Accumulating research indicates that, in addition to their well-established palliative effects on cancer-related symptoms, cannabinoids can actively suppress tumor growth. This antitumor activity is mediated by the modulation of critical cellular signaling pathways involved in the regulation of cancer cell proliferation and survival. Furthermore, cannabinoids have been shown to inhibit angiogenesis and impair cell proliferation across various tumor types [15]. It has been shown that both natural cannabinoid ligands and synthetic ligands reduce tumor cell proliferation, including that of prostate tumor cells [11,15]. Specifically, endocannabinoids exert potent antiproliferative effects across a wide spectrum of cancer types, including breast, brain, skin, thyroid, prostate, and colorectal carcinomas [16]. For instance, elevated levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported to inhibit the invasion of PC-3 and DU-145 prostate cancer cells; conversely, suppressing 2-AG synthesis or pharmacologically blocking its binding to CB₁ receptors with antagonists stimulates cell invasiveness [17]. Furthermore, Olea-Herrero et al. demonstrated that the CB₂ agonist JWH-015 significantly reduced tumor growth in vivo and exhibited robust antiproliferative effects in PC-3 cells in vitro [11].

Prior literature indicates that the expression levels of both CB₁ and CB₂ receptors are significantly elevated in human prostate cancer cells compared to their normal counterparts. Pharmacological administration of a dual CB₁/CB₂ agonist has been shown to reduce proliferation, migration, and invasion in prostate cancer cells, while simultaneously inducing apoptosis and triggering cell cycle arrest at the G0/G1 phase. Furthermore, this treatment leads to a marked downregulation in the protein expression of key oncogenic markers, including prostate-specific antigen (PSA), androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor (VEGF) [10,13].

The selective CB₂ agonist JWH-133 has been demonstrated to exert potent antiproliferative and antiangiogenic effects in non-small cell lung cancer (NSCLC) cells in vitro [18]. Consistently, Preet et al. reported that JWH-133 effectively inhibits tumor growth and lung metastasis in NSCLC models [18]. Beyond lung malignancies, JWH-133 has been shown to induce apoptosis in the breast cancer cell line MDA-MB-231 in vitro, while concurrently suppressing cell proliferation, migration, and significantly reducing tumor progression and metastasis in vivo [19]. Furthermore, independent in vivo investigations confirmed that JWH-133 administration markedly decreases breast tumor burden, lung metastases, and tumor-associated angiogenesis, thereby slowing tumor growth and promoting apoptotic cell death [20].

Notably, CB₂ receptor expression levels in astrocytomas have been directly correlated with tumor malignancy, with the administration of JWH-133 completely halting the growth of stage IV human astrocytoma cells in murine models [21]. Similarly, local application of JWH-133 has been demonstrated to attenuate angiogenesis, restrict tumor growth, and stimulate apoptosis in non-melanoma skin cancer in vivo [22].

Although JWH-133 modulated apoptosis-related gene expression, Annexin V/PI staining revealed no significant induction of phenotypic cell death in prostate cancer cells. This discrepancy suggests that the observed transcriptional changes were insufficient to trigger the final apoptotic cascade. Instead, our functional assays demonstrated that JWH-133 profoundly suppressed cell migration and colony formation. Rather than acting as a direct cytotoxic drug at the tested concentrations, these findings indicate that JWH-133 primarily impairs the aggressive and metastatic potential of cancer cells, serving as a promising anti-invasive therapeutic candidate.

In the present study, JWH-133 treatment significantly suppressed wound closure in PC-3 cells, whereas DU-145 and LNCaP cells demonstrated more limited migratory sensitivity under the tested experimental conditions. These findings suggest that the biological efficacy of JWH-133 varies across prostate cancer cell lines possessing distinct molecular profiles. Furthermore, the differential phenotypes observed between androgen-dependent and androgen-independent lines may reflect variations in downstream signaling pathways, receptor expression landscapes, or intrinsic proliferative behaviors. The differential effects of JWH-133 on migration and gene expression may be attributed to the distinct molecular characteristics of the prostate cancer cell lines, including androgen sensitivity, AR signaling, p53 status, and PTEN alterations. LNCaP cells are androgen-sensitive and AR-positive, whereas PC-3 and DU145 cells are androgen-independent. Moreover, PC-3 cells are PTEN- and p53-deficient, while DU145 cells harbor mutant p53. Consistent with these molecular differences, JWH-133 significantly inhibited migration only in PC-3 cells. qPCR analysis also revealed increased TP53 expression in PC-3 cells, whereas DU145 cells exhibited decreased TP53 and increased CDKN1A (p21) expression, suggesting that JWH-133 may activate distinct molecular pathways depending on the genetic background of each cell line. Consequently, further mechanistic investigations are warranted to elucidate the precise molecular underpinnings of these cell-line-specific responses.

The differential gene expression patterns observed between PC3 and DU-145 cells suggest that cellular responses to JWH-133 may vary depending on the molecular characteristics of each prostate cancer cell line. While several apoptosis- and cell-cycle-related genes demonstrated altered expression following treatment, the biological significance of these transcriptional changes requires further investigation at the protein and functional levels.

