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  • br expression Error bars represent the mean SD n

    2020-08-12


    expression. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the solvent control group).
    4. DISCUSSION
    Increasing evidence supports that statins possess anti-cancer prop-erties; however, in PCa, a broad range of sensitivity to fluvastatin was observed (Figure 3A). In order to advance statins as anti-cancer agents for the treatment of PCa, it is crucial to understand what features 
    distinguish the subset of prostate tumors that are responsive to statins and/or identify effective statin-drug combinations to increase their therapeutic potential.
    Sensitivity to fluvastatin was inversely associated with the ability to induce the expression of SREBP2 target genes following fluvastatin exposure. When HMGCR activity is inhibited by statins, intracellular
    124 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/).
    www.molecularmetabolism.com
    Figure 4: Inhibition of the sterol-regulated feedback loop of the MVA pathway potentiates fluvastatin-induced cell death in PCa cell lines. (A) LNCaP and DU145 C11 BODIPY 581/591 were treated with a range of fluvastatin doses a sub-toxic dose (1 mM) of 25-hydroxycholesterol (25-HC) for 72 h, and cell viability was determined using an MTT assay. Error bars represent the mean SD, n ¼ 3, *p < 0.05 (Student t test, unpaired, two-tailed). (B) Fluvastatin IC50 values for LNCaP and DU145 cells treated with fluvastatin alone or in
    combination with 1 mM 25-HC. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (Student t test, unpaired, two-tailed). (C) LNCaP cells expressing inducible shRNAs against SREBF2 were induced for 72 h with 1 mg/mL doxycycline and protein was isolated to assay for SREBP2 expression by immunoblotting. (D) LNCaP shScramble and shSREBF2 cells
    were treated with 1 mg/mL doxycycline for 56 h and then EtOH or 10 mM fluvastatin for an additional 16 h in the presence of 1 mg/mL doxycycline. RNA was isolated to assay for HMGCS1 expression by qRT-PCR. mRNA expression data are normalized to RPL13A expression. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (Student t test, unpaired,
    two-tailed). (E) LNCaP shScramble and shSREBF2 cells were treated with a range of fluvastatin doses in the presence of 1 mg/mL doxycycline for 72 h, and cell viability was determined using an MTT assay. The IC50 values are plotted. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the shScramble control). (F) LNCaP shScramble and shSREBF2 cells were treated with EtOH or 10 mM fluvastatin for 72 h in the presence of 1 mg/mL doxycycline. Protein was then isolated to assay for PARP cleavage by immunoblotting.
    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/). 125 www.molecularmetabolism.com
    Original Article
    Figure 5: Dipyridamole inhibits fluvastatin-induced SREBP activation and potentiates fluvastatin-induced apoptosis in PCa cell lines. (A) PCa cell lines were treated with a range of fluvastatin doses a sub-lethal dose (5 mM) of dipyridamole for 72 h, and cell viability was determined using an MTT assay. The IC50 values are plotted. Error bars represent the mean þ SD, n ¼ 3e5, *p < 0.05 (Student t test, unpaired, two-tailed). (B) LNCaP and DU145 cells were treated with solvent controls, 10 mM fluvastatin, 5 mM dipyridamole (DP) or the combination 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 cells after 72 h of treatment and immunoblotting was performed to assay for PARP cleavage. (C) LNCaP cells were treated with 10 mM fluvastatin 5 mM DP for 8 h, and protein was isolated to assay for SREBP1 and SREBP2 expression and cleavage (activation) by immunoblotting. (D) SREBP1 and SREBP2 cleavage (cleaved/full-length) was quantified by densitometry and normalized to
    Actin expression. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test, where each group was compared to the solvent controls group). (E) LNCaP cells were treated with 10 mM fluvastatin 5 mM DP for 16 h, and RNA was isolated to assay for HMGCR, HMGCS1, INSIG1 and SCD expression by qRT-PCR. mRNA expression data are normalized to RPL13A expression. Error bars represent the mean þ SD, n ¼ 3, *p < 0.05 (one-way ANOVA with Bonferroni’s multiple