There is an unmet need to find new therapeutic approaches that can improve the prognosis and survival of patients suffering from malignant gliomas, especially GBM. Among the potential therapeutic targets for these conditions are the mitochondrial protein PINK1 and GPR55 receptors, as the modulation of these has been shown to affect the progression of these cancers in various models [48,49,50]. Additionally, in recent years, ECS has emerged as a major area of interest in multiple pathologies, including cancer, due to its ubiquitous distribution and the findings of ECS-related disorders in cancer [51,52].

In this context, the molecular docking study (Table 2 and Table 3 and Figure 1 and Figure 2) provides valuable insights into the binding affinities and interactions of selected compounds with the PINK1 and GPR55 proteins. Key compounds, such as CBC, P, and THC, exhibited strong and consistent binding to these targets, highlighting their potential as therapeutic modulators of PINK1 and GPR55, both of which are involved in various cellular and pathological processes.

For PINK1, it is worth noting that OSI demonstrated the highest binding affinity, particularly with the Vina scoring function (−9.30 kcal/mol). Its interaction profile includes strong hydrophobic contacts with residues such as Ile162, Leu369, and Met318, along with crucial electrostatic interactions with Asp366 and Lys164. These interactions likely stabilize the OSI-PINK1 complex, reinforcing its potential as a potent modulator of PINK1 activity. OSI, also known as AZD9291, is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of non-small cell lung cancer (NSCLC) with a T790M mutation in EGFR. It demonstrated significant efficacy against various cancers in both preclinical and clinical trials [53]. Since EGFR is one of the most frequently mutated genes in GBM, we evaluated its potential as a therapeutic target.

Additionally, P demonstrated strong binding affinities, achieving the highest Autodock score (−9.75 kcal/mol). Its interaction with Tyr321, through hydrogen bonding, along with hydrophobic contacts involving Val381 and Arg276, underscores its high binding potential. Similarly, THC (−7.35 kcal/mol) exhibited a well-balanced interaction profile, forming hydrogen bonds and hydrophobic interactions with residues such as Gln327 and Val170.

Compounds like CBG and CBD demonstrated moderate binding affinities, primarily interacting with residues associated with PINK1’s catalytic activity. In contrast, TMZ and CisPt displayed weaker binding affinities, suggesting the direct limited modulation of PINK1 by these agents, consistent with the canonical mechanism of action of alkylating agents.

For GPR55, CBC and P emerged as top-performing ligands. CBC exhibited the strongest binding affinity with Vina (−9.20 kcal/mol) and ranked highly across other scoring functions. Its key interactions include hydrophobic contacts with residues such as Phe102 and Leu148, along with a hydrogen bond with Val149, suggesting its potential as a potent GPR55 modulator. OSI also demonstrated high binding affinities, forming hydrogen bonds with Gly189 and engaging in hydrophobic interactions with residues like Phe159 and Leu148. Similarly, P and CBG consistently interacted via hydrophobic contacts across the GPR55 ligand-binding pocket. In contrast, CisPt, despite forming hydrogen bonds with Asn280 and Cys281, exhibited weaker binding affinities, limiting its potential as a GPR55 modulator. Additionally, compounds like BCP and TMZ displayed minimal direct interactions with the receptor’s active site, indicating a lower modulatory potential.

The docking results reveal distinct interaction profiles between PINK1 and GPR55 for the tested compounds. CBC and P emerge as strong modulators of GPR55, and THC and CBDA display better affinities for PINK1. This differential affinity could guide the design of selective or dual-targeted therapeutic agents based on disease-specific requirements.

Interestingly, in silico analyses suggest that P and CBC are promising candidates for modulating pathways involving PINK1 and GPR55. Their interactions with key residues, through both hydrophobic and electrostatic forces, emphasize the importance of optimizing ligand structures in enhancing these interactions. The diversity in binding modes further suggests potential for tailoring compounds to achieve specificity for one target over the other.

While these docking simulations provide significant insights, the absence of crystallized structures for PINK1 and GPR55 introduces uncertainties. Although AlphaFold-generated models are reliable, they remain predictive rather than experimentally validated. To strengthen these findings, future studies should incorporate experimental approaches such as site-directed mutagenesis, X-ray crystallography, or cryo-electron microscopy.

