umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cannabinoids as modulators of cancer cell viability, neuronal differentiation, and embryonal development
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology. (Jacobsson)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Effekter av cannabinoider på cancerceller, neuronal differentiering och embryonal utveckling (Swedish)
Abstract [en]

Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis.

In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied.  In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production.

In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients.

Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist).

In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2012. , 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1474
Keyword [en]
Cannabinoids, cell viability, neuronal differentiation, colorectal cancer, chick embryo
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi; Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-51560ISBN: 978-91-7459-358-7 (print)OAI: oai:DiVA.org:umu-51560DiVA: diva2:488475
Public defence
2012-02-24, Sal E04, by 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-02-03 Created: 2012-01-26 Last updated: 2012-02-27Bibliographically approved
List of papers
1. Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil
Open this publication in new window or tab >>Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil
2009 (English)In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 63, no 4, 691-701 p.Article in journal (Refereed) Published
Abstract [en]

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 [3H]-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, α-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.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2009
Keyword
Cannabinoids, Polyunsaturated fatty acids, 5-Fluorouracil, Colorectal cancer, Cytotoxicity
National Category
Pharmacology and Toxicology
Research subject
Toxicology; biokemisk farmakologi
Identifiers
urn:nbn:se:umu:diva-50488 (URN)10.1007/s00280-008-0788-5 (DOI)
Available from: 2011-12-11 Created: 2011-12-11 Last updated: 2017-12-08Bibliographically approved
2. High tumour cannabinoid CB(1) receptor immunoreactivity negatively impacts disease-specific survival in stage II microsatellite stable colorectal cancer
Open this publication in new window or tab >>High tumour cannabinoid CB(1) receptor immunoreactivity negatively impacts disease-specific survival in stage II microsatellite stable colorectal cancer
Show others...
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 8, 1-11 p.Article in journal (Refereed) Published
Abstract [en]

Background: There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB(1) receptor immunoreactive intensity (CB(1)IR intensity) is associated with disease severity and outcome.

Methodology/Principal Findings: CB(1)IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB(1)IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB(1)IR <2 and >= 2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB1IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB(1)IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB(1)IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB(1)IR upon disease specific survival. The 5 year probabilities of event-free survival were: 8565 and 66+/-8%; tumour interior, 86+/-4% and 63+/-8% for the CB(1)IR<2 and CB(1)IR >= 2 groups, respectively.

Conclusions/Significance: The level of CB(1) receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB(1)IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients.

Place, publisher, year, edition, pages
San Francisco, CA: Public Library of Science, 2011
National Category
Biomedical Laboratory Science/Technology Biological Sciences
Identifiers
urn:nbn:se:umu:diva-47396 (URN)10.1371/journal.pone.0023003 (DOI)
Available from: 2011-09-23 Created: 2011-09-20 Last updated: 2017-12-08Bibliographically approved
3. Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells
Open this publication in new window or tab >>Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells
Show others...
2013 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 87, no 11, 1939-1951 p.Article in journal (Refereed) Published
Abstract [en]

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211(with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 µM and 0.6 µM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects, and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm2. The CB-induced cytotoxicity, that appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs, produce seemingly unspecific cytotoxic effects in the P19 EC cells.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2013
Keyword
cannabinoids, polyunsaturated fatty acids, embryonal carcinoma cells, cytotoxicity, oxidative stress
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi
Identifiers
urn:nbn:se:umu:diva-51550 (URN)10.1007/s00204-013-1051-3 (DOI)000325700300005 ()
Available from: 2012-01-31 Created: 2012-01-26 Last updated: 2017-12-08Bibliographically approved
4. The effects of cannabinoids on the viability and differentiation of neurons derived from retinoic acid-induced  P19 embryonal carcinoma cells
Open this publication in new window or tab >>The effects of cannabinoids on the viability and differentiation of neurons derived from retinoic acid-induced  P19 embryonal carcinoma cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cannabinoids and cannabinoid receptors play an important role in development and differentiation of the nervous system, but the mechanisms behind that role have not been fully elucidated. We have examined the effects of synthetic and endogenous cannabinoids and related polyunsaturated fatty acids upon mouse embryonal carcinoma P19 stem cell viability - before, during and after retinoic acid (RA)-induced neural differentiation. Experiments were also performed to investigate whether the cannabinoids affect the differentiation of P19-derived neurons by measuring the development and growth of neurites and intracellular acetylcholinesterase activity.

