School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639, USA.
Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, CO 80639, USA.
Biomolecules. 2022 Jun 20;12(6):854. doi: 10.3390/biom12060854.
Cholangiocarcinoma (CCA) is a rare and highly lethal disease with few effective treatment options. Cannabinoids, cannabidiol (CBD) and cannabigerol (CBG) are non-psychedelic components extracted from cannabis. These non-psychoactive compounds have shown anti-proliferative potential in other tumor models; however, the efficacy of CBD and CBG in CCA is unknown. Furthermore, two cell death pathways are implicated with CBD resulting in autophagic degeneration and CBG in apoptosis. HuCC-T1 cells, Mz-ChA-1 cells (CCA cell lines) and H69 cells (immortalized cholangiocytes), were treated with CBD and CBG for 24 to 48 h. The influence of these cannabinoids on proliferation was assessed via MTT assay. Apoptosis and cell cycle were evaluated via Annexin-V apoptosis assay and propidium iodide, respectively. The expression of proliferation biomarker Ki-67, apoptosis biomarker BAX, and autophagic flux biomarkers LC3b and LAMP1 were evaluated via immunofluorescence. Cell migration and invasion were evaluated via wound healing assay and trans-well migration invasion assays, respectively. The colony formation was evaluated via colony formation assay. In addition, the expression of autophagy gene LC3b and apoptosis genes BAX, Bcl-2, and cleaved caspase-3 were evaluated via Western blot. CBD and CBG are non-selective anti-proliferative agents yielding similar growth curves in CCA; both cannabinoids are effective, yet CBG is more active at lower doses. Low doses of CBD and CBG enhanced immortalized cholangiocyte activity. The reduction in proliferation begins immediately and occurs maximally within 24 h of treatment. Moreover, a significant increase in the late-stage apoptosis and a reduction in the number of cells in S stage of the cell cycle indicates both CBD and CBG treatment could promote apoptosis and inhibit mitosis in CCA cells. The fluorescent expression of BAX and LC3b was significantly enhanced with CBD treatment when compared to control. LAMP1 and LC3b colocalization could also be observed with CBD and CBG treatment indicating changes in autophagic flux. A significant inhibition of migration, invasion and colony formation ability was shown in both CBD and CBG treatment in CCA. Western blot showed an overall decrease in the ratio of anti-apoptotic protein Bcl-2 with respect to pro-apoptotic protein BAX with CBG treatment. Furthermore, CBD treatment enhanced the expression of Type II cell death (autophagic degeneration) protein LC3b, which was reduced in CBG-treated CCA cells. Meanwhile, CBG treatment upregulated Type I cell death (programmed apoptosis) protein cleaved caspase-3. CBD and CBG are effective anti-cancer agents against CCA, capable of inhibiting the classic hallmarks of cancer, with a divergent mechanism of action (Type II or Type I respectively) in inducing these effects.
胆管癌（CCA）是一种罕见且致命的疾病，治疗选择有限。大麻素、大麻二酚（CBD）和大麻萜酚（CBG）是从大麻中提取的非致幻成分。这些非精神活性化合物在其他肿瘤模型中显示出抗增殖潜力；然而，CBD 和 CBG 在 CCA 中的疗效尚不清楚。此外，有两种细胞死亡途径与 CBD 相关，导致自噬退化，与 CBG 相关则导致细胞凋亡。HuCC-T1 细胞、Mz-ChA-1 细胞（CCA 细胞系）和 H69 细胞（永生化胆管细胞）用 CBD 和 CBG 处理 24 至 48 小时。通过 MTT 测定评估这些大麻素对增殖的影响。通过 Annexin-V 凋亡测定和碘化丙啶分别评估细胞凋亡和细胞周期。通过免疫荧光评估增殖生物标志物 Ki-67、凋亡生物标志物 BAX 和自噬通量生物标志物 LC3b 和 LAMP1 的表达。通过划痕愈合试验和 Transwell 迁移侵袭试验分别评估细胞迁移和侵袭。通过集落形成试验评估集落形成。此外，通过 Western blot 评估自噬基因 LC3b 和凋亡基因 BAX、Bcl-2 和裂解的 caspase-3 的表达。CBD 和 CBG 是非选择性的抗增殖剂，在 CCA 中产生相似的生长曲线；两种大麻素均有效，但 CBG 在较低剂量时更活跃。低剂量的 CBD 和 CBG 增强了永生化胆管细胞的活性。增殖的减少立即开始，并在治疗后 24 小时内达到最大值。此外，晚期凋亡的显著增加和细胞周期 S 期细胞数量的减少表明 CBD 和 CBG 治疗可促进 CCA 细胞的凋亡和抑制有丝分裂。与对照相比，用 CBD 处理后 BAX 和 LC3b 的荧光表达明显增强。用 CBD 和 CBG 处理也可以观察到 LAMP1 和 LC3b 的共定位，表明自噬通量发生变化。在 CBD 和 CBG 处理中均显著抑制 CCA 的迁移、侵袭和集落形成能力。Western blot 显示，与促凋亡蛋白 BAX 相比，CBG 处理时抗凋亡蛋白 Bcl-2 的比值总体降低。此外，CBD 处理增强了 II 型细胞死亡（自噬退化）蛋白 LC3b 的表达，而 CBG 处理的 CCA 细胞中 LC3b 的表达减少。同时，CBG 处理上调了 I 型细胞死亡（程序性凋亡）蛋白 cleaved caspase-3。CBD 和 CBG 是针对 CCA 的有效抗癌药物，能够抑制癌症的经典特征，其诱导这些作用的作用机制不同（分别为 II 型或 I 型）。
