Cannabis has been widely reported to provide therapeutic benefits in the management of symptoms such as pain, inflammation, anorexia, mood disorders, and vomiting. Recently, increasing attention has been directed toward its potential role in cancer therapy, particularly due to its ability to alleviate chemotherapy-related side effects and possibly enhance treatment efficacy in resistant cases [15]. Nevertheless, concerns regarding potential drug–drug interactions should be carefully evaluated to determine whether cannabis affects the pharmacological properties of conventional anticancer drugs [16]. Although previous studies have demonstrated the effects of cannabis on the efficacy of several chemotherapy drugs, such as cisplatin, tamoxifen, doxorubicin, and paclitaxel, the molecular effects of combined administration of sorafenib and cannabis have not yet been reported. This study is the first to comprehensively investigate the drug–drug interaction between cannabis and sorafenib, and to characterize the transcriptomic effects of this combination in HepG2 cells, revealing associated biological responses and molecular pathways.

The cytotoxic analysis demonstrated that cannabis extract exhibited an IC₅₀ value approximately 0.15 mg/mL after 24 h of exposure, corresponding to THC and CBD concentrations of 84.1 µM and 7.72 µM, respectively. When compared to other studies, we found that the recent IC₅₀ is higher than that reported in previous studies. It has been suggested that differences in extract composition, particularly the relative proportions of THC and CBD as well as potential variations in cancer types, could reflect distinct effects on cancer treatment [16]. The different mechanisms of action by CBD and THC depend on the ratio between CBD and THC in cannabinoids [17]. Co-administration of tamoxifen with both recreational cannabis (THC:CBD ratio of 9:1) and medicinal cannabis (THC:CBD ratio of 1:3) formulations led to a reduction in tamoxifen’s efficacy, indicating that the simultaneous treatment of a breast cancer cell line with different cannabinoid formulations and tamoxifen results in a diminished anti-proliferative activity of tamoxifen [18]. Moreover, the pro-tumor effect of cannabis was related to the expression of cannabinoid receptors (CB), both CB1 and CB2; then, the cancer cells which originated from tissues with low cannabinoid receptor expression could be less sensitive to cannabinoids [19,20]. Blasco-Benito et al., 2018 [21], reported that cannabis exhibited dual effects depending on the anticancer agent. No synergy was observed with paclitaxel or epirubicin, whereas cisplatin combined with cannabinoid extract showed a synergistic inhibition of cancer cell growth.

The potential interaction between cannabis extract and sorafenib in HepG2 cells was evaluated using the Combenefit platform. The combination treatment at IC₅₀ concentrations demonstrated a synergistic reduction in cell viability, as indicated by both the Highest single agent (HSA) and Bliss independence models. The HSA model provides a conservative baseline comparison without assuming mechanistic interaction [22], whereas the Bliss model predicts additive effects for compounds acting through independent mechanisms [23,24]. Hence, these findings indicate that the interaction between sorafenib and cannabis was predominantly synergistic, likely driven by distinct yet complementary mechanisms, with partial overlap in their modes of action. Mechanistically, sorafenib functions as a multikinase inhibitor targeting receptor tyrosine kinases and downstream serine/threonine kinases, resulting in inhibition of cell proliferation and angiogenesis, as well as induction of apoptosis [25]. In contrast, cannabinoids exerted anticancer effects through activation of CB1 and CB2 receptors, leading to suppression of cell growth, induction of cell cycle arrest, and activation of apoptotic pathways via multiple signaling cascades [26]. Previous studies reported synergistic anti-tumor effects when cannabinoids were combined with tyrosine kinase inhibitors, such as the synergistic effect of CBD and dasatinib through modulation of the PI3K-AKT pathway [27] as well as inhibition of human epidermal growth factor receptor (HER2) signaling [28]. Taken together, our findings suggest that cannabis extract enhanced sorafenib efficacy through both complementary and partially overlapping mechanisms.

