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Polyisoprenylated methylated protein methyl esterase is both sensitive to curcumin and overexpressed in colorectal cancer: implications for chemoprevention and treatment.

Amissah F, Duverna R, Aguilar BJ, Poku RA, Lamango NS - Biomed Res Int (2013)

Bottom Line: Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL.In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001).The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.

ABSTRACT
Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with a K(i) of 0.3 μM (IC50 = 12.4 μM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yielded K M values of 23.6 ± 2.7 and 85.3 ± 15.3 μM in the absence or presence of 20 μM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50 of 22.6 μg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001). The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents.

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(a) Curcumin induced degeneration of human colorectal cancer Caco-2 cells. Human colorectal cancer Caco-2 cells were cultured and seeded in 96-well plates at a density of 2 × 104 as described in the methods. At 72 h after treatment with varying concentrations of curcumin, cell viability was measured by fluorescence using the resazurin reduction assay. Each data point represents the mean ± SEM of 4 wells. The data are representative of 3 separate experiments (EC50 = 22.0 μg/mL). (b) PMPMEase activity in degenerating curcumin-treated human colorectal cancer Caco-2 cells. Cells were cultured to 80% confluence, lysed, and incubated with the indicated concentrations of curcumin as described in the methods. The residual PMPMEase activity was then determined using RD-PNB as the substrate. Each point represents the mean ± SEM (n = 3). The data are representative of 3 separate experiments (IC50 = 22.6 μg/mL).
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fig4: (a) Curcumin induced degeneration of human colorectal cancer Caco-2 cells. Human colorectal cancer Caco-2 cells were cultured and seeded in 96-well plates at a density of 2 × 104 as described in the methods. At 72 h after treatment with varying concentrations of curcumin, cell viability was measured by fluorescence using the resazurin reduction assay. Each data point represents the mean ± SEM of 4 wells. The data are representative of 3 separate experiments (EC50 = 22.0 μg/mL). (b) PMPMEase activity in degenerating curcumin-treated human colorectal cancer Caco-2 cells. Cells were cultured to 80% confluence, lysed, and incubated with the indicated concentrations of curcumin as described in the methods. The residual PMPMEase activity was then determined using RD-PNB as the substrate. Each point represents the mean ± SEM (n = 3). The data are representative of 3 separate experiments (IC50 = 22.6 μg/mL).

Mentions: Malfunctions of polyisoprenylated proteins contribute to aberrant signaling resulting in the progression of several cancers. For example, Ras mutations that occur in over 50% of colorectal cancer cases are associated with a more aggressive disease [58]. Compounds that inhibit PMPMEase may contribute to the prevention or treatment of colorectal cancer. Cytotoxicity studies with Caco-2 cells using curcumin have yielded interesting results, and several mechanisms have been proposed for the apoptotic effects [30, 59–61]. In a study using curcumin and celecoxib, Lev-Ari and coworkers [59] proposed a synergistic inhibition of the COX-2 pathway for the inhibition of cell growth in HT-29 and IEC18-K-ras cells that express high levels of COX-2. However, no plausible conclusion could account for similar additive growth inhibition seen in colorectal cancer cell lines that expressed low or no COX-2 activity (Caco-2 and SW-480) [59]. We therefore sought to understand if the inhibitory effect of curcumin on PMPMEase could account for its anticancer effects using Caco-2 cells. Treatment of Caco-2 cells with curcumin resulted in the concentration-dependent inhibition of both cell viability (EC50 of 60 μM or 22.0 μg/mL) and cellular PMPMEase activity (IC50 of 61 μM or 23 μg/mL) (Figure 4). The loss of cell viability due to PMPMEase inhibition has been demonstrated in a wide variety of cell lines in our laboratory, including human neuroblastoma (SH-SY5Y) cells, human lung cancer (A549 and H460) cells, human triple negative breast cancer MDA-MB-231 cells, human pancreatic (BxPC-3) cells, and human prostate cancer (LNCaP) cells [33–35, 62]. Moreover, the EC50 for cell viability of 22 μg/mL obtained in this study is less than the mean curcumin level of 48.4 μg/g detected in human colorectal tissue biopsies after daily oral doses of 2.35 g curcuminoids [63], bearing in mind a tissue density of about 1.06 g/mL.