We performed additional analysis of BBB permeation and toxicity in selected compounds shown in Figure 3 and Table 4. The boiled egg diagram (Figure 3) provides a detailed analysis of the pharmacokinetic properties of the compounds based on their WLOGP (lipophilicity) and TPSA (topological polar surface area). Compounds 1, 2, 3, 5, 6, and 7 are located within the yellow (yolk) region, indicating a high probability of penetrating the brain via the BBB while also being in the white region, which suggests good gastrointestinal absorption. Compound 4 is in the white region but outside the yellow region, indicating gastrointestinal absorption but reduced brain penetration. Compound 8 (OSI, in blue) is a PGP+ substrate, meaning that active efflux by the P-glycoprotein limits its brain penetration despite being in the white region. Compounds 9 (TMZ) and 10 (CisPt), while positioned outside the yellow region and showing limited potential for BBB penetration due to high TPSA values, are widely used as antitumor agents in the clinical market. Notably, compounds 9 and 10 are extensively employed in glioblastoma treatment despite their pharmacokinetic limitations regarding BBB penetration, and this is likely due to their efficacy in targeting tumor cells and their established therapeutic profiles. This analysis highlights compounds 1–7 as having favorable absorption profiles, with some also showing brain penetration potential, while compounds 8, 9, and 10 underscore the importance of considering both pharmacokinetics and therapeutic context when evaluating drug efficacy.

Table 4 presents the in silico toxicity profiles of various compounds analyzed, including cannabinoids (CBC, CBG, CBD, CBDA, and THC), P, BCP, and reference drugs OSI and TMZ. Notably, none of the compounds exhibited AMES toxicity or hERG channel inhibition, indicating low mutagenic and cardiotoxic potential. However, hepatotoxicity was identified in P, OSI, and TMZ, highlighting a potential risk for liver damage with these compounds. Skin sensitization was observed exclusively with CBG and P, suggesting a selective allergenic response. Importantly, carcinogenicity was absent across all compounds, except for TMZ, which is a known chemotherapeutic agent with established carcinogenic risks. These findings underscore the relatively safe toxicity profiles of cannabinoids compared to synthetic drugs, although specific risks, such as hepatotoxicity and skin sensitization, warrant further investigation for some compounds.

On the other hand, Figure 4, Figure S1, and Table S1 provide insights into the immunohistochemical (IHC) analysis conducted in parallel with the bioinformatic study. The expression patterns of PINK1, GPR55, and mutant p53 were evaluated in glioblastoma patient samples, revealing that PINK1 was detected in 65% of cases (13 positive and 7 negative), GPR55 was highly expressed in 95% (19 positive and 1 negative), and p53 mutations were present in 30% (6 positive and 14 negative). Notably, the clinical data from the Colombian patient cohort align with global epidemiological trends, showing a higher prevalence of glioblastoma in men compared to women and a peak incidence in individuals aged 50–60 years. The presence of p53 mutations in 30% of cases is also consistent with worldwide statistics, reinforcing the relevance of this cohort in glioblastoma research. Further details on patient demographics and molecular profiles are summarized in Table S1, while Figure S1 illustrates the tissue-processing methodology. These findings highlight the potential implications of PINK1 and GPR55 in glioblastoma and support their relevance as biomarkers in the disease’s pathophysiology.

