Molecular Cancer Therapeutics recent issues

Novel therapeutic strategy for osteosarcoma targeting osteoclast differentiation, bone-resorbing activity, and apoptosis pathway

Akiyama, T., Dass, C. R., Choong, P. F.M. — 2008-11-11

Osteosarcoma is the most common bone sarcoma, which mainly affects adolescents and young adults. Although the combination of modern surgery and systemic chemotherapy has improved osteosarcoma treatment dramatically, no substantial change in survival has been seen over the past 20 years. Therefore, novel therapeutic strategies for osteosarcoma are required if the 35% of patients with fatal metastases are to be successfully treated. Recently, osteoclasts have drawn attention as a therapeutic target in various bone disorders including osteosarcoma. The osteoclast is the sole cell that resorbs bone and is central in pathologic situations, where bone destruction is intricately involved. Osteosarcoma cells are of the osteoblastic lineage, the latter of which is characterized by cells secreting the osteoclast-inducing factor, receptor activator of nuclear factor-B ligand. Hence, osteosarcoma is a better candidate for osteoclast-targeted therapy than other primary and metastatic bone tumors. The rapid progress on the molecular mechanism regulating osteoclast has propelled a development of new therapeutic approaches. In this review article, we present the prospects of osteoclast-targeted therapy as a novel treatment strategy for osteosarcoma. Receptor activator of nuclear factor-B-Fc, osteoprotegerin, bisphosphonates, and Src inhibitor are shown as positive candidates and can control various aspects of osteoclast function. This review article will attempt to discuss these issues in term. [Mol Cancer Ther 2008;7(11):3461–9]

Dithiolethiones for cancer chemoprevention: where do we stand?

Zhang, Y., Munday, R. — 2008-11-11

Dithiolethiones are a well-known class of cancer chemopreventive agents; the key mechanism of action of dithiolethiones involves activation of Nrf2 signaling and induction of phase II enzymes. In the past, attention has been focused mainly on 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz), which showed ability as a wide-spectrum inhibitor of chemical carcinogenesis in preclinical models. However, clinical trials of oltipraz have shown questionable efficacy, and at the high doses employed in such studies, significant side effects were observed. Dithiolethiones that are markedly more effective and potent than oltipraz in both induction of phase II enzymes and inhibition of chemical carcinogenesis in preclinical studies have been identified, and these compounds have shown pronounced organ specificity in vivo. Further investigation of these compounds may lead to development of effective and safe agents for cancer prevention in humans. [Mol Cancer Ther 2008;7(11):3470–9]

Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

2008-11-11

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G₁, with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype. [Mol Cancer Ther 2008;7(11):3480–9]

Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors

2008-11-11

In developing inhibitors of the LIM kinases, the initial lead molecules combined potent target inhibition with potent cytotoxic activity. However, as subsequent compounds were evaluated, the cytotoxic activity separated from inhibition of LIM kinases. A rapid determination of the cytotoxic mechanism and its molecular target was enabled by integrating data from two robust core technologies. High-content assays and gene expression profiling both indicated an effect on microtubule stability. Although the cytotoxic compounds are still kinase inhibitors, and their structures did not predict tubulin as an obvious target, these results provided the impetus to test their effects on microtubule polymerization directly. Unexpectedly, we confirmed tubulin itself as a molecular target of the cytotoxic kinase inhibitor compounds. This general approach to mechanism of action questions could be extended to larger data sets of quantified phenotypic and gene expression data. [Mol Cancer Ther 2008;7(11):3490–8]

HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancer cells

2008-11-11

Tumor cells with genomic amplification of MET display constitutive activation of the MET tyrosine kinase, which renders them highly sensitive to MET inhibition. Several MET inhibitors have recently entered clinical trials; however, as with other molecularly targeted agents, resistance is likely to develop. Therefore, elucidating possible mechanisms of resistance is of clinical interest. We hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in MET-amplified cancer cells by reactivating key survival pathways. Treatment of MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752 abrogated MEK/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling, resulting in cyclin D1 loss and G₁ arrest. PHA-665752 also inhibited baseline phosphorylation of epidermal growth factor receptor (EGFR) and HER-3, which are transactivated via MET-driven receptor cross-talk in these cells. However, MET-independent HER kinase activation using EGF (which binds to and activates EGFR) or heregulin-β1 (which binds to and activates HER-3) was able to overcome the growth-inhibitory effects of MET inhibition by restimulating MEK/MAPK and/or PI3K/AKT signaling, suggesting a possible escape mechanism. Importantly, dual inhibition of MET and HER kinase signaling using PHA-665752 in combination with the EGFR inhibitor gefitinib or in combination with inhibitors of MEK and AKT prevented the above rescue effects. Our results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-"addicted" cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3. [Mol Cancer Ther 2008;7(11):3499–508]

Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes

2008-11-11

The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer. [Mol Cancer Ther 2008;7(11):3509–18]

Sorafenib inhibits signal transducer and activator of transcription 3 signaling associated with growth arrest and apoptosis of medulloblastomas

