Molecular Cancer Therapeutics current issue

In vitro differential sensitivity of melanomas to phenothiazines is based on the presence of codon 600 BRAF mutation

2008-06-19

The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. We previously sequenced 24 cancer genes in those cell lines. Eleven of the genes were found to be mutated in three or more of the lines. Using a pharmacogenomic approach, we analyzed the relationship between drug activity and mutations in those 11 genes (APC, RB1, KRAS, NRAS, BRAF, PIK3CA, PTEN, STK11, MADH4, TP53, and CDKN2A). That analysis identified an association between mutation in BRAF and the antiproliferative potential of phenothiazine compounds. Phenothiazines have been used as antipsychotics and as adjunct antiemetics during cancer chemotherapy and more recently have been reported to have anticancer properties. However, to date, the anticancer mechanism of action of phenothiazines has not been elucidated. To follow up on the initial pharmacologic observations in the NCI-60 screen, we did pharmacologic experiments on 11 of the NCI-60 cell lines and, prospectively, on an additional 24 lines. The studies provide evidence that BRAF mutation (codon 600) in melanoma as opposed to RAS mutation is predictive of an increase in sensitivity to phenothiazines as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay (Wilcoxon P = 0.007). That pattern of increased sensitivity to phenothiazines based on the presence of codon 600 BRAF mutation may be unique to melanomas, as we do not observe it in a panel of colorectal cancers. The findings reported here have potential implications for the use of phenothiazines in the treatment of V600E BRAF mutant melanoma. [Mol Cancer Ther 2008;7(6):1337–46]

Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies

Garcia, J. A., Danielpour, D. — 2008-06-19

The mammalian target of rapamycin (mTOR) is a protein kinase that regulates protein translation, cell growth, and apoptosis. Recently, there has been an enormous increase in our understanding on molecular mechanisms underlying the therapeutics of rapamycin in cancer. Alterations in the pathway regulating mTOR occur in many solid malignancies including prostate, bladder, and kidney cancer; in vitro and in vivo models of prostate and bladder cancer have established the importance of the mTOR pathway in control of cancer progression and metastasis. Temsirolimus (Torisel) and everolimus (RAD-001), two ester analogues of rapamycin, as well as rapamycin itself have clear antitumor activity in in vitro and in vivo models and are under clinical trial investigations for prostate and bladder cancer. Phase II and III trials have already established the clinical efficacy of temsirolimus in renal cancer, and current renal trials are evaluating the combined effects of vascular endothelial growth factor and mTOR inhibition. Ongoing studies in prostate and bladder cancer will soon define the activity and safety profiles of everolimus and temsirolimus. Recent molecular advances have uncovered a startling complexity in the macromolecular function of mTOR complexes, with the identification of new mTOR partners (raptor, rictor, FKBP38, PRAS40, and mSIN1), putative cancer therapeutic/prognostic targets for future clinical trials. [Mol Cancer Ther 2008;7(6):1347–54]

Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth

Yan, Z., Zou, H., Tian, F., Grandis, J. R., Mixson, A. J., Lu, P. Y., Li, L.-Y. — 2008-06-19

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers. [Mol Cancer Ther 2008;7(6):1355–64]

Modulation of the expression of the invasion-suppressor CRMP-1 by cyclooxygenase-2 inhibition via reciprocal regulation of Sp1 and C/EBP{alpha}

2008-06-19

Collapsin response mediator protein-1 (CRMP-1) controls neural development and axonal growth but also acts as a cancer invasion suppressor. In this study, we investigated the transcriptional regulation of CRMP-1 expression. Using a serial deletion strategy, we identified a basal promoter region between nucleotides -100 and -180 in the 5' flanking region of CRMP-1 (nucleotides -1,920 to +50) that contains multiple putative Sp1 and C/EBP sites. Site-directed mutagenesis and deletion analysis revealed that the two C/EBP sites, from nucleotides -122 to -133 and from nucleotides -101 to -113, are the most important regulatory elements. Gel-shift and antibody supershift assays showed that Sp1 protein was also present at this C/EBP site, which overlaps with a Sp1 site. Overexpression of Sp1 decreased CRMP-1 promoter activity and protein expression, whereas overexpression of C/EBP produced the opposite effect. Chromatin immunoprecipitation assays confirmed that Sp1 and C/EBP compete for binding at the overlapping C/EBP and Sp1 sites and reciprocally regulate CRMP-1 expression. Overexpression of cyclooxygenase-2 (COX-2) decreased CRMP-1 mRNA and protein expression. Conversely, the COX-2 inhibitor, celecoxib, induced a dose-dependent increase in CRMP-1 expression. COX-2 inhibition also decreased Sp1-DNA complex formation and inhibited cell invasion. We conclude that transcription of the invasion suppressor, CRMP-1, is reciprocally regulated at the promoter region by C/EBP and Sp1. COX-2 inhibitors increase CRMP-1 expression by inhibiting Sp1-DNA complex formation and enhancing DNA binding of C/EBP at the promoter. [Mol Cancer Ther 2008;7(6):1365–75]

Eps8 decreases chemosensitivity and affects survival of cervical cancer patients

Chen, Y.-J., Shen, M.-R., Chen, Y.-J., Maa, M.-C., Leu, T.-H. — 2008-06-19

The oncoprotein Eps8 facilitates proliferation in fibroblasts and colon cancer cells. However, its role in human cervical cancer is unclear. By immunohistochemical staining and Western blotting, aberrant Eps8 expression was observed in cervical carcinoma compared with normal cervical epithelial cells. Clinicopathologic analysis of 45 patients indicated that Eps8 expression was associated with parametrium invasion and lymph node metastasis, two major poor prognostic factors for early-stage cervical cancer. Kaplan-Meier analysis of cervical cancer specimens also indicated an inverse relationship between the level of Eps8 and the patients' survival rate. Using small interfering RNA of eps8, we observed reduced proliferation and tumorigenesis in Eps8-attenuated HeLa and SiHa cells cultured in dishes or inoculated in mice. Furthermore, diminished Eps8 impeded G₁-phase progression in HeLa and SiHa cells that might be attributable to reduced expression of cyclins D1, D3, and E, elevated accumulation of p53 and its downstream target p21^Waf1/Cip1, and suppressed hyperphosphorylation of retinoblastoma. Alteration of these cell cycle–related proteins could be reversed by ectopic Eps8, implicating that the effect of Eps8 on the mentioned cell cycle modulators was specific. Notably, the augmented expression of p53 by diminished Eps8 was at least due to its decreased turnover rate. Concurrent with p53 up-regulation and the decrement of Src and AKT activity, Eps8-attenuated HeLa and SiHa cells exhibited increased chemosensitivity to cisplatin and paclitaxel. Together, our findings implicate the involvement of Eps8 in chemoresistance and show its importance in prognosis of cervical cancer patients. [Mol Cancer Ther 2008;7(6):1376–85]