Figure 5, Figure 6 and Figure 7 provide evidence of the cytotoxic potential of cannabinoids and Piper nigrum bioactives relative to glioblastoma cell lines, including MO3.13 (oligodendrocyte precursor cells) and SH-SY5Y (neuroblastoma). The results indicate that in almost all cell lines (U87MG, T98G, MO3.13, and SH-SY5Y), both Piper nigrum derivatives and cannabis compounds exhibit dose-dependent cytotoxicity, reducing cell viability at higher concentrations. Table 5 summarizes the IC₅₀ values obtained in this study, with some data similar to those reported by other laboratories. Tamara Lah and collaborators, who evaluated CBG and CBD in glioblastoma, demonstrated that CBG exhibit significant effects on reducing cell viability in glioblastoma cell lines, with IC₅₀ values at 28.1 ± 1.1 μM, similarly to THC (IC₅₀ 27.9 ± 1.8 μM). CBD, however, showed greater cytotoxicity with an IC₅₀ of 22.0 ± 2.1 μM, which was significantly more effective than CBG and THC [12,13]. The analysis of our study reveals significant variability in the cytotoxicity of the compounds, with some showing higher potency across specific cell lines. CBC and CBG demonstrated notable activity in the T98G and CCF-STGG1 cell lines, with IC₅₀ values ranging from 1.56 ng/μL to 3,12 ng/μL (4.93 μM to 9.91 μM). PiperOH exhibited cytotoxicity, particularly in the U87MG, SH-SY5Y and T98G cell lines, showing effective results as low as 6.25 ng/μL. CBDA, in comparison to CBD, showed moderate activity in most cell lines with less pronounced effects than CBG and CBC. When comparing CBDA, CBG, and CBC with TMZ (200–250 μM [54,55]) and CisPt (>125 μM [56]), for cancer therapy, the potential of these compounds, especially in glioblastoma and other chemotherapy-resistant tumors, becomes evident due to their effectiveness at lower doses compared to the established standards. In contrast, compounds like PiperOH and PiperEO displayed non-cytotoxic effects (NC*) in some cell lines, indicating their limited or absent anticancer activity in these settings.

Figure 6 shows the potential additive effect of CisPt with CBG compared to CBD in the astrocytoma cell line CCFSTTG1. Preliminary results suggest that CBG may enhance the therapeutic efficacy of drugs, potentially expanding treatment options in oncology. This is particularly relevant for aggressive cancers such as glioblastoma, where therapeutic resistance and poor prognosis remain major challenges.

Furthermore, consistent with our findings, data from the Human Protein Atlas suggest a potential correlation between PINK1 and GPR55 expression profiles and the treatment responses observed in this study (Supplementary Materials Figure S1). Specifically, PINK1 expression follows the order U87MG > T98G > CCF-STTG1 > SH-SY5Y, while data for the MO3.13 cell line remain inconclusive. Regarding GPR55, Figure S5b shows negligible expression in the cell lines evaluated in this study. However, previous reports indicate considerable GPR55 expression in SH-SY5Y [55,56], T98G [57], and U87MG [58] cells and other models [59,60], suggesting its potential role in glioblastoma pathophysiology and therapeutic responses.

Moreover, the immunofluorescence analysis (IF) (Figure S4) of PINK1 and GPR55 in U87MG and T98G cells revealed that treatment with cannabinoids such as CBG and CBD reduced fluorescence intensity, indicating lower PINK1 expression. This effect correlates with mitochondrial membrane potential assessments (Figure 8), where a decrease was observed in astrocytoma cell lines following treatment with these cannabinoids, which aligns with the cytotoxicity results obtained via MTT assays. Notably, TMZ and P had differential effects on mitochondrial potential depending on the astrocytoma subtype. It is worth mentioning that, although CBC induces cell death, it does not appear to alter the intensity of PINK1 or GPR55 expression in U87MG and T98G cell lines, suggesting that its cytotoxic effect may be mediated through a different mechanism. Additionally, immunofluorescence analysis (Figure S4) revealed that the cannabinoid receptor type 1 (CB1) is widely expressed in U87MG glioblastoma cells. Interestingly, while cannabinoids such as CBG also induce cell death, they do not reduce CB1 expression, indicating that their effects on glioblastoma cells may involve alternative or complementary pathways beyond CB1 modulation.

While these results suggest a possible trend, further studies are needed to confirm their clinical significance. To our knowledge, this is the first published IHC report on GPR55 and PINK1 expression in glioblastoma patient samples, providing novel insights into their potential relevance in glioblastoma pathophysiology.

