This study provides the first experimental evidence that CBD exerts direct cytotoxic and cytostatic effects in human RCC cell models (Caki-1 and 769-P) in a concentration-dependent manner. A key finding, consistent with previous cannabinoid research [5,7,9,27,28], was the marked attenuation of CBD-induced cell death when assays were conducted under serum-supplemented conditions compared to serum-free conditions. Although FBS is routinely included in in vitro cancer studies, serum reduction or withdrawal during drug exposure is frequently employed to minimize confounding effects of growth factors [10,29,30]. In the present study, the absence of serum substantially exacerbated CBD cytotoxicity, as evidenced by a pronounced decrease in IC₅₀ values from 14.8 to 20.1 µM in serum-supplemented media to approximately 6.8 µM under serum-free conditions. This enhanced cytotoxicity likely reflects a combination of pharmacokinetic and cellular stress-related factors. CBD is known to bind extensively to plasma proteins (~95%) [31], thereby limiting the freely available fraction capable of cellular uptake and interaction with intracellular targets. At the same time, serum deprivation itself imposes metabolic and oxidative stress, alters redox homeostasis, and reshapes cellular bioenergetics, potentially sensitizing cells to additional insults [32,33,34,35]. Accordingly, part of the enhanced cytotoxicity observed under serum-free conditions may arise from synergistic stress responses rather than CBD-specific mechanisms alone, underscoring the need for cautious interpretation of data generated under non-physiological conditions.

Although the effective micromolar concentrations of CBD revealed in the present study fall within ranges commonly reported in other preclinical studies [16], the concentrations required to elicit cytotoxicity under serum-supplemented conditions (IC₅₀ values up to ~20 µM) raise important translational considerations. Clinical pharmacokinetic studies have shown that oral administration of high CBD doses (1500 mg/day for 7 days or a single dose of 20 mg/kg) results in maximal plasma concentrations of only 3.2–5.2 µM [36,37], highlighting the limited systemic exposure achievable even at high dosing regimens. In addition, the oral bioavailability of CBD in humans is relatively low (<10–20%), largely due to its poor aqueous solubility and significant first-pass hepatic metabolism [38,39], resulting in inconsistent and often subtherapeutic plasma levels. Together, these pharmacokinetic constraints emphasize the importance of conducting in vitro studies under physiologically relevant, serum-containing conditions to avoid overestimating therapeutic potential.

Once absorbed, CBD’s highly lipophilic and redox-active chemical structure (Supplementary Figure S1) enables efficient cellular uptake and direct modulation of intracellular processes critical for cell survival and proliferation [4,16], beyond classical receptor-mediated mechanisms. From a mechanistic perspective, our data point to modulation of intracellular redox homeostasis as a central component of CBD action in RCC cells. CBD altered intracellular ROS/RNS levels in a time- and concentration-dependent manner. At early time points, CBD reduced ROS/RNS levels, consistent with its recognized antioxidant activity, which may involve direct scavenging of reactive species and/or transient activation of cellular antioxidant defenses [1,40,41]. In contrast, prolonged exposure resulted in a progressive increase in ROS/RNS, indicating a shift toward a pro-oxidant state. These temporal dynamics suggest an initial adaptive antioxidant response, followed by redox imbalance as metabolic and mitochondrial stress accumulate. Although sustained ROS/RNS accumulation following CBD treatment is consistent with pro-oxidant mechanisms reported in other cancer models [4], the present study does not establish a direct causal relationship between ROS induction and cell death in RCC cells. Consequently, any involvement of mitochondrial dysfunction, apoptotic signaling, or related stress pathways should be regarded as inferential and hypothesis-generating rather than mechanistically demonstrated. Nevertheless, this later pro-oxidant shift is biologically relevant, as excessive ROS/RNS production is known to disrupt mitochondrial membrane potential and activate caspase-dependent apoptotic pathways. These observations are compatible with prior reports showing that CBD triggers mitochondrial dysfunction, ER stress, and activation of PI3K/Akt/mTOR and MAPK/ERK signaling pathways, ultimately leading to apoptosis and autophagy [3]. In agreement with this proposed mechanism, CBD-induced ROS accumulation has been shown to be required for apoptosis in breast cancer cells, as pharmacological ROS blockade inhibited both apoptotic and autophagic responses [42]. Across multiple cancer models, including breast cancer, leukemia, and glioblastoma, CBD has been reported to disrupt mitochondrial membrane potential, elevate cytosolic and mitochondrial Ca²⁺ levels, and promote early ROS generation, events that collectively activate intrinsic apoptotic signaling pathways [8,16,43,44,45]. These previously described mechanisms are consistent with the redox imbalance and metabolic stress responses observed in RCC cells in the present study, supporting a broader role for CBD as a context-dependent modulator of cancer cell redox biology.

