Inhibition of Myeloma Cell Function by Cannabinoid-Enriched Product Associated With Regulation of Telomere and TP53

Plant Material and Extraction, Compound Preparation, Cannabinoid Content Analysis

BRF1-A is a product that contains enriched cannabinoids and was provided by the Breslin Research Foundation located in Taos, NM. The product was derived from a Cannabis sativa chemotype that was cultivated in southern Colorado to have a high cannabidiol (CBD) content, which is known as “3× Spectrum.” The dried flowers, seeds, and leaves of this plant were extracted using CO₂ under standardized parameters at room temperature to produce a crude extract. The crude extract was then refined through a winterization process, which resulted in a refined CO₂ extract suspended to 2.2× its weight of 95% ethanol 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-µm sterile nylon filter. The ethanol was then separated from the winterized CO₂ extract using rotary evaporation, followed by further solvent removal in a vacuum oven, resulting in an amber-colored oil of high viscosity. The filtered solution was added to a fused quartz glass breaker and imprinted using the QBITS Technology, which yielded BRF1A. BRF1-A was analyzed by LC-MS at Marigold Botanicals in Taos, NM, to determine its cannabinoid content and terpene profile. The results of the analysis showed that this extract contained 32.91% cannabidiolic acid CBDa, 28.08% CBD, 1.52% Delta 9-Tetrahydrocannabinol (Delta 9 THC), and 0% Delta 9-Tetrahydrocannabinolic acid (THCA-A). The terpene composition was found to be 1.393%, with the predominant terpenes being alpha-bisabolol (0.627%), beta-caryophyllene (0.505%), alpha-humulene (0.165%), and beta-myrcene (0.096%). Based on the data obtained through the analysis of the concentrated CO₂ extract, the estimated cannabinoid content in the BRF1A product was found to be 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.

IgE, IgG Myeloma Cell Culture and IgE, IgG Level Measurement Via ELISA

The cell line referred to as human U266 cells (RRID:CVCL_0566) are myeloma cells that produce IgE. These cells were purchased from the American Type Culture Collections (Rockville, MD, USA) and were derived from the B cell lymphocyte of a 53-year-old patient with myeloma. In addition to the U266 cells, the ARH-77 cell line (RRID:CVCL_1072) was also used in the experiment. These cells were also purchased from the American Type Culture Collection (Rockville, MD, USA).¹⁴U266 and ARH-77 cells were cultured as previously described by Yang et al¹⁵in a media composed of RPMI 1640 medium supplemented, 10% FBS, 1 mM sodium pyruvate, 1 × 10⁻⁵ M β-ME and 0.5% penicillin-streptomycin and incubated at 37°C under 5% CO₂. U266 at 1 × 10⁶ cells/mL were incubated with BRF1A at 0.25 µM, 0.5 µM, 1.0 µM and 0.1% DMSO for the untreated control in a 24-well plate for 1, 3, and 5 days. Similarly, ARH77 cells at 1 × 10⁶ cells/mL were incubated with BRF1A at 1.0 µM and 0.1% DMSO for the untreated control in a 24-well plate for 1, 3, and 5 days. The levels of immunoglobulin E and G were determined according to previously published methods by Yang et al¹⁵by harvesting the supernatant obtained from the culture and using immunoglobulin E and G ELISA kit purchased from Mabtech, Inc. (Cincinnati, OH, USA).

Human ARH-77 and U266 Cell Viability Via Trypan Blue Exclusion Assay

The cell viability of Human U266 (RRID: CVCL_0566) and ARH-77 (RRID: CVCL_1072) was determined by using trypan blue exclusion assay as previously described.¹⁶A total of 10 µL from the cell culture was mixed with 10 µL of trypan blue dye purchased from Sigma Aldrich (Burlington, MA, USA). The mixture was loaded on a hemocytometer and counted. The percentage of live cells in the total number of cells was calculated to determine cell viability.

