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  • br www molecularmetabolism com br Figure Fluvastatin can be

    Figure 2: Fluvastatin can be measured in the mouse prostate. Male NOD/SCID mice were treated with PBS or 50 mg/kg/day fluvastatin by oral gavage for 4 consecutive days. 2 h after the last treatment, serum samples were collected, the mice were euthanized and prostate and liver tissues were harvested. Fluvastatin concentrations were quantified by HPLC-MS/MS. Error bars represent the mean SD, n ΒΌ 5 mice per group.
    3.3. Inhibition of the sterol-regulated feedback loop of the MVA pathway potentiates fluvastatin-induced cell death in PCa cell lines Given that fluvastatin sensitivity seemed to be inversely associated with the ability of PCa (-)-Epigallocatechin gallate to activate SREBP2 and upregulate the expression of sterol metabolism genes in response to fluvastatin treatment, we next evaluated whether inhibition of the sterol-regulated feedback loop of the MVA pathway potentiated the cytotoxic effects of fluvastatin. Given that the addition of 25-HC prevented the upregulation of HMGCR and HMGCS1 mRNA expression in response to fluvastatin treatment (Figure 3F), we tested whether 25-HC could sensitize PCa cells to flu-vastatin. Treatment of LNCaP and DU145 cells with a sub-toxic con-centration of 25-HC significantly decreased the IC50 value of fluvastatin, suggesting that inhibition of SREBP2 activation can potentiate the cytotoxic effects of fluvastatin (Figure 4AeB). As a complementary approach, we knocked down SREBP2 in LNCaP cells using two inde-pendent doxycycline-inducible shRNAs (Figure 4C). SREBP2 knockdown abrogated fluvastatin-induced HMGCS1 expression and significantly decreased the IC50 value of fluvastatin (Figure 4DeE). Moreover, treatment of LNCaP cells with fluvastatin in the presence of SREBP2 knockdown resulted in increased apoptosis, as evidenced by increased PARP cleavage (Figure 4F). Collectively, these data suggest that inhib-iting the sterol-regulated feedback loop of the MVA pathway is a viable approach to potentiate statin-induced PCa cell death.
    3.4. Dipyridamole inhibits fluvastatin-induced SREBP activation and potentiates fluvastatin-induced apoptosis in PCa cell lines There is significant interest in targeting the SREBP family of tran-scription factors in PCa, as reactivation of lipogenesis has been shown to promote disease progression [17]. In addition to SREBP2, the master transcriptional regulator of fatty acid metabolism (SREBP1) has also been implicated as a viable therapeutic target in PCa [37]. Small molecule inhibitors, such as fatostatin, have been identified to inhibit both SREBP1 and SREBP2 and exhibit anti-cancer activity in vivo [37]; however, fatostatin has yet to be evaluated in clinical trials. More recently, our lab identified that the drug dipyridamole, which is currently approved as an anti-platelet agent, can also inhibit statin-induced SREBP2 activation [38]. Given that inhibiting SREBP2 poten-tiated statin-induced cell death in PCa cells (Figure 4), dipyridamole could potentially offer an immediately-available option to increase the therapeutic window of statins as anti-PCa agents.
    To evaluate whether dipyridamole treatment could sensitize PCa cells 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 [38]. 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).