The relevance of this redox-perturbing mechanism is further underscored by the distinctive metabolic features of RCC. RCC exhibits profound metabolic reprogramming, including glutamine addiction, and elevated glutathione-based antioxidant capacity, enabling tumor cells to withstand oxidative stress and resist therapy [46,47,48,49]. By driving ROS/RNS overproduction, CBD may overwhelm this enhanced antioxidant buffering capacity and thereby exploit a metabolic vulnerability intrinsic to RCC, consistent with pro-oxidant and pro-apoptotic mechanisms described in other cancer types [3,42].

A major finding of our study is the comparable sensitivity of malignant RCC cells and non-tumoral HK-2 renal epithelial cells to CBD, indicating limited tumor selectivity under the in vitro conditions employed. However, this apparent lack of selectivity must be interpreted within the constraints of the experimental model used. The present study was conducted in two-dimensional (2D) monocultures, which are suitable for hypothesis-generating investigations but do not recapitulate key features of the in vivo renal microenvironment, including tissue architecture, cell–cell interactions, drug metabolism, and exposure dynamics [50,51]. As a result, cytotoxic responses observed in immortalized renal epithelial cells grown in 2D culture may not reliably predict renal toxicity in vivo. Indeed, despite the relatively high concentrations of CBD required to elicit cytotoxic effects in vitro, clinical studies have consistently reported favorable tolerability profiles, even at high oral doses (up to 1500 mg/day) [36,37,52,53], including in individuals with mild to severe renal impairment [54]. These studies have not identified significant safety concerns, serious adverse events, or clinically relevant laboratory abnormalities. Together, these observations suggest that systemic toxicity, including renal toxicity, may not directly mirror in vitro cytotoxicity profiles, highlighting the importance of cautious interpretation when extrapolating preclinical findings to clinical contexts.

To translate these mechanistic insights into meaningful therapeutic potential for RCC, several limitations must be addressed in future studies. First, CBD’s poor bioavailability and extensive serum binding necessitate the development of improved delivery platforms, such as lipid nanoparticles or nanoemulsions, to enhance systemic exposure and tumor accumulation. Targeted delivery approaches, including ligand-conjugated nanoparticles or renal tumor-specific carriers, may further improve selectivity while minimizing off-target toxicity [55,56,57]. Second, the present study relies primarily on metabolic (MTT) and redox-based assays, which provide useful information but cannot reliably differentiate between growth arrest, reduced metabolic activity, and true cell death. Additional functional validation, including apoptosis markers, cell cycle analysis, and clonogenic survival assays, will be required to strengthen biological interpretation and reduce reliance on metabolic readouts alone. Accordingly, the present findings should be regarded as preliminary and hypothesis-generating, providing a foundation for deeper mechanistic investigation. Third, validation in more physiologically relevant preclinical models is required. Advanced systems such as 3D cultures, organoids, co-culture systems incorporating stromal and immune components and patient-derived xenografts are required to validate these findings and to better assess the translational relevance and therapeutic potential of CBD in RCC. In this context, the impact of hypoxic conditions—an intrinsic feature of RCC driven by VHL/HIF dysregulation—on CBD responsiveness remains an important open question. Finally, the high concentrations required to achieve anticancer effects in vitro, together with the lack of tumor selectivity and attenuation of activity under serum-containing conditions, indicate that CBD is unlikely to be clinically viable as a standalone monotherapy for RCC. Future studies should therefore prioritize strategies aimed at improving tumor specificity, including formulation-based approaches (e.g., nanoparticles or liposomes) and rational combination regimens that permit dose reduction. In particular, CBD may hold promise in combination with established RCC therapies, such as tyrosine kinase inhibitors or immunotherapies, by potentiating oxidative stress and apoptotic signaling, potentially enabling effective antitumor activity at lower, clinically achievable CBD concentrations and representing a promising avenue for future investigation.