Doxycycline inhibited mitochondrial action in SH-SY5Y cells, starting at a concentration of 6.25 µg/mL (p ≤ 0.05). The drug at 12.5 µg/mL inhibited DNA synthesis and induced cell membrane breakdown. At 50 µg/mL, a decrease in cell proliferation and lysosomal activity was observed (Figure 1).

The presence of CBD in the doxycycline mixtures reduced the drug concentrations required to elicit effects on most of the endpoints studied. Exposure of neuronal cells to both mixtures resulted in inhibition of DNA synthesis at a lower drug concentration (1.56 µg/mL) compared to doxycycline alone (50 µg/mL). Cell membrane disruption, as well as inhibition of lysosomal activity and cell proliferation, occurred at two-fold lower drug concentrations in the DOX + C2 mixture (which contained a higher concentration of CBD)—6.25 µg/mL and 25 µg/mL, respectively—compared to the DOX + C1 mixture and doxycycline alone (Figure 1). No effect of either DOX + C mixture on mitochondrial activity was observed in SH-SY5Y cells compared to doxycycline alone.

An increase in free radical production in cultured neuronal cells (SH-SY5Y), accompanied by cytotoxicity, was observed starting from the lowest doxycycline concentration (1.56 µg/mL) also in both tested mixtures with CBD (Figure 2).

The IC₅₀ values for doxycycline and its two mixtures with CBD (DOX + C1 and DOX + C2) in SH-SY5Y neuronal cells revealed that the lowest IC₅₀ values were observed in the MTT and BrdU assays. In all assays, IC₅₀ values decreased with increasing CBD concentration in the mixtures. In the BrdU assay, a significant reduction in IC₅₀ values were observed for both mixtures, falling below the lowest tested concentration (1.56 µg/mL), compared to doxycycline alone (Table 1).

A decrease in HepG2 cell viability was observed after exposure to higher doxycycline concentrations compared to SH-SY5Y neuronal cells. The drug inhibited DNA synthesis in HepG2 cells at 12.5 µg/mL. Increasing the drug concentration to 25 µg/mL inhibited lysosomal activity, while a further increase to 50 µg/mL impaired mitochondrial function, disrupted membrane integrity, and reduced cell proliferation (Figure 1).

A low concentration of CBD in the DOX mixture (DOX + C1) increased the drug concentration required to inhibit HepG2 cell proliferation to 200 µg/mL (Figure 1). In contrast, the presence of CBD reduced the DOX concentration required to induce mitochondrial dysfunction and cell membrane damage to 25 µg/mL—half the concentration needed to achieve the same effect with DOX alone. No significant effect of CBD in combination with DOX on DNA synthesis or lysosomal activity was observed in HepG2 cells compared to treatment with doxycycline alone.

It should be noted that a significant increase in free radical production was observed in cultured HepG2 cells following exposure to doxycycline at 12.5 µg/mL, as well as in the tested mixtures with CBD (Figure 2).

The lowest IC₅₀ values for DOX and its two mixtures with CBD (DOX + C1 and DOX + C2) were observed in the NRU assay, compared to the BrdU assay in HepG2 liver cells.

In both methods, an increase in IC₅₀ values was observed only for the DOX + C1 mixture. In contrast, a decrease in IC₅₀ values was noted for the DOX + C2 mixture in four assays—MTT, NRU, LDH, and BrdU—compared to the DOX + C1 mixture (Table 1).

The highest sensitivity to DOX was exhibited by kidney cells (HEK-293). The low concentration of the drug (3.12 µg/mL) inhibited DNA synthesis. At a higher concentration of 6.25 µg/mL, it inhibited both mitochondrial and lysosomal activity and caused degradation of the cell membrane. At 12.5 µg/mL, it inhibited kidney cell proliferation (Figure 1).

The presence of CBD in both DOX + C1 and DOX + C2 mixtures increased the doxycycline concentrations required to inhibit mitochondrial activity, reaching 50 µg/mL and 12.5 µg/mL, respectively, compared to doxycycline alone (Figure 1). However, a high concentration of CBD in the DOX + C2 mixture reduced the effective doxycycline concentration needed to inhibit DNA synthesis to 1.56 µg/mL, compared to doxycycline alone and the DOX + C1 mixture. CBD in both tested mixtures did not affect lysosomal activity, cell membrane integrity, or cell proliferation compared to doxycycline alone (Figure 1).