2008-11-11

Medulloblastomas are the most frequent malignant brain tumors in children. Sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in a variety of tumor cells. Sorafenib inhibited proliferation and induced apoptosis in two established cell lines (Daoy and D283) and a primary culture (VC312) of human medulloblastomas. In addition, sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both cell lines and primary tumor cells. The inhibition of phosphorylated STAT3 (Tyr⁷⁰⁵) occurs in a dose- and time-dependent manner. In contrast, AKT (protein kinase B) was only decreased in D283 and VC312 medulloblastoma cells and mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2) were not inhibited by sorafenib in these cells. Both D-type cyclins (D1, D2, and D3) and E-type cyclin were down-regulated by sorafenib. Also, expression of the antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was decreased and correlated with apoptosis induced by sorafenib. Finally, sorafenib suppressed the growth of human medulloblastoma cells in a mouse xenograft model. Together, our data show that sorafenib blocks STAT3 signaling as well as expression of cell cycle and apoptosis regulatory proteins, associated with inhibition of cell proliferation and induction of apoptosis in medulloblastomas. These findings provide a rationale for treatment of pediatric medulloblastomas with sorafenib. [Mol Cancer Ther 2008;7(11):3519–26]

Anticancer effects of tanshinone I in human non-small cell lung cancer

2008-11-11

Tanshinones are the major bioactive compounds of Salvia miltiorrhiza Bunge (Danshen) roots, which are used in many therapeutic remedies in Chinese traditional medicine. We investigated the anticancer effects of tanshinones on the highly invasive human lung adenocarcinoma cell line, CL1-5. Tanshinone I significantly inhibited migration, invasion, and gelatinase activity in macrophage-conditioned medium-stimulated CL1-5 cells in vitro and also reduced the tumorigenesis and metastasis in CL1-5-bearing severe combined immunodeficient mice. Unlike tanshinone IIA, which induces cell apoptosis, tanshinone I did not have direct cytotoxicity. Real-time quantitative PCR, luciferase reporter assay, and electrophoretic mobility shift assay revealed that tanshinone I reduces the transcriptional activity of interleukin-8, the angiogenic factor involved in cancer metastasis, by attenuating the DNA-binding activity of activator protein-1 and nuclear factor-B in conditioned medium-stimulated CL1-5 cells. Microarray and pathway analysis of tumor-related genes identified the differentially expressed genes responding to tanshinone I, which may be associated with the Ras-mitogen-activated protein kinase and Rac1 signaling pathways. These results suggest that tanshinone I exhibits anticancer effects both in vitro and in vivo and that these effects are mediated at least partly through the interleukin-8, Ras-mitogen-activated protein kinase, and Rac1 signaling pathways. Although tanshinone I has a remarkable anticancer action, its potential anticoagulant effect should be noted and evaluated. [Mol Cancer Ther 2008;7(11):3527–38]

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells

Khwaja, F. S., Quann, E. J., Pattabiraman, N., Wynne, S., Djakiew, D. — 2008-11-11

The p75 neurotrophin receptor (p75^NTR) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75^NTR expression in several prostate cancer cell lines leading to p75^NTR-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75^NTR levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75^NTR-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75^NTR before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75^NTR levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75^NTR by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75^NTR protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75^NTR-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. [Mol Cancer Ther 2008;7(11):3539–45]

A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death

2008-11-11

Evasion of death receptor ligand-induced apoptosis is an important contributor to cancer development and progression. Therefore, molecules that restore sensitivity to death receptor stimuli would be important tools to better understand this biological pathway and potential leads for therapeutic adjuncts. Previously, the small-molecule N-[4-chloro-3-(trifluoromethyl)phenyl]-3-oxobutanamide (fasentin) was identified as a chemical sensitizer to the death receptor stimuli FAS and tumor necrosis factor apoptosis-inducing ligand, but its mechanism of action was unknown. Here, we determined that fasentin alters expression of genes associated with nutrient and glucose deprivation. Consistent with this finding, culturing cells in low-glucose medium recapitulated the effects of fasentin and sensitized cells to FAS. Moreover, we showed that fasentin inhibited glucose uptake. Using virtual docking studies with a homology model of the glucose transport protein GLUT1, fasentin interacted with a unique site in the intracellular channel of this protein. Additional chemical studies with other GLUT inhibitors and analogues of fasentin supported a role for partial inhibition of glucose transport as a mechanism to sensitize cells to death receptor stimuli. Thus, fasentin is a novel inhibitor of glucose transport that blocks glucose uptake and highlights a new mechanism to sensitize cells to death ligands. [Mol Cancer Ther 2008;7(11):3546–55]

Involvement of c-FLIP and survivin down-regulation in flexible heteroarotinoid-induced apoptosis and enhancement of TRAIL-initiated apoptosis in lung cancer cells