At the mechanistic level, the combined effects of sorafenib and cannabinoids appeared to join in the induction of cellular stress. These include oxidative stress, mitochondrial dysfunction, and disruption of cellular homeostasis, ultimately leading to apoptosis and ferroptosis [29,30]. Cannabinoid-derived compounds such as β-caryophyllene showed the potential enhancement of sorafenib cytotoxicity by inhibiting drug efflux and increasing intracellular drug accumulation [31]. Combination treatments with cannabinoids and cisplatin have also been associated with suppression of DNA replication and repair, as well as induction of cell cycle arrest [32]. Notably, synergistic interactions with tyrosine kinase inhibitors, including cabozantinib, have been linked to activation of p53 signaling and endoplasmic reticulum (ER) stress, further promoting apoptotic cell death [33]. Likewise, chemotherapy in the class of platinum-based drugs exerted the induction of ROS in the mitochondria of gastric cancer cells leading to loss of mitochondrial membrane potential. As a consequence, the accumulation of misfolded proteins triggered the unfold protein response in an attempt to maintain cellular homeostasis [34]. Given that both sorafenib and cannabinoids are metabolized by cytochrome P450, family 3, subfamily A (CYP3A) enzymes, pharmacokinetic interactions may additionally contribute to the observed effects [35,36]. These findings support a multifaceted mechanism underlying the interaction, while highlighting the importance of transcriptomic analysis to further elucidate system-level responses.

RNA-sequencing analysis revealed that the combination treatment induced a distinct transcriptional profile, characterized by upregulation of stress- and apoptosis-related genes, for example, SAT1 and MTHFD2, and downregulation of genes involved in proliferation, metabolism, and tumor progression such as SLC2A1, MCM3, PLOD2, and ENC1. These gene expression changes are consistent with suppression of metabolic reprogramming, inhibition of cell proliferation and angiogenesis, and activation of cell death pathways. For instance, upregulation of SAT1 affected the reduction in polyamine levels resulting in the inhibition of tumor growth through AKT/GSK3β/β-catenin signaling [37,38], while downregulation of AGT, MCM3, PLOD2, and ENC1 has been reported to associate with decreased tumor proliferation, invasion, and epithelial–mesenchymal transition [39,40,41,42,43]. Importantly, these gene-level alterations are highly consistent with pathway-level enrichment identified by GSEA and ShinyGO, collectively indicating coordinated suppression of metabolic programs and activation of stress- and apoptosis-related signaling. Consistent with these gene-level alterations, GO enrichment analysis further demonstrated that the combination treatment prominently activates biological processes related to transcriptional regulation and stress adaptation. Notably, enrichment of integrated stress response (ISR) signaling, cytokine and chemokine receptor binding, and histone acetyltransferase activity was uniquely observed in the combination group. The ISR represents a key adaptive mechanism to cellular stress such as hypoxia, nutrient deprivation, and endoplasmic reticulum stress resulting in shifting from pro-survival to pro-apoptotic signaling under sustained stress conditions [44,45]. In parallel, cytokine and chemokine signaling pathways reflect dynamic regulation of tumor–microenvironment interactions, influencing processes such as inflammation, immune response, angiogenesis, and metastasis [46]. Importantly, enrichment of histone acetyltransferase activity which serves as key processes in DNA repair, apoptosis, and tumor progression highlights a role for epigenetic regulation in mediating the observed transcriptional reprogramming [47,48]. Consistent with previous studies, both cannabinoids and sorafenib were reported to modulate epigenetic landscapes associated with inflammation and stress responses [49,50,51]. Hence, the integration of gene expression and GO enrichment findings suggests that the combination treatment induced a coordinated anticancer response involving metabolic suppression, activation of stress and immune-related signaling, and epigenetic reprogramming. These multi-layered mechanisms likely contribute to the enhanced anti-proliferative and pro-apoptotic effects observed in HepG2 cells.