An increase in free radical (ROS) production was observed in cultured kidney cells (HEK-293), starting from the lowest tested concentration of doxycycline, as well as in both tested mixtures. The presence of CBD in both mixtures induced stronger ROS production than doxycycline alone at the two lowest concentrations (1.56 and 3.125 µg/mL) (Figure 3).

The lowest IC₅₀ values for the drug and its two mixtures with CBD were observed in the BrdU assay compared to the MTT, NRU, TPC, and LDH assays in HEK-293 kidney cells. A statistically significant increase in the IC₅₀ values was observed for the DOX + C1 mixture in the MTT, TPC, and LDH assays. In contrast, a decrease in IC₅₀ values was observed for the DOX + C2 mixture in the same assays (Table 1).

The nature of the interaction between the drug and CBD depended on the cell type as well as the CBD and doxycycline concentration in each mixture. The CI values for the DOX + CBD mixtures are presented in Table 1. Figure 2 shows the CI values calculated across a wide range of cytotoxicity levels (from 20% to 90%) for both combinations.

The decrease in IC₅₀ values for both mixtures compared to the drug alone indicated a synergistic interaction in the MTT and LDH assays in SH-SY5Y cells (Table 1). Strong synergistic effects were observed in neuronal cells at high drug concentrations, whereas additive effects predominated at low doxycycline concentrations in both mixtures across the MTT, NRU, TPC, and LDH assays (Figure 3). Regardless of the drug concentrations, a strong synergistic effect was observed in the BrdU assay for both mixtures (Figure 3).

The increase in IC₅₀ values for both mixtures suggests additive and antagonistic interactions in the NRU and BrdU assays, respectively, for HepG2 cells (Table 1). Regardless of the drug concentration, the DOX + C1 mixture exhibited an additive interaction in NRU and BrdU assays (Figure 3). Synergistic effects were observed in HepG2 cells at high drug concentrations, whereas additive effects predominated at low doxycycline concentrations in DOX + C1 mixture across the MTT and LDH assays. In TPC assay, additive and antagonistic effects were shown at low and higher concentrations, respectively. In contrast, the interaction pattern of the DOX + C2 mixture varied with drug concentration. At low DOX concentrations, an antagonistic effect was observed in the NRU assay and an additive effect in the MTT, LDH, TPC and BrdU assay. At higher concentrations of drug, the antagonistic (protective) effect was particularly evident in the BrdU and TPC assays (Figure 2). This protective effect is supported by a rise in IC₅₀ value in the BrdU assay and the shift in the DOX concentration in the TPC assay, indicating the influence of CBD on drug cytotoxicity. In the MTT and LDH assays, an additive effect was observed for both mixtures at low drug concentrations, whereas at higher concentrations, a synergistic effect was noted when DOX was combined with CBD.

In HEK-293 cells, the nature of the interaction depended on the concentration of CBD in the mixture. Antagonistic effects were observed at low drug concentrations, whereas additive effects predominated at higher doxycycline concentrations in both mixtures across the MTT. In the NRU assay, additive effects were observed in the DOX + C1 mixture, whereas in the DOX + C2 mixture, additive effects predominated at low drug concentrations, but synergistic effects prevailed at higher doxycycline concentrations. In TPB, an additive effect was observed for both mixtures. In LDH assay, additive and antagonistic effects were shown at low and higher concentrations, respectively, in both mixtures. The DOX + C1 mixture exhibited an additive effect in the BrdU assays. An antagonistic effect was observed in the remaining assays in the DOX + C2 mixture (Table 1, Figure 3).

Exposure to DOX and its two mixtures with CBD induced morphological changes in SH-SY5Y, HepG2, and HEK-293 cells (Table 2). Drug treatment resulted in cell aggregation followed by detachment from the adhesive surface. The DOX + CBD mixtures caused cell shrinkage, leaving only thin cytoplasmic connections between adjacent cells. Additionally, cell rounding and separation were observed (Table 2).

