Cannabinoid Derived Product is a Potential Novel Therapeutic for Papillary Thyroid Carcinoma
New York Medical College, Valhalla, USA
Breslin Research Foundation, Taos, NM, USA
General Nutraceutical Technology LLC, Elmsford, NY, USA
Westchester Medical Center, Valhalla NY, USA
Abstract
Rationale:
Papillary thyroid carcinoma (PTC) is the most common thyroid cancer that typically affects women ages 20 to 50, presenting as an asymptomatic neck mass. Treatment with total or partial thyroidectomy shows an excellent prognosis. However, investigation of non-invasive therapeutic options with minimal adverse effects is ongoing. This study seeks to investigate the K1 cell line, which consists of PTC cells obtained from metastatic tumors of well-differentiated PTC.
Objective:
Our investigation focuses on a cannabinoid-based product (named BRF1-A) and its potential anti-cancer effects through modulation of gene expression. We investigated its effects on gene expression of p53, c-Myc, and BCL-2 in K1 papillary thyroid cancer cells.
Methods:
BRF1A was co-cultured with K1 cell line (1 × 106 cells/ml) and incubated at 37°C under 5% CO2 for 24 and 48 hours. After the culture time points, the cells were harvested, and cell viability was determined via trypan blue exclusion assay. Using qRT-PCR, we determined the effect on the gene expression of TP53, c-Myc, and BCL-2.
Results:
Results show that the BRF1A decreased the viability of K1 PTC cells in a dose and time-dependent manner. Within 24 hours, the cannabinoid- containing product increased the gene expression of TP53 and decreased the gene expression of BCL-2 and c-Myc in K1 PTC cells.
Conclusion:
The results suggest that the cannabinoid-containing product BRF1A interacts as a potential regulator in well-differentiated thyroid cancer with the upregulation of p53 and downregulation of BLC-2 and c-Myc. Further in vitro and in vivo studies are needed to understand the exact mechanism and therapeutic potential of the cannabinoid-containing products in papillary thyroid cancer.
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Keywords: cannabinol, senescence, tetrahydrocannabinol, cannabidiol, cancer suppressor genes
Article notes
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Received 2024 Sep 7; Accepted 2025 Mar 21; Collection date 2025.
Introduction
Papillary thyroid carcinoma (PTC) is the most frequent type of malignant epithelial growth of thyroid tissue, with an excellent prognosis following surgical intervention. 1 Novel investigations with the use of human thyroid cancer cell lines focus on the genetic alterations that determine tumorigenesis and disease progression to improve the management of PTC. Driver mutations in PTC such as BRAF, 2 RAS, 3 and RET-PTC 2 have been identified, as well as a less common p53 mutation. Mutations in p53 are well-known features in malignancy that have been linked to oncogenic effects such as genomic instability, support of proliferation and survival, and metastasis. 4 In thyroid cancer, the loss of function of p53 is linked to cancer progression from well-differentiated PTC to poorly differentiated thyroid cancer and possibly anaplastic thyroid carcinoma. 5 An inappropriate increase in transcription of Wnt-mediated signaling pathways has been linked to unrestrained growth of cells and tumor development. 6 In thyroid cancer, the disruption of the Wnt cell pathway is thought to be related to malignant cell transformation leading to remarkable upregulation of c-myc, resulting in enhancement of tumor growth and drug resistance. 6 Previous studies revealed that upregulation of BCL-2 occurs at later stages of tumorigenesis and is involved in chemotherapy resistance in some forms of carcinoma. 7 In thyroid cancer, the downregulation of BCL-2 is associated with the loss of differentiation leading to more aggressive malignancies. 8
Cannabinoids have shown effectiveness in inhibiting different types of cancer, with research dating back to the 1970s. 9 Furthermore, cannabinoid compounds such as dronabinol and nabilone have been approved for the treatment of cancer-related side effects. In vitro and in vivo studies show that cannabinoids can effectively modulate tumor growth. 10 However, apoptotic properties of cannabinoids are largely dependent on the cancer type and drug dose/concentration. 11 Prior investigations have shown that endocannabinoids have inhibitory effects on the proliferation of breast and prostate cancer, 12 as well as the targeting of the hallmark signaling pathways involved in cancer. 13 In thyroid cancer, cannabinoids have shown anti-proliferative and pro-apoptopic effects in murine models. 13 While previous research showed promising results, it is limited to murine models. Continued investigation is pertinent to understand the effect of cannabinoids on gene expression in relation to their impact on the cell cycle and tumorigenesis. Our investigation focuses on the cannabinoid-based product, BRF1A, and its potential anti-cancer effects by modulation of gene expression in the K1 cell line.
