Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Biopharmacy, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary.
Eur J Pharm Sci. 2024 Dec 1;203:106920. doi: 10.1016/j.ejps.2024.106920. Epub 2024 Sep 30.
Despite the targeted- and immunotherapies used in the past decade, survival rate among patients with metastatic melanoma remains low, therefore, melanoma is responsible for the majority of skin cancer-related deaths. The ongoing investigation of natural antitumor agents, the nonpsychoactive cannabinoid, cannabigerol (CBG) found in Cannabis sativa is emerging as a promising candidate. CBG offers a potential therapeutic role in the treatment of melanoma demonstrating cell growth inhibition in some tumors. Its low water solubility and bioavailability hinder the potential effectiveness. To address these challenges, a modified CBG, namely LE-127/2 was synthesized by Mannich-type reaction. The aim was to investigate the effect of this novel compound on cell proliferation as well as the mechanism of cell death with a particular focus on autophagy and apoptosis. Human cutan melanoma cell lines, WM35, A2058 and WM3000 were utilized for the present study. Cell proliferation of the cells after the treatment with LE-127/2, parent CBG or vemurafenib was assessed by Cell Titer Blue Assay. Cells were treated with a 1.25-80 µM of the above-mentioned compounds, and it was found that at 20 μM of all drugs showed a comparable effective inhibition of cell proliferation, however, vemurafenib and CBG proved to be more effective than LE-127/2. In addition, clonogenic cell survival assays were performed to examine the inhibitory effect of LE-127/2 on the colony formation ability of melanoma cell lines. Cells treated with 20 µM of LE-127/2 for 14 days showed about a 50% suppression of clonogenic cell survival. LE-127/2 exerted the most intensive inhibition on A2058 cell colonies. Furthermore, notably, LDH cytotoxicity assay performed on HaCaT cell line, proved LE-127/2 to be cytotoxic only at higher concentration, such as 80 μM, while the parent CBG was cytotoxic at concentration as low as 5 μM, suggesting that the new CBG derivative as a drug candidate may be applied in human pharmacotherapy without causing a substantial damage in intact epidermal cells. Analysis of protein expression revealed the impact of LE-127/2 on the expression of basic proteins (LC-3, Beclin-1 and p62) involved in the process of autophagy in the three different melanoma cell lines studied. Elevated expression of these proteins was detected as a result of LE-127/2 (20 µM) treatment. LE-127/2 also induced the expression of some proteins involved in apoptosis, and it is particularly noteworthy the increased level of cleaved PARP. Based on the results obtained, it can be concluded that LE-127/2 induced autophagy could lead to the inhibition of cell proliferation and death in melanoma cells.
尽管在过去十年中使用了靶向治疗和免疫疗法，转移性黑色素瘤患者的存活率仍然很低，因此黑色素瘤是导致大多数皮肤癌相关死亡的原因。目前正在研究天然抗肿瘤药物，在大麻中发现的非精神活性大麻素大麻萜酚（CBG）作为一种有前途的候选药物正在出现。CBG 在治疗黑色素瘤方面具有潜在的治疗作用，在一些肿瘤中表现出细胞生长抑制作用。其低水溶性和生物利用度阻碍了其潜在的有效性。为了解决这些挑战，通过曼尼希型反应合成了一种改良的 CBG，即 LE-127/2。目的是研究这种新型化合物对细胞增殖的影响以及细胞死亡的机制，特别关注自噬和细胞凋亡。本研究采用人皮肤黑色素瘤细胞系 WM35、A2058 和 WM3000。通过 Cell Titer Blue 分析评估细胞在用 LE-127/2、母体 CBG 或 vemurafenib 处理后的细胞增殖情况。用 1.25-80 μM 上述化合物处理细胞，发现所有药物在 20 μM 时均表现出相当有效的细胞增殖抑制作用，但 vemurafenib 和 CBG 比 LE-127/2 更有效。此外，还进行了集落形成细胞存活测定，以检查 LE-127/2 对黑色素瘤细胞系集落形成能力的抑制作用。用 20 μM 的 LE-127/2 处理 14 天的细胞显示出约 50%的克隆形成细胞存活抑制。LE-127/2 对 A2058 细胞集落的抑制作用最为强烈。此外，值得注意的是，在 HaCaT 细胞系上进行的 LDH 细胞毒性测定表明，LE-127/2 仅在较高浓度（如 80 μM）时才具有细胞毒性，而母体 CBG 在低至 5 μM 的浓度时就具有细胞毒性，这表明新的 CBG 衍生物作为候选药物可能在人类药物治疗中应用，而不会对完整的表皮细胞造成实质性损害。蛋白质表达分析显示，LE-127/2 对三种不同黑色素瘤细胞系中参与自噬过程的基本蛋白（LC-3、Beclin-1 和 p62）的表达有影响。用 20 μM 的 LE-127/2 处理后，这些蛋白质的表达水平升高。LE-127/2 还诱导了一些参与细胞凋亡的蛋白质的表达，值得注意的是，cleaved PARP 的水平增加。基于所获得的结果，可以得出结论，LE-127/2 诱导的自噬可能导致黑色素瘤细胞增殖和死亡的抑制。
