John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA 90404, USA.
Int J Oncol. 2010 Dec;37(6):1591-7. doi: 10.3892/ijo_00000813.
The role of glucosylceramide synthase (GCS) in regulating ceramide-induced apoptosis has been widely studied. The purpose of this investigation was to evaluate the role of P-glycoprotein (P-gp) in regulating ceramide cytotoxicity by using C6-ceramide. To accomplish this, we employed HeLa cells with conditional expression of the multidrug resistance gene 1/P-gp. HeLa cells expressing P-gp (P-gp/on cells) challenged with [14C]C6-ceramide (6 µM), synthesized 4.5-fold the amount of C6-glucosylceramide (GC) compared to HeLa cells with suppressed expression of P-gp (P-gp/off cells), whereas the generated levels of C6-sphingomyelin were almost equal (33 and 29% of intracellular 14C, respectively). Tamoxifen, a P-gp antagonist, decreased the C6-GC levels from 3.5-1.0% in the P-gp/off and from 17-2.8% of the total lipid 14C levels in the P-gp/on cells. Tamoxifen did not inhibit cell-free C6-GC synthesis in the P-gp/off or P-gp/on homogenates. However, a specific GCS inhibitor, ethylenedioxy-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (ethylenedioxy-P4), blocked synthesis by 90%. In the cytotoxicity assays, the P-gp/off cells were sensitive to C6-ceramide and the P-gp/on cells were resistant. Resistance to C6-ceramide in the P-gp/on cells was reversed by tamoxifen but not by ethylenedioxy-P4. Experiments in another cervical cancer model showed that multidrug-resistant P-gp-rich KB-V1 cells synthesized 3-fold more C6-GC from C6-ceramide than the parental, P-gp-poor KB-3-1 cells, and whereas tamoxifen had no effect on the C6-GC synthesis in the KB-3-1 cells, it inhibited synthesis by 70% in the KB-V1 cells. This study demonstrates that P-gp potentiates C6-ceramide glycosylation and if antagonized augments C6-ceramide sensitivity, both features previously ascribed to GCS. We propose that P-gp can be an effective target for enhancing short-chain ceramide cytotoxicity in the treatment of drug-resistant cancer.
糖基神经酰胺合酶 (GCS) 在调节神经酰胺诱导的细胞凋亡中的作用已得到广泛研究。本研究旨在通过使用 C6-神经酰胺来评估 P-糖蛋白 (P-gp) 在调节神经酰胺细胞毒性中的作用。为了实现这一目标，我们使用了条件表达多药耐药基因 1/P-gp 的 HeLa 细胞。与 P-gp 表达受抑制的 HeLa 细胞 (P-gp/off 细胞) 相比，表达 P-gp 的 HeLa 细胞 (P-gp/on 细胞) 在受到 [14C]C6-神经酰胺 (6 µM) 挑战时，合成的 C6-葡糖神经酰胺 (GC) 量增加了 4.5 倍，而生成的 C6-神经酰胺水平几乎相等 (分别为细胞内 14C 的 33%和 29%)。他莫昔芬是一种 P-gp 拮抗剂，可使 P-gp/off 细胞中的 C6-GC 水平从 3.5%降至 1.0%，使 P-gp/on 细胞中的总脂质 14C 水平从 17%降至 2.8%。他莫昔芬不能抑制 P-gp/off 或 P-gp/on 匀浆中的细胞外 C6-GC 合成。然而，一种特定的 GCS 抑制剂，乙二氧基-1-苯基-2-十六烷酰氨基-3-吡咯烷-1-丙醇 (乙二氧基-P4)，可阻断 90%的合成。在细胞毒性测定中，P-gp/off 细胞对 C6-神经酰胺敏感，而 P-gp/on 细胞具有抗性。P-gp/on 细胞对 C6-神经酰胺的耐药性被他莫昔芬逆转，但不能被乙二氧基-P4 逆转。在另一个宫颈癌模型中的实验表明，多药耐药的 P-gp 丰富的 KB-V1 细胞从 C6-神经酰胺合成的 C6-GC 是亲本 P-gp 缺乏的 KB-3-1 细胞的 3 倍，而他莫昔芬对 KB-3-1 细胞中的 C6-GC 合成没有影响，但抑制了 KB-V1 细胞中的合成 70%。本研究表明，P-gp 增强了 C6-神经酰胺的糖基化作用，如果被拮抗，会增强 C6-神经酰胺的敏感性，这两个特征以前都归因于 GCS。我们提出，P-gp 可以成为增强治疗耐药性癌症的短链神经酰胺细胞毒性的有效靶点。
