INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1735-1741, 2014
Ubiquitin-proteasomal degradation of antiapoptotic survivin facilitates induction of apoptosis in prostate cancer cells by pristimerin Yong Bo Liu1, Xiaohua Gao1, Dorrah Deeb1, Chris Brigolin2, Yiguan Zhang3, Jiajiu Shaw3, Kirit Pindolia2 and Subhash C. Gautam1 Departments of 1Surgery, 2Medical Genetics and 3Internal Medicine, Henry Ford Health System, Detroit, MI 48202, USA Received April 29, 2014; Accepted June 17, 2014 DOI: 10.3892/ijo.2014.2561 Abstract. Pristimerin (PM), a quinonemethide triterpenoid, is a promising anticancer agent with potent antiproliferative and apoptosis-inducing activities against cancer cell lines. However, the anticancer activity and mechanisms of PM in prostate cancer cells have not been adequately investigated. Here we report that the degradation of survivin plays an important role in the antiproliferative and proapoptotic effects of PM in carcinoma of the prostate (CaP) cell lines. Treatment with PM inhibited proliferation and induced apoptosis in LNCaP and PC-3 cells as characterized by the loss of cell viability and an increase in Annexin V-binding and cleavage of PARP-1, respectively. The antiproliferative and apoptosis-inducing effects of PM were associated with the inhibition of cell cycle regulatory proteins, antiapoptotic survivin and members of the Bcl-2 family. Data showed that response to PM is regulated by survivin since overexpression of survivin rendered CaP cells resistant to PM. Furthermore, downregulation of survivin by PM was mediated through the ubiquitin-proteasomal degradation. Together, these data demonstrate that pristimerin inhibits proliferation and induces apoptosis in CaP cells by abolishing survivin through the ubiquitin-proteasome pathway.
Correspondence to: Dr Subhash C. Gautam, Surgical Research-4D, Henry Ford Health System, One Ford Place, Detroit, MI 48202, USA E-mail: [email protected]
and identification of bioactive components from medicinal plants have led to the synthesis of potent anticancer drugs, including Vinca alkaloids, taxol, camptothecan, etoposide and retinoids. Triterpenoids are members of a large family of structurally related compounds known as cyclosqualenoids that are widely distributed in nature. Pristimerin (PM) is a quinonemethide triterpenoid present in various plant species in the Celastraceae and Hippocrateaceae families (1,2). PM and related compounds have shown anti-inflammatory, antioxidant and antimalarial activities (3-5). PM has also shown potent antiproliferative and apoptosis-inducing activity in glioma, leukemia, breast, lung and prostate cancer cell lines (6-9). Induction of apoptosis by PM involves generation of reactive oxygen species (ROS), activation of caspases, mitochondrial dysfunction, inhibition of nuclear factor κ B (NF-κ B), Akt and MAP kinases (10-13). In addition, PM also inhibits proteasome activity, tumor cell migration and angiogenesis (8,14,15). Carcinoma of the prostate is the most commonly diagnosed cancer in American males and the second ranked cause of cancer related mortality (16). An estimated 233,000 new cases of prostate cancer will be diagnosed and 29,480 deaths are expected to occur from this disease in the United States in 2014 (17). Current therapies (e.g., androgen deprivation, radical prostatectomy, radiotherapy or brachytherapy) while effective in treating localized prostate cancer have limited efficacy against advanced disease and metastatic hormone-refractory disease remains incurable (18-20). Since the incidence of CaP increases with advancing age, prostate cancer is expected to be an increasingly greater problem as life expectancy improves. In a previous report we have shown that PM induces apoptosis in CaP cell lines through a ROS-dependent Bcl-2 degradation pathway (21). In the present study, we demonstrate that induction of apoptosis in CaP cells by PM is associated with inhibition of cell cycle regulatory proteins and proteasomal degradation of antiapoptotic survivin, a member of the inhibitors of apoptosis (IAP) family.