Human U266 Cell Proliferation Via CCK-8 Assay

CCK8 assay is a rapid and simple method to measure living cell activity. Cell Counting Kit-8 was utilized to assess cell cytotoxicity following the manufacturer’s guidelines (Dojindo, Rockville, MD, USA) and previously published methods.^15,17–19 U266 cells were cultured as mentioned earlier, then incubated with BRF1A at concentrations of 0.25 µM, 0.5 µM, 1.0 µM, and 0.1% DMSO (untreated control) in a 24-well plate for 1, 3, and 5 days. Similarly, ARH77 cells were cultured and incubated with BRF1A at concentrations of 1.0 µM and 0.1% DMSO (untreated control) in a 24-well plate for 1, 3, and 5 days. Then, we transferred 100 μL of cell suspension into a 96-well plate. Next, 10 μL of CCK8 solution was added to each well of the plate and incubated for 1 hour. Finally, the optical density was measured using a microplate reader at 450 nm.

Real-Time Polymerase Chain Reaction

The quantitative real-time polymerase chain reaction was used to measure the effect of BRF1A on the gene expression of myeloma cells based on previous studies by Rafat et al²⁰and Yang et al.¹⁵We cultured U266 and ARH-77 cells as previously described in the cell culture methods. Human U266 cells at a concentration of 1.0 × 106 cells/mL were cultured with BRF1A at a concentration of 1 µM and incubated at different time points of 24 and 48 hours. After incubation, the cells were harvested and washed with PBS by centrifuging at 1000 rpm for 10 minutes. We isolated RNA from the harvested cells using the QIAGEN RNeasy kit purchased from Redwood City, CA. Transfer Buffer RLT into Eppendorf tubes containing cell pellets, vortex for 2 minutes, and centrifuge for 3 minutes. Extract DNA by transferring lysate to Allprep DNA spin columns and spinning at 50 000 rpm for 30 seconds. Add 70% ethanol to the flow-through and mix gently. Extract RNA by transferring 700 μL of sample to the RNeasy spin column, washing with Buffer RW1 and Buffer RPE, and spinning at maximum speed. Add 60 μL of RNA-free water to the spin column membrane and centrifuge for 1 minute at 10 000 rpm. We quantified the RNA using the NanoDrop™ 2000/2000c Spectrophotometer. Next, we generated cDNA from the isolated RNA using the reverse transcription RevertAid RT Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA) according to the instructions provided by the manufacturer. Thin-walled and reaction tubes were chilled on ice, as was nuclease-free water. RNA (up to 1 µg) and the cDNA primer were mixed in nuclease-free water on ice to a final volume of 5 µL per RT reaction. The tube was then placed into a preheated 70°C heat block for 5 minutes and immediately chilled on ice for 5 minutes. Then, each tube was spun to collect all the condensate. The reverse transcription reaction mix was prepared with ImProm-II™ 5X Reaction Buffer, MgCl2, dNTP, Recombinant RNasin^® Ribonuclease Inhibitor, ImProm-II™ Reverse Transcriptase, and nuclease-free water, bringing the final volume to 15 μL. A total of 15 µL of the reverse transcription reaction mix was added to each reaction tube containing the 5 µL RNA and primer mix, resulting in a final volume of 20 µL. The RT reaction was completed using PCR at 25°C for 5 minutes, 42°C for 60 minutes, and 70°C for 15 minutes. The RT-PCR amplification was conducted using the Maxima™ SYBR Green qPCR Master Mix (2×) kit (ThermoFisher, Waltham, MA, USA) with epsilon germline primers or GAPDH primers. We measured the mRNA expression of our target genes immunoglobulin E heavy chain (IgEH), immunoglobulin G heavy chain (IgGH), Tel (Telomere), hTERT (human telomerase reverse transcriptase), tumor protein 53 (TP53), cellular myelocytomatosis (cMyc) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) using the Maxima SYBR Green qPCR Master Mix kit obtained from Fisher Scientific, Pittsburgh, Pennsylvania. The primer sequences of the targets used in the study were purchased from Integrated DNA Technologies (Coralville, IA, USA) and are listed in Supplemental Table 2.

