Ping Lei
@tamus.edu
Institute of Biosciences and Technology, Texas A&M Health Science Center
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Xinyi Liu, Indira Jutooru, Ping Lei, KyoungHyun Kim, Syng-ook Lee, Lisa K. Brents, Paul L. Prather, and Stephen Safe
American Association for Cancer Research (AACR)
Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid–dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor–dependent, thus identifying a new cellular target for this anticancer agent. Mol Cancer Ther; 11(7); 1421–31. ©2012 AACR.
Satya S. Pathi, Ping Lei, Sandeep Sreevalsan, Gayathri Chadalapaka, Indira Jutooru, and Stephen Safe
Informa UK Limited
Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (<5 mM) is due, in part, to reactive oxygen species-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1–3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.
Sudhakar Chintharlapalli, Sabitha Papineni, Syng-Ook Lee, Ping Lei, Un Ho Jin, Steven I. Sherman, Libero Santarpia, and Stephen Safe
Wiley
Methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate (CDODA‐Me) and the corresponding 2‐trifluoromethyl analog (CF3DODA‐Me) are derived synthetically from the triterpenoid glycyrrhetinic acid, a major component of licorice. CDODA‐Me and CF3DODA‐Me inhibited growth of highly invasive ARO, DRO, K‐18, and HTh‐74 thyroid cancer cells and this was due, in part, to decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in these cells. CDODA‐Me and CF3DODA‐Me also decreased expression of Sp‐dependent genes, such as survivin and vascular endothelial growth factor (VEGF), and induced apoptosis. In addition, pituitary tumor‐transforming gene‐1 (PTTG‐1) protein and mRNA levels were also decreased in thyroid cancer cells treated with CDODA‐Me or CF3DODA‐Me and this was accompanied by decreased expression of PTTG‐1‐dependent c‐Myc and fibroblast growth factor‐2 (FGF‐2) genes. RNA interference studies against Sp1, Sp3, and Sp4 proteins showed that in thyroid cancer cells, PTTG‐1 was an Sp‐dependent gene. This study demonstrates for the first time that drugs, such as CDODA‐Me and CF3DODA‐Me, that decrease Sp protein expression also downregulate PTTG‐1 in thyroid cancer cells and therefore have potential for clinical treatment of thyroid cancer and other endocrine neoplasias where PTTG‐1 is a major pro‐oncogenic factor. © 2011 Wiley‐Liss, Inc.
Sudhakar Chintharlapalli, Sabitha Papineni, Ping Lei, Satya Pathi, and Stephen Safe
Springer Science and Business Media LLC
BackgroundBetulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells.MethodsThe effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression.ResultsBA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10.ConclusionsThese results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.
Indira Jutooru, Gayathri Chadalapaka, Ping Lei, and Stephen Safe
Elsevier BV
Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFκB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFκB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFκB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFκB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.
Jingjing Guo, Sudhakar Chintharlapalli, Syng-ook Lee, Sung Dae Cho, Ping Lei, Sabitha Papineni, and Stephen Safe
Springer Science and Business Media LLC
Purpose1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) substituted in the phenyl ring with a para-, t-butyl, trifluoromethyl (DIM-C-pPhCF3) or phenyl (DIM-C-pPhC6H5) group activate peroxisome proliferator-activated receptor γ (PPARγ) in several cancer cell lines, and DIM-C-pPhCF3 also activates the orphan receptor Nur77. In this study, we have examined the effects of 5,5′-dihydroxy, 5,5′-dimethyl, 5,5′-dibromo, 5,5′-dinitro and 5,5′-dimethoxyindole ring-substituted analogs of DIM-C-pPhC6H5 on their activity as PPARγ agonists.MethodsVarious substituted C-DIM analogs were used to investigate their growth-inhibitory activities and activation of PPARγ-mediated transactivation in colon and pancreatic cancer cells. Their structure-dependent induction of putative PPARγ-responsive genes/proteins including p21, KLF-4 and caveolin1 were also determined by Western and Northern blot analysis.ResultsIntroduction of the 5,5′-dihydroxy and 5,5′-dimethyl substituents enhanced activation of PPARγ in colon and pancreatic cancer cells. However, activation of p21 in Panc28 pancreatic cancer cells and induction of caveolin-1 and KLF4 in colon cancer cells by the C-DIM compounds were structure- and cell context-dependent.ConclusionsThe results demonstrate that DIM-C-pPhC6H5 and indole ring-substituted analogs are selective PPARγ modulators.
Indira Jutooru, Gayathri Chadalapaka, Sandeep Sreevalsan, Ping Lei, Rola Barhoumi, Robert Burghardt, and Stephen Safe
Elsevier BV
Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, < or =5 microM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 microM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide.
Shu Zhang, Ping Lei, Xinyi Liu, Xiangrong Li, Kelcey Walker, Leela Kotha, Craig Rowlands, and Stephen Safe
Bioscientifica
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.
