bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells
Authors: Edwards Lincoln A. , Verreault Maite , Thiessen Brian , Dragowska Wieslawa H. , Hu Yanping , Yeung Juliana H.F. , Dedhar Shoukat , Bally Marcel B.
Published: March 2006

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase–regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V–positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy. [Mol Cancer Ther 2006;5(3):645–54]
Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo
Authors: Zhang Sheng-Zhou , Pan Fei-Yan , Xu Jian-Feng , Yuan Jun , Guo Shi-Ying , Dai Gu , Xue Bin , Shen Wei-Gan , Wen Chuan-Jun , Zhao Dong-Hong , Li Chao-Jun
Published: March 2006

c-Met is highly expressed and constitutively activated in various human tumors. We employed adenovirus-mediated RNA interference technique to knock down c-Met expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. Among the five hepatocellular carcinoma and one normal human liver cell lines we analyzed, c-Met was highly expressed and constitutively tyrosine phosphorylated in only MHCC97-L and HCCLM3 hepatocellular carcinoma cells. Knockdown of c-Met could inhibit MHCC97-L cells proliferation by arresting cells at G0-G1 phase. Soft agar colony formation assay indicated that the colony forming ability of MHCC97-L cells decreased by ∼70% after adenovirus AdH1-small interfering RNA (siRNA)/met infection. In vivo experiments showed that adenovirus AdH1-siRNA/met inhibited the tumorigenicity of MHCC97-L cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of hepatocellular carcinoma in which c-Met is overexpressed.
Cyclooxygenase inhibitors in urinary bladder cancer:in vitroandin vivoeffects
Authors: Mohammed Sulma I. , Dhawan Deepika , Abraham Shaji , Snyder Paul W. , Waters David J. , Craig Bruce A. , Lu Ming , Wu Lan , Zheng Rong , Stewart Jane , Knapp Deborah W.
Published: March 2006

More than 14,000 people die from invasive transitional cell carcinoma (TCC) of the urinary bladder yearly in the United States. Cyclooxygenase (COX)-inhibiting drugs are emerging as potential antitumor agents in TCC. The optimal in vitro or in vivo systems to investigate COX inhibitor antitumor effects have not been defined. The purpose of this study was to determine COX-1 and COX-2 expression and antitumor effects of COX inhibitors in human TCC cell lines (HT1376, RT4, and UMUC3 cells) and xenografts derived from those cell lines. COX-2 expression (Western blot, immunocytochemistry) was high in HT1376, modest in RT4, and absent in UMUC3 cells in vitro. Similarly, COX-2 expression was noted in RT4 but not UMUC3 xenografts. COX-2 expression in HT1376 xenografts was slightly lower than that observed in vitro. None of four COX inhibitors evaluated (celecoxib, piroxicam, valeryl salicylate, and NS398) reduced TCC growth in standard in vitro proliferation assays at concentrations that could be safely achieved in vivo (≤5 μmol/L). Higher celecoxib concentrations (≥50 μmol/L) inhibited proliferation and induced apoptosis in all three cell lines. Celecoxib or piroxicam treatment in athymic mice significantly delayed progression of HT1376 xenografts, which express COX-2, but not UMUC3 xenografts that lack COX-2 expression. In conclusion, standard in vitro assays were not useful in predicting COX inhibitor antitumor effects observed in vivo. Athymic mice bearing TCC xenografts provide a useful in vivo system for COX inhibitor studies. Results of this study provide justification for further evaluation of COX inhibitors as antitumor agents against TCC. [Mol Cancer Ther 2006;5(2):329–36]
An oncolytic adenovirus expressing soluble transforming growth factor-β type II receptor for targeting breast cancer:in vitroevaluation
Authors: Wang Zhen-Guo , Zhao Wenli , Ramachandra Murali , Seth Prem
Published: March 2006

