understanding colorectal tumour biology, there is still a need to improve the 5-year survival rate of patients with colorectal cancer as approximately 40% of patients presenting with advanced disease will remain resistant to therapy. One of the major contributing factors in resistance to therapy is the failure of colorectal tumour cells to undergo apoptosis. Hence there is an urgent need to develop novel therapeutic approaches that can target apoptosis-resistant cells. To this end, we investigated the potential efficacy of the endogenous cannabinoid anandamide to induce cell death in apoptosis-resistant colon cancer cells. Here, for the first time, we show that anandamide can induce cell death in the apoptosis-resistant HCT116 Bax-/- colorectal cell line. Importantly, we provide direct genetic evidence that this induction of cell death is dependent on COX-2 expression. Interestingly, increased COX-2 expression also sensitised the SW480 colorectal cancer cell line (low endogenous COX-2) to anandamide-induced death, whereas COX-2 suppression by RNAi inhibited anandamide-induced cell death in the HCA7 colorectal cancer cell line (high endogenous COX-2 expression). This COX-2-dependent death was independent of cannabinoid receptor engagement (CB1 or CB2), and not a direct consequence of reactive oxygen species (ROS) formation. This study demonstrates a novel utilisation for COX-2 expression, targeting apoptotic defective colorectal cancer cells for destruction by anandamide. As COX-2 is not expressed in the normal colorectal epithelium, but highly expressed in colorectal tumours and apoptosis resistance contributes to treatment failure, these data suggest that anandamide has the potential to be an effective therapeutic in colorectal cancer.”

http://www.ncbi.nlm.nih.gov/pubmed/20514410

Effects of anandamide on polyamine levels and cell growth in human colon cancer cells.

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Abstract

“BACKGROUND:

Anandamide (AEA) is an endogenous agonist for cannabinoid receptor CB1-R and seems to be involved in the control of cancer growth. Polyamines are compounds that play an important role in cell proliferation and differentiation. Our aim was to investigate the effect of AEA on the polyamine levels (putrescine, spermidine and spermine) and cell growth of three human colon cancer cell lines, positive for CB1-R.

MATERIALS AND METHODS:

After AEA treatment of DLD-1, HT-29 and SW620 cells, polyamine analysis was performed by high-performance liquid chromatography (HPLC) and cell growth was measured by 3-(4,5 di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. CB1 gene expression was determined using reverse transcription and polymerase chain reaction (RT-PCR).

RESULTS:

AEA significantly reduced polyamine levels and cell proliferation dose-dependently when the tested cell lines were exposed for 24 h and 48 h. This inhibitory effect was mediated by CB1-R, since SR 1411716A, a selective CB-1 receptor antagonist, was able to entirely antagonize the effect of AEA. CB1-R mRNA levels were enhanced after AEA treatment in DLD-1 cells, whereas no induction was found in HT-29 and SW620 cells.

CONCLUSION:

It appears that mechanisms by which AEA may affect growth of colon cancer cells involve a decrease in cell proliferation rate by reducing the polyamine levels.”

http://www.ncbi.nlm.nih.gov/pubmed/20682986

Alternative targets within the endocannabinoid system for future treatment of gastrointestinal diseases.

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Abstract

“Many beneficial effects of herbal and synthetic cannabinoids on gut motility and inflammation have been demonstrated, suggesting a vast potential for these compounds in the treatment of gastrointestinal disorders. These effects are based on the so-called ‘endocannabinoid system’ (ECS), a cooperating network of molecules that regulate the metabolism of the body’s own and of exogenously administered cannabinoids. The ECS in the gastrointestinal tract quickly responds to homeostatic disturbances by de novo synthesis of its components to maintain homeostasis, thereby offering many potential targets for pharmacological intervention. Of major therapeutic interest are nonpsychoactive cannabinoids or compounds that do not directly target cannabinoid receptors but still possess cannabinoid-like properties. Drugs that inhibit endocannabinoid degradation and raise the level of endocannabinoids are becoming increasingly promising alternative therapeutic tools to manipulate the ECS.”

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174079/

Induction of apoptosis by cannabinoids in prostate and colon cancer cells is phosphatase dependent.

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Abstract

“AIM:

We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases.