Key words: pristimerin, prostate cancer, apoptosis, survivin, ubiquitin,
Materials and methods
Introduction Herbal remedies are commonly used in traditional medicine to treat and prevent human diseases including cancer. Numerous plant derived flavonoids and phenolic/polyphenolic compounds with antioxidant and anti-inflammatory activities are currently used by cancer patients as dietary supplements to complement chemotherapy. In fact, isolation
proteasome
Reagents. PM was purchased from Sigma Chemicals (St. Louis, MO). Anti-PARP-1, anti-Bcl-2, anti-Bcl-xL and
1736
Liu et al: Pristimerin and prostate cancer
anti-survivin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The 96 AQueous One Solution Proliferation Assay System was from Promega (Madison, WI). Annexin V-FITC apoptosis detection kit was purchased from BD Pharmingen (San Diego, CA). Stock solution of PM (100 mM) was prepared in DMSO and all test concentrations were prepared by diluting stock solution in tissue culture medium. Cell lines. LNCaP and PC-3 human prostate cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). LNCaP were grown in RPMI-1640 supplemented with FBS and penicillin/streptomycin. PC-3 cells were grown in F-12K nutrient mixture (Gibco BRL, Rockville, MD) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 25 mM HEPES buffer. Both cell lines were cultured at 37˚C in a humidified atmosphere consisting of 5% CO2 and 95% air, and maintained by subculturing cells twice a week. Measurement of cell viability (MTS assay). Tumor cells (1x104) in 100 µl of tissue culture medium were seeded into each well of a 96-well plate. After 24‑h incubation to allow cells to adhere, cells were treated with PM at concentrations ranging from 0 to 5 µM. Cultures were incubated for additional 72 h and cell viability was then determined by the colorimetric MTS assay using CellTiter 96 AQueous One Solution Proliferation Assay System from Promega. This assay measures the bioreduction of the tetrazolium compound MTS by intracellular dehydrogenases in the presence of electron-coupling reagent phenazine methosulfate. MTS and phenazine methosulfate were added to the culture wells, and cultures were incubated for 2 h at 37˚C. The absorbance, which is directly proportional to the number of viable cells in the cultures, was measured at 490 nm using a microplate reader. Apoptosis assay. Apoptosis was assessed by the binding of Annexin V-FITC to phosphotidylserine, which is externalized to the outer leaflet of the plasma membrane early during induction of apoptosis. Briefly, untreated cells and cells treated with PM were resuspended in the binding buffer provided in the Annexin V-FITC apoptosis detection kit II (BD Biosciences, San Diego, CA) and allowed to react with 5 µl of Annexin V-FITC reagent and 5 µl of propidium iodide for 30 min at room temperature in the dark. Stained cells were analyzed by flow cytometry using Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI). The induction of apoptosis by PM was confirmed by the cleavage of PARP-1 by western blot analysis. Western blot analysis. Cell lysates were prepared by detergent lysis [1% Triton-X 100 (v/v), 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 10% glycerol, 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A and 10 µg/ml 4-2-aminoethyl-benzenesulfinyl f luoride]. Lysates were clarified by centrifugation at 14,000 x g for 10 min at 4˚C, and protein concentrations were determined by Bradford assay. Samples (50 µg) were boiled in an equal volume of sample buffer [20% glyc-
erol, 4% SDS, 0.2% bromophenol blue, 125 mM Tris-HCl (pH 7.5), and 640 mM 2-mercaptoethanol] and separated on 10% SDS-polyacrilamide gels. Proteins resolved on the gels were transferred to nitrocellulose membranes and probed with antibodies against proteins of interest followed by HRP-conjugated secondary antibody. Immune complexes were visualized by chemiluminescence. Protein bands were imaged and band densities analyzed using the NIH/Scion image analysis software. DNA transfection. For expression of HA tagged-survivin, semi-confluent cultures of PC-3 cells in 60 mm2 cell culture dishes were transfected with 10 µg of empty or HA-survivin expression vector (pcDNA3-HA-survivin) (CH3 BioSystems, Amherst, NY) using Lipofectamine Plus reagent. After incubation for 36 h, overexpression of survivin in transfected cells was confirmed by immunoblotting. Immunoprecipitation. After treatment with PM (5 µM) for 6 h cells were washed with cold PBS and lysed in NP‑40 cell lysis buffer (Invitrogen, Camarillo, CA) supplemented with 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatinin, and 10 µg/ml 4-2-aminoethyl-benzenesulfinyl fluoride for 30 min on ice. Supernatants were collected after centrifugation at 14,000 x g for 10 min and protein concentration was determined. Each sample (400 µg protein) in 200 µl of antibody binding buffer containing anti-HA antibody was incubated for 1 h at room temperature followed by incubation with protein A agarose beads for 1 h. Immune complexes were washed two times with lysis buffer and analyzed for ubiquitin by western blot analysis. Statistical analysis. Data are expressed as mean ± SD. The difference between control and treatment groups was determined using Dunnett's multiple comparison test. Differences with p