In recent years, adenoviruses that selectively replicate in tumor cells have been developed. However, there is a tremendous need to improve their anticancer efficacy. We wish to investigate whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of soluble form of transforming growth factor-β type II receptor (sTGFβRII) offers a therapeutic advantage. We chose to target TGF-βs because they play a pivotal role in late-stage tumorigenesis by enhancing tumor invasion and metastasis. A sTGFβRII cDNA was cloned in conditionally replicating adenoviral vector rAd-sTRII and in a replication-deficient adenovirus Ad-sTRII. Infection of MDA-MB-231 breast cancer cells with rAd-sTRII or Ad-sTRII followed by Western blot analysis indicated the expression of diffused glycosylated forms of sTGFβRII that were also secreted into the extracellular medium. The secreted proteins were shown to bind with TGF-β and antagonize TGF-β–induced p38 mitogen-activated protein kinase activity. However, marked differences in the replication potential of rAd-sTRII and Ad-sTRII were observed in breast tumor cells. Infection of MDA-MB-231 cells with rAd-sTRII resulted in cytotoxicity and significant increase in the adenoviral titers that were comparable with a wild-type adenovirus dl309. However, Ad-sTRII was much less toxic to the tumor cells, and the viral titers of Ad-sTRII remained relatively unchanged. These results suggest that the infection of breast tumor cells with conditionally replicating adenoviral vector rAd-sTRII produced sTGFβRII that can abrogate TGF-β signaling while maintaining the replication potential of the virus, indicating that rAd-sTRII could be a potential anticancer agent. [Mol Cancer Ther 2006;5(2):367–73]
Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons
Authors: Yamada Hiroshi Y. , Gorbsky Gary J.
Published: March 2006

Mitotic spindle poisons (e.g., Taxol and vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning, we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases associated with diverse cellular activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison–mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] resulted in a decrease of measurable cellular proteasome activity, indicating that OP-TRIP1(88-406) had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. Small interfering RNA (siRNA)–mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons. [Mol Cancer Ther 2006;5(1):29–38]
Sensitization of TRAIL–resistant cells by inhibition of heat shock protein 90 with low-dose geldanamycin
Authors: Ma Yulin , Lakshmikanthan Vijayabaskar , Lewis Ronald W. , Kumar M. Vijay
Published: March 2006

Due to its specificity and effectiveness, tumor necrosis factor-α–related apoptosis-inducing ligand (TRAIL) is being tested for cancer therapy. Inhibition of the function of heat shock protein 90 (HSP90) is under clinical trials for cancer therapy. However, some cancer cells are resistant to TRAIL, and at the dose required for inducing apoptosis, geldanamycin, a drug that inhibits HSP90 function, has shown adverse effects. Therefore, our working plan was to identify a sublethal dose of geldanamycin and combine it with TRAIL to induce apoptosis in TRAIL-resistant prostate cancer cells. Treatment of LNCaP with 250 nmol/L geldanamycin inhibited HSP90 function but did not induce significant apoptosis. However, combination of geldanamycin and TRAIL induced highly significant apoptosis in TRAIL-resistant LNCaP cells. In addition to inducing caspase activity and apoptosis, treatment with geldanamycin and TRAIL decreased inhibitor of κB (IκB) kinase (IKK) complex proteins, IKKα, IKKβ, and IKKγ. The loss of IKK affected IκBα/nuclear factor-κB (NF-κB) interaction and reduced nuclear transport of NF-κB, resulting in reduced NF-κB activity. Our data show increase in apoptosis using low, suboptimal dose of geldanamycin when used with TRAIL. These results provide a means to alleviate two problems: resistance to TRAIL and adverse effects of high-dose geldanamycin. [Mol Cancer Ther 5006;5(1):170–8]
Silencing of survivin gene by small interfering RNAs produces supra-additive growth suppression in combination with 17-allylamino-17-demethoxygeldanamycin in human prostate cancer cells
Authors: Paduano Francesco , Villa Raffaella , Pennati Marzia , Folini Marco , Binda Mara , Daidone Maria Grazia , Zaffaroni Nadia
Published: March 2006

Survivin is an antiapoptotic gene, which is overexpressed in most human tumors and involved in mitotic checkpoint control. Recent evidence points to an essential role for heat shock protein 90 (Hsp90) in survivin function regulation. Although the survivin-Hsp90 association may promote tumor cell proliferation, it may also suggest new opportunities for the design of novel anticancer approaches. We evaluated the effect of small interfering RNA (siRNA)–mediated inhibition of survivin on the proliferative potential of prostate cancer cells and their sensitivity to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Human androgen-independent prostate cancer cell lines (DU145 and PC-3) were transfected with four 21-mer double-stranded siRNAs (100 nmol/L) directed against different portions of survivin mRNA. After transfection, cells were collected and analyzed for survivin mRNA and protein expression, cell proliferation rate, ability to undergo apoptosis, and sensitivity to 17-AAG. Transfection of prostate cancer cells with siRNAs induced a variable extent of inhibition of survivin mRNA expression (39–60% compared with controls), which was paralleled by a 38% to 75% reduction in survivin protein abundance. The three siRNAs able to induce the greatest inhibition of survivin expression also significantly reduced cell proliferation and enhanced the rate of apoptosis, with a concomitant increase in caspase-9 activity. Sequential treatment with siRNA and 17-AAG induced supra-additive antiproliferative effects in all cell lines, with an enhanced caspase-9-dependent apoptotic response. These findings suggest that combined strategies aimed at interfering with the survivin-Hsp90 connection may provide novel approaches for treatment of androgen-independent prostate cancer. [Mol Cancer Ther 2006;5(1):179–86]
Sulindac sulfide–induced apoptosis is enhanced by a small-molecule Bcl-2 inhibitor and by TRAIL in human colon cancer cells overexpressing Bcl-2
Authors: Sinicrope Frank A. , Penington Robert C.
Published: March 2006

Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) that induces apoptosis in cultured colon cancer cells and in intestinal epithelia in association with its chemopreventive efficacy. Resistance to sulindac is well documented in patients with familial adenomatous polyposis; however, the molecular mechanisms underlying such resistance remain unknown. We determined the effect of ectopic Bcl-2 expression upon sulindac-induced apoptotic signaling in SW480 human colon cancer cells. Sulindac sulfide activated both the caspase-8-dependent and mitochondrial apoptotic pathways. Ectopic Bcl-2 attenuated cytochrome c release and apoptosis induction compared with SW480/neo cells. Coadministration of sulindac sulfide and the small-molecule Bcl-2 inhibitor HA14-1 increased apoptosis induction and enhanced caspase-8 and caspase-9 cleavage, Bax redistribution, and cytochrome c and second mitochondria-derived activator of caspase release. Given that sulindac sulfide activated caspase-8 and increased membrane death receptor (DR4 and DR5) protein levels, we evaluated its combination with the endogenous death receptor ligand tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Coadministration of sulindac sulfide and TRAIL cooperatively enhanced apoptotic signaling as effectively as did HA14-1. Together, these data indicate that HA14-1 or TRAIL can enhance sulindac sulfide–induced apoptosis and represent novel strategies for circumventing Bcl-2-mediated apoptosis resistance in human colon cancer cells.
Cross-talk between DNA damage and cell survival checkpoints during G2 and mitosis: pharmacologic implications
Authors: Bozko Przemyslaw , Sabisz Michal , Larsen Annette K. , Skladanowski Andrzej
Published: March 2006

In this study, we wanted to clarify the role of survivin-mediated survival signaling during G2 and M in tumor cells treated with DNA-damaging agents. As a cellular model, we selected MOLT-4 human T-cell lymphoblastic leukemia cells that overexpress survivin and nonfunctional p53. Treatment with melphalan, a classic DNA-damaging agent, led to the induction of the DNA damage checkpoint and growth arrest in the G2 phase of the cell cycle. Checkpoint abrogation by caffeine was accompanied by mitotic entry and rapid apoptotic cell death, whereas cells remaining in G2 remained viable during the same time interval. Unexpectedly, when the spindle checkpoint was activated following G2 abrogation, two different effects could be observed. If the microtubules of the melphalan-treated cells were destabilized by nocodazole, cells became arrested in prometaphase with low survivin levels and entered apoptosis. In contrast, if the microtubules of the melphalan-treated cells were stabilized by taxol, cells were still arrested in prometaphase, but apoptotic execution was inhibited. This effect is, most likely, directly mediated by survivin itself given its well-established antiapoptotic functions. In conclusion, depending on the way the spindle checkpoint was activated in cells with damaged DNA, cells could be either protected by survivin or die during mitosis. We suggest that the efficacy of DNA damage checkpoint abrogators used in combination with DNA-damaging agents may critically depend on whether DNA damage is able to invoke spindle checkpoint response and to activate survivin-associated survival signaling during mitosis. [Mol Cancer Ther 2005;4(12):2016–25]
Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures
Authors: Mahadevan Daruka , Spier Catherine , Della Croce Kimiko , Miller Susan , George Benjamin , Riley Chris , Warner Stephen , Grogan Thomas M. , Miller Thomas P.
Published: March 2006

