Cytotoxicity Potency of Cannabinoids Is by Serum Supplementation: Implications for In Vitro Cancer Studies
Department of Physiology, Faculty of Health Sciences University of Pretoria Pretoria South Africa
Department of Pharmacology, Faculty of Health Sciences University of Pretoria Pretoria South Africa
* Correspondence:Vanessa Steenkamp (vanessa.steenkamp@up.ac.za)
ABSTRACT
Background
Fetal calf serum (FCS) is a key supplement in cell culture, providing nutrients, and growth factors that support cancer cell proliferation. Studies suggest that compound cytotoxicity profiles in vitro vary depending on FCS concentration in culture medium. The study aimed to examine how extracellular conditions influence the in vitro response of cancer cells to phytocannabinoids, with particular emphasis on serum supplementation.
Methods and Results
Cells were exposed to CBD and THC in media containing 0.5% or 10% FCS for 24–72 h. Cell viability was assessed using the sulforhodamine B assay and live‐cell imaging. Significantly enhanced cytotoxic effects of CBD and THC were noted under low‐serum conditions (0.5% FCS) compared to standard conditions (10% FCS), particularly after 72 h incubation. THC demonstrated greater cytotoxicity than CBD in SiHa cells, while both compounds showed similar effects in HeLa cells.
Conclusion
The study demonstrates that serum concentration critically modulates the cytotoxic potency of cannabinoids. Reduced FCS enhances cannabinoid efficacy by limiting protein binding, emphasizing the need to optimize serum conditions for accurate in vitro cytotoxicity assessments.
1Introduction
Cervical cancer remains one of the most prevalent malignancies affecting women worldwide, with a particularly high burden in low‐ and middle‐income countries [1]. Despite advancements in the treatment regime, the disease continues to pose significant challenges, particularly in cases of drug resistance and disease recurrence [2]. Consequently, there is growing interest in alternative therapeutic strategies, including cannabinoids, which have demonstrated anticancer potential in preclinical studies [3].
Cannabinoids, a diverse class of compounds derived from the Cannabis plant, have gained significant attention for their potential therapeutic applications [4]. Among them, cannabidiol (CBD) and tetrahydrocannabinol (THC) are the most studied, with CBD recognized for its anti‐inflammatory, analgesic, and neuroprotective properties [5], whereas THC is known for its psychoactive effects and potential anticancer activity [6]. Emerging research suggests that cannabinoids may influence various cellular pathways, including apoptosis, oxidative stress, and immune modulation, making them promising candidates for treating conditions such as chronic pain, epilepsy, and various cancers [7, 8].
The endocannabinoid system (ECS) plays a crucial role in various physiological processes, including immune regulation, pain sensation, and cell proliferation [9]. This system comprises endogenous cannabinoids, their receptors (CB1 and CB2), and associated enzymes [9]. Dysregulation of the ECS has been implicated in the development and progression of various cancers, sparking interest in exploring cannabinoid receptor agonists as potential anti‐cancer therapies [9].
Cell culture media plays a vital role in maintaining and growing cells in vitro by providing essential nutrients, growth factors, and environmental conditions necessary for cell survival and proliferation [10]. The composition of culture media varies depending on the specific cell type and research requirements, and it typically includes antibiotics to prevent bacterial contamination, l‐glutamine as an energy source, and additional components such as pH regulators, nonessential amino acids, sodium pyruvate, and growth factors to support cell growth, proliferation, and differentiation [10]. A key supplement commonly used in cell culture media is fetal calf serum (FCS), which supplies essential nutrients, hormones, and proteins necessary for optimal cell function [11].
Recent studies have suggested that FCS may significantly influence the cytotoxic effects of cannabinoids on cancer cells, raising concerns about its impact on cannabinoid research in cancer treatment [12, 13, 14]. The high protein content in FCS, particularly albumin, has been shown to bind to cannabinoids, effectively reducing their bioavailability and limiting their interaction with cellular targets [15]. To address this, researchers investigating the anticancer properties of cannabinoids have adjusted experimental conditions by using reduced FCS concentrations in culture media [13, 14]. This modification aims to minimize binding effects and provide a more accurate assessment of the true cytotoxic potential of cannabinoids against cancer cells.