To further investigate whether the Piper nigrum extract modulates the cytotoxic profile of cannabinoids (CBD and CBG) when used concomitantly, we focused on the CCF-STTG1 cell line (Figure 6). Interestingly, Piper nigrum did not exhibit cytotoxic effects in the CCF-STTG1 cells; instead, both PiperEO and PiperOH increased cell viability. Moreover, the concomitant use of cannabinoids with Piper nigrum derivatives resulted in a complete or apparent blockade of cannabinoid-induced cytotoxicity in CCF-STTG1 cells (Figure 7a,b) in contrast to the T98G and U87MG models. This suggests a distinct interaction between these compounds in this specific cellular context.

To determine whether purified P from Piper nigrum was responsible for this antagonistic effect, we assessed its impact on cell viability. While P alone had no effect or even increased cell viability at high doses, previous studies have reported its cytotoxicity in various cancer models [61,62]. However, when used concomitantly with cannabinoids, F did not appear to alter the cytotoxic response, suggesting that another component of the Piper nigrum fraction may be responsible for this effect, warranting further investigation.

Figure 7b further explores the cellular effects of these treatments, highlighting morphological changes indicative of apoptosis. Cells treated with CBD or CBG exhibited classic apoptotic features, such as membrane blebbing and cellular shrinkage. Notably, the addition of Piper nigrum extracts to CBG or CBD treatment reduced these apoptotic effects, reinforcing the observed modulation of glioma cell responses by these compounds.

The CCF-STTG1 cell line, classified as a grade IV mixed astrocytoma according to the WHO 2021 classification of CNS tumors, presents a unique context for interpreting these results [63]. One of the main findings lies in the pro-viability effects of Piper nigrum derivatives observed in this cell line, contrasted with their cytotoxic effects in other models. In the CCF-STTG1 cell model, Piper nigrum derivatives do not appear to exert direct cytotoxic effects and instead attenuate the activity of cannabinoids. One possible explanation is that these compounds mitigate oxidative stress and mitochondrial damage induced by cannabinoids before the activation of cell death. Interestingly, Figure 8C shows that treatment with PiperOH increases mitochondrial potential, possibly as a protective mechanism to prevent mitochondrial depolarization and safeguard the cell from apoptosis. This suggests that PiperOH may play a role in preserving mitochondrial function under stress conditions induced by cannabinoids, and this specific cell subtype could have a distinct vulnerability that Piper nigrum could modulate, potentially opening new avenues for targeted therapeutic approaches.

Another potential mechanism involves the interference of Piper nigrum derivatives with ABC transporter family members [64,65]. Which could affect the bioavailability and intracellular transport of cannabinoids in these cells.

Additionally, the apparent attenuation or blockade of the cytotoxic effects of cannabinoids in the CCF-STTG1 model suggests a potential mechanistic overlap. Based on in vitro results and molecular docking analyses, a plausible explanation is that Piper nigrum derivatives modulate PINK1 function by competing for its binding, thereby altering its role in mitochondrial homeostasis and cellular phosphoproteome regulation. This competitive interaction could mitigate the pro-apoptotic effects typically induced by cannabinoids, particularly in a tumor context with complex metabolic dynamics, such as that of the CCF-STTG1 cell line.

To further elucidate this phenomenon, complementary biochemical studies, such as Western blotting (WB) and PCR assays, are currently underway within our research group. These investigations aim to clarify the interaction between PINK1 and GPR55 modulation, mitochondrial dynamics, and the observed effects on cell viability. Understanding these mechanisms could pave the way for more precise and effective therapeutic strategies, particularly for glioblastoma and other CNS tumors.

Building on the molecular interaction data from docking studies and the in vitro viability results discussed earlier, additional insights were gained through data mining across multiple transcriptomic and expression profiling databases, including UALCAN, GLIOVIS, TCGA, GEPIA, and the Human Protein Atlas. These platforms revealed patterns mainly of PINK1 expression in glioblastoma tissues compared to normal brain samples. In glioblastoma datasets from UALCAN and TCGA, PINK1 expression was consistently downregulated, aligning with the hypermethylation observed in Figure S6. This suggests that PINK1 is epigenetically silenced in glioblastoma, potentially impairing mitochondrial quality control and contributing to tumor progression. The comparison between normal and primary tumor tissue samples shows an extremely significant difference in PINK1 expression, with a p-value of 1.11 × 10⁻¹⁶. This suggests that PINK1 expression is significantly lower in primary tumors (n = 156) compared to normal tissues (n = 5). This drastic reduction in expression may indicate a crucial role of PINK1 in glioblastoma biology (as a potential indicator of tumor progression). This aligns with previous findings that PINK1 supports cellular survival by maintaining mitochondrial quality control. However, its downregulation in glioblastoma may reflect an adaptive response to the tumor microenvironment’s metabolic demands, potentially favoring tumorigenesis. These findings highlight PINK1 as a promising biomarker and therapeutic target, particularly for strategies aimed at reversing epigenetic silencing. GPR55 data were not included, as no significant results were observed.