To glean biological differences and similarities of peripheral T-cell lymphoma–not otherwise specified [PTCL-NOS] to diffuse large B-cell lymphoma (DLBCL), a transcriptosome analysis was done on five PTCL-NOS and four DLBCL patients and validated by quantitative real-time reverse transcription-PCR on 10 selected genes. Normal peripheral blood T cells, peripheral blood B cells, and lymph node were used as controls. The resultant gene expression profile delineated distinct “tumor profile signatures” for PTCL-NOS and DLBCL. Several highly overexpressed genes in both PTCL-NOS and DLBCL involve the immune network, stroma, angiogenesis, and cell survival cascades that make important contributions to lymphomagenesis. Inflammatory chemokines and their receptors likely play a central role in these complex interrelated pathways: CCL2 and CXCR4 in PTCL-NOS and CCL5 and CCR1 in DLBCL. Highly overexpressed oncogenes unique to PTCL-NOS are SPI1, STK6, α-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to both lymphomas are MAFB, MET, NF-κB2, LCK, and LYN. Several tumor suppressors are also down-regulated (TPTE, MGC154, PTCH, ST5, and SUI1). This study illustrates the relevance of tumor-stroma immune trafficking and identified potential novel prognostic markers and targets for therapeutic intervention. [Mol Cancer Ther 2005;4(12):1867–79]
Using bioinformatics and genome analysis for new therapeutic interventions
Authors: Mount David W. , Pandey Ritu
Published: March 2006

The genome era provides two sources of knowledge to investigators whose goal is to discover new cancer therapies: first, information on the 20,000 to 40,000 genes that comprise the human genome, the proteins they encode, and the variation in these genes and proteins in human populations that place individuals at risk or that occur in disease; second, genome-wide analysis of cancer cells and tissues leads to the identification of new drug targets and the design of new therapeutic interventions. Using genome resources requires the storage and analysis of large amounts of diverse information on genetic variation, gene and protein functions, and interactions in regulatory processes and biochemical pathways. Cancer bioinformatics deals with organizing and analyzing the data so that important trends and patterns can be identified. Specific gene and protein targets on which cancer cells depend can be identified. Therapeutic agents directed against these targets can then be developed and evaluated. Finally, molecular and genetic variation within a population may become the basis of individualized treatment.
Genes to vaccines for immunotherapy: how the molecular biology revolution has influenced cancer immunology
Authors: Laheru Dan A. , Pardoll Drew M. , Jaffee Elizabeth M.
Published: March 2006

Recent advances in our understanding of the complex signaling pathways involved in immune system regulation, along with analyses of genetic differences between tumors and their normal cellular counterparts, have accelerated development of immune-based strategies for cancer treatment and prevention. More clinically relevant animal models have shown that successful immune-based strategies will require the integration of interventions that target specific tumor antigens with regulators of the antitumor immune response. Immunotherapy for cancer is at a critical crossroad, as therapeutics designed to target cancer-associated antigens and regulatory signaling molecules enter clinical trials. We outline here a paradigm for early-stage clinical development of immunotherapy combinations that use vaccines to drive tumor antigen-specific responses while simultaneously targeting immune regulatory pathways.
Antitumor efficacy of a urokinase activation–dependent anthrax toxin
Authors: Rønø Birgitte , Rømer John , Liu Shihui , Bugge Thomas H. , Leppla Stephen H. , Kristjansen Paul E.G.
Published: March 2006

Previously, we have generated a potent prodrug consisting of modified anthrax toxins that is activated by urokinase plasminogen activator (uPA). The cytotoxicity of the drug, PrAg-U2 + FP59, is dependent on the presence of receptor-associated uPA activity. Local intradermal administration of PrAg-U2 + FP59 adjacent to the tumor nodules in mice with transplanted solid tumors had a potent antitumor effect. In succession of these experiments, we have now investigated the systemic antitumor efficacy of PrAg-U2 + FP59. C57Bl/6J mice bearing syngenic tumors derived from B16 melanoma, T241 fibrosarcoma, or Lewis lung carcinoma cells were treated with different mass ratios and doses of PrAg-U2 + FP59. Tumor volumes were recorded daily by caliper measurements. In some experiments, dexamethasone was coadministered. Our data show a significant antitumor effect of systemic administration of PrAg-U2 + FP59 in three syngenic tumor models. Optimal antitumor effect and low toxicity was obtained with a 25:1 mass ratio between the two components (PrAg-U2 and FP59). The experiments show that PrAg-U2 + FP59 displays a clear dose-response relationship with regard to both antitumor efficacy and systemic toxicity. Dose-limiting toxicity seemed to be due to activation of the prodrug by uPA and its receptor in the intestinal mucosa. Concurrent treatment with dexamethasone was found to prevent dose-limiting toxicity. Taken together, these data indicate that uPA-activated toxins may be promising candidates for targeted therapy of human cancers that overexpress uPA and its receptor. [Mol Cancer Ther 2006;5(1):89–96]
WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway
Authors: Kosakowska-Cholody Teresa , Cholody W. Marek , Monks Anne , Woynarowska Barbara A. , Michejda Christopher J.
Published: March 2006