2008-11-11

The flexible heteroarotinoid, SHetA2, is a novel compound with apoptosis-inducing and anticancer activities in vitro and in vivo. Our previous research showed that up-regulation of death receptor 5 plays a critical role in the mechanism of SHetA2-induced apoptosis in human lung cancer cells. The hypothesis of this study was that the mechanism of SHetA2-induced apoptosis requires modulation of additional proteins critical for regulation of apoptosis, including cellular FLICE-inhibitory protein (c-FLIP), survivin, X-linked inhibitor of apoptosis, Bcl-2, Bcl-X_L, Bax, and Bim. Western blot analysis showed that c-FLIP and survivin were substantially reduced in all of the tested cell lines exposed to SHetA2 compared with other proteins that were reduced only in a subset of the cell lines tested. Strikingly, overexpression of c-FLIP, but not survivin, protected cells from SHetA2-induced apoptosis and enhancement of TRAIL-initiated apoptosis, although knockdown of endogenous survivin did slightly sensitize cells to SHetA2-induced apoptosis. Consistent with these results, small interfering RNA-mediated reduction of c-FLIP was more effective than survivin down-regulation in triggering apoptosis in these cell lines. SHetA2 increased ubiquitination of c-FLIP and the consequent degradation was abrogated by the proteasome inhibitor MG132. Although SHetA2 treatment led to increased c-Jun phosphorylation, the JNK inhibitor SP600125 did not prevent c-FLIP down-regulation by SHetA2. Thus, it appears that SHetA2 down-regulates c-FLIP levels by facilitating its ubiquitin/proteasome-mediated degradation independent of JNK activation. Collectively, the present study indicates that, in addition to death receptor 5 up-regulation, c-FLIP down-regulation is another important component of flexible heteroarotinoid (SHetA2)-induced apoptosis as well as enhancement of TRAIL-induced apoptosis. [Mol Cancer Ther 2008;7(11):1–10] [Mol Cancer Ther 2008;7(11):3556–65]

The flavonoid kaempferol sensitizes human glioma cells to TRAIL-mediated apoptosis by proteasomal degradation of survivin

Siegelin, M. D., Reuss, D. E., Habel, A., Herold-Mende, C., von Deimling, A. — 2008-11-11

Resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) limits its potential as a drug for cancer therapy. Here, we report that kaempferol, a bioactive plant flavonoid, sensitizes U251 and U87 glioma cells to TRAIL-mediated apoptosis. In contrast, U373 cells are not affected by kaempferol treatment. Treatment of kaempferol alone for 24 h did not induce apoptosis in the cell lines. We provide evidence that TRAIL-induced apoptosis is partially driven by kaempferol-mediated reduction of survivin protein levels. On kaempferol treatment, proteasomal degradation of survivin was observed. Inhibition of proteasomal degradation with MG132 in kaempferol-treated cells restored survivin protein levels in both glial cell lines. Consequently, overexpression of survivin attenuated TRAIL-kaempferol–induced apoptosis. In addition, we show that kaempferol mediates down-regulation of phosphorylated Akt, thereby further reducing survivin protein level. Furthermore, the blockage of the serine/threonine kinase Akt activity by kaempferol is important for inhibition of survivin because active phosphorylated Akt enhances the stability of survivin. However, we also show that the combined treatment of TRAIL and kaempferol induces cleavage (activation) of caspase-8, thereby exerting a proapoptotic effect independent of survivin known not to inhibit caspase-8 activation. Other effects induced by kaempferol were suppression of X-linked inhibitor of apoptosis proteins as the antiapoptotic members of the Bcl-2 family, Bcl-2, Bcl-xL, and Mcl-1 in a concentration-dependent manner. In summary, we showed that suppression of survivin is an essential mechanism in TRAIL-kaempferol–mediated apoptosis. [Mol Cancer Ther 2008;7(11):3566–74]

Targeting multiple pathways in gliomas with stem cell and viral delivered S-TRAIL and Temozolomide

Hingtgen, S., Ren, X., Terwilliger, E., Classon, M., Weissleder, R., Shah, K. — 2008-11-11

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells. However, its short half-life, poor delivery, and TRAIL-resistant tumor cells have diminished its clinical efficacy. In this study, we explored whether novel delivery methods will represent new and effective ways to treat gliomas and if adjuvant therapy with the chemotherapeutic agent temozolomide would enhance the cytotoxic properties of TRAIL in glioma lines resistant to TRAIL monotherapy. We have engineered adeno-associated virus (AAV) vectors encoding recombinant secreted TRAIL (S-TRAIL) and bioluminescent-fluorescent marker fusion proteins and show that AAV-delivered S-TRAIL leads to varying degrees of killing in multiple glioma lines, which correspond with caspase-3/7 activation. In vivo, dual bioluminescent imaging revealed efficient delivery of therapeutic AAV vectors directly into the tumor mass, which induced marked attenuation of tumor progression. Treatment of glioma cells with the chemotherapeutic agent temozolomide alone lead to a significant accumulation of cells in G₂-M phase, activated the cell cycle checkpoint protein Chk1, and increased death receptor expression in a time-dependent manner. Furthermore, combined treatment with AAV-S-TRAIL or neural stem cell-S-TRAIL and temozolomide induced cell killing and markedly up-regulated proapoptotic proteins in glioma cells least sensitive to TRAIL. This study elucidates novel means of delivering S-TRAIL to gliomas and suggests combination of clinically relevant temozolomide and S-TRAIL may represent a new therapeutic option with increased potency for glioblastoma patients. [Mol Cancer Ther 2008;7(11):3575–85]

Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity

2008-11-11

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87 cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87 cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87 cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, β-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87 cells with a decrease in G₁ and increase in S and G₂ fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87 cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. [Mol Cancer Ther 2008;7(11):3586–97]

A RNA antagonist of hypoxia-inducible factor-1{alpha}, EZN-2968, inhibits tumor cell growth

