Figure 1.
Comparison of the effects of hydrophilic-pravastatin and hydrophobic-simvastatin on normal hepatocytes and cancer cells.
The effects of pravastatin and simvastatin on liver hepatocytes and tumor cells was determined by MTT assay and are represented as a percent of the control absorbance at a wavelength of 540 nm. All data were performed at 72 h except for Panc 28 which was responsive at 24 h. Data shown are from representative experiments (n = 8; except colon, n = 4). Values are expressed as mean+SD. * p<0.05, significant difference between control and simvastatin or pravastatin groups. ¶ p<0.05, significant difference between simvastatin and pravastatin groups. # p<0.05, significant difference between well differentiated and poorly differentiated cell types.
Figure 2.
Simvastatin induced shape change and mitochondrial redistribution in cancer cells at early time points.
PC-3 prostate cells and Panc 28 pancreatic cells were treated with 10 uM simvastatin and stained with calcein AM (green), MitoTracker CM-H2XRos, a reduced, non-fluorescent dye that fluoresces (red) upon oxidation, and DAPI, a staining dye that emits (blue) when bound to DNA. These images of PC-3 and Panc 28 were acquired via fluorescence microscopy and illustrate that changes in cell shape occurred within 6 hours becoming more extensive by 24 h. These shape changes were accompanied by a significant redistribution of mitochondria.
Figure 3.
Simvastatin but not pravastatin dose-dependently induces morphological changes and apoptotic behavior in responsive tumor cells.
Image analysis of cells stained with three fluorescent markers calcein AM (green), DAPI (blue), and MitoTracker Red CM-H2XRos (red), was performed as described in the methods section. Tumor cells were examined at 72 h except in the case of Panc 28 which were processed at 24 h.
Figure 4.
3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression varies between cells but the organic anion transporter (OATP) is expressed exclusively in normal liver.
A) HMGCR was examined by Western analysis for its expression as follows: liver (normal hepatocytes, lane 1; HepG2, lane 2; Hep3B, lane 3), pancreas (Capan 1, lane 4; Mia Paca, lane 5), prostate (LnCap, lane 6; PC-3, lane 7), lung (Calu3, lane 8; Calu6, lane 9), breast (MCF-7, lane 10; SkBr3, lane 11), colon (Cacao 2, lane 12; HCT116, lane 13), bladder (U9, lane 14; U14, lane 15), or squamous cell carcinoma (SCCP9, lane 16; SCCM7, lane 17). These data illustrate that HMGCR expression does not correspond to drug response. B) Total RNA isolated from human liver tissue (lane L) or cells and analyzed for expression of a 565 bp amplimers from OATP. The numeric sequence of PCR samples is the same as described for protein in A. GAPDH primers were used on the same series of mRNA samples to determine the quality and loading consistency of PCR products. Only whole liver (L) and hepatocytes (1) expressed multiple OATP amplimers by PCR. Note: PCR amplimers observed in pancreatic samples (lane 6) were not present when other HMGCR primer sets were used. C) Western analysis revealed OATP protein only in the human liver hepatocytes in lane 1. Tumor cell total protein was examined in the same sequence as in A and did not reveal any OATP protein.
Figure 5.
Incorporation and growth response of normal hepatocytes or tumor cells to hydrophilic-pravastatin or hydrophobic-simvastatin.
A & B) Total ion chromatography methods were developed to attain critical separation profiles showing pravastatin and simvastatin. C) Mass spectroscopy was performed by using deuterated standards as internal controls for separation to distinguish between statins. Statins were detected by using electrospray-negative ionization and monitoring by magnetic resonance microscopy. Fragmentation of the statins were performed using argon as the collision gas at a collision cell pressure of 2.1×10−3 torr. D) LS/MS/MS determination of statins. Monolayers of normal hepatocytes or prostate cancer cells (PC-3) were placed in fresh serum-free medium before the addition of 10 µM pravastatin or simvastatin. Cell culture medium and cells were collected 6 h after treatment. The statins were subjected to solid-phase extraction and analyzed for the presence of pravastatin or simvastatin by LC/MS/MS analysis. Data represent two determinations run in duplicate. E) Effects of pravastain and simvastatin on OATP expressing human hepatocytes were determined by MTT assay and are represented as a percent of the control absorbance at a wavelength of 540 nm. Data shown are from representative experiments (n = 8). These data illustrate that both pravastatin and simvastatin suppressed the growth of hepatocytes to nearly the same extent. Values are expressed as mean+SD. * p<0.05, significant difference between control and simvastatin or pravastatin groups. ¶ p<0.05, significant difference between simvastatin and pravastatin groups.
Figure 6.
OATP and HMGCR redistribution, loss of p-caveolin and clustering of p-cofilin in statin treated cells.
A) OATP immunofluorescent labeling (green) is observed diffusely distributed over the surface of untreated hepatocytes but becomes more perinuclear in cells treated with hydrophilic-pravastatin or lipophilic-simvastatin. Cells were counterstained for actin using alexa-594-phalloidin (red) and nuclear DNA using DAPI (blue). Actin redistribution occurs in conjunction with cellular elongation. B) HMGCR immunofluorescent labeling (left column, green) was diffusely distributed within untreated hepatocytes but became more perinuclear in cells treated with hydrophilic-pravastatin or lipophilic-simvastatin. Cells were counterstained for actin using alexa-594-phalloidin (red) and nuclear DNA using DAPI (blue). P-Tyr14-caveolin (green) expression was lost following statin treatment (middle column). P-Ser3-cofilin (green) formed clusters following statin treatment (right column).