Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells

Abstract

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Figure 1. Simvastatin induces cell death in glioblastoma, non-small lung cancer cell, and breast cancer cell lines.

(A,B) U87 cells were treated with simvastatin (1, 2.5, 5, 10 or 20 μM) and cell viability was assessed 48 and 96 hrs thereafter by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.001). (C,D) A549 cells were treated with simvastatin (1, 2.5, 5, 10 or 20 μM) and cell viability was assessed 48 and 96 hrs thereafter by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as percentage of corresponding time point control and represent the means ± SD of 15 replicates in 3 independent experiments (**P < 0.01; ***P < 0.001). (E,F) MDA-MB-231 cells were treated with simvastatin (1, 2.5, 5, 10 or 20 μM) and cell viability was assessed 48 and 96 hrs thereafter by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (***P < 0.001). (G) U87, A549, and MDA-MB231 cells treated with 10 μM simvastatin for 60 hrs were then photographed under phase contrast microscopy settings. Arrows indicate partially detached cells with condensed morphology.

Figure 2. Simvastatin induces intrinsic apoptosis in glioblastoma, non-small lung cancer cell, and breast cancer cell lines.

Percent sub-G1 (A) U87, (B) A549, (C) MDA-MB-231 abundance induced by simvastatin (10 μM) or DMSO solvent control after 60 hrs. Results represent the means ± SD of 9 replicates in three independent experiments. *P < 0.05; and ***P < 0.001 compared to time-matched control. Representative figures of the flow cytometry histogram for U87, A549 and MDA-MB-231 are shown (D). Effects of simvastatin (10 μM) treatment (36 hrs) on caspase-8, caspase-3/-7, and caspase-9 enzymatic activity, as detected by Caspase-Glo^® luminometric assay in U87 (E), A549 (F), MDA-MB-231 (G). Caspase activity normalized to that measured for solvent-only treated cultures is represented on the Y-axis. The data represent mean ± SD of triplicate experiments performed on 3 independent experiments. U87, A549, and MDA-MB231 cells were treated with 10 μM simvastatin for 18 and 36 hrs. Control cells were treated with media and vehicle control (DMSO). Cells were stained with TMRM, Hoechst, and imaged by standard fluorescence techniques. Simvastatin (10 μM, 18 hrs) did not significantly change TMRM fluorescence intensity in U87, A549, and MDA-MB231 cells (H–K),while simvastatin (10 μM, 36 hrs) significantly decreased TMRM florescence intensity in U87 (P < 0.01), A549 (P < 0.05), and MDA-MB231 (P < 0.001) (L–O) which indicates the decrease of mitochondrial membrane potential in simvastatin-treated cells. Data represent the average values from triplicates of three independent experiments.

Figure 3. Simvastatin-induced cell death in glioblastoma, non-small lung cancer cell, and breast cancer cell lines is independent of cholesterol.

2.5, and 5 mM MEV or, 25, 50 μM cholesterol, were added to the cells 4 hrs prior to treatment with simvastatin (10 μM, 96 hrs). Cell death was measured by MTT assay in U87 (A,B), A549 (C,D), and MDA-MB-231 (E,F). For each experiment control cells were treated with simvastatin solvent (DMSO) alone (control) or with both DMSO and the appropriate solvent (i.e. ethanol for “mevalonate control”). Results are expressed as mean ± SD of 9 replicate in 3 independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001). U87, A549, and MDA-MB231 cells were treated with simvastatin (10 μM) and after 36 hrs total and de novo cholesterol content in cells were measured. Both total and de novo cholesterol were significantly decreased in U251 cells (G,H) after simvastatin treatment. However, there was no significant change in the amount of total and de novo cholesterol for A549 (I,J) and MDA-MB231 cells (K,L). For each experiment control cells were treated with DMSO. Results are expressed as mean ± SD of 3 replicate 3 independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 4. Simvastatin-induced cell death in glioblastoma, non-small lung cancer cell, and breast cancer cell lines dependents on FPP and GGPP.

7.5, 15 μM FPP, or 7.5, 15 μM GGPP were added to the cells 4 hrs prior to treatment with simvastatin (10 μM, 96 hrs) on cell death, measured by MTT assay in U87 (A,B), A549 (C,D), and MDA-MB-231 (E,F). For each experiment control cells were treated with simvastatin solvent (DMSO) alone (control) or with both DMSO and the appropriate solvent for each cholesterol precursor (i.e. DMSO for “FPP control” and “GGPP control”). Results are expressed as mean ± SD of 9 replicate in 3 independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 5. Simvastatin promotes cytosolic localization of RhoA, CDC42, and Rac1/2/3.

U87, A549, and MDA-MB231 were treated with simvastatin (10 μM, 12, 24, 36 hrs) and the abundance of RhoA, cdc42, and Rac1/2/3 in membrane and cytosolic fractions obtained from U251 (A), A549 (C), and MDA-MB-231 (E). GAPDH and Pan-Cadherin abundance was also assessed to control for loading in cytosolic and membrane fractions, and to confirm lack of cytosolic contamination in membrane fractions. Data are typical of 3 independent experiments using different primary cultures. Cropped representative of blots have been showed. G-LISA assay was done to evaluate the GTP-bound Rho protein in U251, A549, and MDA-MB231. Different conditions were tested for 36 hrs including starvation, Simva. (10 μM), Mev (2.5 mM), FPP, and GGPP (15 μM), Simva. + Mev, Simva. + FPP, and Simva. + GGPP. Simva. significantly increased GTP-bound Rho in U251 cells (B) while Mev, FPP, and GGPP co-treatment decreased GTP-bound Rho compared to control. none of the treatments significantly change GTP-bound protein in A549 (D) and MDA-MB231 (F) cells. For each experiment a positive control provided in the kit was used and control cells were treated with the reagent in the kit. Results are expressed as mean ± SD of 2 replicates in an independent experiment (***P < 0.001).

Figure 6. FTi-277 and GGTi-298 induces differential cell death in cancer cells.

U87, A549, and MDA-MB231 cells were treated with FTi-277 (0–40 μM, 36, 60 hrs) (A–F) and GGTi-298 (0–40 μM, 36, 60 hrs) (G–L) and the cytotoxic effects were measured using MTT assay. For each experiment control cells were treated with GGTi solvent (DMSO) and FTi-277 solvent (distilled water) alone (control). Results are expressed as mean ± SD of 15 replicate in 3 independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 7. Summary of the mechanism involved in statin-induced cell death in cancer cells. MEV cascade inhibitors induce the intrinsic apoptotic pathway which is regulated by gernaylgenralyation of small Rho GTPAse protein.