Beyond cannabinoids, serum proteins have been shown to influence the bioavailability of various drugs in vitro [16, 17]. Many chemotherapeutic agents, such as platinum‐based drugs (e.g., cisplatin and 5‐fluorouracil), also exhibit altered efficacy depending on serum concentration, as high levels of albumin can reduce drug delivery to cancer cells [18, 19]. These findings highlight the broader implications of serum‐drug interactions in cell culture research. Understanding these interactions is crucial for optimizing cell culture conditions in drug testing, ensuring that observed cytotoxic effects are not artificially modulated by serum components. The present study investigates how variations in serum concentration affect the cytotoxic responses of cervical cancer and non‐tumorigenic cells to cannabinoids.
2Materials and Methods
2.1Test Compounds
The cannabinoids, THC and CBD, were procured from LECO Africa (Pty) Ltd. (South Africa), and cisplatin from Sigma‐Aldrich (St. Louis, USA). The stock solution of the positive control, cisplatin (20 mM), was prepared using dimethyl sulfoxide (DMSO). The CBD stock solution (3.225 mM) was dissolved in methanol and diluted in the respective cell culture media to obtain a 20 μM working solution. This solution was further diluted to generate test concentrations ranging from 0.039 to 10 μM. The stock solution of THC (3.184 mM) was dissolved in methanol (1 mL) and diluted in the respective cell culture media to obtain a 60 μM working solution. This solution was further diluted to obtain a concentration range of 0.234–30 μM, which was used in the experimental procedures.
2.2Cell Culture and Maintenance
The HeLa (CCL‐2) cervical cancer cell line was procured from the American Type Culture Collection (ATCC; Virginia, USA). The SiHa (HTB‐35) cervical cancer cell line was gifted by the Pan African Cancer Research Institute (PACRI, University of Pretoria, Pretoria, South Africa). The MCF‐12A human non‐tumorigenic mammary epithelial cell line (CRL‐10782) was purchased from Highveld Biological (Pty) Ltd. (Sandringham, Johannesburg, South Africa).
Both the cervical cancer cells were grown in 75 cm2 cell culture flasks (Greiner Bio‐One, Kremsmünster, Austria) containing Dulbecco's Modified Eagle Medium (DMEM, Capricorn Scientific, Ebsdorfergrund, Germany), supplemented with 10% FCS (56°C for 30 min) and 1% penicillin–streptomycin (Sigma‐Aldrich, St. Louis, USA) in an incubator (5% CO2 and 37°C). The MCF‐12A cells were grown under the same conditions; however, the growth medium consisted of a 1:1 mixture of DMEM and Hams F12 medium (Capricorn Scientific, Ebsdorfergrund, Germany), containing 20 ng/mL epidermal growth factor, 100 ng/mL cholera toxin, 10 μg/mL insulin, and 500 ng/mL hydrocortisone, supplemented with 10% heat‐inactivated FCS (56°C for 30 min), penicillin (100 μg/L), streptomycin (100 μg/L), and fungizone (250 μg/L).
Cells were grown to a confluence of ~80%. Once confluency was reached, the cell culture flasks were rinsed with phosphate buffered saline (PBS) and the cells were detached from the flasks using trypsin/versene (Sigma‐Aldrich, St. Louis, USA). Cells were centrifuged at 300g for 5 min, and counted using the trypan blue exclusion assay (0.1% w/v). Cells were thereafter diluted with DMEM to the seeding density required for the assays.
2.3Cytotoxicity Determination
Cytotoxicity of the test compounds was assessed using the sulforhodamine B (SRB) staining assay as described by Vichai and Kirtikara [20] with minor changes to volumes used. The SRB stains cellular proteins, which can be extracted and quantified to assess cell viability and growth. The HeLa, SiHa, and MCF‐12A cells were seeded in 96‐well plates at a density of 5000 cells per well and incubated for 24 h at 37°C and 5% CO2 to allow for attachment. Each plate contained three sets of controls: negative control (NC; cells propagated in growth medium), the vehicle control (VC, growth media containing 0.01% methanol), and the positive control (30 μM Cisplatin).