{alpha}-Catenin overrides Src-dependent activation of {beta}-catenin oncogenic signaling

2008-06-19

Loss of -catenin is one of the characteristics of prostate cancer. The catenins ( and β) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of β-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where β-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, β-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of β-catenin invariably are altered in cancer. Although a wealth of information is available about β-catenin deregulation during oncogenesis, much less is known about how or whether -catenin regulates β-catenin functions. In this study, we show that -catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of β-catenin. In -catenin-null prostate cancer cells, reexpression of -catenin increased cell-cell adhesion and decreased β-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of β-catenin is a major mechanism for decreased β-catenin interaction with E-cadherin in -catenin-null cells. -Catenin attenuated the effect of Src phosphorylation by increasing β-catenin association with E-cadherin. We also show that -catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that -catenin is a key regulator of β-catenin transcriptional activity and that the status of -catenin expression in tumor tissues might have prognostic value for Src targeted therapy. [Mol Cancer Ther 2008;7(6):1386–97]

Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes

Shkreta, L., Froehlich, U., Paquet, E. R., Toutant, J., Elela, S. A., Chabot, B. — 2008-06-19

Inducing an apoptotic response is the goal of most current chemotherapeutic interventions against cancer. However, little is known about the effect of chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have tested 20 of the mainstream anticancer drugs for their ability to influence the production of Bcl-x splice isoforms. We find that many drugs shift splicing toward the proapoptotic Bcl-x_S splice variant in 293 cells. The drugs modulate splicing decisions most likely through signaling events because the splicing switch is not compromised by inhibiting de novo protein synthesis or the activity of caspases. Several drugs also shift Bcl-x splicing in cancer cell lines (MCF-7, HeLa, PC-3, PA-1, and SKOV-3), but the set of active drugs varies between cell lines. We also examined the effect of anticancer agents on the alternative splicing of 95 other human apoptotic genes in different cell lines. Almost every drug can alter a subset of alternative splicing events in each cell line. Although drugs of the same class often influence the alternative splicing of the same units in individual cell lines, these units differ considerably between cell lines, indicating cell line–specific differences in the pathways that control splicing. [Mol Cancer Ther 2008;7(6):1398–409]

Trichostatin A up-regulates p73 and induces Bax-dependent apoptosis in cisplatin-resistant ovarian cancer cells

2008-06-19

Several studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells. [Mol Cancer Ther 2008;7(6):1410–9]

Recombinant, refolded tetrameric p53 and gonadotropin-releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells

LaFevre-Bernt, M., Wu, S., Lin, X. — 2008-06-19

The p53 tumor suppressor is mutated in over 50% of human cancers. Mutations resulting in amino acid changes within p53 result in a loss of activity and consequent changes in expression of genes that regulate DNA repair and cell cycle progression. Replacement of p53 using protein therapy would restore p53 function in p53-deficient tumor cells, with a consequence of tumor cell death and tumor regression. p53 functions in a tetrameric form in vivo. Here, we refolded a wild-type, full-length p53 from inclusion bodies expressed in Escherichia coli as a stable tetramer. The tetrameric p53 binds to p53-specific DNA and, when transformed into a p53-deficient cancer cell line, induced apoptosis of the transformed cells. Next, using the same expression and refolding technology, we produced a stable tetramer of recombinant gonadotropin-releasing hormone-p53 fusion protein (GnRH-p53), which traverses the plasma membrane, slows proliferation, and induces apoptosis in p53-deficient, GnRH-receptor–expressing cancer cell lines. In addition, we showed a time-dependent binding and internalization of GnRH-p53 to a receptor-expressing cell line. We conclude that the GnRH-p53 fusion strategy may provide a basis for constructing an effective cancer therapeutic for patients with tumors in GnRH-receptor–positive tissue types. [Mol Cancer Ther 2008;7(6):1420–9]

ISG15 as a novel tumor biomarker for drug sensitivity

2008-06-19

Tumor cells are known to exhibit highly varied sensitivity to camptothecins (CPT; e.g., irinotecan and topotecan). However, the factors that determine CPT sensitivity/resistance are largely unknown. Recent studies have shown that the ubiquitin-like protein, IFN-stimulated gene 15 (ISG15), which is highly elevated in many human cancers and tumor cell lines, antagonizes the ubiquitin/proteasome pathway. In the present study, we show that ISG15 is a determinant for CPT sensitivity/resistance possibly through its effect on proteasome-mediated repair of topoisomerase I (TOP1)-DNA covalent complexes. First, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 (major E2 for ISG15) in breast cancer ZR-75-1 cells decreased CPT sensitivity, suggesting that ISG15 overexpression in tumors could be a factor affecting intrinsic CPT sensitivity in tumor cells. Second, the level of ISG15 was found to be significantly reduced in several tumor cells selected for resistance to CPT, suggesting that altered ISG15 regulation could be a significant determinant for acquired CPT resistance. Parallel to reduced CPT sensitivity, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 in ZR-75-1 cells resulted in increased proteasomal degradation of CPT-induced TOP1-DNA covalent complexes. Taken together, these results suggest that ISG15, which interferes with proteasome-mediated repair of TOP1-DNA covalent complexes, is a potential tumor biomarker for CPT sensitivity. [Mol Cancer Ther 2008;7(6):1430–9]

Chk1 and Chk2 are differentially involved in homologous recombination repair and cell cycle arrest in response to DNA double-strand breaks induced by camptothecins

Huang, M., Miao, Z.-H., Zhu, H., Cai, Y.-J., Lu, W., Ding, J. — 2008-06-19

Camptothecins (CPT) activate S or G₂-M arrest and the homologous recombination (HR) repair pathway in tumor cells. In this process, both checkpoint kinases 1 and 2 (Chk1 and Chk2, respectively) are activated, but their differential roles, especially in the coordination of checkpoint and repair control, and potential clinic relevance remain to be fully elucidated. In this study, the repairable double-strand breaks were induced in human colon cancer HCT116 cells by 1-h exposure to 25 or 100 nmol/L CPT and its novel derivative chimmitecan. The cellular disposal of double-strand breaks was reflected as the progressive dispersal of -H2AX foci, reduction of "comet" tails, dynamic activation of RAD51-mediated HR repair, and reversible G₂-M arrest. In this model, the differential kinetics of Chk1 and Chk2 activation was characterized by the progressively increased phosphorylation of Chk2 until 72 h, the degradation of Chk1, and the disappearance of phosphorylated Chk1 48 h after drug removal. Using RNA interference, we further showed that Chk2 was essential to G₂-M arrest, whereas Chk1 was mainly required for HR repair in CPT-treated HCT116 cells. Moreover, Chk2, rather than Chk1, predominated over the control of cell survival in this model. The differential roles of Chk1 and Chk2 in regulating HR repair and G₂-M phase arrest were also confirmed in HT-29 colon cancer cells. Together, these findings systematically dissect the differential roles of Chk1 and Chk2 in a favorable model pursuing CPT-driven DNA damage responses, providing critical evidence to further explore checkpoint modulation, especially Chk2 inhibition as a therapeutic strategy in combination with CPT. [Mol Cancer Ther 2008;7(6):1440–9]