WMC-79 is a synthetic agent with potent activity against colon and hematopoietic tumors. In vitro, the agent is most potent against colon cancer cells that carry the wild-type p53 tumor suppressor gene (HCT-116 and RKO cells: GI50 <1 nmol/L, LC50 ∼40 nmol/L). Growth arrest of HCT-116 and RKO cells occurs at the G1 and G2-M check points at sublethal concentrations (10 nmol/L) but the entire cell population was killed at 100 nmol/L. WMC-79 is localized to the nucleus where it binds to DNA. We hypothesized that WMC-79 binding to DNA is recognized as an unrepairable damage in the tumor cells, which results in p53 activation. This triggers transcriptional up-regulation of p53-dependent genes involved in replication, cell cycle progression, growth arrest, and apoptosis as evidenced by DNA microarrays. The change in the transcriptional profile of HCT-116 cells is followed by a change in the levels of cell cycle regulatory proteins and apoptosis. The recruitment of the p53-dependent apoptosis pathway was suggested by the up-regulation of p53, p21, Bax, DR-4, DR-5, and p53 phosphorylated on Ser15; down-regulation of Bcl-2; and activation of caspase-8, -9, -7, and -3 in cells treated with 100 nmol/L WMC-79. Apoptosis was also evident from the flow cytometric studies of drug-treated HCT-116 cells as well as from the appearance of nuclear fragmentation. However, whereas this pathway is important in wild-type p53 colon tumors, other pathways are also in operation because colon cancer cell lines in which the p53 gene is mutated are also affected by higher concentrations of WMC-79.
Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation
Authors: Paz Keren , Brennan Laura A. , Iacolina Michelle , Doody Jacqueline , Hadari Yaron R. , Zhu Zhenping
Published: March 2006

Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human single-domain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules.
Determination of the importance of the stereochemistry of psorospermin in topoisomerase II–induced alkylation of DNA and in vitro and in vivo biological activity
Authors: Fellows Ingrid M. , Schwaebe Michael , Dexheimer Thomas S. , Vankayalapati Hariprasad , Gleason-Guzman Mary , Whitten Jeffrey P. , Hurley Laurence H.
Published: March 2006

Psorospermin is a natural product that has been shown to have activity against drug-resistant leukemia lines and AIDS-related lymphoma. It has also been shown to alkylate DNA through an epoxide-mediated electrophilic attack, and this alkylation is greatly enhanced at specific sites by topoisomerase II. In this article, we describe the synthesis of the two diastereomers of O5-methyl psorospermin and their in vitro activity against a range of solid and hematopoietic tumors. The diastereomeric pair (±)-(2′R,3′R) having the naturally occurring enantiomer (2′R,3′R) is the most active across all the cell lines and shows approximately equal activity in both drug-sensitive and drug-resistant cell lines. In subsequent studies using all four enantiomers of O5-methyl psorospermin, the order of biological potency is (2′R,3′R) > (2′R,3′S) = (2′S,3′R) > (2′S,3′S). This order of potency is also found in the topoisomerase II–induced alkylation of O5-methyl psorospermin and can be rationalized by molecular modeling of the psorospermin-duplex binding complex. Therefore, this study defines the optimum stereochemical requirements for both the topoisomerase II–induced alkylation of DNA and the biological activity by psorospermin and its O5-methyl derivatives. Finally, (2′R,3′R) psorospermin was found to be as effective as gemcitabine in slowing tumor growth in vivo in a MiaPaCa pancreatic cancer model. In addition, (2′R,3′R) psorospermin in combination with gemcitabine was found to show an at least additive effect in slowing tumor growth of MiaPaCa.
A β-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy
Authors: Harding Fiona A. , Liu Amy D. , Stickler Marcia , Razo O. Jennifer , Chin Regina , Faravashi Nargol , Viola Wendy , Graycar Tom , Yeung V. Pete , Aehle Wolfgang , Meijer Daan , Wong Stephanie , Rashid M. Harunur , Valdes Ana M. , Schellenberger Volker
Published: March 2006

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. β-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of β-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. β-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The β-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of β-lactamase variant with adjuvant. The β-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.
Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment
Authors: Westfall Suzanne D. , Skinner Michael K.
Published: March 2006