The antitumor effects of cannabinoids go beyond classical apoptosis and cell cycle regulation, triggering a more complex signaling network that encompasses regulated cell death (RCD) and metabolic intervention pathways. Recent findings demonstrate that natural compounds with cannabinoid-like effects render cancer cells susceptible to death, particularly by inhibiting PI3K/AKT/mTOR signaling and triggering ferroptosis via the p53/GPX4/SLC7A11 axis. This is directly consistent with cannabinoids’ ability to disrupt the redox balance in cancer cells and increase lipid peroxidation [23]. Furthermore, a study emphasize that forms of inflammatory cell death, such as pyroptosis, and the enhancement of these processes through autophagy inhibition, hold strategic importance in boosting tumor immunity. The multifaceted effects of cannabinoids on autophagy and metabolic reprogramming offer a potential therapeutic foundation for targeting these new-generation RCD pathways and open the door to a synergistic approach in future cancer treatments [24].

Furthermore, a study demonstrates that cannabinoids exhibit a multidimensional antitumor effect by targeting metabolic reprogramming in cancer cells. It appears that cannabinoids disrupt the biosynthetic and energy requirements of cancer cells, particularly by inhibiting critical pathways such as glycolysis (the Warburg effect), the pentose phosphate pathway, and fatty acid synthesis. These compounds trigger oxidative stress and mitochondrial damage by increasing ROS production, which in turn leads to apoptosis via caspase activation. At the same time, they induce metabolic stress in cancer cells through AMPK activation and inhibition of the PI3K/Akt/mTOR signaling pathway, thereby triggering cell cycle arrest and autophagy mechanisms. In particular, their effects on key regulators such as p53 and HIF-1α disrupt the tumor’s adaptation to the hypoxic microenvironment, thereby reducing its metastatic potential and drug resistance [25].

Taken together, the present findings indicate that JWH-133 influences multiple phenotypic responses in prostate cancer cells, including viability, colony formation, migration-related behavior, and transcriptional regulation of selected genes. However, the underlying molecular mechanisms responsible for these effects remain unclear. In particular, the contribution of CB₂ receptor-mediated signaling pathways requires further investigation through receptor expression analyses and protein-level functional studies.

Several limitations of the present study should be acknowledged. First, CB₂ receptor expression levels were not directly evaluated in the prostate cancer cell lines used in this study. In addition, protein-level analyses were not performed to confirm the functional significance of the observed transcriptional alterations. Furthermore, the use of MRC-5 cells as a non-cancerous reference model may not fully reflect the biological characteristics of normal prostate epithelial cells. Therefore, further investigations incorporating receptor-level characterization, protein-based functional assays, and non-malignant prostate epithelial cell models are warranted to fully elucidate the biological efficacy of JWH-133 in prostate cancer. The other limitations are the exclusively in vitro design, lack of in vivo validation, absence of pharmacokinetic considerations, lack of long-term resistance studies and absence of combination therapy investigations In addition, although JWH-133 is widely recognized as a highly selective CB₂ receptor agonist, the present study did not directly evaluate CB₂ receptor expression in the prostate cancer cell lines or investigate receptor dependency using pharmacological antagonists or genetic approaches. Therefore, the observed antiproliferative and pro-apoptotic effects cannot be conclusively attributed to CB₂ receptor activation, and the possibility of CB₂-independent or off-target mechanisms cannot be excluded. These findings should therefore be interpreted with caution. Future studies should determine CB₂ receptor expression in the experimental models and confirm receptor involvement through the use of selective CB₂ antagonists, such as AM630, or genetic knockdown/knockout strategies. Such experiments will be essential to establish whether the biological effects of JWH-133 are mediated specifically through CB₂ receptor signaling and to further clarify the underlying molecular mechanisms.

Further empirical evidence comes from Zhang et al., who found that treatment with JWH-133 induced cell death in breast cancer cell lines such as MDA-MB-231 and enhanced the efficacy of other therapeutic approaches, including photodynamic therapy, pointing to a synergistic effect that may be beneficial for treatment [26]. Similarly, Caffarel et al. noted that cannabinoids could reduce the progression of breast cancer driven by the ErbB2 oncogene through Akt signaling inhibition, highlighting a critical pathway in tumorigenesis [20].

Interestingly, the effects of JWH-133 may vary based on the cancer model. Singh et al. reported that the interaction of cannabinoid treatment with opioid medications can influence pain and cancer progression; however, the specific anti-cancer properties of JWH-133 remain significant [27]. Although Aso et al.’s work focused primarily on neuroprotection through CB₂ activation in models of neurodegeneration, it suggests some overlap between cannabinoid receptor activation and pathways of cancer biology [28].

Conflict arises from the findings by Blanton et al., which indicate that JWH-133 could promote tumor growth under certain conditions, particularly when used in immunocompromised models. Their study revealed an increase in ectopic ovarian tumor growth with JWH-133 administration, suggesting the context of use and immune system status can significantly affect outcomes associated with cannabinoid therapy [29]. This highlights the necessity for further research to clarify the precise effects and interactions of JWH-133 across various cancer types and biological contexts.

In conclusion, the present study demonstrates that JWH-133 influences several cellular responses in prostate cancer cell lines, including cell viability, colony-forming capacity, migration-related behavior, and transcriptional regulation of selected genes. The observed effects differed among prostate cancer cell lines, suggesting cell-line-dependent responses to JWH-133 treatment. However, the precise molecular mechanisms underlying these effects remain unclear and require further investigation through receptor-level and protein-based functional analyses.