To determine the effect that FCS has on the toxicity of the compounds on the cells, medium containing 0.5% or 10% FCS was used in the assays. The cells were treated with varying concentrations of CBD (0.039–10 μM) or THC (0.234–30 μM) for a period of 24, 48, and 72 h. After the exposure period, 50 μL of a 50% trichloroacetic acid (TCA; Sigma‐Aldrich, USA) solution was added to each well for fixation of the cells, and the plates were incubated at 4°C for 24 h. Thereafter, plates were washed three times with slow‐running tap water and dried in an oven (EcoTherm, Labotec, South Africa) set at 40°C–45°C. A volume of 100 μL of 0.057% SRB solution (Sigma‐Aldrich, St. Louis, USA) was added to each well, and the plates were incubated at room temperature for 30 min in the dark. Thereafter, unbound dye was removed by washing the plates three times with 150 μL of 1% acetic acid solution (Sigma‐Aldrich, St. Louis, USA) and dried in the oven. The bound dye was extracted from fixed cells by adding 200 μL of 10 mM Tris‐base solution (Sigma‐Aldrich, St. Louis, USA) to each well. Plates were placed on a gyratory shaker (VRN‐200, Gemmy Industrial Corporation, Taiwan) for 1 h to solubilize the protein‐bound dye. After the dye has solubilized, the optical density (OD) was measured at 540 nm (reference: 630 nm) using an ELX 800 microplate reader (BioTek instruments Inc., Highland Park, USA). All values were blank‐subtracted, and the cell density (%) calculated using the following formula:where “OD sample” refers to the corrected optical density of the sample and “OD negative” is the corrected optical density of the negative control.
2.4Live‐Dead Staining
Fluorescein diacetate (FDA; Sigma‐Aldrich, St. Louis, USA) was used to stain the cells to assess cell viability. Metabolically active, viable cells are visualized as a green fluorescence. The HeLa, SiHa, and MCF‐12A cells were seeded and treated as described above. Thereafter, the cells were washed three times with 0.1 M PBS before being immersed in staining solution. The staining solution consisted of 4 μg/mL propidium iodide (PI, Sigma‐Aldrich, USA) and 5 μg/mL FDA in PBS. The cells were exposed to the dye for 4 min in the dark. Thereafter, 100 μL of PBS was added to each well. The stained cells were then examined using a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss Inc., Oberkochen, Germany), utilizing filter sets for FDA fluorescence and a ×20 magnification. A composite image was generated using ImageJ software version 1.54.
2.5Statistical Analysis
Microsoft Excel 2019 was used to capture raw data and statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Experiments were carried out on three occasions, each in triplicate (n = 9). All data are expressed as the mean ± standard deviation (SD). For cytotoxicity studies, the logarithmic drug concentration was plotted against the relative response (compared to the negative control). The cytotoxic range of each drug was calculated using a nonlinear regression curve fit (log 3 vs. relative cell number) with a robust fit. Statistical significance was considered as p < 0.05.
3Results
3.1 CBD Suppressed HeLa, SiHa and MCF‐12A Viability in a Time and Serum‐Dependent Manner
CBD exhibited variable cytotoxic effects, which were dependent on both the specific cell line and the experimental conditions (Figure 1). In HeLa cells, the IC50 values were 1.62, 4.40, and 2.34 μM after 24, 48, and 72‐h incubation periods, respectively. There was no consistent temporal progression, indicating that the compound's efficacy fluctuated rather than steadily increased or decreased over time (Figure 1A–C, Table 1). A gradual reduction in IC50 values was observed in SiHa cells (Figure 1D–F) with increasing incubation time. A similar time‐dependent trend was observed for MCF‐12A cells (Figure 1G–I) with the most pronounced effects occurring after 72 h of incubation. Notably, the IC50 values were significantly (p ≤ 0.0001) lower in the medium containing 0.5% FCS compared to medium containing 10% FCS (Table 1).