Integrated analysis of GSEA and the ShinyGO database showed that pathway enrichment analysis revealed that key signaling networks, including TNF/NF-κB, MAPK, JAK/STAT, ROS, and p53 pathways, were activated across the treatments, with stronger and more coordinated activation observed in the combination group. This pattern suggests that the combination amplifies shared stress and apoptotic signaling rather than inducing entirely distinct pathways. In the sorafenib-treated cells, we found significantly upregulated stress- and inflammation-associated pathways (TNF/NF-κB, MAPK, JAK/STAT, and TGF-β signaling). Despite activation of these signaling pathways related to tumor progression and chemoresistance, it likely reflected compensatory and adaptive responses rather than pro-tumorigenic effects under therapeutic stress. At the cellular level, some cells might be induced to undergo apoptosis, while other cells might activate NF-κB/STAT3/PI3K-driven resistance programs to promote survival [52]. In addition, the simultaneous activation of stress-related pathways and suppression of metabolic processes by the ShineyGo database reflects a dual response involving cytotoxic effects and adaptive signaling which suggests suppression of cellular metabolism leading to reduced cancer cell growth and survival [53].

NF-κB plays crucial roles in cellular stress responses and inflammation [54]. The couple function between NF-κB and p53 might be simultaneously activated by DNA damage or cytokines resulting in the induction of cell death. This suggests that NF-κB contributes more to the execution phase of apoptosis rather than its initiation [55]. In addition, NF-κB was induced by DNA-damaging agents through receptor-independent mechanisms resulting in responses to diverse forms of genotoxic stress, including SN1-type DNA methylation induced by temozolomide, DNA crosslinking caused by cisplatin, and double-strand breaks generated by ionizing radiation [56]. Moreover, NF-κB expression was upregulated in HT-29 cells following treatment with chemotherapeutic agents such as fluorouracil, oxaliplatin, or paclitaxel [57]. Similarly, previous studies showed that adenosine induced G0/G1 cell cycle arrest in hepatocellular carcinoma cells, accompanied by increased caspase-3 activity and upregulation of p53. Concurrently, NF-κB expression was elevated, while the anti-apoptotic protein Bcl-2 was downregulated [58].

Activation of the MAPK and JAK/STAT pathways in response to sorafenib likely reflects compensatory survival signaling. The cancer cells could activate alternative pathways to counteract sorafenib effects. Particularly, inhibition of RAF kinases by sorafenib may paradoxically reactivate downstream MAPK/ERK signaling resulting in restoration of MAPK activity which occurs in a dose-dependent manner and contributes to continued cancer cell survival [59]. While the MAPK pathway generally drives cell proliferation and survival, the MAPK branches known as JNK and p38 were predominantly activated by environmental stressors, cytokines, and UV radiation to mediate apoptosis [60,61]. Similarly, activation of the JAK/STAT pathway supports cell survival under therapeutic pressure. These adaptive responses are further supported by the activation of additional signaling pathways, including PI3K/Akt and receptor tyrosine kinases, highlighting the dynamic rewiring of signaling networks that limits the efficacy of sorafenib in hepatocellular carcinoma [62,63]. Notably, a previous study reported that the short-chain fatty acid sodium propionate inhibited JAK2–STAT3 activation, induced cell cycle arrest, and promoted ROS generation, which in turn activated p38 and triggered apoptosis [64]. Notably, p53 and apoptosis pathways were also markedly upregulated following sorafenib treatment. These findings are consistent with our experimental observations demonstrating increased ROS generation and apoptosis. The concurrent upregulation of both MAPK and p53 signaling pathways in our study reflects a paradoxical feature of tumor biology, in which pro-survival (MAPK) and tumor-suppressive (p53) mechanisms were activated simultaneously. It is suggested that cancer cells experience high levels of intrinsic and treatment-induced stress such as DNA damage, ROS, and metabolic pressure, leading to the simultaneous engagement of both survival and stress-induced cell death pathways [65,66,67,68]. These findings assumed that sorafenib exerts anti-tumor effects through metabolic suppression and oxidative stress induction, while simultaneously triggering compensatory survival pathways that may limit its therapeutic efficacy.