Exposure of SH-SY5Y cells to DOX increased the proportion of apoptotic and necrotic cells. In contrast, treatment with DOX–CBD mixtures reduced both populations compared to the drug alone. This effect coincided with a decrease in the total cell number, suggesting a synergistic cytotoxic interaction (Table 2). Similarly, in HepG2 cells, doxycycline induced apoptosis and necrosis, whereas both DOX + C mixtures reduced the proportion of apoptotic cells relative to doxycycline treatment alone (Table 2). These findings indicate that CBD may attenuate DOX-induced apoptosis in HepG2 cells. In HEK-293 cells, doxycycline and the DOX + C1 mixture increased apoptotic and necrotic cell fractions, while the DOX + C2 mixture slightly reduced both parameters (Table 2).

Monitoring studies have shown that doxycycline is one of the most frequently detected antibiotics in both food and the environment. Although it is widely used in medicine, exposure to low concentrations of this drug promotes the development of antibiotic resistance and contributes to adverse effects in humans. Studies conducted on various human cell lines have demonstrated that extended incubation (96 h) with doxycycline, at concentrations ranging from 0.1 to 10 µg/mL, leads to cytotoxic effects through increased lactate production and decreased oxygen consumption. A concentration of 1 µg/mL has been observed to inhibit proliferation and induce apoptotic cell death in the tested cell cultures [43]. In our studies, we observed these effects after only 72 h of exposure in human cell cultures.

Doxycycline exhibits significant pharmacokinetic properties, including the ability to penetrate all organs and tissues, allowing it to reach therapeutic concentrations even in the central nervous system [44]. Neurotoxic effects of doxycycline have been observed during sclerotherapy procedures [44,45,46,47]. Based on available studies, the neurotoxicity of this drug depends on both the duration of exposure and the type of neuronal cells involved. For instance, studies using the rat neuronal cell line PC12 revealed no signs of neurotoxicity after 24 h of exposure to doxycycline at a concentration of 20 µM (10.26 µg/mL) [48]. Interestingly, in SH-SY5Y human neuroblastoma cells, doxycycline at concentrations ranging from 4 ng/mL to 4.4 µg/mL demonstrated a protective effect after 24 h of exposure by reducing the production of reactive oxygen species (ROS) [49]. However, another study reported that a 24 h incubation with doxycycline at higher concentrations up to 4 µg/mL decreased SH-SY5Y cell density—likely indicating a toxic effect—although the effect was minimal at 1 µg/mL [50]. Following a 48 h exposure, the IC₅₀ value for doxycycline in SH-SY5Y cells was determined to be 3.7 ± 0.1 µM (1.9 µg/mL) [51]. Additionally, some studies have reported that doxycycline exhibits antitumor activity in neuronal cells [51,52]. In our study, 72 h exposure of SH-SY5Y neuronal cells to 6.25 µg/mL doxycycline decreased cell viability, mainly due to the inhibition of mitochondrial activity. The calculated IC₅₀ value for doxycycline, depending on the cellular parameter analyzed, ranged from 9.8 to >200 µg/mL. An increase in free radical production, accompanying cytotoxicity, was observed even at the lowest tested concentration (1.56 ug/mL). Moreover, exposure to doxycycline was associated with a marked increase in apoptotic and necrotic cell populations in the culture. Clear morphological changes in SH-SY5Y cells were observed after exposure to the drug.

Among the numerous contraindications and adverse effects associated with doxycycline use, liver and kidney dysfunction are frequently reported. Studies have indicated a correlation between long-term doxycycline use and the severity of liver dysfunction [53,54,55]. In vitro studies using various hepatoma cell lines have shown that doxycycline can induce several effects indicative of potential hepatotoxicity, primarily related to mitochondrial damage and oxidative stress. Exposure of HepG2 cells to 20 µM (10.3 µg/mL) doxycycline resulted in reduced cell viability due to inhibited mitochondrial activity, cell membrane damage, and increased production of reactive oxygen species (ROS) [56]. Another study demonstrated that doxycycline induces lipid accumulation, mitochondrial membrane depolarization, and a dose-dependent increase in ROS levels, with concentrations of approximately 20–25 µM (10.3–13.0 µg/mL) causing about a 50% reduction in HepG2 cell viability [55,57]. The exposure (72 h) also resulted in mitochondrial inhibition, with an IC₅₀ of 20 µM (10.3 µg/mL) [58]. In contrast, studies using human pluripotent stem cell-derived hepatocytes showed that 32 µM (16.5 µg/mL) doxycycline depolarized the mitochondrial membrane and caused significant lipid accumulation, although ROS levels were not significantly elevated [58,59]. Our results are consistent with the studies cited above. We observed that exposure to doxycycline at a concentration of 12.5 µg/mL primarily inhibited DNA synthesis in HepG2 cells, accompanied by increased ROS production. The calculated IC₅₀ values, depending on the cellular parameter evaluated, ranged from 13.4 to >200 µg/mL. As in neuronal cells, exposure to doxycycline led to an increase in the number of apoptotic and necrotic cells, along with visible changes in HepG2 cell morphology.