Targeting of sonic hedgehog-GLI signaling: a potential strategy to improve therapy for mantle cell lymphoma

2008-06-19

Mantle cell lymphoma (MCL) has one of the worst clinical outcomes among the B-cell lymphomas, with a median survival of only 3 to 4 years. Therefore, a better understanding of the underlying mechanisms that regulate MCL proliferation/survival is needed to develop an effective therapy. Because sonic hedgehog (Shh)-GLI signaling has been shown to be important in the proliferation and survival of several cancers, and no such information is available for MCL, this study was undertaken. Our results show that the molecules associated with Shh-GLI signaling, such as PTCH and SMO receptors, and GLI1 and GLI2 target transcription factors were expressed in the human MCL cell lines and primary MCL cells from patients. Perturbation of this signaling in the presence of exogenous Shh/cyclopamine significantly (P < 0.001) influenced the proliferation of JVM2 MCL cells. Furthermore, down-regulation of GLI transcription factors using antisense oligonucleotides not only resulted in significantly (P < 0.001) decreased proliferation of the MCL cells but also significantly (P < 0.05) increased their susceptibility to chemotherapeutic drug, doxorubicin. Also, down-regulation of GLI decreased cyclin D1 and BCL2 transcript levels, which suggests that these key molecules might be regulated by GLI in MCL. Thus, our results indicate a significant role for Shh-GLI signaling in the proliferation of MCL, and molecular targeting of GLI is a potential therapeutic approach to improve the treatment for MCL. [Mol Cancer Ther 2008;7(6):1450–60]

Potent synergy of dual antitumor peptides for growth suppression of human glioblastoma cell lines

2008-06-19

Molecular targeting agents have become formidable anticancer weapons, which show much promise against the refractory tumors. Functional peptides are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms a basis for potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. Here, we examine growth suppression efficiency of human glioblastomas by dual-peptide targeting. We did simultaneous introduction of two tumor suppressor peptides (p14^ARF and p16^INK4a or p16^INK4a and p21^CIP1 functional peptides) compared with single-peptide introduction using Wr-T-mediated peptide delivery. Wr-T-mediated transport of both p14^ARF and p16^INK4a functional peptides (p14-1C and p16-MIS, respectively) into human glioblastoma cell line, U87EGFR, reversed specific loss of p14 and p16 function, thereby drastically inhibiting tumor growth by >95% within the first 72 h, whereas the growth inhibition was ~40% by p14 or p16 single-peptide introduction. Additionally, the combination of p16 and p21^CIP1 (p21-S154A) peptides dramatically suppressed the growth of glioblastoma line Gli36EGFR, which carries a missense mutation in p53, by >97% after 120 h. Significantly, our murine brain tumor model for dual-peptide delivery showed a substantial average survival enhancement (P < 0.0001) for peptide-treated mice. Wr-T-mediated dual molecular targeting using antitumor peptides is highly effective against growth of aggressive glioblastoma cells in comparison with single molecule targeting. Thus, jointly restoring multiple tumor suppressor functions by Wr-T-peptide delivery represents a powerful approach, with mechanistic implications for development of efficacious molecular targeting therapeutics against intractable human malignancies. [Mol Cancer Ther 2008;7(6):1461–71]

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198

2008-06-19

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G₂-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1 levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors. [Mol Cancer Ther 2008;7(6):1472–82]

Insulin-like growth factor-I receptor blockade by a specific tyrosine kinase inhibitor for human gastrointestinal carcinomas

2008-06-19

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. In this study, we sought to evaluate the effect of a new tyrosine kinase inhibitor of IGF-IR, NVP-AEW541, on the signal transduction and the progression of GI carcinomas. We assessed the effect of NVP-AEW541 on signal transduction, proliferation, survival, and migration in four GI cancer cells: colorectal adenocarcinoma HT29, pancreatic adenocarcinoma BxPC3, esophageal squamous cell carcinoma TE1, and hepatoma PLC/PRF/5. The effects of NVP-AEW541 alone and with chemotherapy were studied in vitro and in nude mouse xenografts. We also analyzed the effects of NVP-AEW541 on insulin signals and hybrid receptor formation between IGF-IR and insulin receptor. NVP-AEW541 blocked autophosphorylation of IGF-IR and both Akt and extracellular signal-regulated kinase activation by IGF but not by insulin. NVP-AEW541 suppressed proliferation and tumorigenicity in vitro in a dose-dependent manner in all cell lines. The drug inhibited tumor as a single agent and, when combined with stressors, up-regulated apoptosis in a dose-dependent fashion and inhibited mobility. NVP-AEW541 augmented the effects of chemotherapy on in vitro growth and induction of apoptosis. Moreover, the combination of NVP-AEW541 and chemotherapy was highly effective against tumors in mice. This compound did not influence hybrid receptor formation. Thus, NVP-AEW541 may have significant therapeutic utility in human GI carcinomas both alone and in combination with chemotherapy. [Mol Cancer Ther 2008;7(6):1483–93]

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor, exerts antimyeloma effects by DNA damage and mitochondrial signaling

2008-06-19

Histone deacetylase inhibitors have emerged as promising anticancer drugs. Using an unbiased ultrahigh throughput screening system, a novel mercaptoketone-based histone deacetylase inhibitor series was identified that was optimized to the lead compound, KD5170. KD5170 inhibited the proliferation of myeloma cell lines and the viability of CD138⁺ primary myeloma cells by induction of apoptosis, accompanied by an increase of acetylation of histones and activation of caspase-3, caspase-8, and caspase-9. Treatment with KD5170 caused a loss of mitochondrial membrane potential resulting in release of apoptogenic factors such as cytochrome c, Smac, and apoptosis-inducing factor. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells as evidenced by the up-regulation of heme oxygenase-1 and H2A.X phosphorylation. Combination of KD5170 with proteasome inhibitor bortezomib or tumor necrosis factor–related apoptosis-inducing ligand synergistically enhanced the antimyeloma activity. We further found that resistance of myeloma cells to KD5170 was associated with activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway under treatment with KD5170. Pretreatment with the mitogen-activated protein kinase inhibitor U0126 restored sensitivity to KD5170, suggesting that the combination of KD5170 with U0126 could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited and survival was prolonged. Histone acetylation was increased in spleen and tumor tissues of animals treated with KD5170. Our data indicate that KD5170 has potent antimyeloma activity in vitro and in vivo, which is mediated by DNA damage and mitochondrial signaling and subsequent induction of apoptosis. [Mol Cancer Ther 2008;7(6):1494–505]

