Abstract
Although, cisplatin is one of the most effective broad-spectrum anticancer drugs, prolonged cisplatin treatment often results in development of chemoresistance and subsequent therapeutic failure. Dysregulation of the taurine transporting systems i.e., the taurine transporter (TauT) and volume regulated anion channels (VRACs) have been assigned important roles in apoptotic cell death, proliferation and cell survival, as well as in development of multi-drug resistance. However, the molecular mechanisms contributing to the altered TauT and VRAC regulation in cancer cells are incompletely understood. Cisplatin resistance correlates with a reduction in the volume regulated anion current and taurine release mediated by VRACs, as well as an improved cellular accumulation of taurine through TauT. In human ovarian A2780 cancer cells, for instance, cisplatin resistance is associated with an absent swelling-induced taurine release and inability to volume regulate. The dismissed taurine release was due to an almost absent leucin-rich-repeat containing 8A (LRRC8A) total protein expression. LRRC8A is an important component of VRACs. Cellular taurine contributes to the intracellular pool of organic osmolytes. Moreover, taurine has in various rodent models been denoted protective roles against cisplatin-induced side effects as nephrotoxicity, hepatotoxicity and cardiotoxicity.
This dissertation includes one review on the role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance (articles I), as well as four original articles (articles II, III, IV and V), which focuses on the role of TauT and the VRAC mediating swelling-induced taurine release in development of cisplatin resistance. In article II, we show that siRNA-mediated silencing of LRRC8A and pharmacological inhibition of VRAC protects human ovarian A2780 and alveolar A549 carcinoma cells against cisplatin-induced apoptosis, through an impaired expression of p53, MDM2 and p21Waf1/Cip1, as well as a reduced Caspase-9/-3 activation. Moreover, we show that termination of prolonged cisplatin treatment or vector-mediated overexpression of LRRC8A restores cisplatin sensitivity. In article III, we show that the VRAC mediating swelling-induced taurine release is also responsible for the cellular uptake of cisplatin. Hypoosmotic cell swelling and membrane depolarization, which stimulates the activity of VRACs, increase the cellular accumulation of cisplatin in A2780 and ELA cells.
Article IV focusses on in vitro evaluation of the enantiomeric R- and S-1,1’-binaphthyl-2,2’-diaminodichlorido-Pt(II) complexes (denoted S- and R-[Pt(DABN)Cl2]) in human Burkitt lymphoma cells with emphasis on cellular accumulation, cytotoxicity, DNA binding, and ability to induce apoptosis. We demonstrate that cisplatin-resistant Gumbus cells are not cross-resistant to S- and R-[Pt(DABN)Cl2]. Furthermore, we show that S-[Pt(DABN)Cl2] is slightly more toxic compared to R-
[Pt(DABN)Cl2], even though the cellular accumulation and calf thymus-DNA-binding of R[Pt(DABN)Cl2] is significantly better than S-[Pt(DABN)Cl2]. We find that the cellular accumulation of S- and R-[Pt(DABN)Cl2] are much greater compared to cisplatin and involves a VRAC independent mechanism. Furthermore, we show that S- and R-[Pt(DABN)Cl2] induces apoptosis and G0/G1 cell cycle arrest.
Finally, article V focusses on the involvement of growth-associated signaling in the regulation of swelling-induced taurine release and the effect of cisplatin on TauT and the LRRC8A/D-dependent VRAC activity. We demonstrate that cisplatin reduces the activity of TauT. Furthermore, we find that the PI3K/PTEN/mTORC2/Akt cascade is important for the swelling-induced taurine release. Cisplatin leads to a transient activation of Akt, and it seems that the PI3K/PTEN/Akt pathway interferes with cisplatin-mediated effects on the LRRC8A/D-dependent VRAC.
Collectively, the results presented in this PhD thesis provide new insights into the molecular mechanisms underlying development of cisplatin resistance. Furthermore, elucidation of this topic may contribute to development of new therapeutic treatment strategies against tumors which has become resistant to cisplatin.
This dissertation includes one review on the role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance (articles I), as well as four original articles (articles II, III, IV and V), which focuses on the role of TauT and the VRAC mediating swelling-induced taurine release in development of cisplatin resistance. In article II, we show that siRNA-mediated silencing of LRRC8A and pharmacological inhibition of VRAC protects human ovarian A2780 and alveolar A549 carcinoma cells against cisplatin-induced apoptosis, through an impaired expression of p53, MDM2 and p21Waf1/Cip1, as well as a reduced Caspase-9/-3 activation. Moreover, we show that termination of prolonged cisplatin treatment or vector-mediated overexpression of LRRC8A restores cisplatin sensitivity. In article III, we show that the VRAC mediating swelling-induced taurine release is also responsible for the cellular uptake of cisplatin. Hypoosmotic cell swelling and membrane depolarization, which stimulates the activity of VRACs, increase the cellular accumulation of cisplatin in A2780 and ELA cells.
Article IV focusses on in vitro evaluation of the enantiomeric R- and S-1,1’-binaphthyl-2,2’-diaminodichlorido-Pt(II) complexes (denoted S- and R-[Pt(DABN)Cl2]) in human Burkitt lymphoma cells with emphasis on cellular accumulation, cytotoxicity, DNA binding, and ability to induce apoptosis. We demonstrate that cisplatin-resistant Gumbus cells are not cross-resistant to S- and R-[Pt(DABN)Cl2]. Furthermore, we show that S-[Pt(DABN)Cl2] is slightly more toxic compared to R-
[Pt(DABN)Cl2], even though the cellular accumulation and calf thymus-DNA-binding of R[Pt(DABN)Cl2] is significantly better than S-[Pt(DABN)Cl2]. We find that the cellular accumulation of S- and R-[Pt(DABN)Cl2] are much greater compared to cisplatin and involves a VRAC independent mechanism. Furthermore, we show that S- and R-[Pt(DABN)Cl2] induces apoptosis and G0/G1 cell cycle arrest.
Finally, article V focusses on the involvement of growth-associated signaling in the regulation of swelling-induced taurine release and the effect of cisplatin on TauT and the LRRC8A/D-dependent VRAC activity. We demonstrate that cisplatin reduces the activity of TauT. Furthermore, we find that the PI3K/PTEN/mTORC2/Akt cascade is important for the swelling-induced taurine release. Cisplatin leads to a transient activation of Akt, and it seems that the PI3K/PTEN/Akt pathway interferes with cisplatin-mediated effects on the LRRC8A/D-dependent VRAC.
Collectively, the results presented in this PhD thesis provide new insights into the molecular mechanisms underlying development of cisplatin resistance. Furthermore, elucidation of this topic may contribute to development of new therapeutic treatment strategies against tumors which has become resistant to cisplatin.
| Originalsprog | Engelsk |
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| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
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| Antal sider | 190 |
| Status | Udgivet - 2018 |