Interestingly, the Kaplan–Meier survival analysis in Figure 9 using Gliovis reveals a subtype-dependent relationship between PINK1 expression and patient prognosis. In proneural glioblastoma subtypes, high PINK1 expression correlates with poorer survival, suggesting a pro-oncogenic role in specific molecular contexts. Conversely, in mesenchymal and classical subtypes, no significant association is observed, highlighting the complexity of PINK1’s role in glioblastoma biology. This context-dependent variability underscores the need for stratifying glioblastoma patients based on molecular profiles when considering PINK1-targeted therapies.

Figure 10 and Figure 11 expand the analysis of PINK1’s role in cancer, emphasizing its tumor-specific variability and prognostic potential. The Kaplan–Meier survival analysis in Figure 9 reveals that PINK1 expression levels correlate with patient outcomes in a tumor-type-dependent manner. In malignancies such as kidney renal clear cell carcinoma (KIRC), high PINK1 expression is associated with better survival rates, indicating a protective role. Conversely, in lung squamous cell carcinoma (LUSC) and PAAD, elevated PINK1 expression corresponds to poorer survival outcomes, suggesting a pro-oncogenic function in these contexts. These findings underscore PINK1’s dual role in cancer biology, acting as either a tumor suppressor or promoter depending on the tumor microenvironment and molecular landscape.

It is worth mentioning that some studies point to PINK1 as a potential biomarker [66] and therapeutic agent. For example, some studies demonstrate that PINK1 depletion in the non-small cell lung cancer (NSCLC) A549 cell line via shRNA reduced cancer cell proliferation, increased cell death, decreased ATP production, inhibited mitophagy, and increased ROS levels and caspase-9-dependent apoptosis [67]. These findings suggest that PINK1 depletion disrupts energy metabolism and increases sensitivity to glycolysis inhibitors. In this context, targeting the accelerated metabolic activity of tumor cells could be a promising therapeutic strategy while minimizing damage to non-malignant tissues. Furthermore, in breast cancer, PINK1 expression has been significantly correlated with histological grade, and its depletion induces a distinct proteomic profile, particularly impacting energy metabolism pathways. These results support an oncogenic role for PINK1 in breast cancer, offering new insights into its potential regulatory function [68].

The boxplot in Figure 11 further highlights the significant differences in PINK1 expression between tumor and normal tissues across various cancer types, including GBM, LGG, KIRP, and others. In GBM and PAAD, PINK1 expression is notably downregulated in tumor samples, aligning with its reported epigenetic silencing in these malignancies. In contrast, its upregulation in renal cancers, such as KIRC and KIRP, suggests that PINK1 may have context-dependent protective effects.

These data reflect PINK1’s potential to function as a molecular switch, influenced by the tumor’s genetic background, metabolic demands, and microenvironmental factors. For instance, in glioblastoma, the loss or gain of PINK1 may contribute to mitochondrial dysfunction and altered mitophagy, affecting tumor progression. Meanwhile, in renal cancers, its upregulation might enhance mitochondrial integrity, supporting cellular resistance against metabolic stress.

The prognostic variability of PINK1 demonstrated in Figure 10 and Figure 11 underscores the importance of integrating molecular profiling into cancer treatment strategies. Beyond glioblastoma, these insights reveal opportunities to explore PINK1 as a therapeutic target or biomarker in a broader range of cancers. However, its dual role necessitates further investigation to delineate the mechanisms underlying its protective versus pro-oncogenic functions.