2008-11-11

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the -subunit of HIF-1 (HIF-1), which occurs in response to hypoxia or activation of growth factor pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1 protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits the expression of HIF-1 mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC₅₀, 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor cell growth. Additionally, down-regulation of HIF-1 protein by EZN-2968 led to reduction of its transcriptional targets and of human umbilical vein endothelial cell tube formation. In vivo, administration of EZN-2968 to normal mice led to specific, dose-dependent, and highly potent down-regulation of endogenous HIF-1 and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program. [Mol Cancer Ther 2008;7(11):3598–608]

Salirasib (farnesyl thiosalicylic acid) for brain tumor treatment: a convection-enhanced drug delivery study in rats

2008-11-11

Our aim was to assess the ability of convection-enhanced drug delivery (CED), a novel approach of direct delivery of drugs into brain tissue and brain tumors, to treat brain tumors using salirasib (farsnesyl thiosalicylic acid). CED was achieved by continuous infusion of drugs via intracranial catheters, thus enabling convective distribution of high drug concentrations over large volumes while avoiding systemic toxicity. Several phase II/III CED-based trials are currently in progress but have yet to overcome two major pitfalls of this methodology (the difficulty in attaining efficient CED and the significant nonspecific neurotoxicity caused by high drug doses in the brain). In this study, we addressed both issues by employing our previously described novel CED imaging and increased efficiency methodologies to exclusively target the activated form of the Ras oncogene in a 9L gliosarcoma rat model. The drug we used was salirasib, a highly specific Ras inhibitor shown to exert its suppressive effects on growth and migration of proliferating tumor cells in in vitro and in vivo models, including human glioblastoma, without affecting normal tissues. The results show a significant decrease in tumor growth rate in salirasib-treated rats relative to vehicle-treated rats as well as a significant correlation between CED efficacy and tumor growth rate with no observed toxicity despite drug concentrations an order of magnitude higher than previously detected in the brain. The results show that CED of salirasib is efficient and nontoxic for the treatment of glioblastoma in a rat model, thus suggesting that it may be considered for clinical application. [Mol Cancer Ther 2008;7(11):3609–16]

Psorospermin structural requirements for P-glycoprotein resistance reversal

Carey, S. S., Gleason-Guzman, M., Gokhale, V., Hurley, L. H. — 2008-11-11

Resistance to chemotherapy reduces its effectiveness, resulting in increased mortality. Psorospermin, a natural product, is a topoisomerase II–directed DNA alkylating agent active against multidrug-resistant (MDR) cell lines, including multiple myeloma. In this study, the mechanism of the P-glycoprotein (P-gp) modulation activity of psorospermin and that of its associated pharmacophore were examined. Flow cytometry shows that doxorubicin-resistant multiple myeloma cells (8226/D40) pretreated with psorospermin enhance intracellular retention of doxorubicin compared with control (75% versus 38%). Because the overexpression of P-gp is the primary cause of drug resistance in the 8226/D40 cells, psorospermin-induced sensitization was likely due to mdr1/P-gp expressional or functional inhibition. As shown by PCR and Western blot, neither transcription of mdr1 nor translation of P-gp was down-regulated by psorospermin treatment. Therefore, the mechanism of psorospermin-induced resistance reversal is most likely through a direct interaction between psorospermin and P-gp. Furthermore, because only the (2'R,3'R) isomer of psorospermin showed any resistance reversal activity, the side chain of psorospermin is apparently a crucial moiety for resistance reversal. By understanding the mechanism of psorospermin-induced MDR modulation, psorospermin and similar compounds can be combined with other chemotherapies to treat resistant cancers. [Mol Cancer Ther 2008;7(11):3617–23]

Bortezomib is ineffective in an orthotopic mouse model of pancreatic adenocarcinoma

2008-11-11

The purpose of the present study was to evaluate the potency of the proteasome inhibitor bortezomib ± gemcitabine in vitro and in vivo in pancreatic carcinoma. It could be shown that bortezomib induced apoptosis and inhibited proliferation of pancreatic carcinoma very efficiently in vitro. In contrast, in an orthotopic pancreatic adenocarcinoma mouse model, gemcitabine treatment inhibited tumor growth, whereas bortezomib promoted it. Bortezomib-treated animals showed significantly higher tumor burden compared with gemcitabine-treated and control animals, although bortezomib was locally active and induced a decrease of proteasome activity, which was most pronounced following the simultaneous administration of gemcitabine. Also, tumor progression was not caused by immunosuppression as a result of proteasome inhibition. Interestingly, anti-CD31 staining of tumors showed that angiogenesis was significantly increased in the tumors of bortezomib-treated mice compared with the tumors of control animals. In addition, bortezomib resulted an increase of pericytes, vascular endothelial growth factor, RGS-5, and hypoxia-inducible factor-1 in the tumor. Although this study supports efficacy of bortezomib against pancreatic carcinoma in vitro, it strongly indicates that bortezomib therapy has a significant tumor-promoting effect in vivo by induction of angiogenesis. The data are in accordance with the complete failure of bortezomib in a phase II trial for this indication. Choosing the right schedule of gemcitabine and bortezomib showed some synergistic effects, but the gain might not be big enough to compensate the potentially detrimental effects. [Mol Cancer Ther 2008;7(11):3624–31]

Role of repair protein Rad51 in regulating the response to gefitinib in human non-small cell lung cancer cells

