br To evaluate whether dipyridamole treatment
To evaluate whether dipyridamole treatment could sensitize PCa Pam3CSK4 to fluvastatin, we treated PC-3, LNCaP, DU145 and VCaP cells with
fluvastatin alone or in combination with a sub-lethal, physiologically-achievable dose of dipyridamole . Treatment of LNCaP, DU145 and VCaP cells with dipyridamole significantly lowered the IC50 value of fluvastatin in these cell lines (Figure 5A). Furthermore, combining fluvastatin and dipyridamole, at doses that had a minimal effect when used as single agents, resulted in significantly increased apoptosis in both LNCaP and DU145 cells (Figure 5B). Cell death in response to the fluvastatin and dipyridamole combination was fully rescued by the addition of MVA (Supplementary Fig. 4).
Interestingly, the fluvastatin IC50 value of PC-3 cells remained unaffected by dipyridamole co-treatment, which is consistent with the observation that PC-3 cells failed to upregulate sterol metabolism gene expression in response to fluvastatin (Figures 3E, 5A, Supplementary Fig. 5).
Treatment of less statin-sensitive PCa cell lines with dipyridamole abrogated fluvastatin-induced cleavage and activation of SREBP2 and upregulation of lipid metabolism gene expression (Figure 5CeE, Supplementary Fig. 6). Given that activation of SREBP1 is post-translationally regulated by the same mechanism as SREBP2, we evaluated whether dipyridamole could also inhibit SREBP1. Indeed, fluvastatin-induced cleavage of SREBP1 was also inhibited by dipyr-idamole, an effect that was previously undocumented for this clinically-approved agent (Figure 5CeD).
3.5. The combination of fluvastatin and dipyridamole delays prostate tumor growth
Given that both fluvastatin and dipyridamole are clinically-approved and poised for repurposing, we evaluated whether the fluvastatin-dipyridamole combination was effective at delaying tumor growth in vivo. We treated NOD/SCID mice harboring established LNCaP xenografts orally with fluvastatin and/or intraperitoneally (i.p.) with dipyridamole. The combination of fluvastatin and dipyridamole significantly decreased tumor volumes, while each agent alone had no effect compared to vehicle-treated mice (Figure 6A). After 12 days of treatment, LNCaP tumors from mice treated with both fluvastatin and dipyridamole had increased TUNEL staining compared to vehicle-treated mice, suggesting that the drug combination induced apoptosis in vivo (Figure 6B). We further evaluated the drug combi-nation in a clinically-relevant patient-derived xenograft (PDX) model of androgen-sensitive PCa. Consistent with the LNCaP xenograft results, the fluvastatin-dipyridamole drug combination significantly decreased tumor volumes and final tumor weights in the PDX model (Figure 6CeD).
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/). 123 www.molecularmetabolism.com
Figure 3: Sensitivity to HMGCR inhibition is inversely associated with fluvastatin-induced SREBP2 activation in PCa cell lines. (A) PCa cell lines were treated with a range of fluvastatin doses for 72 h, and cell viability was determined using an MTT assay. Error bars represent the mean SD, n ¼ 3e5. (B) PC-3 cells were treated with fluvastatin 200 mM MVA for 72 h, fixed in ethanol and assayed for DNA fragmentation (% pre-G1 population) as a marker of cell death by propidium iodide staining. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). Protein was also isolated from PC-3 cells after 72 h of treatment and immunoblotting was performed to assay for PARP cleavage. (C) Schematic representation of the MVA pathway and its sterol-regulated feedback loop. Depletion of cholesterol following statin-mediated inhibition of HMGCR results in the cleavage and activation of SREBP2 and upregulation of the MVA pathway enzymes HMGCR and HMGCS1. (D) PCa cell lines were treated with 10 mM fluvastatin for 8 h. Protein was then isolated and lysates were analyzed for statin-induced SREBP2 activation by immunoblotting. Both the full-length (inactive) and cleaved forms of SREBP2 were detected. (E) PC-3 cells were treated with 1 or 5 mM fluvastatin for 16 h, and RNA was isolated to assay for HMGCR and HMGCS1 expression by qRT-PCR. mRNA expression data are normalized to RPL13A expression. Error bars represent the mean þ SD, n ¼ 3. (F) LNCaP cells were treated with 5 mM fluvastatin 1 mM 25-hydroxycholesterol (25-HC) for 16 h, and RNA was isolated to assay for HMGCR and HMGCS1 expression by qRT-PCR. mRNA expression data are normalized to RPL13A