Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 2. Survey of selected cell lines for drug response, prodrug hydrolysis, and CES2 expression. A, drug sensitivity. The cytotoxicity of LY2334737 and gemcitabine was measured and the ratio of EC50 values was calculated as described in Materials and Methods. Data are the mean SEM of 2 or more independent experiments measured in quadruplicate. B, CES transcript levels. Values for CES1 and CES2 were measured by qRT-PCR and normalized to GADPH. CES2 data are the mean SEM of quadruplicates and represent 2 independent experiments, whereas the CES1 data are the mean of quadruplicates from one experiment. C, Western blot analysis. Proteins of cell lysates (40 mg/lane) or rhCES (1 ng/lane) were separated by SDS-PAGE and immunoblotted with anti-CES1, CES2, or b-actin antibody as described in Materials and Methods. Molecular weight markers (kDa) are indicated on the left. D, LY2334737 hydrolysis. Cell lysates were incubated for 18 hours at 37C with 500 nmol/L [3H]LY2334737 and 1 mmol/L THU to block CDA deamination of gemcitabine as described in Materials and Methods. Human liver S9 lysate served as a control. Data are the mean SEM of 1 to 7 independent determinations measured in duplicate. Statistical analyses were conducted on samples with 3 or more independent measurements. *, significantly different from HCT-116 hydrolysis (P 0.05). ns, not significantly different from HCT-116 hydrolysis (P > 0.05).

Article Snippet: Gemcitabine hydrochloride, LY2334737, and a hemiP-toluenesulfonic acid hemihydrate salt of LY2334737 were prepared by Eli Lilly and Company (3).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Molecular Weight, Incubation, Blocking Assay, Control

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 3. Characterization of CES-transfected HCT-116 cells. A, Western blots ofrhCES and transfectant cell lysates (40 mg/lane) were separated by SDS-PAGE; blots were developed using antibodies to CES1, CES2, or b-actin as described in Materials and Methods. The blot was probed with the anti-CES antibody corresponding to the isoform that was loaded and is indicated on the right. Molecular weight markers on the left. B, LY2334737 dose response of transfectants. Cells were grown for 3 days in the presence of drug. Data are the mean SEM of quadruplicates from one representative experiment. Symbols for the transfectants with their EC50 are: (!) mock transfectant (2.33 mmol/L), (&) CES1 transfectant (1.98 mmol/L), and (o) CES2 transfectant (0.16 mmol/L). Gemcitabine EC50 values were 0.012 to 0.016 mmol/L (data not shown). C, correlation of CES2 transcript with LY2334737 cytotoxicity of HCT-116 transfectants. EC50 values for LY2334737 and gemcitabine were determined in 5-day cytotoxicity assays and their ratios calculated. Expression was measured by qRT-PCR. Values are the mean SEM of quadruplicates and representative of 2 independent experiments. The line was fitted by linear regression (r2 ¼ 0.94).

Article Snippet: Gemcitabine hydrochloride, LY2334737, and a hemiP-toluenesulfonic acid hemihydrate salt of LY2334737 were prepared by Eli Lilly and Company (3).

Techniques: Transfection, Western Blot, SDS Page, Molecular Weight, Expressing, Quantitative RT-PCR

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 4. Properties of SK-OV-3 CES2 knockdown. A, CES2 expression in SK-OV-3 parent and CES2 knockdown (KD). qRT-PCR measurements are the mean SEM of quadruplicate determinations and represent 2 independent experiments. B, Western blot analysis. Proteins in cell lysates were separated by SDS- PAGE electrophoresis and stained with anti-CES2 and anti-b-actin antibodies as described in Materials and Methods. C, IHC labeling. Expression was quantified by positive pixel count. CES2 protein immunolabeling levels were 66.8% in parental SK-OV-3 and 6% in the CES2 knockdown. D, effect of CES2 inhibition by shRNA knockdown or loperamide on cytotoxicity of LY2334737 and gemcitabine. SK-OV-3 parent (&; &) and knockdown (*; o) cells were grown 5 days in the absence (&; o) or presence (&; *) of a noncytotoxic concentration of loperamide (10 mmol/L), a CES2 inhibitor. Data are the average range of duplicate determinations from one experiment and are representative of 4 independent experiments. For LY2334737 cytotoxicity, the drug response of SK- OV-3 cells with CES2 neutralization, either by knockdown or chemical inhibition or both, were not significantly different from one another but were significantly different than the drug response of CES2-expressing parental SK-OV-3 cells (P 0.001). For gemcitabine, the EC50 values ranged between 24 to 34 nmol/L.

Article Snippet: Gemcitabine hydrochloride, LY2334737, and a hemiP-toluenesulfonic acid hemihydrate salt of LY2334737 were prepared by Eli Lilly and Company (3).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Electrophoresis, Staining, Labeling, Immunolabeling, Inhibition, shRNA, Concentration Assay, Neutralization

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 5. Efficacy of LY2334737 in a xenograft model bearing mock or CES2- HCT-116 transfectants. Mice (20/group) were treated by oral gavage QD 21 with vehicle (& mock; & CES2) or 3.2 mg/kg LY2334737 (~ mock; D CES2). Mice (10/group) received 80 mg/kg gemcitabine HCl i. p Q3D 4 (* mock; o CES2). Dosing began on day 20. Tumor growth was significantly different (***, P 0.001) between LY2334737-treated CES2 transfectant versus mock transfectant, and was significantly different than vehicle-treated CES2 transfectant (P 0.01). Tumor growth of mock and CES2 transfectants was not significantly different from each other when treated with vehicle (P > 0.05) or gemcitabine (P > 0.5).

Article Snippet: Gemcitabine hydrochloride, LY2334737, and a hemiP-toluenesulfonic acid hemihydrate salt of LY2334737 were prepared by Eli Lilly and Company (3).