Another adverse effect of DOX is renal impairment. Recent clinical case reports have indicated the occurrence of acute interstitial nephritis following doxycycline administration. The drug’s nephrotoxicity is likely related to its tendency to accumulate in renal tissue [60,61]. In vitro studies have shown that doxycycline at a concentration of 20 µg/mL reduces the viability of HEK-293 cells, although it does not induce apoptosis. The IC₅₀ value for doxycycline was reported to be 11.85 µM (6.05 µg/mL) after 48 h of exposure [51]. Additional studies demonstrated that doxycycline at 20 µg/mL also inhibited DNA synthesis in normal HK-2 cells without triggering apoptosis. However, cells of neoplastic origin were more sensitive to the drug compared to normal cells. Doxycycline significantly inhibited protein synthesis and mitochondrial complex activity at a concentration of 5 µg/mL, while a concentration of 10 µg/mL induced apoptosis in renal cancer cells [62]. Doxycycline exerts mitochondrial effects due to the structural similarity between bacterial and mitochondrial ribosomes. Furthermore, it inhibits the activity of matrix metalloproteinases (MMPs), which have a significant role in cancer progression, including renal cell carcinoma [63]. However, the precise mechanism underlying doxycycline’s cytotoxic effects on renal cancer cells remains unclear. Our study demonstrated that 72 h exposure of normal HEK-293 cells to 3.125 µg/mL doxycycline resulted in nephrotoxic effects, primarily through inhibition of DNA synthesis. Cytotoxic effects were accompanied by increased ROS production, even at the lowest tested concentration. The calculated IC₅₀ values for doxycycline, depending on the cellular parameter assessed, ranged from 8.9 to 30.4 µg/mL. In contrast to previous reports, we observed a significant increase in apoptotic and necrotic cells in culture following exposure. These cytotoxic effects were also associated with noticeable morphological changes in HEK-293 cells. Our research shows that DOX can cause undesirable effects in humans at the cellular level. Therefore, the challenge was to minimize this effect of the drug. Doxycycline mustn’t cause harmful interactions with other drugs or substances administered concurrently [64].

The ethnological, economic, and well-documented therapeutic relevance of Cannabis sativa has driven growing scientific interest in its bioactive compounds.

In the present study, cannabidiol (CBD) exhibited a synergistic effect in SH-SY5Y neuroblastoma cells, enhancing the cytotoxicity of doxycycline (DOX). Our findings further indicate that the combined DOX + CBD treatment exacerbates mitochondrial dysfunction in SH-SY5Y cells, leading to elevated ROS generation and subsequent cell death. Both compounds are known to disrupt the mitochondrial membrane potential (MMP), promoting cytochrome c release and activation of the caspase cascade (caspase-9 → caspase-3) in cancer cells. Although a detailed mechanistic analysis was not performed, pronounced DNA fragmentation was observed following DOX + CBD exposure, as revealed by BrdU incorporation and Hoechst 33342 staining, suggesting caspase-3–dependent apoptosis. Future studies should explore the potential involvement of apoptosis-inducing factor (AIF) translocation and PARP cleavage, both of which are indicative of caspase-mediated cell death. While Hoechst/PI staining supported apoptosis-related changes, definitive discrimination between apoptosis and necrosis requires further validation using Annexin V/PI assays and the detection of cleaved PARP and caspase-3. These analyses will be incorporated into future experiments to confirm and extend the present findings. Numerous reports have confirmed the synergistic potential of CBD in cancer therapy [65,66,67,68,69,70,71,72]. In paclitaxel-based chemotherapy, CBD has demonstrated dual functionality—alleviating pain while enhancing the drug’s therapeutic efficacy [66]. A similar synergistic antiproliferative effect has been observed with CBD in combination with anticancer drugs such as mitoxantrone or cisplatin in melanoma cells [67]. Moreover, studies in canine urothelial cancer have shown that CBD combined with mitoxantrone or vinblastine further enhances the antitumor response [68]. During anticancer therapy, the administration of CBD has been reported to protect healthy cells and improve their function [69,70,71].

