Apoptosis is programmed cell death, cell suicide. Cannabinoids come from cannabis. This science says: “We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases.
“The anticancer activities of phytochemicals and their synthetic derivatives is complex and dependent on tumor type; however, several studies have shown that an important component of their activity is associated with induction of specific phosphatases which in turn inhibit kinase signaling pathways that are overexpressed in many tumor types. For example, several phytochemical anticancer agents, including betulinic acid and other polycyclic terpenoids, inhibit signal transducer and activator of transcription 3 (STAT3) activation and this has been linked to induction of the protein tyrosine phosphatase, non-receptor type 6 (PTPN6) (1–7). Curcumin, resveratrol and calcitriol (vitamin D analog) also induce expression of the dual specificity phosphatase 10 (DUSP10) mRNA in prostate cancer cell lines (8, 9) and this response has been linked to inhibition of p38 stress kinase activity.
Cannabinoids have long been used for ameliorating the debilitating effects of cytotoxic anticancer drugs; however, these compounds also exhibit antitumorigenic activity against multiple tumor types (9–13) and are already in clinical trials for treatment of brain tumors (e.g. gliomas). The mechanisms of action of cannabinoids are complex and dependent on ligand structure and cell context, and the effects are both receptor (CB1 and/or CB2) dependent and independent. Like many other phytochemical anticancer agents, cannabinoids induce growth inhibitory, proapoptotic and antimetastatic responses which are accompanied by modulation of several kinase activities (14–25). We hypothesized that cannabinoids may also induce phosphatases and the first objective of this study was to investigate the effects of the cannabinoids WIN 55,512-22 (WIN) and cannabidiol (CBD) on apoptosis in colon and prostate cancer cells. The second objective was to investigate the induction of phosphatases and the role of the cannabinoid (CB) receptors (CB1 and CB2) and the phosphatase inhibitor sodium orthovanadate (SOV) on cannabinoid-induced apoptosis.”
Induction of Apoptosis by Cannabinoids in Prostate and Colon Cancer Cells Is Phosphatase Dependent
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.
Discussion
Cannabinoids have emerged as an important new class of anticancer drugs that bind cannabinoid receptors and activate several downstream pathways leading to inhibition of cancer cell proliferation, and induction of apoptosis (10–13). Their mechanisms of action are complex and dependent on cell context and ligand structure and there are examples of cannabinoid-mediated activities that are receptor dependent and independent (14–24). Several studies show that cannabinoids such as WIN, Δ9-tetrahydrocannabinol (ΔTHC) and CBD modulate kinase activities in cancer cell lines and this includes inhibition of membrane-bound and intracellular kinases, resulting in their dephosphorylation. The mechanisms associated with cannabinoid-induced inactivation of kinases is unknown; however, several different classes of anticancer drugs induce dual specificity and protein tyrosine phosphatases which selectively inactivate phosphokinases that play a role in cancer cell growth and survival (1–8, 27–33).
Previous findings on the effects of cannabinoids on activated phosphokinases are highly variable in different cancer cell lines and dependent on the structure of the cannabinoid and cell context. WIN inhibited growth and induced apoptosis in LNCaP cells and this was due in part to sustained phosphorylation of p42/44 MAPK and lower p-AKT expression (22). The phytochemical ΔTHC inhibited growth and induced apoptosis in SW480 cells and this was accompanied by reduced expression of phospho-p42/p44 MAPK and phospho-AKT (19).