Techniques: Transfection

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 2. Survey of selected cell lines for drug response, prodrug hydrolysis, and CES2 expression. A, drug sensitivity. The cytotoxicity of LY2334737 and gemcitabine was measured and the ratio of EC50 values was calculated as described in Materials and Methods. Data are the mean SEM of 2 or more independent experiments measured in quadruplicate. B, CES transcript levels. Values for CES1 and CES2 were measured by qRT-PCR and normalized to GADPH. CES2 data are the mean SEM of quadruplicates and represent 2 independent experiments, whereas the CES1 data are the mean of quadruplicates from one experiment. C, Western blot analysis. Proteins of cell lysates (40 mg/lane) or rhCES (1 ng/lane) were separated by SDS-PAGE and immunoblotted with anti-CES1, CES2, or b-actin antibody as described in Materials and Methods. Molecular weight markers (kDa) are indicated on the left. D, LY2334737 hydrolysis. Cell lysates were incubated for 18 hours at 37C with 500 nmol/L [3H]LY2334737 and 1 mmol/L THU to block CDA deamination of gemcitabine as described in Materials and Methods. Human liver S9 lysate served as a control. Data are the mean SEM of 1 to 7 independent determinations measured in duplicate. Statistical analyses were conducted on samples with 3 or more independent measurements. *, significantly different from HCT-116 hydrolysis (P 0.05). ns, not significantly different from HCT-116 hydrolysis (P > 0.05).

Article Snippet: [3H] LY2334737 (21 Ci/mmol; purity of 97%) was custom synthesized by GE (Amersham).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Molecular Weight, Incubation, Blocking Assay, Control

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 3. Characterization of CES-transfected HCT-116 cells. A, Western blots ofrhCES and transfectant cell lysates (40 mg/lane) were separated by SDS-PAGE; blots were developed using antibodies to CES1, CES2, or b-actin as described in Materials and Methods. The blot was probed with the anti-CES antibody corresponding to the isoform that was loaded and is indicated on the right. Molecular weight markers on the left. B, LY2334737 dose response of transfectants. Cells were grown for 3 days in the presence of drug. Data are the mean SEM of quadruplicates from one representative experiment. Symbols for the transfectants with their EC50 are: (!) mock transfectant (2.33 mmol/L), (&) CES1 transfectant (1.98 mmol/L), and (o) CES2 transfectant (0.16 mmol/L). Gemcitabine EC50 values were 0.012 to 0.016 mmol/L (data not shown). C, correlation of CES2 transcript with LY2334737 cytotoxicity of HCT-116 transfectants. EC50 values for LY2334737 and gemcitabine were determined in 5-day cytotoxicity assays and their ratios calculated. Expression was measured by qRT-PCR. Values are the mean SEM of quadruplicates and representative of 2 independent experiments. The line was fitted by linear regression (r2 ¼ 0.94).

Article Snippet: [3H] LY2334737 (21 Ci/mmol; purity of 97%) was custom synthesized by GE (Amersham).

Techniques: Transfection, Western Blot, SDS Page, Molecular Weight, Expressing, Quantitative RT-PCR

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 4. Properties of SK-OV-3 CES2 knockdown. A, CES2 expression in SK-OV-3 parent and CES2 knockdown (KD). qRT-PCR measurements are the mean SEM of quadruplicate determinations and represent 2 independent experiments. B, Western blot analysis. Proteins in cell lysates were separated by SDS- PAGE electrophoresis and stained with anti-CES2 and anti-b-actin antibodies as described in Materials and Methods. C, IHC labeling. Expression was quantified by positive pixel count. CES2 protein immunolabeling levels were 66.8% in parental SK-OV-3 and 6% in the CES2 knockdown. D, effect of CES2 inhibition by shRNA knockdown or loperamide on cytotoxicity of LY2334737 and gemcitabine. SK-OV-3 parent (&; &) and knockdown (*; o) cells were grown 5 days in the absence (&; o) or presence (&; *) of a noncytotoxic concentration of loperamide (10 mmol/L), a CES2 inhibitor. Data are the average range of duplicate determinations from one experiment and are representative of 4 independent experiments. For LY2334737 cytotoxicity, the drug response of SK- OV-3 cells with CES2 neutralization, either by knockdown or chemical inhibition or both, were not significantly different from one another but were significantly different than the drug response of CES2-expressing parental SK-OV-3 cells (P 0.001). For gemcitabine, the EC50 values ranged between 24 to 34 nmol/L.

Article Snippet: [3H] LY2334737 (21 Ci/mmol; purity of 97%) was custom synthesized by GE (Amersham).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Electrophoresis, Staining, Labeling, Immunolabeling, Inhibition, shRNA, Concentration Assay, Neutralization

Journal: Clinical Cancer Research

Article Title: Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1184

Figure Lengend Snippet: Figure 5. Efficacy of LY2334737 in a xenograft model bearing mock or CES2- HCT-116 transfectants. Mice (20/group) were treated by oral gavage QD 21 with vehicle (& mock; & CES2) or 3.2 mg/kg LY2334737 (~ mock; D CES2). Mice (10/group) received 80 mg/kg gemcitabine HCl i. p Q3D 4 (* mock; o CES2). Dosing began on day 20. Tumor growth was significantly different (***, P 0.001) between LY2334737-treated CES2 transfectant versus mock transfectant, and was significantly different than vehicle-treated CES2 transfectant (P 0.01). Tumor growth of mock and CES2 transfectants was not significantly different from each other when treated with vehicle (P > 0.05) or gemcitabine (P > 0.5).

Article Snippet: [3H] LY2334737 (21 Ci/mmol; purity of 97%) was custom synthesized by GE (Amersham).

Techniques: Transfection