MATERIALS AND METHODS:

The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown.

RESULTS:

CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent.

CONCLUSION:

Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.”

http://www.ncbi.nlm.nih.gov/pubmed/22110202

Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.

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Abstract

“BACKGROUND & AIMS:

Two G-protein-coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function.

METHODS:

The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments.

RESULTS:

CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1-lysophosphatidic acid 1 heteromeric receptor complex.

CONCLUSIONS:

CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity.”

http://www.ncbi.nlm.nih.gov/pubmed/16083701

Cannabinoid Receptor Activation Induces Apoptosis through Tumor Necrosis Factor α–Mediated Ceramide De novo Synthesis in Colon Cancer Cells

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Abstract

“PURPOSE:

Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB1 and CB2, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation.

EXPERIMENTAL DESIGN:

Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB1 agonist arachinodyl-2′-chloroethylamide and the CB2 agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-alpha production were evaluated. The knockdown of TNF-alpha mRNA was obtained with the use of selective small interfering RNA.

RESULTS:

We show that the CB1 receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB2 receptor. The activation of the CB1 and, more efficiently, of the CB2 receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB2 agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-alpha mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation.

CONCLUSIONS:

The present study shows that either CB1 or CB2 receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-alpha acts as a link between cannabinoid receptor activation and ceramide production.”

http://clincancerres.aacrjournals.org/content/14/23/7691.long

Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil.

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Abstract

“Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [(3)H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, alpha-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.”

http://www.ncbi.nlm.nih.gov/pubmed/18629502

Interaction of endocannabinoid system and steroid hormones in the control of colon cancer cell growth.

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Abstract

“Increasing evidence suggest the role of the cannabinoid receptors (CBs) in the control of cell survival or death and signaling pathways involved in tumor progression. Cancer cell lines are characterized by a subtle modulation of CB levels which produces a modified responsiveness to specific ligands, but the molecular mechanisms underlying these events are poorly and partially understood. We previously provided evidence that the endocannabinoid (EC) anandamide (AEA) exerts anti-proliferative effect likely by modulation of the expression of genes involved in the cellular fate. In this study we focused on the role of the CB1 receptor, ECs, and steroids in the mechanisms involved in colorectal cancer (CRC) cell growth inhibition in vitro. We demonstrated that, in DLD1 and SW620 cells, 17β-estradiol induced a specific and strong up-regulation of the CB1 receptor by triggering activation of the CB1 promoting region, localized at the exon 1 of the CNR1 gene. Moreover, treatment of DLD1 and SW620 cells with Met-F-AEA, a stable AEA-analogous, or URB597, a selective inhibitor of FAAH, induced up-regulation of CB1 expression by co-localization of PPARγ and RXRα at the promoting region. Finally, increased availability of AEA, of both exogenous and endogenous sources, induced the expression of estrogen receptor-beta in both cell lines. Our results partially elucidated the role of EC system in the molecular mechanisms enrolled by steroids in the inhibition of colon cancer cell growth and strongly suggested that targeting the EC system could represent a promising tool to improve the efficacy of CRC treatments.”

http://www.ncbi.nlm.nih.gov/pubmed/21412772

Possible endocannabinoid control of colorectal cancer growth.

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Abstract

“BACKGROUND & AIMS:

The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1). the levels of endocannabinoids, cannabinoid CB(1) and CB(2) receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (2). the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation.

METHODS:

Tissues were obtained from 21 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB(1), CB(2), and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-2 and DLD-1) were used to test antiproliferative effects.

RESULTS:

All tissues and cells analyzed contain anandamide, 2-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and 2-fold higher in adenomas and CRCs than normal mucosa. Anandamide, 2-AG, and the CBR agonist HU-210 potently inhibit CaCo-2 cell proliferation. This effect is blocked by the CB(1) antagonist SR141716A, but not by the CB(2) antagonist SR144528, and is mimicked by CB(1)-selective, but not CB(2)-selective, agonists. In DLD-1 cells, both CB(1) and CB(2) receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-2 cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-2 cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids.

CONCLUSIONS:

Endocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents.”

http://www.ncbi.nlm.nih.gov/pubmed/12949714