Since there is no peer-reviewed information on the effect of cannabis and cannabis compounds on OCSC development and properties, we sought to summarize the known effects of phytocannabinoids on genetic pathways and biological processes related to CSCs and OCSCs. Indeed, several of the major phytocannabinoids have been shown to affect signaling pathways that are mainly involved with OC stemness.

CBD, cannabidivarin (CBDV; Table 1), and the 2,3-epoxy derivative of CBD exhibited, in a dose-dependent manner, considerable inhibitory activity against the Wnt/β-catenin pathway [70]. In general, enhanced Wnt/β-catenin pathway activity is a hallmark of the stemness and drug resistance of OCSC and other cancers [46,47,49]. In addition, in vivo intraperitoneal administration of “cannabis smoke” (in the form of condensate cannabis resin extract collected in a smoking machine and dissolved in olive oil) inhibited ALDH activity in a rat model [71]. ALDH activity, induced by the Wnt/β-catenin pathway, is another marker closely associated with the OCSC state [44,47,61]. In this case, it might be that inhibition of the Wnt/β-catenin signaling pathway promoted the inhibition of ALDH activity (Figure 1).

THC (via CB1 activation) and CBD were shown to inhibit Akt phosphorylation in colorectal cancer cells and to induce apoptosis in these cells via inhibition of the PI3K-Akt survival signaling cascade (Figure 1; [72,73]). In contrast, CBD and THC were suggested to promote PI3K/Akt signaling and thereby downregulate the expression of GSK3-β, a serine/threonine kinase and an inhibitor of the Wnt/β-catenin pathway in various glaucoma-related models [74]. However, these trends of upregulation of PI3K/Akt signaling by CBD or THC were primarily shown in non-cancerous cells (e.g., mesenchymal stem cells derived from gingiva; non-cancerous mice brain [74]) and are opposite to what was reported for cancer cells [72,73]. Perhaps the activity of CBD or THC on malignant and non-malignant cells differs with their effect on the PI3K/Akt and related pathways.

As detailed above, the HH-GLI signaling pathway is involved with OC and OCSC drug resistance and upregulation of the ABC transporters’, ABCB1 and ABCG2, gene expression [44]. THC is a direct inhibitor of the HH-GLI signaling pathway in vitro, and in mice that bear a subthreshold defect in HH signaling. The inhibitory activity of the HH-GLI pathway by THC was not mediated via the CB1 receptor (Figure 1; [75]). HH-GLI pathway inhibition by phytocannabinoids may suggest the alteration of ABC transporter expression and activity by these compounds. Increased sensitivity to THC-induced hypothermia was evident in ABC transporter knockout mice in comparison to wild type mice [76], further solidifying a connection between the THC and ABC transporter expression or activity. In contrast, using the in vitro bidirectional transport assay, it was shown that neither CBCA nor CBC inhibited the ABC transporter activity. CBCA only served as a substrate for the ABCB1 transporter (Figure 1; [77]). CBD is also not a substrate of the ABC transporters ABCB1 or ABCG2 [78].

CBD treatment downregulated the expression of CD44 in human gingival mesenchymal stem cells in vitro (Figure 1; [79]). A reduction in the number of CD44+ and CD133+ cells was also obtained with CBD treatment of cisplatin-resistant non-small cell lung cancer compared to controls. This treatment suppressed additional CSC-associated properties, including sphere formation and protein expression of Snail, Nanog, and Vimentin in cell lines [80]. The apoptotic activity of CBD on the cell lines was mediated via the TRPV2 receptor. In addition, in a mouse xenograft model of these drug-resistant non-small cell lung cancer cells, CBD treatment reduced tumor progression and metastasis [80].

In lipopolysaccharide (LPS)-stimulated microglia cells, CBD inhibited NADPH oxidase-mediated ROS production and NF-κB-dependent signaling events (Figure 1; [81]). This activity was only slightly reduced by blocking the CB2 receptor and was mainly receptor-independent [81]. This inhibitory activity of CBD on NF-κB-dependent signaling events might accord with its inhibitory effect on CD44+ expression or display, as the NF-κB pathway is activated in CD44+ OCSCs [44,59].

CBD and other phytocannabinoids induce endoplasmic reticulum stress [31,32,82] and, subsequently, in MDA-MB231 breast cancer cells, inhibit AKT and mTOR signaling (Figure 1; [82]). In these cells, CBD inhibited the association between BCL-2 and beclin1 and induced Bax elevation (Figure 1; [82]). It also activated caspase-8 and led to the cleavage of beclin-1 [82]. This cleavage product translocases to mitochondria and enhances the release of cyt c to the cytosol [82]. In support, in cisplatin-resistant non-small cell lung cancer cells, an increase in cleaved caspase-3 and cyt c release was evident upon CBD treatments [80]. As indicated above, the release of cyt c to the cytoplasm activates caspase and ensures apoptosis (Figure 1; [65,82]).

CBD was demonstrated to inhibit the expression of ID1 in breast cancer cells, which maintains cancer stemness [64], and, as a direct result, leads to anti-metastatic activity [83]. CBD also inhibited the expression of ID1 in head and neck, prostate, and salivary gland cancers [84]. In vivo, mice with advanced metastatic progression, once treated with the CBD analog O-1663, had a high survival rate, even beyond that of the group treated with CBD [13,83].

Lastly, CBD was shown to inhibit EMT and induce reversion to a non-invasive phenotype in breast cancer cells [33]. CBD treatment blocked cell migration and inhibited the progression of the IL-1β/IL-1RI/β-catenin signaling pathway [33]. The treatment re-localized β-catenin and E-cadherin to the adherent junctions and re-established the epithelial organization lost by the IL-1β-induced dispersion of the cells. It also prevented the nuclear translocation of β-catenin and decreased the overexpression of several genes, ID1 proteins included [33]. Here too, CBD treatment inhibited AKT activation (Figure 1; [33]).