Polyisoprenylated methylated protein methyl esterase is both sensitive to curcumin and overexpressed in colorectal cancer: implications for chemoprevention and treatment.

Amissah F, Duverna R, Aguilar BJ, Poku RA, Lamango NS - Biomed Res Int (2013)

(a) Curcumin induced degeneration of human colorectal cancer Caco-2 cells. Human colorectal cancer Caco-2 cells were cultured and seeded in 96-well plates at a density of 2 × 104 as described in the methods. At 72 h after treatment with varying concentrations of curcumin, cell viability was measured by fluorescence using the resazurin reduction assay. Each data point represents the mean ± SEM of 4 wells. The data are representative of 3 separate experiments (EC50 = 22.0 μg/mL). (b) PMPMEase activity in degenerating curcumin-treated human colorectal cancer Caco-2 cells. Cells were cultured to 80% confluence, lysed, and incubated with the indicated concentrations of curcumin as described in the methods. The residual PMPMEase activity was then determined using RD-PNB as the substrate. Each point represents the mean ± SEM (n = 3). The data are representative of 3 separate experiments (IC50 = 22.6 μg/mL).
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fig4: (a) Curcumin induced degeneration of human colorectal cancer Caco-2 cells. Human colorectal cancer Caco-2 cells were cultured and seeded in 96-well plates at a density of 2 × 104 as described in the methods. At 72 h after treatment with varying concentrations of curcumin, cell viability was measured by fluorescence using the resazurin reduction assay. Each data point represents the mean ± SEM of 4 wells. The data are representative of 3 separate experiments (EC50 = 22.0 μg/mL). (b) PMPMEase activity in degenerating curcumin-treated human colorectal cancer Caco-2 cells. Cells were cultured to 80% confluence, lysed, and incubated with the indicated concentrations of curcumin as described in the methods. The residual PMPMEase activity was then determined using RD-PNB as the substrate. Each point represents the mean ± SEM (n = 3). The data are representative of 3 separate experiments (IC50 = 22.6 μg/mL).
Mentions: Malfunctions of polyisoprenylated proteins contribute to aberrant signaling resulting in the progression of several cancers. For example, Ras mutations that occur in over 50% of colorectal cancer cases are associated with a more aggressive disease [58]. Compounds that inhibit PMPMEase may contribute to the prevention or treatment of colorectal cancer. Cytotoxicity studies with Caco-2 cells using curcumin have yielded interesting results, and several mechanisms have been proposed for the apoptotic effects [30, 59–61]. In a study using curcumin and celecoxib, Lev-Ari and coworkers [59] proposed a synergistic inhibition of the COX-2 pathway for the inhibition of cell growth in HT-29 and IEC18-K-ras cells that express high levels of COX-2. However, no plausible conclusion could account for similar additive growth inhibition seen in colorectal cancer cell lines that expressed low or no COX-2 activity (Caco-2 and SW-480) [59]. We therefore sought to understand if the inhibitory effect of curcumin on PMPMEase could account for its anticancer effects using Caco-2 cells. Treatment of Caco-2 cells with curcumin resulted in the concentration-dependent inhibition of both cell viability (EC50 of 60 μM or 22.0 μg/mL) and cellular PMPMEase activity (IC50 of 61 μM or 23 μg/mL) (Figure 4). The loss of cell viability due to PMPMEase inhibition has been demonstrated in a wide variety of cell lines in our laboratory, including human neuroblastoma (SH-SY5Y) cells, human lung cancer (A549 and H460) cells, human triple negative breast cancer MDA-MB-231 cells, human pancreatic (BxPC-3) cells, and human prostate cancer (LNCaP) cells [33–35, 62]. Moreover, the EC50 for cell viability of 22 μg/mL obtained in this study is less than the mean curcumin level of 48.4 μg/g detected in human colorectal tissue biopsies after daily oral doses of 2.35 g curcuminoids [63], bearing in mind a tissue density of about 1.06 g/mL.

Bottom Line: Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL.In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001).The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.

ABSTRACT
Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with a K(i) of 0.3 μM (IC50 = 12.4 μM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yielded K M values of 23.6 ± 2.7 and 85.3 ± 15.3 μM in the absence or presence of 20 μM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50 of 22.6 μg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001). The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents.

Show MeSH
Related in: MedlinePlus