Western Blotting

Rafat et al²⁰utilized western blotting to investigate telomerase inhibition in primary AML and KG-1a cells. Similarly, we assessed protein expression of the targets regulated by BRF1A; Western blot analysis was performed. Human U266 cells (3.0 × 10⁶ cells/mL) were cultured with BRF1A at 1.0 µM for 48 hours. The cells were harvested and supernatant removed by centrifugation at 3000 rpm for 5 minutes. The cells’ suspension was washed with PBS at 3000 rpm for 5 minutes. Protein extraction was done through cell lysis, using RIPA lysis buffer, according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). To extract the protein, we added 100 to 200 µL of RIPA lysis buffer and vortex vigorously while incubating on ice for about 1 hour. Then, we centrifuged the mixture at 14 000 rpm for 20 minutes at 4°C. The isolated protein was used for standard western blot analysis. We ran the gel using electrophoresis with settings at 90 V for 15 min followed by 100 to 110 V for the remaining time. After running the gel, we transferred the proteins to a PVDF membrane. We incubated the membrane overnight with primary antibodies of p-IκBα, p-NFκB, NFκB, XBP-1s, XBP-1u, hTERT, p-TERT, P53, c-Myc, β-actin, and GAPDH. After incubating the membrane overnight with the primary antibodies, we washed the membrane with PBST the next day and incubated the membrane with secondary antibodies for 2 hours. The following primary antibodies used in the study: p-IκBα, p-NFκB, NFκB, XBP-1u, XBP-1s, P53, c-Myc, β-actin, and GAPDH. These antibodies were ordered from Cell Signaling (Danvers, MA, USA) and were used at a 1:1000 dilution. The primary antibody of XBP-1u was purchased from Proteintech (Rosemont, IL, USA) and was used at dilutions ranging from 1:500 to 1:1000. The primary antibody of p-TERT was purchased from Invitrogen (Waltham, MA, USA) and was used at 1:500. The primary antibody of hTERT was purchased from Bioss Antibodies (Woburn, MA, USA) and used at a 1:500 dilution. The secondary antibodies used in the study were ordered from Cell Signaling (Danvers, MA, USA) and were used at a 1:10 000 dilution.

Statistics

We used the GraphPad Prism software (version 9; GraphPad Software, Inc., San Diego, CA, USA) for statistical analyses. To compare pairwise differences, we performed 1-way ANOVA (analysis of variance) followed by Bonferroni correction. A P-value of ≤.05 was considered statistically significant.

BRF1A Inhibits the Excessive Production of IgE U266 Cells in a Dose- and Time-Dependent Manner

The key pathologic function of myeloma cells is over production of immunoglobulins leading to tissue damage.²¹To evaluate the suppressive effect of BRF1A on myeloma cell function, we cultured BRF1A with the IgE-producing myeloma cell line, U266, which is aggressive and resistant to anti-cancer drugs.²²BRF1A was added to U266 culture at various concentrations of 0.25 µM, 0.5 µM, 1.0 µM, and 0.1% DMSO at multiple time points of 1, 3, and 5 days to establish the dose-response and time course of anti-myeloma suppressive effects. Our results showed that the production of IgE was significantly reduced after 1 day of culture in the group that received BRF1A at concentrations of 0.25, 0.5, and 1.0 µM, compared to the untreated culture with 0.1% DMSO (Figure 1A; P < .05 to P < .001). The reduction percentages observed were 23% to 36%, 27% to 61%, and 59% to 64%, respectively. We also found that IgE production in the day 3 and 5 cultures was significantly decreased across the different concentrations of BRF1A compared to the untreated culture at 0.1% DMSO. The IgE reduction percentages in the day 3 culture group were 37% to 49%, 52% to 61%, and 71% to 77% (Figure 1B, P < .001). In the day 5 culture group, we observed a further decrease in IgE reduction percentages of 91% to 92%, 95% to 96%, and 96% to 97% (Figure 1C, P < .001). These results suggest that BRF1A effectively suppressed myeloma cell function in a dose- and time-dependent manner, as evidenced by its significant reduction of IgE production.

Dose and Time-Dependent Effect of BRF1A on Myeloma Cell

BRF1A decreased IgE production in IgE myeloma cells; therefore, we evaluated the effect of BRF1A on cell viability. The cell viability was evaluated at various concentrations of 0.25, 0.5, and 1.0 µM. The cell viability of myeloma cells treated with 0.5 µM concentration of BRF1A ranged from 72% to 75%, at 0.5 µM it ranged from 72% to 74%, and at 1.0 µM it ranged from 65% to 71.1% compared to the untreated culture group, which showed a viability between 75% and 86% (Figure 2A, P < .05). Similarly, on day 3, the cell viability for the culture group treated with 0.25 µM of BRF1A was between 68.2% and 75.5%, for 0.5 µM it was 70.9% to 73.3%, and for 1.0 µM it was 66.8% to 72.2% (Figure 2B, P < .05; P < .01), while the untreated group had viability between 75% and 81%. By day 5, cell viability was further decreased across all treatment groups compared to the untreated culture, with 0.25 µM of BRF1A between 57% and 61%; for 0.5 µM it was 51% to 55%, and for 1.0 µM it was 31% to 41% (Figure 2C, P < .05; P < .001). These findings indicate the potential of BRF1A as an antimyeloma agent. Furthermore, the results suggest that BRF1A exerts its antimyeloma effect within 24 hours and this effect increases with time.

We next used the colorimetric assay CCK-8, which evaluates the cytotoxic effects of anticancer drugs. The results were similar to the cell viability results across all concentrations of 0.25, 0.5, and 1.0 µM as compared to untreated controls (Supplemental Figure 3A-D). This indicates that BRF1A inhibits myeloma cell function in a non- or less-cytotoxic manner.

BRF1A Inhibits Excessive IgG Production in ARH77 Cells Without Cytotoxicity

BRF1A suppressed IgE-producing myeloma function; therefore, we wanted to see if it has a similar suppressive effect in IgG-producing myeloma ARH-77 cells. We tested this by using a concentration of 1.0 µM of BRF1A, which had previously been shown to inhibit IgE production. We cultured the ARH-77 cells with this concentration of BRF1A for 1, 3, and 5 days. Our findings showed that compared to untreated cultures, there was a significant decrease in IgG production on days 1, 3, and 5 (as seen in Figure 3A-C, P < .001). Within 24 hours, the IgG concentration of the cells cultured with BRF1A was decreased to 79 to 144 ng/mL, compared to the untreated culture group where the IgG concentration ranged between 185 and 299 ng/mL (as shown in Figure 1A, P < .05 to P < .001). A subsequent decrease in IgG concentration was observed upon reaching days 3 and 5 of the culture period. In the treatment group, the IgG concentration ranged between 46 and 99 ng/mL on day 3, while the untreated culture group had IgG concentrations between 244 and 316 ng/mL. Similarly, on day 5, the treatment group showed IgG concentrations of 44 to 94 ng/mL, while the untreated group exhibited 278 to 329 ng/mL concentrations. We also observed no significant cytotoxicity in the cells at day 1 and 3 time points (as seen in Figure 4A-C).

Additionally, using CCK-8, we found that BRF1A had a similar effect on IgG-producing myeloma cells as it did on IgE-producing myeloma cells (as seen in Figure 4D-F). Therefore, our results suggest that BRF1A can suppress both IgE- and IgG-producing myeloma cells. This suggests that its suppressive effects are not specific to IgE-producing myeloma cells, thereby highlighting the potential of BRF1A as a therapeutic agent for myeloma.

BRF1A Inhibited the Expression of Immunoglobulin E and G Heavy Chain Genes in Myeloma Cells

We observed a significant decrease in IgE and IgG production when BRF1A was cultured with myeloma cells at a concentration of 1.0 µM on days 1 and 3 of the culture, with minimal change in cell viability. This led us to investigate whether BRF1A regulates the gene expression of IgEH, IgGH which are biomarkers of IgE and IgG myeloma cell function. We cultured BRF1A with myeloma cells at a concentration of 1.0 µM for 24 hours and isolated RNA to measure the mRNA expression of IgEH. Our results revealed a significant decrease in IgEH (P < .001) in the 24-hour culture time point (Figure 5A). We next determined if BRF1A also decreased the gene expression of immunoglobulin G heavy chain (IgGH) in ARH-77 cells, we cultured BRF1A at a similar concentration and time point as U266 cells and isolated RNA to measure mRNA expression. Our results indicate that BRF1A significantly decreased the mRNA expression of IgGH (Figure 5B, P < .001). These results demonstrate that BRF1A suppresses IgG and IgE antibody production in IgG and IgE myeloma cells, respectively, by suppressing the IgEH and IgGH genes. Since BRF-1A similarly inhibited U266 and ARH-77 antibody production and antibody heavy chain expression, and because U266 cells are aggressive and resistant to anti-cancer drugs,²²we further assessed the mechanism focusing on U266 cells in the following studies.