Xinyi Liu, Maen Abdelrahim, Ala Abudayyeh, Ping Lei, and Stephen Safe
American Association for Cancer Research (AACR)
Tolfenamic acid (TA) is a nonsteroidal anti-inflammatory drug that inhibits pancreatic cancer cell and tumor growth through decreasing expression of specificity protein (Sp) transcription factors. TA also inhibits growth of erbB2-overexpressing BT474 and SKBR3 breast cancer cells; however, in contrast to pancreatic cancer cells, TA induced down-regulation of erbB2 but not Sp proteins. TA-induced erbB2 down-regulation was accompanied by decreased erbB2-dependent kinase activities, induction of p27, and decreased expression of cyclin D1. TA also decreased erbB2 mRNA expression and promoter activity, and this was due to decreased mRNA stability in BT474 cells and, in both cell lines, TA decreased expression of the YY1 and AP-2 transcription factors required for basal erbB2 expression. In addition, TA also inhibited tumor growth in athymic nude mice in which BT474 cells were injected into the mammary fat pad. TA represents a novel and promising new anticancer drug that targets erbB2 by decreasing transcription of this oncogene. [Mol Cancer Ther 2009;8(5):OF1–11]
P. Lei, M. Abdelrahim, S. D. Cho, Xinyi Liu and S. Safe
Sung Dae Cho, Sudhakar Chintharlapalli, Maen Abdelrahim, Sabitha Papineni, Shengxi Liu, Jingjing Guo, Ping Lei, Ala Abudayyeh, and Stephen Safe
American Association for Cancer Research (AACR)
Bis(3′-indolyl)methane (DIM) is a metabolite of the phytochemical indole-3-carbinol, and both compounds exhibit a broad spectrum of anticancer activities. We have developed a series of synthetic symmetrical ring-substituted DIM analogues, including 5,5′-dibromoDIM, which are more potent than DIM as inhibitors of cancer cell and tumor growth. In colon cancer cells, 5,5′-dibromoDIM decreased cell proliferation and inhibited G0-G1- to S-phase progression, and this was accompanied by induction of the cyclin-dependent kinase inhibitor p21 in HT-29 and RKO colon cancer cells. Mechanistic studies showed that induction of p21 in both RKO (p53 wild-type) and HT-29 (p53 mutant) cells by 5,5′-dibromoDIM was Krüppel-like factor 4 (KLF4) dependent, and induction of p53 in RKO cells was also KLF4 dependent. Analysis of the p21 promoter in p53-dependent RKO cells showed that 5,5′-dibromoDIM activated p21 gene expression through the proximal GC-rich sites 1 and 2, and chromatin immunoprecipitation assays showed that KLF4 and p53 bound to this region of the promoter, whereas in HT-29 cells unidentified upstream cis-elements were required for induction of p21. 5,5′-DibromoDIM (30 mg/kg/d) also inhibited tumor growth and induced p21 in athymic nude mice bearing RKO cells as xenografts, showing that ring-substituted DIM such as 5,5′-dibromoDIM represent a novel class of mechanism-based drugs for clinical treatment of colon cancer. [Mol Cancer Ther 2008;7(7):2109–20]
Ping Lei, Maen Abdelrahim, Sung Dae Cho, Xingi Liu, and Stephen Safe
American Association for Cancer Research (AACR)
1,1-Bis(3′-indoly)-1-(p-substituted phenyl)methanes (C-DIM) exhibit structure-dependent activation of peroxisome proliferator–activated receptor γ and nerve growth factor–induced Bα (Nur77) and induce receptor-dependent and receptor-independent apoptosis in cancer cells and tumors. In this study, we investigated the activation of apoptosis in pancreatic cancer cells by p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) and structurally related analogues that do not activate either peroxisome proliferator–activated receptor γ or Nur77. The ortho, meta, and para-bromo and -fluoro isomers all activated endoplasmic reticulum (ER) stress-dependent apoptosis in pancreatic cancer cells; however, methylation of the indole N group significantly decreased activity, suggesting that a free N was important for activation of ER stress. Both DIM-C-pPhBr and DIM-C-pPhF resembled the classic ER stress inducer thapsigargin in pancreatic cancer cells and activated ER stress markers, such as glucose-related protein 78 and the c-Jun NH2 kinase pathway, resulting in the induction of CCAAT/enhancer-binding protein homologous protein, death receptor 5, and the extrinsic apoptotic pathway. Moreover, DIM-C-pPhBr also inhibited tumor growth in an orthotopic model for pancreatic cancer, demonstrating the clinical potential for this C-DIM compound in pancreatic cancer chemotherapy. [Mol Cancer Ther 2008;7(10):3363–72]
P. Lei, M. Abdelrahim, S. D. Cho, S. Liu, S. Chintharlapalli, and S. Safe
Oxford University Press (OUP)
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77 and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para >/= meta >/= ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose) polymerase (PARP) that is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with the activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK.