Signal transduction pathways associated with cancer progression and chemotherapeutic resistance are being investigated as molecular targets for chemotherapy. The phosphatidylinositol 3-kinase (PI3K) pathway has been found to be frequently amplified and have increased activity in ovarian cancer. The current study investigates the efficacy of an antagonist of PI3K, LY294002, in inhibiting ovarian cancer cell growth and survival both in vitro and in vivo. The hypothesis tested is that inhibition of PI3K signaling makes ovarian cancer cells susceptible to the effects of platinum-based chemotherapy. Observations show that LY294002 is an effective inhibitor of ovarian cancer cell growth and survival in vitro. Inhibition of PI3K/Akt signaling increased the sensitivity of ovarian cell cultures to the cytotoxic effects of carboplatin. The combined treatment of LY294002 and carboplatin was needed to optimally promote cellular apoptosis and decrease ovarian cancer cell survival in vitro. To extend these observations, a model involving in vivo i.p. growth of human ovarian tumors in a nude mouse was used. LY294002 in combination with carboplatin was more effective in inhibiting ovarian cancer cell xenograft growth than either agent alone. The results of this study suggest that the combined treatment of carboplatin and LY294002 can effectively decrease ovarian tumor progression and support the use of a PI3K inhibitor (e.g., LY294002) as an adjunct platinum-based drug therapy for treatment of ovarian cancer.
Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv–expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion
Authors: Zhang Hongtao , Knutson Keith L. , Hellstrom Karl Erik , Disis Mary L. , Hellstrom Ingegerd
Published: March 2006

Tumor-destructive immune responses can be generated by engaging CD137 (4-1BB) via infusing a monoclonal antibody specific for CD137 or vaccinating with a single-chain Fv (scFv) CD137-expressing whole-cell tumor vaccine. We assessed whether such a vaccine can induce tumor rejection in the neu-transgenic (neu-Tg) mouse breast cancer model and compared the antitumor efficacy of vaccination with the infusion of a CD137-specific antibody. Mammary carcinoma cells (MMC) from a neu-Tg mouse were transfected to stably express surface scFv derived from the anti-CD137 rat hybridoma 1D8 or 3H3. The anti-CD137 scFv-expressing cells were rejected when transplanted into neu-Tg mice by a mechanism that involved both CD4+ and CD8+ T cells, and vaccination with such cells delayed the outgrowth of MMC cells transplanted 3 days previously. T cells from neu-Tg mice that had been vaccinated proliferated and produced IFN-γ when stimulated by MMC but not by antigen-negative variant breast cancer cells that did not express the neu tumor antigen. In addition, antibodies binding to the MMC but not to antigen-negative variant cells were detected in sera from some but not all of the immunized mice. Complete regression of s.c. transplanted MMC tumors was observed in mice repeatedly immunized against MMC-1D8 starting on the day the MMC cells were transplanted. In contrast, repeated administration of either of two different anti-CD137 monoclonal antibodies did not induce complete tumor regression, although tumor growth was delayed. [Mol Cancer Ther 2006;5(1):149–55]
The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells
Authors: Hsi Linda C. , Xi Xiaopei , Wu Yuanqing , Lippman Scott M.
Published: March 2006

DNA methylation by DNA methyltransferases in CpG-rich promoter regions of genes is a well-described component of epigenetic silencing in human cells. Dysregulation of this process in cancer cells may lead to hypermethylation of promoter CpG islands, thus disabling transcription initiation of certain genes, such as tumor suppressor genes. Reversing epigenetic silencing and up-regulating genes involved in preventing or reversing the malignant phenotype has become a new, important targeted approach for cancer prevention and treatment. Therefore, methyltransferase inhibitors (MTI) have emerged recently as promising chemotherapeutic or preventive agents. The potent MTI 5-aza-2-deoxycytidine (5-Azadc) causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. The present study shows that low micromolar concentrations of 5-Azadc induce the expression of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer cells. The expression of 15-LOX-1 correlates with 5-Azadc-induced increases in 13-S-hydroxyoctadecadienoic acid levels, growth inhibition, and apoptosis in these cells. Furthermore, specific inhibition of 15-LOX-1 by pharmacologic means or small interfering RNA significantly reduced the 5-Azadc-induced effects. These novel findings are the first demonstration of a mechanistic link between the induction of 15-LOX-1 by a MTI and apoptosis in cancer cells. This result has important implications for the study of 5-Azadc and other MTIs in the prevention and therapy of colorectal cancer and supports future investigations of the mechanisms by which MTIs up-regulate 15-LOX-1.
Lipid rafts remodeling in estrogen receptor–negative breast cancer is reversed by histone deacetylase inhibitor
Authors: Ostapkowicz Anna , Inai Kunihiro , Smith Leia , Kreda Silvia , Spychala Jozef
Published: March 2006

