Cannabidiol-induced activation of the metallothionein pathway impedes anticancer effects of disulfiram and its metabolite CuET

Abstract

Disulfiram (DSF), an established alcohol-aversion drug, is a candidate for repurposing in cancer treatment. DSF's antitumor activity is supported by preclinical studies, case reports, and small clinical trials; however, ongoing clinical trials of advanced-stage cancer patients encounter variable results. Here, we show that one reason for the inconsistent clinical effects of DSF may reflect interference by other drugs. Using a high-throughput screening and automated microscopy, we identify cannabidiol, an abundant component of the marijuana plant used by cancer patients to mitigate side effects of chemotherapy, as a likely cause of resistance to DSF. Mechanistically, in cancer cells, cannabidiol triggers the expression of metallothioneins providing protective effects by binding heavy metal-based substances including the bis-diethyldithiocarbamate-copper complex (CuET). CuET is the documented anticancer metabolite of DSF, and we show here that the CuET's anticancer toxicity is effectively neutralized by metallothioneins. Overall, this work highlights an example of undesirable interference between cancer therapy and the concomitant usage of marijuana products. In contrast, we report that insufficiency of metallothioneins sensitizes cancer cells toward CuET, suggesting a potential predictive biomarker for DSF repurposing in oncology.

Keywords: CuET; cancer; cannabidiol; disulfiram; metallothionein.

Fig. 1

Cannabidiol (CBD) protects cells from bis‐diethyldithiocarbamate‐copper complex (CuET). (A) Dot plot depicting the results of high‐throughput screening of the chemical library. CuET + mock‐treated controls are in blue, and untreated controls are in yellow. Tested compounds overcoming the threshold of 0.5% positive hits are highlighted in red, green, black, orange, and violet colors. Cells were pretreated with compounds (10 µm) for 17 h and treated with 0.2 µm CuET for 3 h. The screening result represents one experiment (n = 1). (B,C) Microscopy‐based confirmation of the strongest hit. Cells were pretreated with 10 μm CBD for 17 h and treated with 0.2 μm CuET for 3 h and analyzed by microscope including microscopy‐based quantitative analysis (20 µm scale bar) of NPL4‐GFP signal in Triton X‐100 pre‐extracted cells. Combined treatment shows significantly less accumulation of nondissolvable (pre‐extraction resistant) NPL4 protein compared with CuET treatment only (two‐tailed t‐test). Pre‐extraction was performed before the fixation step. The figures show results from one of three independent experiments (n = 3). (D) Cells pretreated with 10 μm CBD for 17 h and treated with 0.2 μm CuET for 3 h accumulate less endogenous NPL4 protein in insoluble fractions compared with CuET treatment only as observed by western blot (WB). The figure shows one of three independent experiments (n = 3). (E) Cells pretreated with 10 μm CBD for 17 h and treated with increasing concentration of CuET for 72 h are more resistant compared with CuET treatment only as observed by the XTT assay. The results represent the mean and standard deviation of three independent experiments (n = 3). (F) WB analysis of K48 polyubiquitinated (Ub K48) proteins reflecting differences in a malfunction of protein degradation in mock, CuET, CBD, and CBD + CuET‐treated cells. For the experiment, the cells were treated for 3h by 0.2 μm CuET. In the combined treatment, CuET was added 17 h after 10 μm CBD. The figure shows one of three independent experiments (n = 3).

Fig. 2

Induction of metallothioneins MT‐1E, MT‐2A expression by cannabidiol (CBD), and bis‐diethyldithiocarbamate‐copper complex (CuET) measured by quantitative polymerase chain reaction (qPCR) A) CBD and CuET increase the expression of MT‐1E mRNA. B) CBD and CuET increase the expression of MT‐2A mRNA. The experimental setup involved pretreatment with 10 μm CBD for 17 h and treatment with 0.2 µm CuET for 3 h. In the combined treatment, CuET was added 17 h after CBD. For both charts, a two‐tailed t‐test was used for P‐value calculation. The result represents the mean and standard deviation of three independent experiments (n = 3).

Fig. 3

Metallothionein level modulates the cellular responses to bis‐diethyldithiocarbamate‐copper complex (CuET) (A) MTF1‐silenced cells are more sensitive to CuET. Cells were treated with increasing concentration of CuET for 72 h and analyzed by XTT assay. The result represents the mean and standard deviation of three independent experiments (n = 3). (B, C) MTF1‐silenced cells accumulate significantly more of the nondissolvable NPL4 after CuET as depicted by microscopic images (20 µm scale bar) and corresponding quantitative microscopic analysis of NPL4‐GFP signal in Triton X‐100 pre‐extracted cells. Cells were treated with 0.2 μm CuET for 3 h. A two‐tailed t‐test was used for P‐value calculation. The results represent one of three independent experiments (n = 3). (D) MTF1‐silenced cells accumulate more K48 polyubiquitinated (Ub K48) proteins after CuET treatment. Cells were treated with 0.2 μm CuET for 3 h and analyzed by western blot (WB). The figure shows one of three independent experiments (n = 3). (E) MT‐2A‐overexpressing cells are more resistant to CuET. Cells were treated with increasing concentrations of CuET for 72 h and analyzed by XTT assay. The result represents the mean and standard deviation of three independent experiments (n = 3). (F, G) MT‐2A‐overexpressing cells accumulate significantly less of the nondissolvable NPL4 as depicted by microscopic images (20 µm scale bar) and corresponding quantitative microscopic analysis of NPL4‐GFP signal in Triton X‐100 pre‐extracted cells. Cells were treated with 0.2 μm CuET for 3 h. A two‐tailed t‐test was used for P‐value calculation. The results represent one of three independent experiments (n = 3). (H) MT‐2A‐overexpressing cells accumulate fewer K48 polyubiquitinated proteins after CuET. Cells were treated with 0.2 μm CuET for 3 h and analyzed by WB. The figure shows one of three independent experiments (n = 3).

Fig. 4

Bis‐diethyldithiocarbamate‐copper complex CuET treatment immobilizes MT‐2A‐GFP. MT‐2A‐GFP protein is immobilized after CuET treatment revealed as depicted by microscopic images after Triton X‐100 based pre‐extraction. Cells were treated with 0.2 and 0.5 μm CuET for 3 h (20 µm scale bar). The figure represents one of two independent experiments (n = 2).

Fig. 5

Concomitant gene silencing with cannabidiol (CBD) treatment and role of metallothionein MT‐2A in response to bis‐diethyldithiocarbamate‐copper complex (CuET) analyzed by XTT assay (A) CBD pretreatment does not protect MTF1‐silenced cells from CuET toxicity. Cells were pretreated with 10 μm CBD for 17 h and treated with 10 μm CBD and increasing concentration of CuET for 72 h. The result represents the mean and standard deviation of three independent experiments (n = 3). (B) MT‐2A‐silenced cells are sensitized to CuET treatment. CBD pretreatment protects MT‐2A‐silenced cells from CuET toxicity only partially. Cells were pretreated with 10 μm CBD for 17 h and treated with 10 μm CBD and increasing concentration of CuET for 72 h. The result represents the mean and standard deviation of three independent experiments (n = 3).