Samsel Leigh, Zaidel Grazyna, Drumgoole Honesty M, Jelovac Danijela, Drachenberg Cinthia, Rhee Juong G, Brodie Angela M H, Bielawska Alicja, Smyth Miriam J
Research Service, Baltimore Veterans Affairs Medical Center, and Department of Pathology, University of Marland School of Medicine, Baltimore, 21201, USA.
Prostate. 2004 Mar 1;58(4):382-93. doi: 10.1002/pros.10350.
Apoptosis is a therapeutic target for the elimination of cancer cells. As elevations in ceramide levels induce apoptosis, there is much excitement about the use of agents that elevate ceramide levels as novel chemotherapeutic agents. Ceramidases are enzymes involved in degradation of ceramide and inhibition of ceramidase has been proposed as a mechanism to increase ceramide levels. This study provides the first insight into the effect of B13, an inhibitor of acid ceramidase, on human prostate cancer cell lines and xenografts.
Cell death was evaluated by the trypan blue assay; apoptosis by the Apo2.7 apoptosis assay; and glutathione levels by HPLC. Tumors were irradiated with a dose of 5 Gy of X-rays (250 kVp, 15 mA, 2 Gy/min) and tumor volume was measured during the course of the experiment. At the conclusion of the experiment, tumor weight was determined and the tumors were evaluated histologically.
B13 is an inducer of cell death, by apoptosis, in cultured prostate cancer cells. LNCaP and PC3 cells have different responsiveness to the enantiomers of B13. In LNCaP cells, the R enantiomer of B13 (10 microM) was significantly more effective than the S enantiomer at inducing cell death as determined by the trypan blue assay, culminating in approximately 90% cell death at 48 hr. In contrast, the same concentration of B13S induced <20% cell death at 48 hr. In PC3 cells, the S enantiomer was a more effective inducer of cell death, culminating in approximately 30% cell death, relative to 14% for B13R in this model. Evaluation of induction of apoptosis by the Apo2.7 mitochondrial assay confirmed that this induction of cell death was by apoptosis. Concurrent with induction of apoptosis, glutathione levels drop in response to B13. Specifically, B13R caused a significant drop in glutathione levels in LNCaP cells, culminating in a reduction to 40% control values at 48 hr. In PC3 cells, in contrast, the drop in glutathione levels was more dramatic, culminating in a drop to 12% control values in response to B13S at 48 hr. The effects of B13R, however, were not significantly different from control values. In in vivo studies using a model of xenografted androgen-insensitive prostate cancer, B13 sensitized the tumors to the effects of radiation, resulting in a significant reduction in tumor volume and weight after treatment with the combination of B13 and radiation. Microscopic evaluation of the tumors indicated that apoptosis was the primary mechanism of this effect.
Targeting ceramide pathways may be a novel treatment strategy for hormone refractory prostate cancer.
细胞凋亡是消除癌细胞的一个治疗靶点。由于神经酰胺水平升高会诱导细胞凋亡，因此人们对使用能提高神经酰胺水平的药物作为新型化疗药物寄予厚望。神经酰胺酶是参与神经酰胺降解的酶，抑制神经酰胺酶被认为是提高神经酰胺水平的一种机制。本研究首次深入探讨了酸性神经酰胺酶抑制剂B13对人前列腺癌细胞系和异种移植瘤的影响。
通过台盼蓝试验评估细胞死亡情况；通过Apo2.7凋亡试验评估细胞凋亡情况；通过高效液相色谱法评估谷胱甘肽水平。用5 Gy的X射线（250 kVp，15 mA，2 Gy/min）照射肿瘤，并在实验过程中测量肿瘤体积。实验结束时，测定肿瘤重量并对肿瘤进行组织学评估。
B13可诱导培养的前列腺癌细胞发生凋亡，从而导致细胞死亡。LNCaP和PC3细胞对B13的对映体有不同的反应性。在LNCaP细胞中，通过台盼蓝试验测定，B13的R对映体（10 microM）在诱导细胞死亡方面比S对映体显著更有效，在48小时时最终导致约90%的细胞死亡。相比之下，相同浓度的B13S在48小时时诱导的细胞死亡<20%。在PC3细胞中，S对映体是更有效的细胞死亡诱导剂，最终导致约30%的细胞死亡，而在该模型中B13R诱导的细胞死亡为14%。通过Apo2.7线粒体试验评估细胞凋亡诱导情况证实，这种细胞死亡诱导是通过凋亡实现的。与细胞凋亡诱导同时发生的是，谷胱甘肽水平因B13而下降。具体而言，B13R导致LNCaP细胞中谷胱甘肽水平显著下降，在48小时时最终降至对照值的40%。相比之下，在PC3细胞中，谷胱甘肽水平的下降更为显著在48小时时，因B13S而最终降至对照值的12%。然而，B13R的作用与对照值无显著差异。在使用异种移植雄激素不敏感前列腺癌模型的体内研究中，B13使肿瘤对辐射的作用敏感，导致在联合使用B13和辐射治疗后肿瘤体积和重量显著减小。对肿瘤的显微镜评估表明，细胞凋亡是这种作用的主要机制。
靶向神经酰胺途径可能是激素难治性前列腺癌的一种新型治疗策略。