Cannabis treatment alone induced a broad cytotoxic transcriptional response, characterized by activation of oxidative stress, inflammatory signaling, and apoptosis, along with suppression of proliferation, metabolism, and DNA repair pathways. Specifically, the upregulation of TNF/NF-κB, TGF-β, and IL-6/JAK–STAT signaling, together with enrichment of ROS and xenobiotic metabolism pathways, indicates that cannabis elicits substantial oxidative and inflammatory stress. This is further supported by activation of p53 signaling and apoptosis, reflecting engagement of programmed cell death mechanisms. These findings are consistent with previous reports showing that cannabinoids induce ROS production and cytokine-mediated signaling, ultimately leading to apoptosis [69]. The p53 and apoptosis pathways are well recognized as key regulators of cell cycle control and cell death in various cancer cells. Several plant-derived compounds, particularly THC and CBD, have been reported to modulate p53 signaling and apoptotic processes through generation of ROS. The ROS play dual roles: (1) as upstream signals that activate p53, and (2) as downstream mediators that promote apoptosis. Furthermore, p53 activation suppress the expression of antioxidant genes, leading to enhanced oxidative stress and ultimately resulting in cell death [70,71]. This is strongly supported by our experimental findings of increased ROS production and apoptosis in cannabis-treated cells. Consistently, the activation of p53 and apoptosis pathways observed in this study supports a ROS-driven cytotoxic mechanism.

The finding of downregulation in cell cycle regulators (G2/M checkpoint, E2F targets), DNA replication and repair pathways, and metabolic processes, including glycolysis and fatty acid metabolism, indicated the impairment of proliferative capacity and the reduction in cellular ability to recover from damage. Previous publications showed that CBD induced anti-tumor effects by causing localized inflammation, oxidative stress, and ER stress leading to apoptosis [72]. Several reports demonstrated that CBD treatment induced cell cycle disturbances, cellular apoptosis, and ER stress in HepG2 and gastric cancer cells [30,73]. Moreover, THC also affected the downregulation of cell cycle progression resulting in an inhibition of cell proliferation. Notably, THC suppressed DNA repair pathways, including mismatch repair and base excision repair, suggesting a reduced capacity for genomic maintenance under stress conditions [74]. In breast cancer cell lines, the activation of CB2 receptor by THC showed the suppression of cell proliferation via the inhibition of cell cycle and induction of apoptosis. Specifically, THC induced cell cycle arrest at the G2/M phase through downregulation of cell division control protein 2 (Cdc2), as evidenced by reduced sensitivity to THC in Cdc2-overexpressing cells [75]. In our study, we found the downregulation of glycolysis, fatty acid metabolism, and peroxisome-associated pathways indicated a global disruption of metabolic cellular function. In contrast, the concurrent activation of PI3K/Akt/mTOR signaling pathways likely represented a compensatory survival response to stress-induced damage, reflecting a dynamic balance between pro-death and pro-survival signaling [76]. A previous study showed that activation of mechanistic target of rapamycin complex 1 (mTORC1) was mediated by ER stress, which induced the inositol-requiring enzyme 1- c-Jun NH2-terminal kinases (IRE1-JNK) pathway and apoptosis. Activation of mTORC1 also reduced Akt phosphorylation, an upstream event in IRE1-JNK signaling that contributed to apoptosis [77]. Besides cell survival response, PI3K/Akt signaling directly and indirectly regulates glucose, lipid, nucleotide, and amino acid metabolism, thereby supporting tumor cell proliferation and survival [78]. The phosphorylation of IRS-1 was reduced by THC leading to promote the PI3K/Akt pathway associated with enhanced glucose uptake, suggesting a link between metabolic modulation and activation of pro-survival signaling pathways [79]. This interplay reflects a dynamic adaptation in which metabolic suppression coexists with activation of survival pathways, ultimately highlighting a balance between stress-induced cytotoxicity and compensatory signaling that may influence treatment response. In addition to PI3K/Akt-mediated metabolic regulation, p53 also serves as a regulator of lipid metabolism, nucleotide biosynthesis, redox homeostasis, and energy production. Specifically, p53 suppressed glycolysis while promoting oxidative phosphorylation through transcriptional control of metabolic genes contributing to the inhibition of tumorigenesis [80]. In the context of the present findings, activation of p53 could contribute to the observed suppression of glycolytic and metabolic pathways, reinforcing its role in mediating anti-proliferative and tumor-suppressive effects.