Materials and Methods
Compound Preparation and Quality Control
A Cannabis sativa chemotype cultivated for a high cannabidiol (CBD) content designated “3× Spectrum” was grown in southern Colorado. Dried 3× Spectrum flowers, seeds, and leaves were extracted under standardized parameters at room temperature with CO2 to yield a crude extract. The biomass was macerated with a whip shredder before undergoing supercritical CO2 extraction on an Apeks Supercritical 5 L “Bambino” machine. The extraction parameters were set at 76°F and 1082 PSI, utilizing a valveless expansion orifice (#22). CO2 was circulated under these conditions over the 3× hemp biomass for 10 hours and 40 minutes. This process yields approximately 10% to 15% crude CO2 extract oil, with roughly 20% of this extract consisting of plant waxes and other undesirable compounds. This crude extract was refined using a winterization process. The entire oil extract was homogenized using a CAT Scientific rotor-stator at approximately 35 000 rpm for 2 to 5 minutes in 95% ethanol. The mixture is prepared at a ratio of 1 part crude CO2 extract oil to 2.2 parts 95% ethanol, which was distilled from organic grapes sourced from organicalcohol.com (1:2.2 ratio of CO2 extract to ethanol). This mixture was then stored in a freezer for 48 hours. After the storage period, the mixture is filtered through 0.2-micron filter paper under a vacuum of 108 mbar. Next, 2.08 ml of this 3× hemp raw CO2 extract and 95% ethanol mixture at a 1:2.2 ratio is added to 120 g of extra virgin olive oil, certified organic and sourced from Greece (Braggs). The combined mixture was poured between 2 beakers 6 times and then transferred into a sealed jar. This process yielded a refined CO2 extract suspended in 2.2× its weight of 95% ethanol. This substance was used to formulate the CBD containing product. The cannabidiol (CBD) containing product was compounded by mixing 2.08 ml of the above-described winterized CO2 extract and 95% ethanol into 132 ml of certified organic extra virgin olive oil. This mixture was filtered through a 0.2-micron sterile nylon filter. The filtered solution was added to a fused quartz glass breaker and imprinted using the QBITS Technology (Supplemental Figure 1). The Quantum Biological Information Transfer System (QBITS) Biotechnology is an innovative platform that uses neuromorphic artificial intelligence to imprint aqueous and lipid substances with information, promoting genetic stability in biological systems. This technology combines various fields including machine learning, quantum computing, and photon pattern emission. It aims to enhance biological coherence and health by stabilizing key genes. The “imprint” process involves bombarding carrier substances with electromagnetic radiation to induce genetic signaling. The resulting “biological information” has a measurable positive effect on biological systems. The cannabinoid containing product was prepared at Breslin Research Foundation (Taos, NM). The ethanol was separated from the winterized CO2 extract using rotary evaporation followed by further solvent removal in a vacuum oven. This process yielded an amber colored oil of high viscosity. This CO2 extracted oil was analyzed for cannabinoid content, terpene profile, and contaminants. The results of this analysis showed this extract to contain: 32.91% cannabidiolic acid CBDa, 28.08%, cannabidiol CBD, 1.52% delta 9-Tetrahydrocannabinol (Delta 9 THC), 0% Delta 9-Tetrahydrocannabinolic acid (THCA-A). The terpene composition is 1.393% in which the predominant terpenes are alpha-bisabolol 0.627%, beta-caryophyllene 0.505%, alpha-humulene 0.165%, beta-myrcene 0.096% (Supplemental Table S1). Extrapolation of the data obtained through the analysis of the concentrated CO2 extract yields an estimated cannabinoid content in the product of 0.44 mg of combined cannabinoids and terpenes per 132 ml of extra virgin olive oil. This equates to approximately 0.0033 mg combined cannabinoids and terpenes per 1 ml of BRF1A. Using the molar mass of CBD (314.47 g/mol) the concentration of cannabinoids in BRF1A was estimated to be 10.65 µM. 14
Papillary Thyroid Cell Culture
The K1 cells (RRID: CVCL_2537) were derived from primary papillary thyroid carcinoma. The cell line was obtained from Millipore Sigma, Burlington, MA. The cells retain the follicular differentiation and express wild type P53 tumor suppressor gene. The cell line was cultured at 37°C under 5% CO2 in complete media containing DMEM (catalog # 12491023) medium supplemented with 10% fetal bovine serum (catalog # A5670701), 1 mmol/l of sodium pyruvate (catalog # 11360070), 1 × 10−5 mol/l of β-mercaptoethanol (catalog # 31350010) and 0.5% penicillin streptomycin (catalog # 15140122) all purchased from ThermoFisher Scientific Inc., Waltham, MA. About 1 × 106 cells were seeded into each well of a 24-well plate. The test compound was added in day 0 at concentrations of 1.0 µM, 0.5 µM, and 0.1% DMSO for the untreated control and incubate for 24 and 72 hours.