Sung Dae Cho, Ping Lei, Maen Abdelrahim, Kyungsil Yoon, Shengxi Liu, Jingjing Guo, Sabitha Papineni, Sudhakar Chintharlapalli, and Stephen Safe
Wiley
1,1‐Bis(3′‐indolyl)‐1‐(p‐methoxyphenyl)methane (DIM‐C‐pPhOCH3) is a methylene‐substituted diindolylmethane (C‐DIM) analog that activates the orphan receptor nerve growth factor‐induced‐Bα (NGFI‐Bα, Nur77). RNA interference studies with small inhibitory RNA for Nur77 demonstrate that DIM‐C‐pPhOCH3 induces Nur77‐dependent and ‐independent apoptosis, and this study has focused on delineating the Nur77‐independent proapoptotic pathways induced by the C‐DIM analog. DIM‐C‐pPhOCH3 induced caspase‐dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl‐2 ratios and release of cytochrome c into the cytosol. DIM‐C‐pPhOCH3 also induced phosphatidylinositol‐3‐kinase‐dependent activation of early growth response gene‐1 which, in turn, induced expression of the proapoptotic nonsteroidal anti‐inflammatory drug‐activated gene‐1 (NAG1) in RKO and SW480 colon cancer cells. Moreover, DIM‐C‐pPhOCH3 also induced NAG‐1 expression in colon tumors in athymic nude mice bearing RKO cells as xenografts. DIM‐C‐pPhOCH3 also activated the extrinsic apoptosis pathway through increased phosphorylation of c‐jun N‐terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5). Thus, the effectiveness of DIM‐C‐pPhOCH3 as a tumor growth inhibitor is through activation of Nur77‐dependent and ‐independent pathways. © 2007 Wiley‐Liss, Inc.
Sung Dae Cho, Kyungsil Yoon, Sudhakar Chintharlapalli, Maen Abdelrahim, Ping Lei, Stanley Hamilton, Shaheen Khan, Shashi K. Ramaiah, and Stephen Safe
American Association for Cancer Research (AACR)
Nerve growth factor-induced Balpha (NGFI-Balpha, Nur77) is an orphan nuclear receptor with no known endogenous ligands; however, recent studies on a series of methylene-substituted diindolylmethanes (C-DIM) have identified 1,1-bis(3'-indolyl)-1-(phenyl)methane (DIM-C-Ph) and 1,1-bis(3'-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) as Nur77 agonists. Nur77 is expressed in several colon cancer cell lines (RKO, SW480, HCT-116, HT-29, and HCT-15), and we also observed by immunostaining that Nur77 was overexpressed in colon tumors compared with normal colon tissue. DIM-C-Ph and DIM-C-pPhOCH3 decreased survival and induced apoptosis in RKO colon cancer cells, and this was accompanied by induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The induction of apoptosis and TRAIL by DIM-C-pPhOCH3 was significantly inhibited by a small inhibitory RNA for Nur77 (iNur77); however, it was evident from RNA interference studies that DIM-C-pPhOCH3 also induced Nur77-independent apoptosis. Analysis of DIM-C-pPhOCH3-induced gene expression using microarrays identified several proapoptotic genes, and analysis by reverse transcription-PCR in the presence or absence of iNur77 showed that induction of programmed cell death gene 1 was Nur77 dependent, whereas induction of cystathionase and activating transcription factor 3 was Nur77 independent. DIM-C-pPhOCH3 (25 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cell xenografts. These results show that Nur77-active C-DIM compounds represent a new class of anti-colon cancer drugs that act through receptor-dependent and receptor-independent pathways.
Ping Lei, Maen Abdelrahim, and Stephen Safe
American Association for Cancer Research (AACR)
1,1-Bis(3′-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu) is a peroxisome proliferator–activated receptor γ (PPARγ) agonist, and treatment of SKOV3 ovarian cancer cells with this compound (5 μmol/L) inhibits cell proliferation, whereas up to 15 μmol/L rosiglitazone had no effect on cell growth. DIM-C-pPhtBu also inhibits G0-G1 to S phase cell cycle progression and this is linked, in part, to PPARγ-dependent induction of the cyclin-dependent kinase inhibitor p21. DIM-C-pPhtBu induces PPARγ-independent down-regulation of cyclin D1 and we therefore further investigated activation of receptor-independent pathways. DIM-C-pPhtBu also induced apoptosis in SKOV3 cells and this was related to induction of glucose-related protein 78, which is typically up-regulated as part of the unfolded protein response during endoplasmic reticulum (ER) stress. Activation of ER stress was also observed in other ovarian cancer cell lines treated with DIM-C-pPhtBu. In addition, DIM-C-pPhtBu induced CCAAT/enhancer binding protein homologous protein through both ER stress and c-jun NH2-terminal kinase–dependent pathways, and CCAAT/enhancer binding protein homologous protein activated death receptor 5 and the extrinsic pathway of apoptosis. These results show that DIM-C-pPhtBu inhibits growth and induces apoptosis in ovarian cancer cells through both PPARγ-dependent and PPARγ-independent pathways, and this complex mechanism of action will be advantageous for future clinical development of these compounds for treatment of ovarian cancer. [Mol Cancer Ther 2006;5(9):2324–38]