Ubiquitin-proteasomal degradation of antiapoptotic survivin facilitates induction of apoptosis in prostate cancer cells by pristimerin Yong Bo Liu1, Xiaohua Gao1, Dorrah Deeb1, Chris Brigolin2, Yiguan Zhang3, Jiajiu Shaw3, Kirit Pindolia2 and Subhash C. Gautam1 Departments of 1Surgery, 2Medical Genetics and 3Internal Medicine, Henry Ford Health System, Detroit, MI 48202, USA Received April 29, 2014; Accepted June 17, 2014 DOI: 10.3892/ijo.2014.2561 Abstract. Pristimerin (PM), a quinonemethide triterpenoid, is a promising anticancer agent with potent antiproliferative and apoptosis-inducing activities against cancer cell lines. However, the anticancer activity and mechanisms of PM in prostate cancer cells have not been adequately investigated. Here we report that the degradation of survivin plays an important role in the antiproliferative and proapoptotic effects of PM in carcinoma of the prostate (CaP) cell lines. Treatment with PM inhibited proliferation and induced apoptosis in LNCaP and PC-3 cells as characterized by the loss of cell viability and an increase in Annexin V-binding and cleavage of PARP-1, respectively. The antiproliferative and apoptosis-inducing effects of PM were associated with the inhibition of cell cycle regulatory proteins, antiapoptotic survivin and members of the Bcl-2 family. Data showed that response to PM is regulated by survivin since overexpression of survivin rendered CaP cells resistant to PM. Furthermore, downregulation of survivin by PM was mediated through the ubiquitin-proteasomal degradation. Together, these data demonstrate that pristimerin inhibits proliferation and induces apoptosis in CaP cells by abolishing survivin through the ubiquitin-proteasome pathway.
Correspondence to: Dr Subhash C. Gautam, Surgical Research-4D, Henry Ford Health System, One Ford Place, Detroit, MI 48202, USA E-mail: [email protected]
and identification of bioactive components from medicinal plants have led to the synthesis of potent anticancer drugs, including Vinca alkaloids, taxol, camptothecan, etoposide and retinoids. Triterpenoids are members of a large family of structurally related compounds known as cyclosqualenoids that are widely distributed in nature. Pristimerin (PM) is a quinonemethide triterpenoid present in various plant species in the Celastraceae and Hippocrateaceae families (1,2). PM and related compounds have shown anti-inflammatory, antioxidant and antimalarial activities (3-5). PM has also shown potent antiproliferative and apoptosis-inducing activity in glioma, leukemia, breast, lung and prostate cancer cell lines (6-9). Induction of apoptosis by PM involves generation of reactive oxygen species (ROS), activation of caspases, mitochondrial dysfunction, inhibition of nuclear factor κ B (NF-κ B), Akt and MAP kinases (10-13). In addition, PM also inhibits proteasome activity, tumor cell migration and angiogenesis (8,14,15). Carcinoma of the prostate is the most commonly diagnosed cancer in American males and the second ranked cause of cancer related mortality (16). An estimated 233,000 new cases of prostate cancer will be diagnosed and 29,480 deaths are expected to occur from this disease in the United States in 2014 (17). Current therapies (e.g., androgen deprivation, radical prostatectomy, radiotherapy or brachytherapy) while effective in treating localized prostate cancer have limited efficacy against advanced disease and metastatic hormone-refractory disease remains incurable (18-20). Since the incidence of CaP increases with advancing age, prostate cancer is expected to be an increasingly greater problem as life expectancy improves. In a previous report we have shown that PM induces apoptosis in CaP cell lines through a ROS-dependent Bcl-2 degradation pathway (21). In the present study, we demonstrate that induction of apoptosis in CaP cells by PM is associated with inhibition of cell cycle regulatory proteins and proteasomal degradation of antiapoptotic survivin, a member of the inhibitors of apoptosis (IAP) family.