2008-11-11

Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can block growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) activation. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas. In this study, we examined the role of Rad51 in regulating the response to gefitinib among different human lung cancer cell lines. The H520 line (human squamous cell carcinoma) was less sensitive to gefitinib compared with the H1650 (human adenocarcinoma) or A549 (human bronchioloalveolar carcinoma) lines. In H1650 and A549 cells but not in H520 cells, gefitinib decreased cellular levels of phospho-ERK1/2 and Rad51 protein and message levels. Moreover, gefitinib decreased Rad51 protein levels by enhancing Rad51 protein instability through 26S proteasome-mediated degradation. Inhibition of endogenous Rad51 levels by si-Rad51 RNA transfection significantly enhanced gefitinib-induced cytotoxicity. In contrast, transfection with constitutively active MKK1 vector could restore both Rad51 protein levels and cell survival inhibited by gefitinib. The MKK1/2-ERK1/2 signaling pathway constitutes the upstream signaling for maintaining Rad51 message and protein levels. Rad51 protein can protect lung cancer cells from cytotoxic effects induced by gefitinib. Suppression of Rad51 may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib. [Mol Cancer Ther 2008;7(11):3632–41]

Activation of toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors

2008-11-11

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-B and inflammatory response–related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression. [Mol Cancer Ther 2008;7(11):3642–53]

Pomegranate extract inhibits NF-{kappa}B

2008-11-11

PGG and prostate cancer apoptosis

2008-11-11

Lapatinib and IGF-I signaling

2008-11-11

ER stress-dependent apoptosis in pancreatic cancer

2008-11-11

Asparagine synthetase is a predictive biomarker of L-asparaginase activity in ovarian cancer cell lines

2008-10-13

We recently used RNA interference to show that a negative correlation of l-asparaginase (l-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and l-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, l-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of l-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical l-ASP activity in ovarian cancer. [Mol Cancer Ther 2008;7(10):3123–8]

Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling

Wilhelm, S. M., Adnane, L., Newell, P., Villanueva, A., Llovet, J. M., Lynch, M. — 2008-10-13

Although patients with advanced refractory solid tumors have poor prognosis, the clinical development of targeted protein kinase inhibitors offers hope for the future treatment of many cancers. In vivo and in vitro studies have shown that the oral multikinase inhibitor, sorafenib, inhibits tumor growth and disrupts tumor microvasculature through antiproliferative, antiangiogenic, and/or proapoptotic effects. Sorafenib has shown antitumor activity in phase II/III trials involving patients with advanced renal cell carcinoma and hepatocellular carcinoma. The multiple molecular targets of sorafenib (the serine/threonine kinase Raf and receptor tyrosine kinases) may explain its broad preclinical and clinical activity. This review highlights the antitumor activity of sorafenib across a variety of tumor types, including renal cell, hepatocellular, breast, and colorectal carcinomas in the preclinical setting. In particular, preclinical evidence that supports the different mechanisms of action of sorafenib is discussed. [Mol Cancer Ther 2008;7(10):3129–40]

A genomic approach to identify molecular pathways associated with chemotherapy resistance

2008-10-13

Resistance to chemotherapy in cancer is common. As gene expression profiling has been shown to anticipate chemotherapeutic resistance, we sought to identify cellular pathways associated with resistance to facilitate effective combination therapy. Gene set enrichment analysis was used to associate pathways with resistance in two data sets: the NCI-60 cancer cell lines deemed sensitive and resistant to specific chemotherapeutic agents (Adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouracil, paclitaxel, and topotecan) and a series of 40 lung cancer cell lines for which sensitivity to cisplatin and docetaxel was determined. Candidate pathways were further screened in silico using the Connectivity Map. The lead candidate pathway was functionally validated in vitro. Gene set enrichment analysis associated the matrix metalloproteinase, p53, methionine metabolism, and free pathways with cytotoxic resistance in the NCI-60 cell lines across multiple agents, but no gene set was common to all drugs. Analysis of the lung cancer cell lines identified the bcl-2 pathway to be associated with cisplatin resistance and the AKT pathway enriched in cisplatin- and docetaxel-resistant cell lines. Results from Connectivity Map supported an association between phosphatidylinositol 3-kinase/AKT and docetaxel resistance but did not support the association with cisplatin. Targeted inhibition of the phosphatidylinositol 3-kinase/AKT pathway with LY294002, in combination with docetaxel, resulted in a synergistic effect in previously docetaxel-resistant cell lines but not with cisplatin. These results support the use of a genomic approach to identify drug-specific targets associated with the development of chemotherapy resistance and underscore the importance of disease context in identifying these pathways. [Mol Cancer Ther 2008;7(10):3141–9]

Class I {beta}-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

2008-10-13

2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 µmol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G₂-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I β-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on β-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions. [Mol Cancer Ther 2008;7(9):3150–9]

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway

2008-10-13

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, -radiation, IFN-, and IFN- in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance. [Mol Cancer Ther 2008;7(10):3160–8]

Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance

2008-10-13

Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 was noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provides preclinical rationale for using Stat3-inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment. [Mol Cancer Ther 2008;7(10):3169–75]

EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance

2008-10-13

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer. [Mol Cancer Ther 2008;7(10):3176–86]

Structural basis for nuclear receptor corepressor recruitment by antagonist-liganded androgen receptor

