Zeng Chenbo, Weng Chi-Chang, Schneider Mark E, Puentes Laura, Riad Aladdin, Xu Kuiying, Makvandi Mehran, Jin Linda, Hawkins William G, Mach Robert H
1Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Room 283 231S. 34th St, Philadelphia, PA 19104 USA.
2Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 USA.
Cell Death Discov. 2019 Jan 28;5:58. doi: 10.1038/s41420-019-0141-2. eCollection 2019.
Sigma-2 receptors have been implicated in both tumor proliferation and neurodegenerative diseases. Recently the sigma-2 receptor was identified as transmembrane protein 97 (TMEM97). Progesterone receptor membrane component 1 (PGRMC1) was also recently reported to form a complex with TMEM97 and the low density lipoprotein (LDL) receptor, and this trimeric complex is responsible for the rapid internalization of LDL. Sigma-2 receptor ligands with various structures have been shown to induce cell death in cancer cells. In the current study, we examined the role of TMEM97 and PGRMC1 in mediating sigma-2 ligand-induced cell death. Cell viability and caspase-3 assays were performed in control, TMEM97 knockout (KO), PGRMC1 KO, and TMEM97/PGRMC1 double KO cell lines treated with several sigma-2 ligands. The data showed that knockout of TMEM97, PGRMC1, or both did not affect the concentrations of sigma-2 ligands that induced 50% of cell death (EC), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 or PGRMC1. Sigma-1 receptor ligands, (+)-pentazocine and NE-100, did not block sigma-2 ligand cytotoxicity, suggesting that sigma-1 receptor was not responsible for sigma-2 ligand cytotoxicity. We also examined whether the alternative, residual binding site (RBS) of 1,3-Di--tolylguanidine (DTG) could be responsible for sigma-2 ligand cytotoxicity. Our data showed that the binding affinities ( ) of sigma-2 ligands on the DTG RBS did not correlate with the cytotoxicity potency (EC) of these ligands, suggesting that the DTG RBS was not fully responsible for sigma-2 ligand cytotoxicity. In addition, we showed that knocking out TMEM97, PGRMC1, or both reduced the initial internalization rate of a sigma-2 fluorescent ligand, . However, concentrations of internalized became identical later in the control and knockout cells. These data suggest that the initial internalization process of sigma-2 ligands does not appear to mediate the cell-killing effect of sigma-2 ligands. In summary, we have provided evidence that sigma-2 receptor/TMEM97 and PGRMC1 do not mediate sigma-2 ligand cytotoxicity. Our work will facilitate elucidating mechanisms of sigma-2 ligand cytotoxicity.
σ-2受体与肿瘤增殖和神经退行性疾病均有关联。最近，σ-2受体被鉴定为跨膜蛋白97（TMEM97）。孕酮受体膜成分1（PGRMC1）最近也被报道与TMEM97和低密度脂蛋白（LDL）受体形成复合物，并且这种三聚体复合物负责LDL的快速内化。已证明具有各种结构的σ-2受体配体可诱导癌细胞死亡。在本研究中，我们研究了TMEM97和PGRMC1在介导σ-2配体诱导的细胞死亡中的作用。在用几种σ-2配体处理的对照、TMEM97基因敲除（KO）、PGRMC1基因敲除和TMEM97/PGRMC1双基因敲除细胞系中进行了细胞活力和半胱天冬酶-3检测。数据显示，敲除TMEM97、PGRMC1或两者均不影响诱导50%细胞死亡（EC）的σ-2配体浓度，这表明这些化合物的细胞毒性作用不是由TMEM97或PGRMC1介导的。σ-1受体配体（+）-喷他佐辛和NE-100不能阻断σ-2配体的细胞毒性，这表明σ-1受体与σ-2配体的细胞毒性无关。我们还研究了1,3-二-对甲苯基胍（DTG）的替代残留结合位点（RBS）是否可能与σ-2配体的细胞毒性有关。我们的数据表明，σ-2配体在DTG RBS上的结合亲和力（ ）与这些配体的细胞毒性效力（EC）不相关，这表明DTG RBS并非完全介导σ-2配体的细胞毒性。此外，我们发现敲除TMEM97、PGRMC1或两者均会降低一种σ-2荧光配体的初始内化速率。然而，对照细胞和基因敲除细胞中内化的 浓度在随后变得相同。这些数据表明，σ-2配体的初始内化过程似乎并不介导σ-2配体的细胞杀伤作用。总之，我们提供了证据表明σ-2受体/TMEM97和PGRMC1不介导σ-2配体的细胞毒性。我们的工作将有助于阐明σ-2配体细胞毒性的机制。