Both synthetic and endogenous cannabinoids as well as related fatty acids produced a concentration-dependent decrease in undifferentiated P19 cell viability, but induction of the neural pathway reduced the sensitivity to the cytotoxic effects, and in differentiated neurons anandamide and related fatty acids showed no cytotoxicity. However, synthetic cannabinoids such as HU 210, HU 211 and WIN 55,212-2 produced cytotoxicity in both undifferentiated and differentiated cells, but there was a right-shifted concentration-effect curve in RA-induced cells and differentiated neurons compared with the undifferentiated cells.

HU 210 produced a time- and concentration-dependent decrease in cell number, percentage of cells expressing neurites, number of neurites per cell and neurite length. Statistically significant inhibition was seen at a concentration of 1 µM to 3 µM, and this was confirmed by the measurement of intracellular acetylcholinesterase activity, an enzyme that is dramatically increased during the differentiation process, where HU 210 significantly decreased the activity after six and nine days of exposure. However, these effects of HU 210 could only be observed in the same concentration range as those affecting neuronal viability. Anandamide, on the other hand, had modest effect on measured markers of neuronal differentiation but decreased the fraction of neurite expressing cells and neurite length after nine days of exposure at a concentration ≥ 10 µM. No effect on the acetylcholinesterase activity was observed.

It is concluded that cannabinoids and related fatty acids have cytotoxic effects in undifferentiated P19 embryonal carcinoma cells, but induction of the neuronal pathway reduces the sensitivity to the cytotoxic effects. The synthetic cannabinoids are more potent than the endogenous cannabinoids and fatty acids in causing cytotoxicity in differentiated neurons, but the CB-induced decrease in neurite formation and acetylcholinesterase activity in RA-induced P19-derived neurons occurs only at concentrations that cause measurable neuronal cell death. 

Keyword
Cannabinoids, P19 EC cells, cell viability, neuronal differentiation, neurite formation, acetylcholinesterase activity
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi
Identifiers
urn:nbn:se:umu:diva-51555 (URN)
Available from: 2012-01-31 Created: 2012-01-26 Last updated: 2012-02-10Bibliographically approved
5. Effects of cannabinoids on the development of chick embryos in ovo
Open this publication in new window or tab >>Effects of cannabinoids on the development of chick embryos in ovo
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have examined the effects of the synthetic cannabinoids HU 210 and HU 211, the plant-derived cannabidiol and the endogenous cannabinoid anandamide on the viability and development of chick embryos. Fertilized White Leghorn chicken eggs were injected with the test compounds or carrier vehicle, via a drilled small hole in the blunt end of the egg, directly into the egg yolk. After nine days of exposure, the embryonal viability and development stages were assessed according to the Hamburger and Hamilton (HH) scale. The potent synthetic cannabinoid receptor agonist HU 210 and the non-psychotropic cannabidiol were embryotoxic at the highest concentrations examined (10 µM and 50 µM, respectively), with no viable embryos (average HH stage 19 ± 3.5 compared to 34 ± 1.1 in the DMSO-treated control group) after the HU 210 injection, and 22% viability (average HH stage 11 ± 4.9) after cannabidiol injections. The effects of HU 210 on the chick embryo were attenuated by 100 µM of the antioxidant α-tocopherol and the cannabinoid receptor antagonist AM251 (1 µM), whereas only α-tocopherol gave a statistically significant protection against the embryotoxic effects of cannabidiol. HU 211, an enantiomer to HU 210 without cannabinoid receptor activity, and anandamide were without any significant effects on the viability and development of the chick embryo at the concentrations examined (up to 10 µM and 50 µM, respectively).

This study shows that exposure to both plant-derived and synthetic cannabinoids during early embryonal development decreased embryonal viability. Extrapolation of data across species is of course difficult, but the data would argue against the use of cannabinoids, be it recreationally or therapeutically, during pregnancy. 

Keyword
cannabinoids, chick embryo, viability, Hamburger-Hamilton scale, α-tocopherol
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:umu:diva-51558 (URN)
Available from: 2012-01-31 Created: 2012-01-26 Last updated: 2012-02-03Bibliographically approved

Open Access in DiVA

Avhandling(33861 kB)951 downloads
File information
File name FULLTEXT01.pdfFile size 33861 kBChecksum SHA-512
0aa47446f4b412b15c28b7af64490c96f658fbf704b27588d234b9f8af2f8c68ed8e314a6276dfd3035463d19d09a3621c16ab810c2c94da61491f2e341bb0ad
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Gustafsson, Sofia
By organisation
Pharmacology
Pharmacology and Toxicology

Search outside of DiVA

GoogleGoogle Scholar
Total: 951 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 6054 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf