The exposed phosphatidylserine on the U266 and RPMI cells membrane surface was detected by Annexin V staining and cytofluorimetric analysis. Briefly, 4 × 10 4 cells/ml were treated with different doses of the appropriate drugs for a maximum of 72 h. Four replicates were used for each treatment. After treatment, the cells were stained with 5 μl of Annexin V FITC (Vinci Biochem, Vinci, Italy) for 10 min at room temperature, washed once with binding buffer (10 mM N- (2- Hydroxyethyl) piperazine-N0-2-ethanesulfonic acid [HEPES]/sodium hydroxide, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and analysed on a FACScan flow cytometer using CellQuest software.
The effect of CBD in blocking cell cycle in G1 phase (38% in U266, 42% in RPMI) was previously proved . So we evaluated the role of THC alone or in combination with CBD in influencing the cell cycle, in both MM cell lines. The cell cycle phases were analysed by propidium iodide (PI) staining and FACS analysis in both cell lines. The results showed that THC was able to induce cell accumulation in the G1 phase, starting from 24 h post-treatment, accompanied by accumulation in the sub-G1 phase (hypodiploid DNA) at 48 h post-treatment, compared with their respective control (Supplementary Figure 1; Figure Figure3). 3 ). The THC-CBD combination was statistically more effective in increasing the G1 cell population and the sub-G1 phase at 24 h post-treatment and in augmenting cell accumulation in the sub-G1 phase at 48 h, compared with THC and CBD  when used alone (Figure (Figure3, 3 , Supplementary Figure 1). This data suggested that the THC-CBD combination was more effective than THC and CBD used as single agents in inducing cell death, in both cell lines.
Total RNA was extracted with the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, PA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reactions (qRT-PCR) for iβ5, CXCR4 and CD147 were performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA). PCR reaction was performed with RT 2 SYBRGreen qPCT mastermix (Qiagen) using 1 μl of cDNA for reaction, following the amplification protocol described in the manufacture’s instruction. RT 2 qPCR Primer assays (Qiagen) were used for target gene amplification. All samples were assayed in triplicates in the same plate. Measurement of GAPDH levels was used to normalize mRNA contents, and target gene levels were calculated by the 2 −ΔΔCt method.
Electrophoresis of DNA was performed to assess DNA fragmentation as an indicator of necrosis and apoptosis. Briefly, 4 × 10 4 cells/ml were treated with the appropriate compounds for 72 h, and the genomic DNA was extracted using a DNA extraction kit (Qiagen, Hilden, Germany). The purified samples were then subjected to electrophoresis on 1.25% agarose gel, stained with ethidium bromide. Ultraviolet spectroscopy at 302 nm was used to obtain the results.
Using PI staining and FACS analysis we also evidenced that THC-CBD combination induces higher necrotic cell death compared with THC and CBD alone, at 48 h post-treatments, in both MM cell lines (Figure (Figure5A). 5A ). Furthermore, we evidenced augmented levels of damaged DNA after addition of THC-CBD combination with respect to the single treatment as demonstrated by genomic DNA fragmentation analysis (Figure (Figure5B). 5B ). We also investigated the presence of γ-H2AX (H2AX), a phosphorylated variant of histone 2A that is associated with DNA double-strand breaks. Immunoblots showed that THC and CBD in both cell lines are able to induce increased levels of the phosphorylated form of H2AX (Figure (Figure5C) 5C ) at 24 h post-treatments, and THC-CBD further improves the H2AX levels respect to the single treatments, in both MM cell lines (Figure (Figure5C 5C ).
To evaluate the potential inhibitory effect of immuno-proteasome inhibitor CFZ on cell viability, U266 and RPMI cell lines were exposed to increasing concentration of CFZ in presence or absence of IFN-γ, and cell viability was evaluated by the MTT assay 72 h post-treatment. As shown (Figure (Figure7A), 7A ), CFZ was able to reduce cell viability in both MM cell lines, with IC50 0.379 μM and 0.012 μM in U266 and RPMI, respectively. Moreover, stimulation with IFN-γ reduced CFZ sensitivity in both cell lines (U266 IC50= 1.426 μM; RPMI IC50= 0.026 μM). To understand the mechanism underlying the effect of CFZ on MM cell viability, we evaluated whether CFZ was able to influence cell cycle progression in MM cell lines. Using PI staining, cell cycle phases were determined in CFZ-treated cells after 24 h of treatment, by FACS analysis. The results showed that CFZ induced a rapid accumulation in sub-G1 phase in MM cell lines (Supplementary Figure 2). These results demonstrated that CFZ was able to induce cell death with minimal effect on cell cycle, in both cell lines. Then, we investigated on the role of caspase-3 in CFZ-induced apoptosis in U266 and RPMI cells. Both cell lines were treated with CFZ for 72 h and western blot analysis was performed to evaluate caspase-3 activation. As shown, CFZ was able to increase cleaved caspase-3 levels in MM cell lines (Figure (Figure7B, 7B , Supplementary Figure 3A). Moreover, the role of caspase-3 in CFZ-induced apoptosis was further confirmed by pre-treating U266 and RPMI cell lines with the caspase-3 inhibitor z-VAD (5 mM) for 1 h prior to treat cells with CFZ for an additional 72 h. FACS analysis demonstrated that CFZ increased Annexin V + cells, while z-VAD reduced CFZ-induced apoptosis in both cell lines (Figure (Figure7C, 7C , Supplementary Figure 3B). In conclusion, these results revealed a pro-apoptotic effect of CFZ in U266 and RPMI cell lines. Both CFZ alone and THC-CBD combination reduce cell viability; therefore, we evaluated the effect of CFZ plus THC-CBD combination on MM cell viability. RPMI and U266 cells were treated with different doses of CFZ (0.9 up to 7.5 nM doses for RPMI, 12.5 up to 100 nM doses for U266) in combination with THC-CBD. The results showed that most of the combinations strongly reduce cell viability compared with single treatments in both cell lines (Figure (Figure7D). 7D ). Furthermore, we evidenced that THC-CBD combination acts synergically (CI<1) with CFZ (50, 25 and 12.5 nM in U266; 7.5 nM in RPMI) to induce cytotoxic effects.
We evaluated a potential role of THC-CBD in regulating the β5i subunit. So, U266 and RPMI cell lines were treated with CBD and THC, after 24 h exposure to IFN-γ (100 U/ml). Using qRT-PCR, we showed that the THC-CBD combination strongly reduces the β5i increased expression level induced by IFN-γ, while low effects were observed with single CBD and THC treatments respect to IFN-γ alone (Figure (Figure6A). 6A ). At protein levels, the expression of the precursor and mature form of β5i was examined by western blot analysis. Results evidenced that the administration of IFN-γ increases both the precursor and the mature form of β5i in MM-treated compared with MM non-treated cells. Moreover, THC and CBD alone had low efficacy in reducing β5i, while the THC-CBD combination impaired the expression of both forms, in U266 and RPMI cell lines (Figure (Figure6B 6B ).
Effect of THC-CBD in regulating the β5i subunit in MM cell lines
U266 and RPMI cells were treated with AM630 (20 μM) alone or in combination with THC 12.5 μM A, C, or with 12.5 μM CBD plus 12.5 μM THC B, D. Cell viability was evaluated by using the MTT assay. Data shown are expressed as mean ± SD of three separate experiments. *p<0.05 vs vehicle treated cells.
CFZ was demonstrated to induce apoptosis in the ANBL-6 cell line, increasing the caspase-3 activity confirmed by the effect of zVAD that blocked CFZ-stimulated apoptosis . Herein, we confirmed the caspase-3 role in CFZ-induced apoptosis, suggesting the caspase-3 driven apoptosis is a common mechanism of action of CFZ in MM cell lines. A mechanism of CFZ resistance determined in MM cells was related to the expression levels of β5i. Moreover, the role of IFN-γ in exchanging the cPTS subunits for iPTS subunits was first demonstrated in J111 leukemia cells . Our findings demonstrated that, in IFN-γ-treated MM cell lines the levels of the β5i subunit increased and this treatment augmented the CFZ resistance. THC-CBD treatments, by reducing β5i subunit both at transcriptional and translational levels, induced inhibition of the CFZ target β5i subunit, indicating cannabinoids as potential drugs for overcoming CFZ resistance mechanisms.
Multiple myeloma (MM) patients are often told that their cancer “is incurable but very treatable.” When I was first diagnosed with multiple myeloma I found this statement to be confusing. When I found a study showing that cannabidiol kills myeloma and enhances Velcade, I understood how MM could be incurable but treatable.
These results showed that Cannabidiol by itself or in synergy with BORT strongly inhibited growth, arrested cell cycle progression and induced MM cells death by regulating the ERK, AKT and NF-κB pathways with major effects in TRPV2+ cells. These data provide a rationale for using Cannabidiol to increase the activity of proteasome inhibitors in MM.”
A selective review of medical cannabis in cancer pain management
Conclusions- Current research shows that there is a potential role for medical cannabis in cancer pain management. However, the scale and quality of studies conducted to date are somewhat limited (12). Therefore, further research is needed to establish the efficacy of medical cannabis, either as an alternative to opiates or as an adjunctive therapy, and to identify the most appropriate methods of administration to achieve optimal therapeutic efficacy with minimal side effects.”