Intrinsically lower AKT, mammalian target of rapamycin, and hypoxia-inducible factor activity correlates with increased sensitivity to 2-deoxy-D-glucose under hypoxia in lung cancer cell lines

Wangpaichitr, M., Savaraj, N., Maher, J., Kurtoglu, M., Lampidis, T. J. — 2008-06-19

Down-regulation by small interfering RNA or absence of hypoxia-inducible factor (HIF-1) has been shown to lead to increased sensitivity to glycolytic inhibitors in hypoxic tumor cells. In surveying a number of tumor types for differences in intrinsic levels of HIF under hypoxia, we find that the reduction of the upstream pathways of HIF, AKT, and mammalian target of rapamycin (mTOR) correlates with increased toxic effects of 2-deoxy-d-glucose (2-DG) in lung cancer cell lines when treated under hypoxia. Because HIF-1 translation is regulated by mTOR, we examined the effects of blocking mTOR under hypoxia with an analogue of rapamycin (CCI-779) in those cell lines that showed increased mTOR and AKT activity and found that HIF-1 down-regulation coincided with increased 2-DG killing. CCI-779, however, was ineffective in increasing 2-DG toxicity in cell lines that did not express HIF. These results support the hypothesis that although mTOR inhibition leads to the blockage of numerous downstream targets, CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1. Overall, our findings show that CCI-779 hypersensitizes hypoxic tumor cells to 2-DG and suggests that the intrinsic expression of AKT, mTOR, and HIF in lung cancer, as well as other tumor types, may be important in dictating the decision on how best to use 2-DG alone or in combination with CCI-799 to kill hypoxic tumor cells clinically. [Mol Cancer Ther 2008;7(6):1506–13]

Antitumor effect of a transducible fusogenic peptide releasing multiple proapoptotic peptides by caspase-3

2008-06-19

We have designed a novel peptide, TK3, composed of three functional domains, a protein transduction domain, a TAT followed by three tandem repeats of a proapoptotic peptide, and a caspase-3 cleavage site, (KLAKLAK)₂-DEVD. TK3 was able to transduce into cells and then activate caspase-3, which in turn cleaved TK3 to release additional (KLAKLAK)₂ peptides. (KLAKLAK)₂ was well transduced by TAT into tumor cells and was able to induce apoptosis in vitro and in vivo. TK3 also induced apoptosis and inhibited angiogenesis in endothelial cells. Further, direct injection of TK3 into established B16F10 melanoma tumors in C57BL/6 mice resulted in almost complete inhibition of the tumor growth. These results suggest that TK3 could be beneficial for the treatment of accessible tumors and used as an adjuvant for cancer therapy. [Mol Cancer Ther 2008;7(6):1514–22]

17-Acetoxyjolkinolide B irreversibly inhibits I{kappa}B kinase and induces apoptosis of tumor cells

2008-06-19

Nuclear factor-B (NF-B) is critically important for tumor cell survival, growth, angiogenesis, and metastasis. One of the key events in the NF-B signaling is the activation of inhibitor of NF-B kinase (IKK) in response to stimuli of various cytokines. We have identified 17-acetoxyjolkinolide B (17-AJB) from a traditional Chinese medicinal herb Euphorbia fischeriana Steud as a novel small-molecule inhibitor of IKK. 17-AJB effectively inhibited tumor necrosis factor-–induced NF-B activation and induced apoptosis of tumor cells. 17-AJB had no effect on binding of tumor necrosis factor- to its receptor or on binding of NF-B to DNA. It inhibited NF-B nuclear translocation. Detailed analysis revealed that the direct target of 17-AJB was IKK. 17-AJB kept IKK in its phosphorylated form irreversibly. This irreversible modification of IKK inactivated its kinase activity, leading to its failure to activate NF-B. The effect of 17-AJB on IKK was specific. It had no effect on other kinases such as p38, p44/42, and JNK. In addition, 17-AJB induced apoptosis in tumor cells. The effects of 17-AJB on apoptosis correlated with inhibition of expression of the NF-B-regulated genes. Taken together, our data suggest that 17-AJB is a novel type NF-B pathway inhibitor. Its unique interaction mechanism with IKK may render it a strong apoptosis inducer of tumor cells and a novel type anticancer drug candidate. [Mol Cancer Ther 2008;7(6):1523–32]

Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer

2008-06-19

MDM2 oncoprotein binds directly to the p53 tumor suppressor and inhibits its function in cancers retaining wild-type p53. Blocking this interaction using small molecules is a promising approach to reactivate p53 function and is being pursued as a new anticancer strategy. The spiro-oxindole MI-43, a small-molecule inhibitor of the MDM2-p53 interaction, was designed and examined for its cellular mechanism of action and therapeutic potential in colon cancer. MI-43 binds to MDM2 protein with a K_i value of 18 nmol/L and is 300 times more potent than a native p53 peptide. MI-43 blocks the intracellular MDM2-p53 interaction and induces p53 accumulation in both normal and cancer cells, with wild-type p53 without causing p53 phosphorylation. Induction of p53 leads to modulation of the expression of p53 target genes, including up-regulation of p21 and MDM2 in normal primary human cells and in colon cancer cells with wild-type p53. Using HCT-116 isogenic colon cancer cell lines differing only in p53 status or RNA interference to knockdown expression of p53 in the RKO colon cancer cell line, we show that the cell growth inhibition and cell death induction by MI-43 is p53 dependent. Furthermore, induction of cell cycle arrest by MI-43 is dependent on p53 and p21. In normal cells, MI-43 induces cell cycle arrest but not apoptosis. This study suggests that p53 activation by a potent and specific spiro-oxindole MDM2 antagonist may represent a promising therapeutic strategy for the treatment of colon cancer and should be further evaluated in vivo and in the clinic. [Mol Cancer Ther 2008;7(6):1533–42]

STAT3 inhibition in prostate and pancreatic cancer lines by STAT3 binding sequence oligonucleotides: differential activity between 5' and 3' ends