Recently, we have found dramatic overexpression of ecto-5′-nucleotidase (or CD73), a glycosylphosphatidylinositol-anchored component of lipid rafts, in estrogen receptor–negative [ER(−)] breast cancer cell lines and in clinical samples. To find out whether there is a more general shift in expression profile of membrane proteins, we undertook an investigation on the expression of selected membrane and cytoskeletal proteins in aggressive and metastatic breast cancer cells. Our analysis revealed a remarkably uniform shift in expression of a broad range of membrane, cytoskeletal, and signaling proteins in ER(−) cells. A similar change was found in two in vitro models of transition to ER(−) breast cancer: drug-resistant Adr2 and c-Jun-transformed clones of MCF-7 cells. Interestingly, similar expression pattern was observed in normal fibroblasts, suggesting the commonality of membrane determinants of invasive cancer cells with normal mesenchymal phenotype. Because a number of investigated proteins are components of lipid rafts, our results suggest that there is a major remodeling of lipid rafts and underlying cytoskeleton in ER(−) breast cancer. To test whether this broadly defined ER(−) phenotype could be reversed by treatment with differentiating agent, we treated ER(−) cells with trichostatin A, an inhibitor of histone deacetylase, and observed reversal of mesenchymal and reappearance of epithelial markers. Changes in gene and protein expression also included increased capacity to generate adenosine and altered expression profile of adenosine receptors. Thus, our results suggest that during transition to invasive breast cancer there is a significant structural reorganization of lipid rafts and underlying cytoskeleton that is reversed upon histone deacetylase inhibition. [Mol Cancer Ther 2006;5(2):238–45]
The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line
Authors: Chattopadhyay Shrikanta , Zhao Rongbao , Krupenko Sergey A. , Krupenko Natalia , Goldman I. David
Published: March 2006

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non–small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced (∼85%) but intracellular pemetrexed levels increased to ∼60% and ∼70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. [Mol Cancer Ther 2006;5(2):438–49]
Class III β-tubulin expression in tumor cells predicts response and outcome in patients with non–small cell lung cancer receiving paclitaxel
Authors: Sève Pascal , Mackey John , Isaac Sylvie , Trédan Olivier , Souquet Pierre-Jean , Pérol Maurice , Lai Raymond , Voloch Alain , Dumontet Charles
Published: March 2006

Both fundamental and clinical studies suggest that class III β-tubulin expression is associated with resistance to taxanes and constitutes a prognostic factor in several solid tumors. In this study, we assessed the prognostic and predictive value of class III β-tubulin in tumors of patients with locally advanced or metastatic non–small cell lung cancer (NSCLC) treated with paclitaxel-based or other regimens that did not include tubulin-binding agents. Expression of class III β-tubulin was examined immunohistochemically in 91 tumor samples obtained before treatment from patients with stage III and IV NSCLC, including 47 who received paclitaxel-based regimens and 44 who received regimens without tubulin-binding agents. Response to chemotherapy, progression-free survival, and overall survival were correlated with the expression of class III β-tubulin protein. The response rate was 37.5% (16 responses among 45 evaluable patients) among patients receiving paclitaxel. Patients whose tumors expressed low levels of class III β-tubulin isotype had a better response rate, longer progression-free survival, and overall survival (P < 0.001, 0.004, and 0.002, respectively), whereas this variable was not found to be predictive in patients receiving regimens without tubulin-binding agents. A multivariate analysis taking into account sex, age, histology, stage, and class III β-tubulin confirmed that low-level class III β-tubulin expression was independently correlated with progression-free survival (P = 0.003) and overall survival (P = 0.003). These findings suggest that the expression levels of class III β-tubulin in tumor cells is predictive of response to therapy and patient outcome in patients with NSCLC receiving paclitaxel-based chemotherapy but is not a general prognostic factor in this patient population. [Mol Cancer Ther 2005;4(12):2001–7]
Novel missense mutation of the DNA topoisomerase I gene in SN-38-resistant DLD-1 cells
Authors: Arakawa Yasuhiro , Suzuki Hideaki , Saito Shinobu , Yamada Hisashi
Published: March 2006