In the present study, an antagonistic interaction between DOX and CBD was observed in hepatic (HepG2) and renal (HEK-293) cell cultures, as evidenced by increased IC₅₀ values for DOX + CBD mixtures. Notably, this protective (antagonistic) effect was observed at low DOX concentrations, whereas higher DOX levels induced a shift toward synergism. These findings suggest that CBD mitigates DOX-induced cytotoxicity in normal cells, reducing organelle damage and apoptosis, while potentiating DOX activity in neuronal tumor cells. The IC₅₀ was dependent on CBD concentration—higher CBD levels were associated with a diminished protective effect. CBD effectively preserved cellular organelle integrity without altering free radical production and displayed anti-apoptotic activity in liver and kidney cell cultures. Previous studies have demonstrated the anti-apoptotic and cytoprotective properties of cannabidiol (CBD), primarily attributed to its antioxidant and anti-inflammatory activities [38,40,73,74,75,76,77,78,79,80]. CBD regulates Nrf2 signaling and enhances the activity of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, thereby reducing oxidative stress and ROS/RNS generation [75,76]. In addition, CBD downregulates pro-inflammatory cytokines such as IL-6 and IFN-α and inhibits the expression of matrix metalloproteinases (MMPs), contributing to reduced cellular inflammation [77]. It also helps maintain ionic homeostasis under stress conditions by modulating Na⁺/Ca²⁺ exchange and promoting calcium storage. The compound supports cell survival, partly through the upregulation of heme oxygenase-1 (HO-1) [78]. Unlike other cannabinoids, CBD exhibits low affinity for classical cannabinoid receptors [78], yet it activates transient receptor potential (TRP) channels, including TRPA1, TRPV1, and TRPV4, even at nanomolar concentrations, which further enhances its cytoprotective effects. The multifaceted effects of CBD are well documented in the literature [65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81]. This compound has demonstrated therapeutic potential in various models of toxic organ injury (e.g., CCl₄, cadmium, alcohol), where it reduced oxidative stress, inhibited inflammatory mediators, and decreased apoptosis [35,36,72,73,74,75,76,77,78,79,80]. In alcohol-induced liver injury, CBD reduced lipid accumulation and oxidative stress, stimulated autophagy, and modulated the inflammatory response. Its administration has also been shown to inhibit apoptosis and promote cell survival [69,78]. When considering interactions between CBD and doxycycline (DOX), the involvement of cytochrome P450 should also be taken into account. Literature data indicate that both compounds are metabolized by different cytochrome P450 isoforms. Regarding cytochrome P450 enzymes, CBD has been reported to be a potent inducer of CYP1A1 expression [81], whereas doxycycline acts as an inducer of CYP3A4 [4,43,44]. Therefore, most interactions involving doxycycline are attributed to its chelating and physicochemical properties rather than to cytochrome P450–mediated metabolism [44].

Analytical standard of doxycycline hyclate (CAS: 24390-14-5; m.w. 512.9 g/mol nr cat. D9891) was purchased from Sigma-Aldrich, Poznan, Poland. Cannabidiol (CAS: 13956-29-1; m.w. 314.5 g/mol nr cat. 85705) was obtained from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Others chemicals used to cell cultures or made tests: triton X-100, hydrogen peroxide, trypan blue, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), neutral red dye (NR), Coomassie brilliant blue R-250 dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, propidium iodide, DCFH-DA, hydrogen peroxide, trypsin-ethylenedinitrilotetraacetic acid (EDTA) and antibiotic solution were purchased from Sigma–Aldrich Co. (Poznan, Poland). All other chemicals were purchased from commercial suppliers and were of the highest available purity.

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed at the corresponding authors.