| Test | Cell line | ||||||
|---|---|---|---|---|---|---|---|
| Compound | HeLa | SiHa | MCF‐12A | ||||
| Incubation period (h) | FCS (%) | ||||||
| 0.5 | 10 | 0.5 | 10 | 0.5 | 10 | ||
| IC50 (μM) | |||||||
| CBD | |||||||
| 24 | 1.62 ± 2.40**** | 25.00 ± 1.50 | 1.18 ± 2.00**** | 19.20 ± 3.04 | 17.00 ± 1.66 | 22.00 ± 1.00 | |
| 48 | 4.40 ± 1.89**** | 30.00 ± 2.50 | 0.50 ± 2.54**** | 68.00 ± 2.97 | 17.00 ± 1.02 | 19.00 ± 1.50 | |
| 72 | 2.34 ± 2.34**** | 23.00 ± 3.01 | 0.50 ± 2.22**** | 4.30 ± 2.09 | 8.20 ± 1.88 | 10.00 ± 2.01 | |
| THC | |||||||
| 24 | 4.50 ± 1.20*** | 7.50 ± 2.23 | 1.60 ± 3.33**** | 12.80 ± 2.56 | 48.00 ± 1.02 | 50.00 ± 2.01 | |
| 48 | 0.80 ± 1.44**** | 10.20 ± 3.03 | 0.80 ± 1.91**** | 55.00 ± 2.22 | 49.00 ± 1.13 | 55.00 ± 1.98 | |
| 72 | 2.30 ± 1.69 | 4.10 ± 3.32 | 1.30 ± 1.80**** | 14.00 ± 1.98 | 28.00 ± 1.45 | 30.00 ± 1.56 | |
3.2 THC Exerted Higher Cytotoxicity Than CBD Under Low Serum Conditions
THC demonstrated marked cytotoxicity in cervical cancer cells, with its effect being dependent on serum concentration and duration of exposure. THC showed increased cytotoxicity under low‐serum conditions (0.5% FCS). In HeLa cells, its cytotoxicity was significantly greater in reduced serum media, with IC50 values of 2.3 μM in media containing 0.5% FCS compared to 4.1 μM in media containing 10% FCS, after 72 h of treatment (Figure 2A–C, Table 1). A comparable cytotoxic response was observed in SiHa cells (Figure 2D–F). In contrast, in the normal breast epithelial cell line (MCF‐12A), THC displayed IC50 values of 28 μM in media containing 0.5% FCS and 30 μM in media containing 10% FCS after 72 h, both of which were significantly (p ≤ 0.0001) higher than the values recorded in cervical cancer cells (Figure 2G–I) (Table 1).
3.3Live Cells Imaging Confirmed Dose‐Dependent Cell Death
The FDA staining images offer a visual representation of the cytotoxicity of CBD, and THC on HeLa, SiHa, and MCF‐12A cells after the 72 h incubation period. This specific time point was selected as it consistently demonstrated the most pronounced drug effects across all cell lines, based on the IC50 data. The effects observed are presented as FDA staining images, for a subset of drug concentrations (0.039, 5, and 10 μM for CBD; 0.0234, 15, and 30 μM for THC) representing low, moderate, and high cytotoxicity.
HeLa, SiHa, and MCF‐12A cells revealed distinct dose‐dependent cytotoxicity of CBD (Figure 3). In HeLa cells, CBD exhibited minimal cytotoxicity at the low concentrations, moderate effects at intermediate concentrations, and marked cell death at the highest concentration tested (10 μM) (Figure 3). A similar trend was observed in SiHa and MCF‐12A cells (Figure 3).
In HeLa cells, THC displayed minimal toxicity at low concentrations, moderate effects at 7.5 μM, and pronounced cytotoxicity at higher doses (15–30 μM) (Figure 4). A similar trend was observed in both SiHa and MCF‐12A cells (Figure 4).
4Discussion
The significant differences in cannabinoid cytotoxic potency between 0.5% and 10% FCS conditions in this study align with previous findings, suggesting enhanced drug sensitivity under low serum conditions [12]. These findings are consistent with studies showing that CBD, for example, exhibited cytotoxic effects on HT‐29 cells when cultured in media containing 0.5% serum, but not in media containing 10% serum [12]. Similarly, Chekarsova et al. demonstrated the synergistic effect of CBD when combined with intermittent serum starvation, likely due to reduced serum protein binding that sequesters lipophilic compounds like CBD [18]. The present study extends these findings, confirming that in low‐serum environments (0.5% FCS) CBD exhibits greater cytotoxicity compared to the standard serum environment (10% FCS).
The observed variations in cytotoxic effects across different cell lines may partly result from differences in cannabinoid receptor expression and albumin uptake mechanisms. Notably, CB1 and CB2 receptors, which are integral components of the ECS, regulate apoptosis, proliferation, and immune responses in cancer, and are highly expressed in cervical carcinoma cell lines such as HeLa and Caski, as well as in tumor biopsy samples [21]. Activation of these receptors by anandamide has been shown to induce apoptosis in these cells. Moreover, CB1 and CB2 receptors are broadly implicated in modulating tumor progression across various cancer types [22].