Hodgson, M. C., Shen, H. C., Hollenberg, A. N., Balk, S. P. — 2008-10-13

Androgen receptor (AR) recruitment of transcriptional corepressors NCoR and SMRT can be enhanced by antagonists such as mifepristone. This study shows that enhanced NCoR binding to the mifepristone-liganded AR is mediated by the NCoR COOH-terminal N1 CoRNR box and that this selectivity is due to charged residues unique to the COOH-terminal CoRNR boxes of NCoR and SMRT. Significantly, these residues are on a helical face adjacent to oppositely charged residues in helix 4 of the AR ligand-binding domain. Mutagenesis of these AR residues in helix 4, as well as mutation of lysine 720 in helix 3 (predicted to interact with the CoRNR box), markedly impaired AR recruitment of NCoR, indicating that N1 CoRNR box binding is being stabilized by these ionic interactions in the AR ligand-binding domain coactivator/corepressor binding site. Finally, results using a helix 12–deleted AR indicate that mifepristone induces allosteric changes in addition to helix 12 displacement that are critical for NCoR binding. These findings show that AR antagonists can enhance corepressor recruitment by stabilizing a distinct antagonist conformation of the AR coactivator/corepressor binding site and support the development of additional antagonists that may be able to further enhance AR recruitment of corepressors. [Mol Cancer Ther 2008;7(10):3187–94]

Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function

Basak, S., Pookot, D., Noonan, E. J., Dahiya, R. — 2008-10-13

Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid hormone receptor family and is very important for the development and progression of prostate cancer. The soy isoflavone genistein has been shown previously to down-regulate AR in androgen-dependent prostate cancer cell lines such as LNCaP. However, the mechanism(s) by which AR is down-regulated by genistein is still not known fully. We show a new mechanism by which genistein inhibits AR protein levels. We show that genistein-treated LNCaP cells exhibit increased ubiquitination of AR, suggesting that AR protein is down-regulated via a proteasome-mediated pathway. AR is normally stabilized by the chaperone activity of the heat shock protein Hsp90. The increased ubiquitination of AR after genistein treatment is attributed to decreased Hsp90 chaperone activity as assessed by its increased functionally inactive acetylated form. Consistent with this result, we find that HDAC6, which is a Hsp90 deacetylase, is inhibited by the antiestrogenic activity of genistein. Hence, in this study, we elucidate a novel mechanism of AR down-regulation by genistein through inhibition of HDAC6-Hsp90 cochaperone function required to stabilize AR protein. Our results suggest that genistein could be used as a potential chemopreventive agent for prostate cancers along with known inhibitors of HDAC6 and Hsp90. [Mol Cancer Ther 2008;7(10):3195–202]

Expression of the fat-1 gene diminishes prostate cancer growth in vivo through enhancing apoptosis and inhibiting GSK-3{beta} phosphorylation

2008-10-13

Epidemiologic studies inclusively indicate that "unhealthy" dietary fat intake is one of the potential risk factors for cancer. In dietary fat, there are two types of polyunsaturated fatty acids (PUFA), -3 (n-3) and -6 (n-6). Numerous studies support that the ratio of n-6/n-3 affects tumorigenesis. It was reported that adenoviral transfer of the fat-1 gene, which converts n-6 to n-3, into breast and lung cancer cells had an antitumor effect in vitro. However, the effects of the fat-1 gene expression on tumor growth in vivo have not been studied and the mechanisms remain unclear. Accordingly, prostate cancer DU145 and PC3 cells were transfected with either the fat-1 gene or a control vector. The cells that expressed the fat-1 gene had a lower n-6/n-3 PUFA ratio compared with the cells that expressed the control vector. The fat-1 gene expression significantly inhibited prostate cancer cell proliferation and invasion in vitro. The fat-1 and control vector-transfected prostate cancer cells were s.c. implanted into severe combined immunodeficient mice for 6 weeks. The fat-1 gene expression significantly diminished tumor growth in vivo, but the control vector had no effect. Finally, we evaluated signaling pathways that may be important for fat-1 gene function. Administration of n-3 PUFA induced caspase-3-mediated prostate cancer cell apoptosis in vitro. The fat-1 gene expression inhibited prostate cancer cell proliferation via reduction of GSK-3β phosphorylation and subsequent down-regulation of both β-catenin and cyclin D1. These results suggest that fat-1 gene transfer directly into tumor cells could be used as a novel therapeutic approach. [Mol Cancer Ther 2008;7(10):3203–11]

Bone marrow and tumor cell colony-forming units and human tumor xenograft efficacy of noncamptothecin and camptothecin topoisomerase I inhibitors

2008-10-13

Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC₉₀ values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC₉₀ values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity. [Mol Cancer Ther 2008;7(10):3212–22]

Human epidermal growth factor receptor (HER-1:HER-3) Fc-mediated heterodimer has broad antiproliferative activity in vitro and in human tumor xenografts

2008-10-13

All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-β1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was ~10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-β1 revealed that RB200 bound HER-1 ligands, including transforming growth factor- and heparin-binding EGF, and HER-3 ligands NRG1- and NRG1-β3. RB200 inhibited EGF- and NRG1-β1–stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid–stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3. [Mol Cancer Ther 2008;7(10):3223–36]