It is worth mentioning that, although no significant correlation between GPR55 and GBM was found in the databases consulted for this study, when comparing the expression data and Kaplan–Meier survival curves between LGG and GBM, significant differences in both GPR55 and PINK1 expression were observed, as shown in the Supplementary Materials (Figure S7). These data were obtained from both the GLIOVIS and GEPIA databases, suggesting a potential role for these proteins in the natural course or progression of the disease. Additionally, while the role of PINK1 in cancer remains controversial [66,69], its possible dual function as a kinase dependent on the tumor subtype is consistent with the existing literature [66,70]. Regarding GPR55, it has been increasingly recognized as a potential oncogene in the carcinogenic development of GBM, as well as in other types of cancer [13,25,28,60].

Additionally, changes in the expression profiles of PINK1 and GPR55 were observed in correlation with survival rates in high-grade vs. low-grade gliomas, suggesting potential alterations in inflammatory and metabolic profiles [39,66,67,68,69,70,71,72]. These changes may correlate with malignancy and the natural progression of the disease, warranting further investigation to unravel this complex interplay.

As a final aspect to mention regarding PINK1, while RNA expression data (nTPM) indicate low levels of PINK1 (Supplementary Material Figure S6), this does not necessarily mean that the protein is absent or not expressed. Our IHC results (Figure 4) revealed that PINK1 was present in neurons and endothelial cells but absent in normal glia, whereas in glioblastoma samples, positive cases (65%) exhibited PINK1 expression. This suggests a possible role in tumor progression, potentially through mitochondrial dynamics, metabolic adaptation, or resistance to apoptosis. Additionally, Kaplan–Meier survival analysis in glioma indicates that lower PINK1 levels correlate with better overall survival (Figure S7 and Figure 9a), further supporting its potential oncogenic role in glioblastoma and highlighting the need for functional validation.

Furthermore, mitochondrial potential assays and immunofluorescence studies showed that compounds such as CBD and CBG led to mitochondrial membrane potential loss and reduced PINK1 expression, correlating with cytotoxic effects in glioblastoma cell lines. These findings indicate the direct and indirect modulation of PINK1, reinforcing the need for further investigation into its regulatory mechanisms in glioblastoma.

Beyond its role as a mitochondrial sensor, PINK1 has additional functions, including calcium [73] and iron homeostasis [74], phosphoproteome regulation [75], and potential immune modulation [76], reflecting its dualistic kinase activity. These diverse roles suggest that PINK1’s oncogenic potential in glioblastoma could be linked to broader cellular signaling pathways beyond mitophagy, emphasizing the need for further investigations to determine its therapeutic relevance in glioblastoma and other cancers.

Finally, it is particularly interesting and valuable to explore the therapeutic implications of cannabinoids in cancers like pancreatic cancer and glioblastoma, given their resistance to conventional treatments and the limited survival rates associated with these cancers. This highlights the urgent need to identify potential vulnerabilities for targeted therapeutic exploitation. To the best of our knowledge, this is the first study to test cannabinoids such as CBC and CBG in various glioma cell lines, correlating them with molecular docking studies and expression data from the IHC of patients and public human databases, specifically focusing on PINK1 and GPR55 expression.

Future studies should aim to further scale these investigations to better understand the underlying molecular pathways. Such work could help determine whether cannabinoids are a viable therapeutic option in oncology. Several preclinical studies already support the potential of plant-derived therapies in gliomas [77,78], and given the current challenges in treating these cancers, there is a pressing need to explore alternative therapies to improve patient outcomes and survival.

Prospective randomized clinical trials should also be conducted to evaluate the use of plant extracts as adjunctive agents in standard treatments. Recent clinical trials such as NCT05753007, NCT05629702, and NCT03529448 underscore the importance of plant-based research in glioblastoma treatment. On the other hand, TTX101, a hydrogel containing piperlongumine from Piper longum, is being tested for post-surgical application to target residual GBM cells due to its pro-apoptotic properties. Meanwhile, clinical trials investigating CBD and THC-based therapies aim to modulate immune responses and reduce inflammation, offering promising alternatives for improving glioma treatment.