Human K1 Cell Viability via Trypan Blue Exclusion Assay
K1 cell viability was measured using trypan blue exclusion assay as previously described. 15 About 10 µl of culture suspension was mixed with equal volume of trypan blue dye (Sigma Aldrich, Burlington, MA). The mixture of trypan blue and culture suspension was loaded into a hemocytometer and cells were counted under a microscope. The percentage of viable cells was calculated as follows: Viable cells (%) = (total number of viable cells)/(total number of cells) × 100.
Real-Time Polymerase Chain Reaction (RT-PCR)
Quantitative real time PCR was used to determine the effect of the cannabinoid-containing compound on gene expression in K1 papillary thyroid cancer cells. We cultured K1 cells to a concentration of 2 × 106 cells/mL with cannabinoid-containing product at a concentration of 1.065 µM for 24 hours. K1 cells were harvested at the culture time point, followed by total RNA isolation using the QIAGEN RNeasy kit catalog # 74104 (Redwood City, CA) per the manufacturer’s instructions. RNA concentrations were measured by using the NanoDrop™ 2000/2000c Spectrophotometer. cDNA was obtained via reverse transcription using RevertAid RT Reverse Transcription Kit (Thermo Fisher, Waltham, MA) per the manufacturer’s instructions. Sterile thin-walled tubes were pre-chilled before preparation of reagents. Reagents used were prepared in thin-walled tubes and reaction tubes on ice with pre-chilled nuclease free water. Respective RNA (up to 1 µg) and cDNA primers were combined in nuclease-free water on ice with a final volume of 5 µl per RT reaction. Tubes were then placed into 70°C preheated heat block for 5 minutes. Tubes were immediately chilled on ice for 5 minutes, then spun to obtain condensate from each respective tube. The prepared reverse transcription reaction mix contained ImProm-II™ 5× Reaction Buffer, MgCl2, dNTP, Recombinant RNasin® Ribonuclease Inhibitor, ImProm-II™ Reverse Transcriptase with Nuclease-Free Water to a final volume of 15 μl. About 15 µl of reverse transcription reaction mix was added to respective reaction tubes containing the 5 µl RNA and primer mix for a final volume of 20 µl. PCR was used to finish the reverse transcription reaction at 25°C for 5 minutes, 42°C for 60 minutes, 70°C for 15 minutes. RT-PCR amplification was performed using Maxima SYBR Green qPCR Master Mix kit catalog # A25780 (Fisher Scientific, Pittsburgh, Pennsylvania) with TP53, BCL-2, c-Myc, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) primers. The primers were synthesized by Sigma-Aldrich Corporation (St. Louis, MO). The sequences from the primers used are listed in Supplemental Table 2.
Statistics
Statistical analyses were conducted using Sigma Stat 3.5 (Systat Software Inc., Chicago, IL) and GraphPad Prism (version 9, GraphPad Software, Inc, San Diego, CA). Pairwise comparisons were analyzed with one-way ANOVA (analysis of variance) followed by Bonferroni correction. For all pairwise comparisons with skewed data, differences between the groups were performed by one-way ANOVA on rank followed by Donne’s method. Two-sided calculation was used to determine P-values, with a value ≤.05 considered statistically significant.
Results
Cannabinoid-Containing Product Decreased the Viability of K1 Cells in a Dose and Time Dependent Manner
We determined the dose and time dependent effect of BRF1A in K1 thyroid cells by culturing BRF1A at a concentration of 0.5 and 1.0 µM for 24 and 48 hours. Our results showed that within 24 hours, the cannabinoid-containing product decreased cell viability compared to the untreated cultured group. The untreated culture group had a cell viability of 71% to 76%, the culture group treated with 0.5 µM had a cell viability of 45% to 50% and the 1.0 µM treated group had cell viability of 29% to 38% (Figure 1A, P < .001). In 48 hours, we saw a further decrease in cell viability compared to the untreated culture group. The cell viability of the untreated group ranged 60% to 62%, the 0.5 and 1.0 µM had cell viability in the range 35% to 39% and 21% to 26%, respectively (Figure 1B, P < .001). Our data showed that the cannabinoid-containing product decreased the cell viability of K1 thyroid cancer cells in a dose-dependent and time-dependent manner.
Cannabinoid-Containing Product Increased TP53 and Suppressed BCL-2, c-Myc mRNA Expression
Due to the relation of the mutations of gene expression of p53, c-Myc, and BCL-2 in the pathogenesis of papillary thyroid cancer, we chose these genes as targets of BRF-1. The cannabinoid-containing product was cultured at a concentration of 1.0 µM with K1 thyroid cancer cells and gene expression of TP53, BCL-2, and c-Myc was measured. Our results showed that the cannabinoid-containing product significantly increased the gene expression of TP53 with a sevenfold increase (Figure 2A, P < .01) and significantly decreased the gene expression of BCL-2 with a 0.4613 fold decrease and c-Myc with a 0.3300 fold decrease (Figure 2B, P < .01 and Figure 2C, P < .05).