FOXO3a mediates the cytotoxic effects of cisplatin in colon cancer cells

Fernandez de Mattos, S., Villalonga, P., Clardy, J., Lam, E. W-F. — 2008-10-13

Cisplatin is a conventional chemotherapeutic agent that binds covalently to purine DNA bases and mediates cellular apoptosis. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action and help to understand the mechanism of drug resistance. In this study, we have investigated the effects of cisplatin in a panel of colon carcinoma cell lines and the involvement of the phosphoinositide-3-kinase/forkhead/winged helix box class O (FOXO) pathway in cisplatin action and resistance. Cisplatin-sensitive and cisplatin-resistant cell lines have been characterized in cell viability, flow cytometry, and clonogenic assays. The main components of the phosphoinositide-3-kinase/protein kinase B pathway, particularly FOXO3a, have been analyzed in sensitive and resistant cells on cisplatin treatment. Interestingly, in sensitive cells, cisplatin induces FOXO3a dephosphorylation and nuclear translocation, and expression of its target genes, whereas in resistant cells the effect of cisplatin on FOXO3a is incomplete. Consistent with this, protein kinase B/FOXO signaling axis modulators triciribine and psammaplysene A sensitize the resistant HT29 cells to cisplatin treatment. Critically, knockdown of FOXO3a expression using small interfering RNA rescues sensitive SW620 cells from cisplatin-induced short- and long-term cell death. Together, our findings suggest that FOXO3a is a relevant mediator of the cytotoxic effects of cisplatin in colon cancer cells. [Mol Cancer Ther 2008;7(10):3237–46]

Role of glutathione S-transferase P1-1 in the cellular detoxification of cisplatin

Peklak-Scott, C., Smitherman, P. K., Townsend, A. J., Morrow, C. S. — 2008-10-13

Cells expressing elevated levels of allelic variants of human glutathione S-transferase P1 (GSTP1) and/or efflux transporters, MRP1 or MRP2, were used to evaluate the role of GSTP1-1 in cisplatin resistance. These studies revealed that GSTP1-1 confers low-level resistance (1.4- to 1.7-fold) to cisplatin-induced cytotoxicity in MCF7 cells. However, expression of MRP1 (MCF7 cells) or MRP2 (HepG2 cells) failed to augment or potentiate GSTP1-1-mediated resistance in either cell line. To understand the mechanism by which variants of GSTP1-1 confer resistance to cisplatin, their relative abilities to catalyze conjugation of cisplatin with glutathione were examined. Enzymes encoded by all three alleles tested, GSTP1a (I₁₀₄A₁₁₃), GSTP1b (V₁₀₄A₁₁₃), and GSTP1c (V₁₀₄V₁₁₃), increased the formation rate of the mono-platinum/glutathione derivative of cisplatin with relative catalytic activities of 1.0 (GSTP1a-1a variant) and 1.8 to 1.9 (GSTP1b-1b and GSTP1c-1c variants). Although these data are consistent with the idea that very low level resistance to cisplatin may be conferred by GSTP1-1-mediated cisplatin/glutathione conjugation, two observations indicate that such catalysis plays a minor role in the protection from cisplatin toxicity. First, the rates of GSTP1-1-mediated conjugation are extremely slow (1.7-2.6 h^–1 at 25°C). Second, despite an 80% to 90% increase in catalysis of cisplatin conjugation by GSTP1b-1b or GSTP1c-1c over GSTP1a-1a, we observed no discernable differences in relative resistances conferred by these alternative variants when expressed in MCF7 cells. We conclude that high-level cisplatin resistance attributed to GSTP1-1 in other studies is not likely due to catalysis of cisplatin conjugation but rather must be explained by other mechanisms, which may include GSTP1-mediated modulation of signaling pathways. [Mol Cancer Ther 2008;7(10):3247–55]

Cisplatin abrogates the geldanamycin-induced heat shock response

2008-10-13

Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH₂-terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1 as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1–mediated Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70^+/+ IC₅₀ = 63.7 ± 14.9 nmol/L and Hsp70^–/– IC₅₀ = 4.3 ± 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1–mediated heat shock response may enhance the efficacy of Hsp90-directed therapy. [Mol Cancer Ther 2008;7(10):3256–64]

ABT-263 and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo

2008-10-13

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-x_L, and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G₀-G₁ arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G₀ cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma. [Mol Cancer Ther 2008;7(10):3265–74]

Antitumor activity and molecular effects of the novel heat shock protein 90 inhibitor, IPI-504, in pancreatic cancer

2008-10-13

Targeting Hsp90 is an attractive strategy for anticancer therapy because the diversity and relevance of biological processes are regulated by these proteins in most cancers. However, the role and mode of action of Hsp90 inhibitors in pancreatic cancer has not been studied. This study aimed to assess the antitumor activity of the Hsp90 inhibitor, IPI-504, in pancreatic cancer and to determine the biological effects of the agent. In vitro, we show that pharmacologic inhibition of Hsp90 by IPI-504 exerts antiproliferative effects in a panel of pancreatic cancer cells in a dose- and time-dependent manner. In pancreatic cancer xenografts obtained directly from patients with pancreas cancer, the agent resulted in a marked suppression of tumor growth. Although known Hsp90 client proteins were significantly modulated in IPI-504-treated cell line, no consistent alteration of these proteins was observed in vivo other than induction of Hsp70 expression in the treated xenografted tumors. Using a proteomic profiling analysis with isotope tags for relative and absolute quantitation labeling technique, we have identified 20 down-regulated proteins and 42 up-regulated proteins on IPI-504 treatment.tumor growth Identical changes were observed in the expression of the genes coding for these proteins in a subset of proteins including HSPA1B, LGALS3, CALM1, FAM84B, FDPS, GOLPH2, HBA1, HIST1H1C, HLA-B, and MARCKS. The majority of these proteins belong to the functional class of intracellular signal transduction, immune response, cell growth and maintenance, transport, and metabolism. In summary, we show that IPI-504 has potent antitumor activity in pancreatic cancer and identify potential pharmacologic targets using a proteomics and gene expression profiling. [Mol Cancer Ther 2008;7(10):3275–84]