Key words: pristimerin, prostate cancer, apoptosis, survivin, ubiquitin,
Materials and methods
Introduction Herbal remedies are commonly used in traditional medicine to treat and prevent human diseases including cancer. Numerous plant derived flavonoids and phenolic/polyphenolic compounds with antioxidant and anti-inflammatory activities are currently used by cancer patients as dietary supplements to complement chemotherapy. In fact, isolation
proteasome
Reagents. PM was purchased from Sigma Chemicals (St. Louis, MO). Anti-PARP-1, anti-Bcl-2, anti-Bcl-xL and
1736
Liu et al: Pristimerin and prostate cancer
anti-survivin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The 96 AQueous One Solution Proliferation Assay System was from Promega (Madison, WI). Annexin V-FITC apoptosis detection kit was purchased from BD Pharmingen (San Diego, CA). Stock solution of PM (100 mM) was prepared in DMSO and all test concentrations were prepared by diluting stock solution in tissue culture medium. Cell lines. LNCaP and PC-3 human prostate cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). LNCaP were grown in RPMI-1640 supplemented with FBS and penicillin/streptomycin. PC-3 cells were grown in F-12K nutrient mixture (Gibco BRL, Rockville, MD) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 25 mM HEPES buffer. Both cell lines were cultured at 37˚C in a humidified atmosphere consisting of 5% CO2 and 95% air, and maintained by subculturing cells twice a week. Measurement of cell viability (MTS assay). Tumor cells (1x104) in 100 µl of tissue culture medium were seeded into each well of a 96-well plate. After 24‑h incubation to allow cells to adhere, cells were treated with PM at concentrations ranging from 0 to 5 µM. Cultures were incubated for additional 72 h and cell viability was then determined by the colorimetric MTS assay using CellTiter 96 AQueous One Solution Proliferation Assay System from Promega. This assay measures the bioreduction of the tetrazolium compound MTS by intracellular dehydrogenases in the presence of electron-coupling reagent phenazine methosulfate. MTS and phenazine methosulfate were added to the culture wells, and cultures were incubated for 2 h at 37˚C. The absorbance, which is directly proportional to the number of viable cells in the cultures, was measured at 490 nm using a microplate reader. Apoptosis assay. Apoptosis was assessed by the binding of Annexin V-FITC to phosphotidylserine, which is externalized to the outer leaflet of the plasma membrane early during induction of apoptosis. Briefly, untreated cells and cells treated with PM were resuspended in the binding buffer provided in the Annexin V-FITC apoptosis detection kit II (BD Biosciences, San Diego, CA) and allowed to react with 5 µl of Annexin V-FITC reagent and 5 µl of propidium iodide for 30 min at room temperature in the dark. Stained cells were analyzed by flow cytometry using Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI). The induction of apoptosis by PM was confirmed by the cleavage of PARP-1 by western blot analysis. Western blot analysis. Cell lysates were prepared by detergent lysis [1% Triton-X 100 (v/v), 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 10% glycerol, 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A and 10 µg/ml 4-2-aminoethyl-benzenesulfinyl f luoride]. Lysates were clarified by centrifugation at 14,000 x g for 10 min at 4˚C, and protein concentrations were determined by Bradford assay. Samples (50 µg) were boiled in an equal volume of sample buffer [20% glyc-
erol, 4% SDS, 0.2% bromophenol blue, 125 mM Tris-HCl (pH 7.5), and 640 mM 2-mercaptoethanol] and separated on 10% SDS-polyacrilamide gels. Proteins resolved on the gels were transferred to nitrocellulose membranes and probed with antibodies against proteins of interest followed by HRP-conjugated secondary antibody. Immune complexes were visualized by chemiluminescence. Protein bands were imaged and band densities analyzed using the NIH/Scion image analysis software. DNA transfection. For expression of HA tagged-survivin, semi-confluent cultures of PC-3 cells in 60 mm2 cell culture dishes were transfected with 10 µg of empty or HA-survivin expression vector (pcDNA3-HA-survivin) (CH3 BioSystems, Amherst, NY) using Lipofectamine Plus reagent. After incubation for 36 h, overexpression of survivin in transfected cells was confirmed by immunoblotting. Immunoprecipitation. After treatment with PM (5 µM) for 6 h cells were washed with cold PBS and lysed in NP‑40 cell lysis buffer (Invitrogen, Camarillo, CA) supplemented with 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatinin, and 10 µg/ml 4-2-aminoethyl-benzenesulfinyl fluoride for 30 min on ice. Supernatants were collected after centrifugation at 14,000 x g for 10 min and protein concentration was determined. Each sample (400 µg protein) in 200 µl of antibody binding buffer containing anti-HA antibody was incubated for 1 h at room temperature followed by incubation with protein A agarose beads for 1 h. Immune complexes were washed two times with lysis buffer and analyzed for ubiquitin by western blot analysis. Statistical analysis. Data are expressed as mean ± SD. The difference between control and treatment groups was determined using Dunnett's multiple comparison test. Differences with p