Irinotecan hydrochloride, a camptothecin derivative, is one of the most effective drugs for colorectal cancer, and SN-38 is its main active metabolite. Development of resistance is a major obstacle to the clinical application of this drug. We established an SN-38-resistant subline from DLD-1 human colon cancer cells by continuous exposure to SN-38 and studied the mechanisms of resistance. The resistant subline (designated as DLDSNR6) had 10- to 100-fold higher resistance to camptothecin derivatives but showed no cross-resistance to doxorubicin, mitomycin C, and etoposide. DLDSNR6 cells carried a missense mutation in one allele of the DNA topoisomerase I gene that substituted glycine for serine at amino acid residue 365 accompanied by loss of the latter part of the remaining wild-type allele. Topoisomerase I expression was equal in DLDSNR6 and DLD-1 cells, but the nuclear extract of DLDSNR6 cells showed lower topoisomerase I catalytic activity. Moreover, exposure to camptothecin caused less accumulation of topoisomerase I-DNA complexes in DLDSNR6 cells than in DLD-1 cells. These findings suggest that the mutation interfered with both the catalytic activity of topoisomerase I and the stability of the ternary complex between topoisomerase I, DNA, and SN-38. This SN-38-resistant DLDSNR6 cell line may be useful for understanding the mechanisms of topoisomerase I function and drug-enzyme interactions. [Mol Cancer Ther 2006;5(3):502–8]
Guggulsterones induce apoptosis and differentiation in acute myeloid leukemia: identification of isomer-specific antileukemic activities of the pregnadienedione structure
Authors: Samudio Ismael , Konopleva Marina , Safe Stephen , McQueen Teresa , Andreeff Michael
Published: March 2006

In this study, the antileukemic effects of three isomeric pregnadienedione steroids [i.e., cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone] were investigated in HL60 and U937 cells as well as in primary leukemic blasts in culture. Our results show that all three compounds inhibited the proliferation of HL60 and U937 cells, with IC50s ranging from 3.6 to 10.9 μmol/L after treatment for 6 days. These growth inhibitory effects correlated with externalization of phosphatidylserine and loss of mitochondrial membrane potential, suggesting that these isomeric steroids induce apoptosis in leukemia cells. z-VAD-fmk prevented phosphatidylserine externalization but not mitochondrial membrane potential loss, indicating that mitochondrial dysfunction occurred in the absence of caspase activation. Interestingly, although all three compounds increased the generation of reactive oxygen species and decreased phosphorylation of extracellular signal-regulated kinase, only cis-guggulsterone induced a rapid depletion of reduced glutathione levels and oxidation of the mitochondrial phospholipid cardiolipin. 16-Dehydroprogesterone and trans-guggulsterone induced differentiation of HL60 and NB4 cells as evidenced by increased surface expression of CD11b and/or CD14, and all three steroids rapidly induced mitochondrial dysfunction and phosphatidylserine externalization of CD34-positive blasts from primary leukemic samples. This study is the first to show that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of apoptosis and differentiation and, more importantly, identifies the pregnadienedione structure as a potential chemotherapeutic scaffold. [Mol Cancer Ther 2005;4(12):1982–92]
Novel human monoclonal antibodies to insulin-like growth factor (IGF)-II that potently inhibit the IGF receptor type I signal transduction function
Authors: Feng Yang , Zhu Zhongyu , Xiao Xiaodong , Choudhry Vidita , Barrett J. Carl , Dimitrov Dimiter S.
Published: March 2006

The insulin-like growth factor (IGF) system plays an important role in a variety of physiologic processes and in diseases such as cancer. Although the role of the IGF system in cancer has been recognized many years ago, components of the system have only recently been targeted and shown to affect cell transformation, proliferation, survival, motility, and migration in tissue cultures and in mouse models of cancer. We have been hypothesizing that targeting IGF-II in addition to blocking its interaction with the IGF receptor type I (IGF-IR) would also allow to block that portion of the signal transduction through the insulin receptor that is due to its interaction with IGF-II. Lowering its level may also not induce up-regulation of its production as for IGF-I. Finally, targeting a diffusable ligand as IGF-II may not require penetration of the antibody inside tumors but could shift the equilibrium to IGF-II complexed with antibody so the ligand concentration would decrease in the tumor environment without the need for the antibody to penetrate the tumor. Here, we describe the identification and characterization of three novel anti-IGF-II fully human monoclonal antibodies. They bound with high (subnanomolar) affinity to IGF-II, did not cross-react with IGF-I and insulin, and potently inhibited signal transduction mediated by the IGF-IR interaction with IGF-II. The most potent neutralizer, IgG1 m610, inhibited phosphorylation of the IGF-IR and the insulin receptor, as well as phosphorylation of the downstream kinases Akt and mitogen-activated protein kinase with an IC50 of the order of 1 nmol/L at IGF-II concentration of 10 nmol/L. It also inhibited growth of the prostate cancer cell line DU145 and migration of the breast cancer line cells MCF-7. These results indicate an immunotherapeutic potential of IgG1 m610 likely in combination with other antibodies and anticancer drugs but only further experiments in mouse models of cancer and human clinical trials could evaluate this possibility. [Mol Cancer Ther 2006;5(1):114–20]