Role of histone deacetylase inhibitor-induced reactive oxygen species and DNA damage in LAQ-824/fludarabine antileukemic interactions

2008-10-13

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 µmol/L) dramatically potentiated apoptosis (≥75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in -H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents. [Mol Cancer Ther 2008;7(9):3285–97]

Gambogic acid mediates apoptosis as a p53 inducer through down-regulation of mdm2 in wild-type p53-expressing cancer cells

2008-10-13

Gambogic acid (GA) is a natural product with potent apoptotic activity. Here, we showed that GA broadly inhibited the growth of cancer cells that expressed wild-type p53 as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-iumbromide assay, ³H-thymidine incorporation analysis, and an in vivo mouse xenograft model. GA induced massive cell apoptosis as judged by Annexin V and propidium iodide dual-staining experiments. Furthermore, we found that GA partially induced cancer cell growth inhibition in a p53-dependent manner because cell survival could be restored after endogenous p53 was attenuated by p53 transcriptional repressor pifithrin- or p53 small interfering RNA. Interestingly, GA had no influence on p53 mRNA synthesis but dramatically enhanced its protein expression. This unique observation could be accounted for by the down-regulation of mdm2 at both mRNA and protein levels. It is concluded that GA enhances p53 protein level through inhibition of mdm2 expression and thereby hampers p53 harboring tumor growth. [Mol Cancer Ther 2008;7(10):3298–305]

Coronarin D, a labdane diterpene, inhibits both constitutive and inducible nuclear factor-{kappa}B pathway activation, leading to potentiation of apoptosis, inhibition of invasion, and suppression of osteoclastogenesis

2008-10-13

Compounds isolated from members of the Zingiberaceae family are traditionally used as a medicine against inflammatory diseases, but little is known about the mechanism. Here, we report the isolation and structural identification of coronarin D [E-labda-8(17),12-diene-15-ol], a labdane-type diterpene, from Hedychium coronarium and delineate its mechanism of action. Because the transcription factor nuclear factor-B (NF-B) is a key mediator of inflammation, apoptosis, invasion, and osteoclastogenesis, we investigated the effect of coronarin D on NF-B activation pathway, NF-B-regulated gene products, and NF-B-regulated cellular responses. The coronarin D inhibited NF-B activation induced by different inflammatory stimuli and carcinogens. This labdane also suppressed constitutive NF-B activity in different cell lines and inhibited IB kinase activation, thus leading to the suppression of IB phosphorylation, degradation, p65 nuclear translocation, and reporter gene transcription. Coronarin D also inhibited the NF-B-regulated gene products involved in cell survival (inhibitor of apoptosis protein 1, Bcl-2, survivin, and tumor necrosis factor receptor-associated factor-2), proliferation (c-myc, cyclin D1, and cyclooxygenase-2), invasion (matrix metalloproteinase-9), and angiogenesis (vascular endothelial growth factor). Suppression of these gene products by the diterpene enhanced apoptosis induced by TNF and chemotherapeutic agents, suppressed TNF-induced cellular invasion, and abrogated receptor activator of NF-B ligand-induced osteoclastogenesis. Coronarin D was found to be more potent than its analogue coronarin D acid. Overall, our results show that coronarin D inhibited NF-B activation pathway, which leads to inhibition of inflammation, invasion, and osteoclastogenesis, as well as potentiation of apoptosis. [Mol Cancer Ther 2008;7(10):3306–17]

Curcumin suppresses constitutive activation of nuclear factor-{kappa}B and requires functional Bax to induce apoptosis in Burkitt's lymphoma cell lines

2008-10-13

We provide evidence that curcumin, a natural compound isolated from rhizomes of plant Curcuma longa, induces apoptosis in several Burkitt's lymphoma cell lines expressing Bax protein (AS283A, KK124, and Pa682PB), whereas it has no effects in cell lines with no Bax expression (BML895 and CA46). Our data show that curcumin treatment results in down-regulation of constitutive activation of nuclear factor-B (NF-B) via generation of reactive oxygen species where it causes conformational changes in Bax protein leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This leads to activation of caspase-9, caspase-3, and poly(ADP)-ribose polymerase cleavage leading to caspase-dependent apoptosis. In addition, curcumin treatment of Burkitt's lymphoma cell lines also causes up-regulation of DR5; however, this up-regulation does not result in apoptosis. Importantly, cotreatment with curcumin and TRAIL induces apoptosis in Bax-deficient cell lines. Taken together, our findings suggest that curcumin is able to induce apoptosis in Bax-positive cell lines, whereas combinations with TRAIL result in apoptosis in Bax-negative cell lines. These findings also raise the possibility that incorporation of curcumin in treatment regimens may provide a novel approach for the treatment of Burkitt's lymphomas and provide the molecular basis for such future translational efforts. [Mol Cancer Ther 2008;7(10):3318–29]