Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

Abstract

Cannabinoids are secondary metabolites stored in capitatesessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and ⌬ 1 -tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca 2؉ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspasedependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.Multicellular organisms such as plants and animals possess cell death-inducing systems to eliminate damaged, superfluous, and ectopic cells. In higher plants, these systems participate in a variety of physiologically important events (e.g. root cap elimination, somatic embryogenesis, xylogenesis, leaf senescence, and defense against microbial pathogens) (1, 2), and therefore plant cell death has attracted a great deal of attention. To reveal the regulatory mechanism of cell death induction, biochemical and molecular examinations have been extensively carried out to date and have demonstrated that, in many cases, plant cell death can be classified into apoptosis or necrosis and that mitochondria control both types of cell death in plants as well as animals (3-5). For example, plant apoptosis, like animal apoptosis, is caused by release of apoptotic proteins (cytochrome c and others) from mitochondria, followed by activation of cellexecuting enzymes (caspases and nucleases) (3, 4). On the other hand, a decrease of the ATP level due to serious mitochondrial dysfunction has been shown to induce necrosis in plants and animals (5, 6).Apoptotic-protein release and mitochondrial dysfunction are often reported to be controlled by mitochondrial membrane permeability, and several systems that regulate this pe...

Elicitation studies in cell suspension cultures of Cannabis sativa L.

“…In H. perforatum plants the phototoxin hypericin accumulats in secretory glands on leaves and flowers (Fields et al, 1990;Zobayed et al, 2006). It has been confirmed that cannabinoids are cytotoxic compounds and thus they should be biosynthesized and accumulated in highly specialized cells such as glandular trichomes (Morimoto et al, 2007).…”

Secondary metabolism in cannabis

“…The THCA synthase reaction produces hydrogen peroxide as well as THCA during the oxidation of Taura et al (1996) trans-CBGA CBGA (Sirikantaramas et al 2004); a toxic amount of hydrogen peroxide could be accumulated together with the cannabinoids which must be secreted into the storage cavity from the glandular hairs to avoid cellular damage itself. Additionally, Morimoto et al (2007) have shown that cannabinoids have the ability to induce cell death through mitochondrial permeability transition in cannabis leaf cells, suggesting a regulatory role in cell death as well as in the defense systems of cannabis leaves. On the other hand, although CBN type cannabinoids have been isolated from cannabis extracts, they are probably artifacts.…”

The inheritance of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free plants

“…Various theories, all relating to the defence against biotic and abiotic stress (Pate 1998), attribute ecological benefits to the presence of cannabinoids which would explain the rarity of the knockout allele. Recently, Morimoto et al (2007) reported on the ability of cannabinoids to induce necrotic cell death in the Cannabis leaf cells. They suggested that these compounds play a role in the plant's defence system and in the process of leaf senescence.…”