Discussion
While there are extensive studies on driver mutations in PTC, there remains limited data focusing on K1 driver mutations in thyroid cancer. As such, there is a need to further identify the driver mutations in K1. Prior studies have shown that cannabinoids have anticancer effect. They are most effective in inducing cancer cell death by apoptosis. The findings of this study demonstrate that the cannabinoid-based product BRF1A exhibits significant anti-cancer properties in K1 papillary thyroid carcinoma (PTC) cells, particularly through the upregulation of the tumor suppressor gene TP53 and the downregulation of oncogenes BCL-2 and c-Myc. These results align with previous research exploring the effects of cannabinoids on cancer cell proliferation and apoptosis.
Several studies have demonstrated that cannabinoids exert anti-proliferative effects on various cancer types. For example, studies conducted by Chakravarti et al 10 showed that cannabinoids induce apoptosis in cancer cells via the modulation of key survival pathways, including the PI3K/Akt and MAPK pathways. Additionally, Sarfaraz et al 9 reported that cannabinoids have significant anti-tumor properties in breast and prostate cancer cells through similar mechanisms. These findings are consistent with the current study, which indicates that BRF1A effectively decreases K1 thyroid cancer cell viability in a dose- and time-dependent manner.
The role of p53 in thyroid cancer is well-documented. McFadden et al 5 highlighted that mutations in TP53 contribute to the progression of well-differentiated PTC to more aggressive forms, such as anaplastic thyroid carcinoma. The upregulation of TP53 observed in this study supports the hypothesis that cannabinoid-based compounds may help restore tumor suppressor activity in thyroid cancer cells. This is consistent with findings from Musa et al, 14 which showed that a cannabinoid-enriched product induced TP53 expression in myeloma cells, leading to reduced cancer cell viability.
Similarly, the observed downregulation of BCL-2 and c-Myc by BRF1A aligns with previous studies demonstrating that these oncogenes are critical drivers of thyroid tumorigenesis and chemotherapy resistance. Sartorius and Krammer 7 identified BCL-2 upregulation as a mechanism for chemotherapy resistance in small cell lung cancer. Additionally, Sanjari et al 6 reported that c-Myc overexpression is associated with increased recurrence rates in PTC. The current study suggests that BRF1A counteracts these oncogenic mechanisms, highlighting its potential as a therapeutic agent.
Limitations
One of the limitations of this study was that it was conducted using a single cell line, K1, which originates from metastatic PTC. While K1 cells are a useful model for studying thyroid cancer, they do not fully represent the heterogeneity of PTC or other subtypes of thyroid carcinoma. Therefore, future studies should include additional PTC cell lines to determine whether the effects of BRF1A are consistent across different genetic backgrounds. Another limitation is the absence of normal thyroid cell lines in this study, which restricts the ability to assess the potential toxicity of BRF1A on non-cancerous cells. Understanding the selectivity of BRF1A is crucial because an effective anti-cancer therapy should ideally target malignant cells while sparing healthy ones. Without this comparison, it remains unclear whether BRF1A has cytotoxic effects on normal thyroid cells, which could limit its therapeutic application. Future research should incorporate normal thyroid cell lines to evaluate the safety profile of BRF1A and its potential effects on non-cancerous tissues. Additionally, this study is a preliminary exploration of BRF1A’s role in K1 thyroid cancer cells. Although the observed upregulation of TP53 and downregulation of BCL-2 and c-Myc suggest promising anti-cancer properties, the precise molecular mechanisms underlying these effects remain unknown. It is unclear whether BRF1A exerts its influence through direct interaction with cannabinoid receptors, modulation of intracellular signaling pathways, or other indirect mechanisms. Further mechanistic studies are needed to clarify the pathways involved, including in vivo studies and investigations into apoptosis, cell cycle regulation, and cannabinoid receptor involvement. Finally, the findings of this study are limited to in vitro conditions, which do not fully replicate the complexities of tumor behavior in a living organism. Factors such as the tumor microenvironment, immune response, and systemic metabolism of BRF1A could significantly impact its therapeutic potential. Future studies should include in vivo models to validate the observations made in cell cultures and assess the pharmacokinetics and bioavailability of BRF1A in a biological system.
Conclusion
Our results suggest that a cannabinoid-containing product, BRF1A, functions as a potential regulator of thyroid cancer cells by upregulating tumor suppressor p53 and downregulating c-Myc and BCL-2. BRF1A may have potential therapeutic benefits in the treatment of thyroid carcinoma and warrants further investigation in vitro and in vivo of its potential mechanism.
Supplemental Material
Acknowledgments
We thank Joseph Breslin in conjunction with Breslin Research Foundation for providing BRF1A for this study.
Footnotes
Footnote Group
References
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