2008-06-19

Signal transducers and activators of transcription (STAT) were originally discovered as components of signal transduction pathways. Persistent aberrant activation of STAT3 is a feature of many malignancies including prostate cancer and pancreatic cancer. One consequence of persistently activated STAT3 in malignant cells is that they depend on it for survival; thus, STAT3 is an excellent molecular target for therapy. Previously, we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. Here, we report that authentic STAT3 binding sequences, identified from published literature, were more effective for inducing apoptosis in prostate cancer cells and pancreatic cancer cells than was oligonucleotide 13410. Moreover, the authentic STAT3 binding sequences showed differing efficacies in the malignant cell lines depending on whether the canonical STAT3 binding sequence was truncated at the 5' or the 3' end. Finally, expression of one STAT3-regulated gene was decreased following treatment, suggesting that STAT3 may regulate the same set of genes in the two types of cancer. We conclude that truncating the 5' end left intact enough of the canonical STAT3 binding site for effective hybridization to the genome, whereas truncation of the 3' end, which is outside the canonical binding site, may have affected binding of required cofactors essential for STAT3 activity, thereby reducing the capacity of this modified oligonucleotide to induce apoptosis. Additional experiments to answer this hypothesis are under way. [Mol Cancer Ther 2008;7(6):1543–50]

Fasudil-induced hypoxia-inducible factor-1{alpha} degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

2008-06-19

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)–dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1 induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1 expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1 protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1 mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1 expression without altering HIF-1 mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1 expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1 expression is through preventing HIF-1 degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1 signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1 signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis. [Mol Cancer Ther 2008;7(6):1551–61]

Generation of adenovirus-mediated anti-CD20 antibody and its effect on B-cell deletion in mice and nonhuman primate cynomolgus monkey

2008-06-19

Therapeutic monoclonal anti-CD20 antibody (Rituxan) is increasingly applied to treat B-cell-related hematologic malignancies and autoimmune disorders with great clinical success, whereas its widespread application is limited by antibody manufacturing capability. Here, we explored a quick and economical adenovirus-mediated anti-CD20 antibody generating system to directly produce anti-CD20 antibody in vivo. We generated a recombinant adenovirus encoding the anti-CD20 antibody gene and found that infection of cells with this recombinant adenovirus led to the generation of anti-CD20 antibody in cells with a similar CD20 binding affinity and specificity as commercial product Rituxan. After one single administration of the anti-CD20-expressing adenoviruses through tail vein at a dose of 1 x 10⁹ plaque-forming units/mouse in nude mice, anti-CD20 antibody in the serum was detectable at day 3, reached to the peak value of 246.34 µg/mL at day 14, and maintained a high serum concentration of >40 µg/mL for 56 days. Furthermore, the in vivo generation of anti-CD20 antibody led a complete elimination of preestablished B-cell lymphoma Raji cells in nude mice, and a single administration of the anti-CD20-expressing adenovirus at a dose of 2.0 x 10⁹ plaque-forming units/kg in cynomolgus monkey led a continuous B-cell deletion in circulation blood and bone marrow. These observations thus suggest that adenovirus-mediated in vivo generation of anti-CD20 antibody may serve as a new strategy to combat B-cell-related hematologic disorders. [Mol Cancer Ther 2008;7(6):1562–8]

Anti-integrin monoclonal antibody CNTO 95 enhances the therapeutic efficacy of fractionated radiation therapy in vivo

Ning, S., Nemeth, J. A., Hanson, R. L., Forsythe, K., Knox, S. J. — 2008-06-19

Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-_v integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin _vβ₃ antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the _v integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors. [Mol Cancer Ther 2008;7(6):1569–78]

Targeting IL-13R{alpha}2-positive cancer with a novel recombinant immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin

Kioi, M., Seetharam, S., Puri, R. K. — 2008-06-19

We have shown previously that high-affinity receptors for interleukin-13 (IL-13R2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13R2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13R2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13R2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13R2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13R2(scFv)-PE38 was 200 µg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13R2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer. [Mol Cancer Ther 2008;7(6):1579–87]

Human {alpha}-defensin-1 inhibits growth of human lung adenocarcinoma xenograft in nude mice

2008-06-19

Human -defensin-1 (HNP1), a small antimicrobial peptide, shows cytotoxicity to tumor cells in vitro and inhibitory activity for pathologic neovascularization in vivo. Here, we did a gene therapy with a plasmid that expresses a secretable form of HNP1 for assaying its antitumor activity. The expression and secretion of HNP1 were determined by reverse transcription-PCR and ELISA in vitro. We found that expression of HNP1 in A549 tumor cells caused significant growth inhibition. This effect is most likely cell autonomous, as a significant amount of recombinant HNP1 protein was found to be accumulated in the cytoplasm by immunohistochemical staining using an anti-HNP1 antibody and the supernatant containing secreted HNP1 failed to produce any noticeable antitumor activity. Flow cytometry and Hoechst 33258 staining showed that the number of apoptotic cells among the A549 cells expressing recombinant HNP1 proteins was significantly greater than that of the nontransfected control cultures, suggesting that this growth-inhibitory activity was due to an apoptotic mechanism triggered by the intracellular HNP1. The antitumor activity of intracellularly expressed HNP1 was also shown in vivo. Decreased microvessel density and increased lymphocyte infiltration were observed in tumor tissue from HNP1-treated mice through histologic analysis. These results indicate that intracellularly expressed HNP1 induces tumor cell apoptosis, which inhibits tumor growth. The antiangiogenesis effect of HNP1 may contribute to its inhibitory activity in vivo, and HNP1 might involve the host immune response to tumor. These findings provide a rationale for developing HNP1-based gene therapy for cancer. [Mol Cancer Ther 2008;7(6):1588–97]

A novel triple-regulated oncolytic adenovirus carrying p53 gene exerts potent antitumor efficacy on common human solid cancers

2008-06-19

Conditionally replicating adenoviruses (CRAd) can replicate specifically in cancer cells and lyse them. The CRAds were widely used in the preclinical and clinical studies of cancer therapy. We hypothesize that more precisely regulated replication of CRAds may further improve the vector safety profile and enhance its antitumor efficacy. Here, a triple-regulated CRAd carrying p53 gene expression cassette, SG600-p53, was engineered. In SG600-p53, the E1a gene with a deletion of 24 nucleotides within CR2 region is controlled under the human telomerase reverse transcriptase (hTERT) promoter, the E1b gene expression is directed by the hypoxia response element (HRE), whereas the p53 gene is controlled by the cytomegalovirus promoter. The precise triple-regulation endows SG600-p53 with enhanced antitumor potential and improved safety profile. The tumor-selective replication of this virus and its antitumor efficacy were characterized in several tumor cell lines in vitro and in xenograft models of human non-small cell lung cancer in nude mice. With the selective replication and oncolysis, it was found by ELISA assay that SG600-p53 expressed p53 efficiently in cancer cells. In NCI-H1299 tumor xenograft models, SG600-p53 displayed a tumor-selective killing capacity. At a dose of 2 x 10⁹ plaque-forming units, SG600-p53 could completely inhibit the tumor growth and more effective than replication-defective Ad-p53. Histopathologic examination revealed that SG600-p53 administration resulted in cancer cell apoptosis. We concluded that the triple-regulated SG600-p53, as a more potent and safer antitumor therapeutic, could provide a new strategy for cancer biotherapy. [Mol Cancer Ther 2008;7(6):1598–603]

