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  • br Sensitivity to HMGCR inhibition

    2022-05-13


    3.2. Sensitivity to HMGCR inhibition is inversely associated with fluvastatin-induced SREBP2 activation in PCa cell lines
    To evaluate the effects of HMGCR inhibition on PCa viability, we treated PCa cell lines with increasing doses of fluvastatin in vitro. We chose to evaluate fluvastatin because we previously demonstrated that flu-vastatin does not interact with P-glycoprotein, a major drug efflux pump associated with drug resistance, at clinically-achievable con-centrations [31]. Fluvastatin also offers a lower potential for druge drug interactions compared to many of the other statins, as it is not metabolized by the cytochrome P450 3A4 (CYP3A4) complex, and therefore foods or the many drugs that can modulate CYP3A4 function will not affect fluvastatin activity [32]. Importantly, while hydrophilic statins (e.g. pravastatin, rosuvastatin) exhibit high hepatoselectivity, lipophilic statins have been measured in extrahepatic tissues such as the LL 37 [33]. It is therefore hypothesized that lipophilic statins, such as fluvastatin, can reach tumors in distant organs, including the prostate. Indeed, we were able to measure fluvastatin in the prostate of NOD/SCID mice after oral delivery, albeit at a concentration approxi-mately 10-fold less than what was measured in the serum (Figure 2). Intriguingly, a range of fluvastatin sensitivities was observed among the four PCa cell lines evaluated (Figure 3A). Fluvastatin exhibited cytotoxic effects in PC-3 cells at low micromolar concentrations similar to those measurable in the mouse prostate, whereas LNCaP, DU145 and VCaP cells were less sensitive to fluvastatin exposure (Figure 3A). Treatment of statin-sensitive PC-3 cells with fluvastatin resulted in cell death, as evidenced by increased DNA fragmentation and PARP cleavage, which was fully rescued by the addition of MVA (Figure 3B). This supports that the apoptotic response in PC-3 cells is due to direct HMGCR inhibition.
    Inhibition of HMGCR activity results in the depletion of intracellular sterol levels, which in turn results in the activation of SREBP2 [30,34]. SREBP2 resides in the endoplasmic reticulum (ER) in its precursor, full-length form. In response to sterol depletion, SREBP2 is escorted to the Golgi apparatus, where it is cleaved. Cleavage of SREBP2 liberates the N-terminal transcription factor, which then translocates to the nucleus to activate the transcription of sterol metabolism genes, including those that encode enzymes of the MVA pathway (Figure 3C). Most normal and cancer cells demonstrate robust SREBP2 activation in response to sterol depletion; however, impair-ment of this sterol-regulated feedback response has been docu-mented in a subset of cancer cells [23,35,36]. We next evaluated SREBP2 activation in PCa cell lines in response to fluvastatin treat-ment. Intriguingly, while increased SREBP2 cleavage was evident after fluvastatin treatment in LNCaP, DU145 and VCaP cells, no fluvastatin-induced SREBP2 cleavage was observed in statin-sensitive PC-3 cells (Figure 3D). In line with this observation, treatment of PC-3
    cells with fluvastatin failed to induce the expression of the SREBP2 target genes HMGCR and HMG-CoA synthase 1 (HMGCS1) after 16 h of treatment (Figure LL 37 3E). In contrast, treatment of LNCaP cells with fluvastatin resulted in the upregulation of both HMGCR and HMGCS1 mRNA expression (Figure 3F). This response was completely abro-gated by the addition of 25-hydroxycholesterol (25-HC), supporting that this restorative feedback mechanism is sterol-regulated in LNCaP cells (Figure 3F).
    122 MOLECULAR METABOLISM 25 (2019) 119e1302019 University Health Network. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).