Although the reported effects of cannabinoids on phosphokinases were highly variable among different cell lines, we observed that WIN and CBD induced mRNA expression of several dual specificity and protein tyrosine phosphatases in LNCaP and SW480 cells (Figures 3 and ?and4).4). Selection of the phosphatases was based on their putative anticarcinogenic activities and on previous reports showing that dual specificity and protein tyrosine phosphatases were induced by several phytochemical anticancer agents including curcumin, resveratrol, ursolic acid, betulinic acid, guaiane sesquiterpenoids, guggulsterone, epigallocatechin-3-gallate and retinoic acid (1–8, 27–33). Previous studies with these compounds primarily focused on induction of a single phosphatase gene, whereas the results of this study showed that WIN and CBD induced mRNA expression of multiple and overlapping phosphatases (Figures 3 and ?and4).4). WIN and CBD induced DUSP1 and DUSP10 after treatment for 24 h in LNCaP cells, whereas DUSP4 induction by CBD and WIN was time dependent (24 and 48 h, respectively) (Figure 3). In SW480 cells, the pattern of phosphatase induction by WIN and CBD differed after treatment for 24 h primarily due to induction of DUSP1 by CBD and not WIN (Figure 4); however, after 48 h, WIN also induced DUSP1 (data not shown). Since both CB receptor antagonists alone induced expression of phosphatase mRNAs (data not shown), the role of the CB receptor in mediating induction of individual phosphatase mRNAs was not further investigated. In addition, the effects of CBD-and WIN-dependent induction of gene expression of dual specificity and protein tyrosine phosphatases on dephosphorylation of phosphokinase proteins was confounded by the parallel decrease of most phosphokinase and total kinase proteins (data not shown). It was apparent in SW480 cells that WIN dramatically reduced AKT phosphorylation without reducing total AKT protein, and this was comparable to the effects reported for ΔTHC in SW480 cells (19). The only other consistent observation was that WIN and CBD enhanced phosphorylation of the p38 stress protein kinase in both cell lines (data not shown). These results demonstrate that WIN-/CBD-induced phosphatases may play a role in kinase inactivation in SW480 cells; however, these results were confounded by the parallel decrease in kinase proteins, and the mechanisms of the latter response are currently being investigated.
CBD and WIN also inhibited LNCaP and SW480 cell growth (Figure 1) and induced apoptosis (Figure 2) in both cell lines, and induction of apoptosis by cannabinoids is an important element of their anticancer activity (6–9). Induction of caspase-dependent PARP cleavage was used as an indicator of apoptosis and it was clear that both WIN and CBD induced PARP cleavage in LNCaP and SW480 cells (Figure 2) and this is consistent with cannabinoid-induced apoptosis in many other cancer cell lines (10–24). Moreover, we also observed that WIN-/CBD-induced PARP cleavage dramatically decreased in cells co-treated with the phosphatase inhibitor SOV (Figure 5A and B), indicating that cannabinoid-induced phosphatases played a role in the induction of SW480 and LNCaP cell death. It should be noted that although induction of PARP cleavage by WIN was maximal after 24 h, there was significant induction within 12 h in LNCaP and SW480 cells (Figure 2). In contrast, induction of phosphatase mRNA levels after treatment with WIN for 12 h was relatively low in SW480 cells, whereas modest but significant induction was observed in LNCaP cells (data not shown). These results suggest that other ‘proapoptotic’ phosphatases may also be induced or activated by WIN and CBD and these are currently being investigated. CB1 and CB2 receptor knockdown by siRNA was used to determine the role of the CB receptors in mediating CB- and WIN-induced proapoptotic activity (cleavage of PARP and caspase-3) and the results demonstrate that the effects of WIN were CB receptor independent, whereas CBD-induced apoptosis was CB receptor dependent (Figure 5).
In summary, this study shows for the first time that cannabinoids induced mRNA expression of several phosphatases in LNCaP and SW480 cells and this was consistent with induction of phosphatases by other phytochemical anticancer drugs (1–8, 27–33). The direct effects of phosphatase induction on reduced phosphokinase protein expression was confounded by a parallel decrease in most (total) kinase proteins. This response may also be important for the anticancer activities of WIN and CBD and is currently being investigated. Previous studies have reported induction of DUSP1 and DUSP6 by cannabinoids in microglial cells and this resulted in inactivation of MAPK; however, effects on apoptosis were not determined (34, 35). The results obtained using the phosphatase inhibitor SOV demonstrate that induction of apoptosis by WIN and CBD was significantly blocked by the phosphatase inhibitor SOV, and this represents a novel proapoptotic pathway induced by cannabinoids. Current studies are focused on identification of specific proapoptotic phosphatases and their mechanisms of induction or activation by cannabinoids.
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.
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