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. 2011 Nov;31(11):3799-807.

Induction of Apoptosis by Cannabinoids in Prostate and Colon Cancer Cells Is Phosphatase Dependent

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Free PMC article

Induction of Apoptosis by Cannabinoids in Prostate and Colon Cancer Cells Is Phosphatase Dependent

Sandeep Sreevalsan et al. Anticancer Res. .
Free PMC article

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.

Conflict of interest statement

Disclosures/Conflicts of Interest: None

Figures

Figure 1
Figure 1
Concentration-dependent effects of WIN and CBD on LNCaP (A, B) and SW480 (C, D) cell proliferation. Cells were treated with DMSO and 2.5–7.5 μM of the synthetic cannabinoid WIN-55,212 (WIN) and 5–15 μM of cannabidiol (CBD) for 72 hr. Cells were counted as described in the Materials and Methods. Results are expressed as mean ± S.E. for three replicate determinations for each treatment group, and significantly (p<0.05) reduced proliferation is indicated (*). Proliferation of cells treated with DMSO (solvent control) was set at 100%. β-Actin served as a loading control for all Western blots.
Figure 2
Figure 2
Induction of apoptosis by the synthetic cannabinoid WIN-55,212 (WIN) and cannabidiol (CBD). Time-dependent effects of WIN and CBD on cleaved poly(ADP)ribose polymerase (PARP) expression in LNCaP (A, B) and SW480 (C, D) cells. LNCaP and SW480 cells were treated with dimethylsulfoxide (DMSO), 7.5 μM WIN (12, 24 and 48 h) and 15 μM CBD (12, 18, and 24 h), and cPARP expression was determined by Western blot analysis of whole-cell lysates as described in the Materials and Methods.
Figure 3
Figure 3
Effects of the synthetic cannabinoid WIN-55,212 (WIN) and cannabidiol (CBD) on phosphatase mRNA levels in LNCaP cells. Induction by WIN (A, B) and CBD (C, D). LNCaP cells were treated with dimethylsulfoxide (DMSO), 7.5 μM WIN and 15 μM CBD for 24 h, and dual specificity phosphatase 1 (DUSP1), dual specificity phosphatase 4 (DUSP4), dual specificity phosphatase 10 (DUSP10), serum prostatic acid phosphate (sACPP), cellular prostatic acid phosphate (cACPP), protein tyrosine phosphatase, non-receptor type 1 (PTPN1), protein phosphatase 2A activator, regulatory subunit 4 (PPP2R4), protein tyrosine phosphatase, receptor type, J (PTPRJ) and protein tyrosine phosphatase, non-receptor type 6 (PTPN6) mRNA levels were determined by real-time PCR as described in the Materials and Methods. Results are expressed as mean ± S.E. for 3 replicate experiments and significant (p<0.05) increases are indicated (*).
Figure 4
Figure 4
Effects of the synthetic cannabinoid WIN-55,212 (WIN) and cannabidiol (CBD) on phosphatase mRNA levels in SW480 cells. Induction by WIN (A, B) and CBD (C, D). SW480 cells were treated with dimethylsulfoxide (DMSO), 7.5 μM WIN and 15 μM CBD for 24 h, and dual specificity phosphatase 1 (DUSP1), dual specificity phosphatase 4 (DUSP4), dual specificity phosphatase 10 (DUSP10), serum prostatic acid phosphate (sACPP), cellular prostatic acid phosphate (cACPP), protein tyrosine phosphatase, non-receptor type 1 (PTPN1), protein phosphatase 2A activator, regulatory subunit 4 (PPP2R4), protein tyrosine phosphatase, receptor type, J (PTPRJ) and protein tyrosine phosphatase, non-receptor type 6 (PTPN6) mRNA levels were determined by real-time PCR as described in the Materials and Methods. Results are expressed as mean ± S.E. for 3 replicate experiments and significant (p<0.05) increases are indicated (*).
Figure 5
Figure 5
Effects of phosphatase inhibitor sodium orthovanadate (SOV) on cannabinoid-induced PARP cleavage in LNCaP (A) and SW480 (B) cells. Cells were pre-treated with SOV followed by treatment with the synthetic cannabinoid WIN-55,212 (WIN) and cannabidiol (CBD) for 24 and 12 h, respectively, and whole-cell lysates were analyzed by Western blot analysis as described in the Materials and Methods. β-Actin served as a loading control for all Western blots. Effects of cannabinoids and CB1 and CB2 receptor knockdown by RNA interference in LNCaP (C) and SW480 (D) cells. Cells were transfected with siRNAs against CB1 and CB2 receptors followed by treatment with CBD for 12 h and whole-cell lysates were examined for expression of cleaved poly(ADP)ribose polymerase (cPARP), cleaved caspase-3, and CB1 and CB2 receptor proteins determined by Western blot analysis as indicated in the Materials and Methods. β-Actin served as a loading control for all Western blot analyses.

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