Pinitol targets nuclear factor-{kappa}B activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis

Sethi, G., Ahn, K. S., Sung, B., Aggarwal, B. B. — 2008-06-19

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-B (NF-B) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-B activation pathway. We found that pinitol suppressed NF-B activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-B activation noted in most tumor cells. The suppression of NF-B activation by pinitol occurred through inhibition of the activation of IB kinase, leading to sequential suppression of IB phosphorylation, IB degradation, p65 phosphorylation, p65 nuclear translocation, and NF-B-dependent reporter gene expression. Pinitol also suppressed the NF-B reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-β–activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IB kinase but not that induced by p65. The inhibition of NF-B activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-B activation pathway, which may explain its ability to suppress inflammatory cellular responses. [Mol Cancer Ther 2008;7(6):1604–14]

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

Wallach-Dayan, S. B., Rubinstein, A. M., Hand, C., Breuer, R., Naor, D. — 2008-06-19

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression. [Mol Cancer Ther 2008;7(6):1615–23]

Biodistribution and kinetics of the novel selective oncolytic adenovirus M1 after systemic administration

2008-06-19

Oncolytic adenoviruses represent a promising novel therapeutic option for the treatment of cancer. Despite their demonstrated safety in human clinical trials, the fundamental properties of oncolytic adenovirus biodistribution, spread, viral persistence, and replication in vivo have not been well characterized. The aim of this study was to evaluate the kinetics of viral distribution, spread, replication, and antitumoral efficacy after i.v. administration of a novel oncolytic mutant M1. This mutant consists of the E1A CR2-deleted Adv5 with a fragment of antisense polo-like kinase 1 (plk1) cDNA inserted into the deleted 6.7K/gp19K region, which combines oncolytic properties with efficient plk1 silencing, as described in our previous reports. In the present study, we established a new human orthotopic gastric carcinoma with a high frequency metastasis mouse model and showed that M1 spread not only in local primary tumors but also in disseminated metastases. M1 could effectively replicate in tumor cells leading to "oncolysis" and was able to eliminate expression of the targeted gene plk1 in human orthotopic gastric carcinoma model mice. Therefore, i.v. administration of M1 could prolong the survival time of tumor-bearing mice. [Mol Cancer Ther 2008;7(6):1624–32]

Evaluation of a chemical library of small-molecule Dishevelled antagonists that suppress tumor growth by down-regulating T-cell factor-mediated transcription

You, L., Xu, Z., Punchihewa, C., Jablons, D. M., Fujii, N. — 2008-06-19

We describe the rational generation of small-molecule agents that suppress tumor cell growth by down-regulating canonical Wnt signaling. We first produced a chemical library of the derivatives of indole-2-ketones and carbinols; we then screened them by using scalable assays of biochemical antagonism of Dishevelled-1 PDZ domain interactions and cell-based assays of Dishevelled-1–driven T-cell factor–mediated transcription. Compounds showing parallel effects in these assays were tested for selective induction of apoptosis in cancer cells. A new compound (24) that met the criteria for high biochemical antagonism, T-cell factor–mediated transcription, and induction of tumor-selective apoptosis was found to significantly suppress the growth of tumor xenografts in mice. [Mol Cancer Ther 2008;7(6):1633–8]

Gambogic acid is an antagonist of antiapoptotic Bcl-2 family proteins

Zhai, D., Jin, C., Shiau, C.-w., Kitada, S., Satterthwait, A. C., Reed, J. C. — 2008-06-19

The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC₅₀), <1µmol/L]. Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer assay. GA functionally inhibited the antiapoptotic Bcl-2 family proteins as shown by experiments using isolated mitochondria in which recombinant purified Bcl-2 family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogues of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax^–/–bak^–/– cells in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of antiapoptotic Bcl-2 family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. [Mol Cancer Ther 2008;7(6):1639–46]

Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells

Li, C., Li, R., Grandis, J. R., Johnson, D. E. — 2008-06-19

Head and neck squamous cell carcinomas (HNSCC) are characterized by resistance to chemotherapy and overexpression of antiapoptotic Bcl-2 family members, including Bcl-X_L and Bcl-2. Molecular targeting of Bcl-X_L and/or Bcl-2 in HNSCC cells has been shown to promote apoptosis signaling and to sensitize cells to chemotherapy drugs, including cisplatin, which is commonly used in the treatment of HNSCC. We report that induction of HNSCC apoptosis by the proteasome inhibitor bortezomib is accompanied by up-regulation of the proapoptotic proteins Bik and Bim, natural cellular inhibitors of Bcl-X_L and Bcl-2. Additionally, bortezomib treatment of HNSCC cells caused up-regulation of antiapoptotic Mcl-1L. Inhibition of Bik or Bim up-regulation using small interfering RNA markedly attenuated bortezomib-induced cell death. By contrast, small interfering RNA–mediated inhibition of Mcl-1L expression resulted in enhanced killing by bortezomib. Further investigation showed that the combination of bortezomib and cisplatin led to synergistic killing of HNSCC cells, with calculated combination indexes well below 1.0. Taken together, these results delineate a novel mechanism of HNSCC killing by bortezomib that involves up-regulation of Bik and Bik. Moreover, our findings suggest that the combination of bortezomib plus cisplatin, or bortezomib plus an inhibitor of Mcl-1L, may have therapeutic value in the treatment of HNSCC. [Mol Cancer Ther 2008;7(6):1647–55]

Molecular mechanisms of the antiangiogenic and antitumor effects of mycophenolic acid

2008-06-19

The relative risk for the development of malignancies following solid organ transplantation seems to be decreased in patients treated with the immunosuppressive agent mycophenolic acid (MPA). However, the molecular mechanisms of the antineoplastic effects of MPA are not completely understood. Here, we report that human endothelial cells and fibroblasts are highly sensitive to MPA treatment. We found that U87 glioblastoma cells were resistant to MPA treatment in vitro. However, U87 tumor growth was markedly inhibited in vivo in BALB/c nude mice, suggesting that MPA exerted its antitumor effects via modulation of the tumor microenvironment. Accordingly, microvascular density and pericyte coverage were markedly reduced in MPA-treated tumors in vivo. Using functional in vitro assays, we showed that MPA potently inhibited endothelial cell and fibroblast proliferation, invasion/migration, and endothelial cell tube formation. To identify the genetic participants governing the antiangiogenic and antifibrotic effects of MPA, we performed genome-wide transcriptional analysis in U87, endothelial and fibroblast cells at 6 and 12 h after MPA treatment. Network analysis revealed a critical role for MYC signaling in endothelial cells treated with MPA. Moreover, we found that the antiangiogenic effects of MPA were organized by coordinated communications between MYC and NDRG1, YYI, HIF1A, HDAC2, CDC2, GSK3B, and PRKACB signaling. The regulation of these "hub nodes" was confirmed by real-time quantitative reverse transcription-PCR and protein analysis. The critical involvement of MYC in the antiangiogenic signaling of MPA was further shown by gene knockdown experiments. Together, these data provide a molecular basis for the antiangiogenic and antifibrotic effects of MPA, which warrants further clinical investigations. [Mol Cancer Ther 2008;7(6):1656–68]

Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis

Singleton, P. A., Garcia, J. G.N., Moss, J. — 2008-06-19

Many patients with cancer receive combinations of drug treatments that include 5-fluorouracil (5-FU) and bevacizumab. Therapeutic doses of 5-FU are often associated with unwanted side effects, and bevacizumab is costly. Therefore, we explored potential agents that can reduce the therapeutic concentration of these drugs. Our data indicate that methylnaltrexone (MNTX), a peripheral antagonist of the µ-opioid receptor, exerts a synergistic effect with 5-FU and bevacizumab on inhibition of vascular endothelial growth factor (VEGF)–induced human pulmonary microvascular endothelial cell (EC) proliferation and migration, two key components in cancer-associated angiogenesis. MNTX inhibited EC proliferation with an IC₅₀ of ~100 nmol/L. Adding 100 nmol/L MNTX to EC shifted the IC₅₀ of 5-FU from ~5 µmol/L to ~7 nmol/L. Further, adding 50 ng/mL MNTX shifted the IC₅₀ of bevacizumab on inhibition of EC migration from ~25 to ~6 ng/mL. These synergistic effects were not observed with naltrexone, a tertiary µ-opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased receptor protein tyrosine phosphatase µ activity, which was independent of µ-opioid receptor expression. Silencing receptor protein tyrosine phosphatase µ expression (small interfering RNA) in human EC inhibited both synergy between MNTX and bevacizumab or 5-FU and increased VEGF-induced tyrosine phosphorylation of Src and p190 RhoGAP with enhanced activation of Akt and the actin cytoskeletal regulatory protein, RhoA, whereas silencing Src, Akt, or RhoA blocked VEGF-induced angiogenic events. Therefore, addition of MNTX could potentially lower the therapeutic doses of 5-FU and bevacizumab, which could improve index. [Mol Cancer Ther 2008;7(6):1669–79]

Arsenic trioxide decreases AKT protein in a caspase-dependent manner

Mann, K. K., Colombo, M., Miller, W. H. — 2008-06-19

Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia but is less successful in other malignancies. To identify targets for potential combination therapies, we have begun to characterize signaling pathways leading to As₂O₃-induced cytotoxicity. Previously, we described the requirement for a reactive oxygen species–mediated, SEK1/c-Jun NH₂-terminal kinase (JNK) pathway to induce apoptosis. AKT inhibits several steps in this pathway; therefore, we postulated that As₂O₃ might decrease its activity. Indeed, As₂O₃ decreases not only AKT activity but also total AKT protein, and sensitivity to As₂O₃ correlates with the degree of AKT protein decrease. Decreased AKT expression further correlates with JNK activation and the release of AKT from the JNK-interacting protein 1 scaffold protein known to assemble the mitogen-activated protein kinase cascade. We found that As₂O₃ regulates AKT protein stability without significant effects on its transcription or translation. We show that As₂O₃ decreases AKT protein via caspase-mediated degradation, abrogated by caspase-6, caspase-8, caspase-9, and caspase-3 inhibitors but not proteosome inhibitors. Furthermore, As₂O₃ enhances the ability of a heat shock protein 90 inhibitor to decrease AKT expression and increase growth inhibition. This suggests that As₂O₃ may be useful in combination therapies that target AKT pathways or in tumors that have constitutively active AKT expression. [Mol Cancer Ther 2008;7(6):1680–7]

Emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase-8-mediated Bid cleavage

Yan, Y., Su, X., Liang, Y., Zhang, J., Shi, C., Lu, Y., Gu, L., Fu, L. — 2008-06-19

AMAD, an emodin azide methyl anthraquinone derivative, was extracted from the nature giant knotweed rhizome of traditional Chinese herbs. Here, we investigated the anticancer activities and signaling pathways implicated in AMAD-induced apoptosis in human breast cancer cell lines MDA-MB-453 and human lung adenocarcinoma Calu-3 cells. AMAD was found to have a potent cytotoxic effect on both cell lines. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V–positive cells in a dose-dependent manner, respectively. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and activated caspases (cysteine aspartase) cascade involving in caspase-8, caspase-9, caspase-3, and poly(ADP-ribose) polymerase cleavage in a concentration-dependent manner. It was noteworthy that AMAD also effectively cleaved Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, and induced the subsequent release of cytochrome c from mitochondria into the cytosol. Furthermore, suppression of caspase-8 activity with Z-IETD-FMK partially inhibited release of cytochrome c and Bid cleavage induced by AMAD, whereas exposure to Z-LETD-FMK, a caspase-9 inhibitor, had no effect. Additionally, there was significant change in other mitochondrial membrane proteins triggered by AMAD, such as Bcl-xl and Bad. It was intriguing that AMAD decreased the generation of reactive oxygen species in both cell lines. DNA-binding assay exhibited apoptosis induced by AMAD was not involved in intercalating to DNA. Taken together, these data suggested that AMAD induced apoptosis via a mitochondrial pathway involving caspase-8/Bid activation in both cell lines. [Mol Cancer Ther 2008;7(6):1688–97]

Sequence-dependent, synergistic antiproliferative and proapoptotic effects of the combination of cytotoxic drugs and enzastaurin, a protein kinase C{beta} inhibitor, in non-small cell lung cancer cells

2008-06-19

Enzastaurin, an acyclic bisindolymaleimide, is a potent and selective competitive inhibitor of protein kinase Cβ, which has been shown to inhibit cancer cell proliferation and angiogenesis in human cancer cell lines. Gemcitabine and pemetrexed are two cytotoxic drugs that are currently used in non-small cell lung cancer (NSCLC) therapy. In this study, we have investigated whether the addition of enzastaurin to gemcitabine or to pemetrexed is able to increase their antitumor activity to establish an effective schedule of combined treatment. The effects on cancer cell proliferation, cell cycle distribution, intracellular mitogenic and antiapoptotic signaling pathways, and induction of apoptosis were evaluated in three different combination sequences (concomitant treatment, sequential treatment with the cytotoxic drug followed by enzastaurin, or sequential treatment with enzastaurin followed by the cytotoxic drug) in a panel of human NSCLC cell lines. The combination of enzastaurin with either gemcitabine or pemetrexed caused different antiproliferative and proapoptotic effects depending on the treatment schedule. A synergistic antiproliferative and proapoptotic activity was only obtained when chemotherapy was followed by treatment with enzastaurin. These effects were accompanied by the arrest of the surviving cancer cells in the S phase, thus limiting their ability to proceed through the cell cycle, and by a maximum inhibition in the activated, phosphorylated forms of Akt and mitogen-activated protein kinase. In contrast, the concomitant treatments or the sequential treatments, in which enzastaurin was given before chemotherapy, resulted in significant antagonistic effects. [Mol Cancer Ther 2008;7(6):1698–707]

Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane in vitro and in vivo using an orthotopic model of pancreatic cancer

Ali, S., Banerjee, S., Ahmad, A., El-Rayes, B. F., Philip, P. A., Sarkar, F. H. — 2008-06-19

Blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase inhibitors is insufficient for effective antitumor activity because of independently activated survival pathways. A multitargeted approach may therefore improve the outcome of anti-EGFR therapies. In the present study, we determined the effects of 3,3'-diindolylmethane (Bioresponse BR-DIM referred to as B-DIM), a formulated DIM with greater bioavailability on cell viability and apoptosis with erlotinib in vitro and in vivo using an orthotopic animal tumor model. BxPC-3 and MIAPaCa cells with varying levels of EGFR and nuclear factor-B (NF-B) DNA-binding activity were treated with B-DIM (20 µmol/L), erlotinib (2 µmol/L), and the combination. Cell survival and apoptosis was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and histone-DNA ELISA. Electrophoretic mobility shift assay was used to evaluate NF-B DNA-binding activity. We found significant reduction in cell viability by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, induction of apoptosis, down-regulation of EGFR phosphorylation, NF-B DNA-binding activity, and expression of antiapoptotic genes in BxPC-3 cells when treated with the combination of erlotinib and B-DIM compared with either agent alone. In contrast, no such effect was observed in MIAPaCa cells by similar treatment. Most importantly, these in vitro results were recapitulated in animal model showing that B-DIM in combination with erlotinib was much more effective as an antitumor agent compared with either agent alone. These results suggest that the utilization of B-DIM could be a useful strategy for achieving better treatment outcome in patients with activated status of EGFR and NF-B in their tumors. [Mol Cancer Ther 2008;7(6):1708–19]

5-Aminolaevulinic acid peptide prodrugs enhance photosensitization for photodynamic therapy

Bourre, L., Giuntini, F., Eggleston, I. M., Wilson, M., MacRobert, A. J. — 2008-06-19

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-l-Phe-ALA-Me (compound 1) and Ac-l-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD₅₀ (2.5 J/cm²) of 25 µmol/L for ALA, 6 µmol/L for 5-aminolaevulinic hexyl ester, and 2.6 µmol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding d-enantiomer, Ac-d-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH. [Mol Cancer Ther 2008;7(6):1720–9]

In vitro and in vivo targeting of hollow gold nanoshells directed at epidermal growth factor receptor for photothermal ablation therapy

2008-06-19

Laser-induced phototherapy is a new therapeutic use of electromagnetic radiation for cancer treatment. The use of targeted plasmonic gold nanoparticles can reduce the laser energy necessary for selective tumor cell destruction. However, the ability for targeted delivery of the currently used gold nanoparticles to tumor cells is limited. Here, we describe a new class of molecular specific photothermal coupling agents based on hollow gold nanoshells (HAuNS; average diameter, ~30 nm) covalently attached to monoclonal antibody directed at epidermal growth factor receptor (EGFR). The resulting anti-EGFR-HAuNS exhibited excellent colloidal stability and efficient photothermal effect in the near-infrared region. EGFR-mediated selective uptake of anti-EGFR-HAuNS in EGFR-positive A431 tumor cells but not IgG-HAuNS control was shown in vitro by imaging scattered light from the nanoshells. Irradiation of A431 cells treated with anti-EGFR-HAuNS with near-infrared laser resulted in selective destruction of these cells. In contrast, cells treated with anti-EGFR-HAuNS alone, laser alone, or IgG-HAuNS plus laser did not show observable effect on cell viability. Using ¹¹¹In-labeled HAuNS, we showed that anti-EGFR-HAuNS could be delivered to EGFR-positive tumors at 6.8% ID/g, and the microscopic image of excised tumor with scattering signal from nanoshells confirmed preferential delivery to A431 tumor of anti-EGFR-HAuNS compared with IgG-HAuNS. The absence of silica core, the relatively small particle size and high tumor uptake, and the absence of cytotoxic surfactant required to stabilize other gold nanoparticles suggest that immuno-HAuNS have the potential to extend to in vivo molecular therapy. [Mol Cancer Ther 2008;7(6):1730–9]

Photodynamic therapy with an endocytically located photosensitizer cause a rapid activation of the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2 terminal kinase with opposing effects on cell survival

Weyergang, A., Kaalhus, O., Berg, K. — 2008-06-19

Photochemical internalization (PCI) is a method for release of endosomally/lysosomally trapped drugs into the cell cytosol. PCI is based on photosensitizers that accumulate in the membranes of endosomes and lysosomes. Light exposure generates reactive oxygen species that cause membrane rupture and subsequently drug release. PCI can be considered as a combination therapy of photodynamic therapy (PDT) and the administrated drug. The present work reports on mitogen-activated protein kinase signaling after PDT with the endocytically located photosensitizer TPPS_2a (meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings) as used for PCI in two cancer cell lines: NuTu-19 and WiDr. Both extracellular signal-regulated kinase (ERK) and p38 were activated immediately after PDT. The photochemically induced ERK phosphorylation was enhanced by epidermal growth factor stimulation to a level above that obtainable with epidermal growth factor alone. Expression of the ERK phosphatase, MAPK phosphatase-1, was increased 2 h after PDT but was not the cause of ERK dephosphorylation observed simultaneously. A transient activation of c-Jun NH₂ terminal kinase was also observed after PDT but only in the NuTu-19 cells. Using suitable inhibitors, it is shown here that the p38 signal is a death signal, whereas c-Jun NH₂ terminal kinase rescues cells after PDT. No direct connection was observed between PDT-induced ERK activation and toxicity of the treatment. The present results document the importance of the mitogen-activated protein kinases in TPPS_2a-PDT-induced cytotoxicity. [Mol Cancer Ther 2008;7(6):1740–50]

Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

Liu, S., Cheng, H., Kwan, W., Lubieniecka, J. M., Nielsen, T. O. — 2008-06-19

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 µmol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models. [Mol Cancer Ther 2008;7(6):1751–61]