BRF1A Suppressed Protein Expression Critical for Myeloma Cell Function and Survival

The transcription factor, NF-ĸB, plays an important role in the production of IgE in plasma B cells. In human B cells, activation of transcription factors of the NF-ĸB family results in the degradation of IκBα and IκBβ. This leads to the eventual release of NF-κB—p50 and NF-κB—p65, which translocate to the nucleus to trigger the transcription of target genes.²³Decreased levels of p-IĸBα indicate that NF-κB is inhibited. XBP-1 is a survival transcription factor for plasma cells, which helps them function under stress. During endoplasmic reticulum (ER) stress, XBP1s translocate into the nucleus and activate target genes encoding ER chaperones and ER-associated degradation (ERAD) components. This, in turn, enhances the capacity of protein folding and degradation, to maintain ER homeostasis.²⁴Therefore, we determined the effect of BRF1A on proteins NFκB, p-NFκB, p-IκBα, XBP-1u, and XBP-1s in U266 cells. We cultured BRF1A at a concentration of 1.0 µM with 3.0 × 10⁶ cells/mL U266 cells for 48 hours to measure protein expression. Our results show that BRF1A significantly inhibited the protein expression of NFκB (P < .01), p-NFκB (P < .05), p-IκBα (P < .01), XBP-1u (P < .05), XBP-1s (P < .01; see Figure 6A-E).

BRF1A Increased the Gene Expression of Telomerase in Myeloma Cells

Next, we determined if BRF1A has any effect on telomerase gene expression given its relevance in cancer progression. The human telomerase is a ribonucleoprotein enzyme made up of at least 2 components, a catalytic subunit known as telomerase reverse transcriptase (hTERT), and a template RNA component called hTR.²⁵This ribonucleoprotein (RNP) enzyme slows down telomere attrition by adding repetitive sequences to chromosome ends, thus maintaining telomere length. To determine the effect of BRF1A on the gene expression of telomerase, we cultured BRF1A at 1.0 µM with 3.0 × 10⁶ cells/mL myeloma cells for 48 hours. Our results revealed that BRF1A increased the expression of hTERT gene (P < .01) after 48 hours (Figure 7A and B).

BRF1A Increased the Protein Expression of hTERT, p-hTERT in Myeloma Cells

The gene expression of telomerase was shown to be increased by product BRF1A. To determine if this regulation is posttranslational, we measured the protein expression of hTERT and p-TERT in U266 cells after treating them with BRF1A at a concentration of 1.0 µM for 48 hours. BRF1A treatment, to determine if the regulation of telomere and telomerase is posttranslational. Our results demonstrated a significant increase in the protein expression of hTERT (P < .05) and p-TERT (P < .05) (Figure 7C and D).

BRF1A Increased the Gene Expression of Tumor Suppressor Gene and Decreased Oncogenes in Myeloma Cells

Next, we determined if BRF1A has any effect on tumor suppressor TP53 gene and oncogene c-Myc. Myc regulates different types of cell functions, therefore it is a target for most anticancer drugs.²⁶Most cancers cause a shutdown in the P53 pathway, therefore increasing its expression will lead to a P53 dependent apoptosis.²⁷We cultured BRF1A at a concentration of 1.0 µM with 3.0 × 10⁶ cells/mL myeloma cells for 48 hours and measured the gene expression of c-Myc and TP53. In 48 hours, BRF1A significantly increased the gene expression of TP53 (P < .01), and significantly decreased the expression of c-Myc (P < .001; Figure 8A and B).

BRF1A Increased TP53 and Decreased c-Myc Protein Expression

BRF1A increased the gene expression of TP53 and suppressed the gene expression of c-Myc. Therefore, we next measured the effect of BRF1A on the protein expression of p53 and c-Myc. We cultured BRF1A at a concentration of 1.0 µM with 3.0 × 10⁶ cells/mL myeloma cells for 48 hours and measured the protein expression of p53 and c-Myc. Our results demonstrated that BRF1A significantly increased the protein expression of p53 (P < .05) and decreased the protein expression of c-Myc (P < .001) (Figure 8C and D). These findings suggest that BRF1A could be a potential therapeutic agent for myeloma treatment.