Anandamide's anti‐cancer effects are not solely mediated by the ECS, but non‐cannabinoid pathways also play a significant role. In HeLa and Caski cervical cancer cells, anandamide induces apoptosis by activating transient receptor potential vanilloid 1 (TRPV1) channels [21]. This pro‐apoptotic effect is inhibited by capsazepine, a TRPV1 antagonist, highlighting TRPV1's role in cervical cancer cell death. TRPV1's influence extends to multiple cancer types, affecting cell fate by regulating calcium influx and subsequent apoptotic signaling pathways [23]. However, TRPV1's expression and function vary significantly depending on the specific cellular context.
Albumin‐binding proteins, such as gp60 and SPARC, are crucial for facilitating albumin uptake, particularly due to their increased expression in cancer cells [24]. This process goes beyond receptor‐mediated signaling. Albumin's dual role as both a nutrient source and a drug release facilitator can impact the observed sensitivity to cannabinoids [15].
The reduced cytotoxicity observed under high‐serum conditions may be due to the strong extracellular binding of cannabinoids to albumin, initially limiting their uptake. However, the heightened sensitivity of cervical cancer cells compared to non‐tumorigenic MCF‐12A cells could be explained by differences in ECS receptor expression, TRPV1 responsiveness, and albumin‐mediated internalization pathways. Notably, albumin‐binding proteins such as gp60 and SPARC, often overexpressed in cancer cells, facilitate albumin uptake beyond receptor‐mediated signaling [24]. Once internalized, albumin undergoes lysosomal degradation, releasing bound compounds like CBD or THC, thus influencing drug delivery and cytotoxicity [25]. This dual function of albumin as both a nutrient source and a drug release facilitator may contribute to the observed differential sensitivity to cannabinoids. Further investigations profiling the expression of CB1, CB2, TRPV1, and albumin‐binding proteins in these cellular models would enhance comprehension of these complex mechanisms.
These findings underscore the importance of considering serum concentration when designing in vitro studies. Low serum conditions may overestimate drug cytotoxicity by increasing the free drug concentration, while high serum conditions might underestimate it [26]. This variability highlights the need for careful consideration of physiological relevance when interpreting in vitro experiments. The observed serum‐dependent effects on cannabinoid cytotoxicity emphasize the need to consider drug‐specific evaluations and the physicochemical properties of test compounds when selecting appropriate in vitro conditions. To improve the translational potential of in vitro studies, researchers should consider optimizing experimental conditions to better mimic the in vivo environment, such as testing drugs at multiple serum concentrations or utilizing advanced models like 3D cell cultures [27]. Additionally, integrating pharmacokinetic modelling with in vitro data and conducting parallel in vitro and in vivo studies could help bridge the gap between controlled studies and real‐world clinical applications [28]. However, translating preclinical findings to clinical settings remains challenging due to individual patient variability, genetic polymorphisms, and disease‐related alterations in protein expression, making careful optimization of experimental conditions essential.
5Conclusion
This study demonstrates the differential cytotoxic effects of CBD and THC on cervical cancer cells (HeLa and SiHa) and non‐tumorigenic breast epithelial cells (MCF‐12A) under varying serum conditions. Cannabinoids showed enhanced cytotoxicity in low serum environments due to reduced protein binding. THC displayed higher potency than CBD in cervical cancer cells, with selectivity for cancerous cells, indicating potential as targeted therapy. The findings highlight the importance of tumor microenvironment in evaluating anticancer agents. It is recommended that further investigation of cannabinoids in cancer therapy, considering physiological conditions and tumor microenvironment for optimal therapeutic efficacy, is undertaken.
Funding
Funding was obtained from the University of Pretoria Research Development Program.
Disclosure
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Ethics Statement
Ethical approval was obtained from the University of Pretoria, Faculty of Health Sciences Ethics Committee with ethics reference no: 207/2024.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
Mr. Keith Ncube is thanked for his assistance with the fluorescence assay and microscopy techniques. The Department of Pharmacology (University of Pretoria) is acknowledged for providing access to their facilities and equipment.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.