Consistent with our observation of metabolic gene downregulation, cannabis was reported to disrupt cancer cell metabolism by depleting metabolites and suppressing metabolic pathways, ultimately reducing cell viability and promoting cell death. Mechanistically, cannabinoids acting through CB1 and CB2 receptors impair glycolytic activity, as reflected by an increased adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio, indicating disruption of cellular energy homeostasis. In addition, cannabinoid-induced autophagy has been linked to early ROS generation, which exacerbates metabolic stress and further compromises energy metabolism, as demonstrated in pancreatic adenocarcinoma models [81,82]. Hence, our findings could propose that cannabis exerted a multi-layered cytotoxic effect by inducing oxidative stress and apoptosis while simultaneously impairing proliferation, metabolism, and DNA repair capacity contributing to potential anticancer activity. The simultaneous induction of oxidative stress and suppression of DNA repair pathways may further exacerbate genomic instability, thereby sensitizing cells to apoptosis.

The combined treatment with sorafenib and cannabis triggered a broad and integrated stress response, involving inflammatory pathways and apoptotic pathways, including TNF/NF-κB, p53, IL2/IL-6-JAK-STAT, and TGF-β, as well as oxidative signaling pathways and ER-associated protein processing. Concurrent activation of PI3K/Akt/mTOR, wingless-related integration site/beta-catenin (Wnt/β-catenin), and Notch pathways likely reflect compensatory survival signaling in response to heightened cellular stress. In parallel, the suppression of proliferative and metabolic programs, as evidenced by downregulation of E2F-driven DNA replication, G2/M cell cycle progression, glycolysis, fatty acid metabolism, peroxisome function, and bile metabolism suggested a shift toward growth arrest and metabolic collapse. This coordinated change assumed that cannabis may improve the effect of sorafenib by blocking cancer cells’ survival mechanisms and increasing its ability to inhibit cell growth. Our findings are supported by previous experimental studies demonstrating that cannabinoid-based combination therapies enhanced cell cycle disruption and apoptosis in cancer cells. Co-treatment of THC, CBD, and cisplatin significantly increased G2/M phase arrest and elevated apoptotic cell populations in cervical cancer cells [83]. Furthermore, the combination of cabozantinib and CBD significantly enhanced apoptotic signaling by activation of stress and apoptosis-related regulators, including phosphorylated Chk2, c-Jun, and p53 [33]. Likewise, this is concordant with our transcriptomic data demonstrating activation of p53 signaling and apoptosis pathways in the combination group.

Although GSEA indicated enrichment of the epithelial–mesenchymal transition (EMT) gene set in the combination treatment, key features of a classical EMT program were not observed. Canonical EMT involves coordinated activation of transcription factors such as SNAI1/2, ZEB1/2, and TWIST1/2, together with upregulation of mesenchymal markers including VIM, CDH2, and FN1 [84,85,86]. In our dataset, these markers showed negative ranking and were not part of the leading-edge subset, indicating limited contribution to the enrichment signal. Instead, the enrichment was primarily driven by genes associated with inflammatory and stress responses, such as CXCL8, CXCL1, JUN, and GADD45A/B, which are known to overlap with EMT-related signatures and can be induced by ROS [87,88] which was consistent with the finding of ROS elevation in the treatment group. Taken together, these findings suggest that the observed EMT enrichment reflects a partial and context-dependent transcriptional response rather than a coordinated EMT phenotype associated with increased invasiveness or metastatic potential.