Volume 117, Issue 4, Pages 838-847 (October 1999) Lovastatin augments sulindac-induced apoptosis in colon cancer cells and potentiates chemopreventive effects of sulindac  Banke Agarwal, Chinthalapally V. Rao, Sanjay Bhendwal, William R. Ramey, Haim Shirin, Bandaru S. Reddy, Peter R. Holt  Gastroenterology  Volume 117, Issue 4, Pages 838-847 (October 1999) DOI: 10.1016/S0016-5085(99)70342-2 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Lovastatin induces apoptosis in colon cancer cells. HCT-116, SW480, and LoVo colon cancer cells were grown to 50% confluence, incubated with lovastatin (■, 0 μmol/L; ▩, 10 μmol/L; ▩, 30 μmol/L) for 48 hours, and harvested for quantification of apoptosis by flow cytometry. Apoptosis was determined as the percentage of subdiploid cells. Data are mean ± SD. P < 0.05 was considered statistically significant (vs. no lovastatin). Similar data were obtained in 3 separate experiments. *Not statistically significant. Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Lovastatin before treatment augments sulindac-induced apoptosis in HCT-116, SW480, and LoVo colon cancer cells. HCT-116 (left panels), SW480 (middle panels), and LoVo (right panels) colon cancer cells were grown to 50% confluence and preincubated with lovastatin for 48 hours. Cells then were incubated for 48 hours with lovastatin with or without 0, 100, 250, and 500 μmol/L sulindac and harvested for quantification of apoptosis by flow cytometry. ■, Apoptosis induced by sulindac alone. Lovastatin before treatment resulted in a dose-dependent augmentation of apoptosis. Because of varying sensitivities to lovastatin-induced apoptosis, different doses of lovastatin were used for HCT-116 (0, 10, and 30 μmol/L), SW480 (0, 5, and 10 μmol/L), and LoVo (0, 10, and 20 μmol/L) cells. Data are mean ± SD. P < 0.05 was considered statistically significant (vs. no lovastatin). Similar data were obtained in 3 separate experiments. *Not statistically significant. (A) Apoptosis quantified as percentage of subdiploid cells. (B) Apoptosis quantified by BrdU/terminal deoxynucleotidyl transferase staining. Similar data were obtained in 3-6 separate experiments. Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Lovastatin before treatment augments sulindac-induced apoptosis in HCT-116, SW480, and LoVo colon cancer cells. HCT-116 (left panels), SW480 (middle panels), and LoVo (right panels) colon cancer cells were grown to 50% confluence and preincubated with lovastatin for 48 hours. Cells then were incubated for 48 hours with lovastatin with or without 0, 100, 250, and 500 μmol/L sulindac and harvested for quantification of apoptosis by flow cytometry. ■, Apoptosis induced by sulindac alone. Lovastatin before treatment resulted in a dose-dependent augmentation of apoptosis. Because of varying sensitivities to lovastatin-induced apoptosis, different doses of lovastatin were used for HCT-116 (0, 10, and 30 μmol/L), SW480 (0, 5, and 10 μmol/L), and LoVo (0, 10, and 20 μmol/L) cells. Data are mean ± SD. P < 0.05 was considered statistically significant (vs. no lovastatin). Similar data were obtained in 3 separate experiments. *Not statistically significant. (A) Apoptosis quantified as percentage of subdiploid cells. (B) Apoptosis quantified by BrdU/terminal deoxynucleotidyl transferase staining. Similar data were obtained in 3-6 separate experiments. Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Lovastatin before treatment reduces the number of viable cells after sulindac treatment. (A) SW480 and (B) HCT-116 cells were grown in 96-well plates and preincubated with lovastatin for 48 hours. Lovastatin with and without sulindac was added, and cells were incubated for an additional 48 hours. ■, 0 μmol/L lovastatin; ▩, 10 μmol/L lovastatin; 2, 30 μmol/L lovastatin. MTT assay then was performed as described in the protocol. The data are presented as the fraction of the optical density of the control wells and plotted as a percentage. Differences in cell viability after sulindac treatment were statistically significant (P < 0.001) between control incubation (no lovastatin) and preincubation with 10 and 30 μmol/L lovastatin at all concentrations of sulindac used. Data are mean ± SD. Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of addition of intermediates in the cholesterol synthetic pathway with lovastatin on sulindac-induced apoptosis. (A) SW480 cells were grown to 50% confluence and preincubated for 48 hours with 15 μmol/L lovastatin with or without isoprenoids (100 μmol/L mevalonate [Mev], 100 μmol/L farnesylpyrophosphate [FPP], or 10 μmol/L geranylgeranylpyrophosphate [GGPP]). Cells were then incubated for 48 hours with 250 μmol/L sulindac with or without lovastatin and isoprenoids and then collected for flow cytometry. Apoptosis was quantified as number of subdiploid cells and is plotted as the fraction of total cell number. Data are mean ± SD. (B) SW480 cells were grown in 96-well plates and preincubated for 48 hours with 30 μmol/L lovastatin alone or with 100 μmol/L mevalonate, 100 μmol/L farnesylpyrophosphate, or 10 μmol/L geranylgeranylpyrophosphate. Sulindac at 100, 250, or 500 μmol/L was then added with or without lovastatin with the isoprenoids, and cells were incubated for 48 hours. MTT assays then were performed as described in the protocol. Data are the fraction of the optical density of control wells and plotted as a percentage (mean ± SD). Similar data were obtained in 3 separate experiments and also in HCT-116 cells (not shown). Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of addition of intermediates in the cholesterol synthetic pathway with lovastatin on sulindac-induced apoptosis. (A) SW480 cells were grown to 50% confluence and preincubated for 48 hours with 15 μmol/L lovastatin with or without isoprenoids (100 μmol/L mevalonate [Mev], 100 μmol/L farnesylpyrophosphate [FPP], or 10 μmol/L geranylgeranylpyrophosphate [GGPP]). Cells were then incubated for 48 hours with 250 μmol/L sulindac with or without lovastatin and isoprenoids and then collected for flow cytometry. Apoptosis was quantified as number of subdiploid cells and is plotted as the fraction of total cell number. Data are mean ± SD. (B) SW480 cells were grown in 96-well plates and preincubated for 48 hours with 30 μmol/L lovastatin alone or with 100 μmol/L mevalonate, 100 μmol/L farnesylpyrophosphate, or 10 μmol/L geranylgeranylpyrophosphate. Sulindac at 100, 250, or 500 μmol/L was then added with or without lovastatin with the isoprenoids, and cells were incubated for 48 hours. MTT assays then were performed as described in the protocol. Data are the fraction of the optical density of control wells and plotted as a percentage (mean ± SD). Similar data were obtained in 3 separate experiments and also in HCT-116 cells (not shown). Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 COX expression in lovastatin-treated cells. LoVo and HCT-116 cells were incubated with lovastatin for 48 hours and then collected and studied as described in Materials and Methods. Lane 1, controls for COX-1 and COX-2; lane 2, untreated LoVo cells; lane 3, LoVo cells treated with 20 μmol/L lovastatin; lane 4, untreated HCT-116 cells; and lane 5, HCT-116 cells treated with 30 μmol/L lovastatin. In LoVo cells, lovastatin treatment results in decreased expression of COX-2 and expression of COX-1 remains undetectable. In HCT-116 cells, COX-1 expression increases and that of COX-2 remains undetectable. Similar results were obtained in 3 separate experiments. Gastroenterology 1999 117, 838-847DOI: (10.1016/S0016-5085(99)70342-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions