bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the Mevalonate Pathway Inhibits the Function of the Epidermal Growth Factor Receptor
Authors: Mantha Angela J. , Hanson Jennifer E.L. , Goss Glenwood , Lagarde Alain E. , Lorimer Ian A. , Dimitroulakos Jim
Published: September 2005

Purpose: The epidermal growth factor receptor (EGFR) is a key regulator of growth, differentiation, and survival of epithelial cancers. In a small subset of tumors, the presence of activating mutations within the ATP binding site confers increased susceptibility to gefitinib, a potent tyrosine kinase inhibitor of EGFR. Agents that can inhibit EGFR function through different mechanisms may enhance gefitinib activity in patients lacking these mutations. Mevalonate metabolites play significant roles in the function of the EGFR; therefore, mevalonate pathway inhibitors may potentiate EGFR-targeted therapies.

Experimental Design: In this study, we evaluated the effect of lovastatin on EGFR function and on gefitinib activity. Effects on EGFR function were analyzed by Western blot analysis using phosphospecific antibodies to EGFR, AKT, and extracellular signal-regulated kinase. Cytotoxic effects of lovastatin and/or gefitinib were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.

Results: Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation by 24 hours that was reversed by the coadministration of mevalonate. Combining lovastatin and gefitinib treatments showed enhanced inhibition of AKT activation by EGF in SCC9 cells. The combination of 10 μmol/L lovastatin and 10 μmol/L gefitinib treatments showed cooperative cytotoxicity in all 8 squamous cell carcinomas, 4 of 4 non–small cell lung carcinoma and 4 of 4 colon carcinoma cell lines tested. Isobologram and flow cytometric analyses of three representative cell lines with wild-type EGFR ATP binding sites confirmed that this combination was synergistic inducing a potent apoptotic response.

Conclusions: Taken together, these results show that targeting the mevalonate pathway can inhibit EGFR function. They also suggest the potential utility of combining these clinically relevant therapeutic approaches.
Differential DNA Hypermethylation of Critical Genes Mediates the Stage-Specific Tobacco Smoke-Induced Neoplastic Progression of Lung Cancer
Authors: Russo Andrea L. , Thiagalingam Arunthathi , Pan Hongjie , Califano Joseph , Cheng Kuang-hung , Ponte Jose F. , Chinnappan Dharmaraj , Nemani Pratima , Sidransky David , Thiagalingam Sam
Published: September 2005

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non–small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer–associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.
Characterization of Chemical Constituents inScutellaria baicalensiswith Antiandrogenic and Growth-Inhibitory Activities toward Prostate Carcinoma
Authors: Bonham Michael , Posakony Jeff , Coleman Ilsa , Montgomery Bruce , Simon Julian , Nelson Peter S.
Published: September 2005

Purpose: Botanical preparations are widely used by patients with prostate cancer. Scutellaria baicalensis, a botanical with a long history of medicinal use in China, was a constituent of the herbal mixture PC-SPES, a product that inhibited prostate cancer growth in both laboratory and clinical studies. Due to the difficulties encountered when evaluating the efficacy of complex natural products, we sought to identify active chemical constituents within Scutellaria and determine their mechanisms of action.

Experimental Design and Results: We used high-performance liquid chromatography to fractionate S. baicalensis and identified four compounds capable of inhibiting prostate cancer cell proliferation; baicalein, wogonin, neobaicalein, and skullcapflavone. Comparisons of the cellular effects induced by the entire extract versus the four-compound combination produced comparable cell cycle changes, levels of growth inhibition, and global gene expression profiles (r2 = 0.79). Individual compounds exhibited antiandrogenic activities with reduced expression of the androgen receptor and androgen-regulated genes. In vivo, baicalein (20 mg/kg/d p.o.) reduced the growth of prostate cancer xenografts in nude mice by 55% at 2 weeks compared with placebo and delayed the average time for tumors to achieve a volume of ∼1,000 mm3 from 16 to 47 days (P < 0.001).

Conclusions: Most of the anticancer activities of S. baicalensis can be recapitulated with four purified constituents that function in part through inhibition of the androgen receptor signaling pathway. We conclude that clinical studies evaluating the efficacy of these agents in the context of chemoprevention or the treatment of prostate cancer are warranted.
Carbamoylphosphonate Matrix Metalloproteinase Inhibitors 3: In vivo Evaluation of Cyclopentylcarbamoylphosphonic Acid in Experimental Metastasis and Angiogenesis
Authors: Reich Reuven , Katz Yiffat , Hadar Rivka , Breuer Eli
Published: September 2005

The spread of malignant tumor cells from a primary neoplasm to distant organs where they multiply and form new foci is the major cause of death from cancer. Despite the different modalities of cancer treatment, no effective curative therapy of metastatic lesions is available. To possess metastatic potential, a cell has to be able to invade the surrounding tissue, spread via lymphatics and/or the bloodstream, extravasate, and multiply at secondary sites. There is increasing evidence for a positive correlation between matrix metalloproteinase-2 (MMP-2) activity and tumor cell invasion. Agents blocking MMP-2 have been shown to prevent tumor cell invasion in vitro and in vivo. Inhibition of MMPs has, therefore, become the focus of considerable interest in connection with a variety of potential therapeutic applications. We have discovered a nontoxic MMP-2–selective inhibitor effective at nanomolar range on recombinant MMP. This compound, cyclopentylcarbamoylphosphonic acid, significantly inhibited cellular invasion and capillary formation in vitro. Further, i.p. or oral administration of the compound significantly reduced lung metastasis formation and s.c. tumor growth in a murine melanoma model. The effect of this novel compound on lung colonization, capillary formation, and s.c. tumor growth indicates that the compound might also be effective in treatment of primary tumor growth in reduction, or at least in prevention, of further tumor growth, thereby reducing the tumor burden of the patient by a nontoxic approach.
(−)-Epigallocatechin Gallate and Polyphenon E Inhibit Growth and Activation of the Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor-2 Signaling Pathways in Human Colon Cancer Cells
Authors: Shimizu Masahito , Deguchi Atsuko , Lim Jin T.E. , Moriwaki Hisataka , Kopelovich Levy , Weinstein I. Bernard
Published: September 2005

Purpose: (−)-Epigallocatechin gallate (EGCG) inhibits activation of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) and multiple downstream signaling pathways in cancer cell lines. In this study we compared the cellular and molecular effects of EGCG with a well-standardized decaffeinated green tea catechin mixture Polyphenon E (Poly E) on human colon cancer cell lines.

Experimental Design and Results: Both EGCG and Poly E preferentially inhibited growth of the Caco2, HCT116, HT29, SW480, and SW837 colon cancer cells when compared with the FHC normal human fetal colon cell line. The EGFR and HER2 proteins were overexpressed and constitutively activated in all of the colon cancer cell lines when compared with the FHC cell line. Treatment of HT29 cells with EGCG or Poly E caused an increase of cells in G1 and induced apoptosis. Both EGCG and Poly E caused a decrease in the phosphorylated forms of EGFR and HER2 proteins, and subsequently caused a decrease in the phosphorylated forms of the extracellular signal-regulated kinase and Akt proteins. Similar effects of these compounds were seen when the cells were stimulated with transforming growth factor α. Reporter assays indicated that both EGCG and Poly E inhibited the transcriptional activity of the activator protein 1 (AP-1), c-fos, nuclear factor κB, and cyclin D1 promoters. The combination of only 1 μg/mL of epicatechin plus 10 μg/mL of EGCG displayed synergistic effects on growth inhibition and induction of apoptosis. Furthermore, when treatment was prolonged for 96 hours, 1 μg/mL of EGCG or Poly E was sufficient to inhibit growth, reduce activation of EGFR and HER2, and induce apoptosis.

Conclusion: Our findings suggest that EGCG or Poly E may be useful in the chemoprevention and/or treatment of colon cancer. Poly E contains about 60% EGCG, yet pure EGCG and Poly E had similar potencies (expressed as μg/ml). Poly E may be preferable because it is easier to prepare and this mixture of catechins may exert synergistic effects.
Single Nucleotide Polymorphism of Pi-Class Glutathione S-Transferase and Susceptibility to Endometrial Carcinoma
Authors: Chan Queeny K.Y. , Khoo Ui-Soon , Ngan Hextan Y.S. , Yang Chong-Qing , Xue Wei-Cheng , Chan Kelvin Y.K. , Chiu Pui-Man , Ip Philip P.C. , Cheung Annie N.Y.
Published: September 2005

Purpose: Endometrial carcinoma is the most common gynecologic cancer in developed countries. Prolonged unopposed estrogen exposure has been identified as the major risk factor. The pi-class glutathione S-transferase (GSTP1) is a phase II metabolic enzyme that is important in the detoxification of a wide range of electrophiles including carcinogenic steroid-hormone intermediates generated through oxidative metabolism. In this study, we aimed at determining the association between the GSTP1 polymorphism and the risk of endometrial carcinoma in a Chinese population.

Experimental Design: Genotyping of 180 cases and 200 age-matched controls were assessed by PCR-RFLP approach and confirmed by direct sequencing.

Results: Statistical analysis showed that patients of valine allele carriers had 2.03-fold of increased risk of developing endometrial carcinoma (P < 0.01). The allele frequencies for the Ile and Val variants between the cancer cases and controls were also significantly different (P < 0.01; odds ratio, 1.59; 95% confidence interval, 1.13-2.23). Such association was shown in endometrial cancers as a group and in type I endometrioid adenocarcinoma but not the type II nonendometrioid adenocarcinoma. In addition, the Val allele was found significantly associated with high-grade endometrial cancer and/or endometrial cancer of deep myometrial invasion (P < 0.01). Interestingly, the relatively low frequency of Val/Val genotype in both the cancer cases and controls, in parallel with the lower incidence of endometrial cancer in Chinese, was observed when compared with those in Caucasians.

Conclusions: Our findings suggested that the GSTP1 Ile105Val polymorphism was associated with an increased risk of endometrial cancer. Further studies may be required to explore the possible significance of these polymorphisms on GSTP1-related metabolism that may affect the susceptibility of Asians to endometrial carcinoma.
Alteration of Gene Expression in Human Hepatocellular Carcinoma with Integrated Hepatitis B Virus DNA
Authors: Tamori Akihiro , Yamanishi Yoshihiro , Kawashima Shuichi , Kanehisa Minoru , Enomoto Masaru , Tanaka Hiromu , Kubo Shoji , Shiomi Susumu , Nishiguchi Shuhei
Published: September 2005

Purpose: Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA.

Experimental Design: We examined 15 cases of HCC infected with HBV by cassette ligation–mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis.

Results: The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes.

Conclusions: HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site–specific expression of such genes during hepatocarcinogenesis.
Antivascular Therapy for Orthotopic Human Ovarian Carcinoma through Blockade of the Vascular Endothelial Growth Factor and Epidermal Growth Factor Receptors
Authors: Thaker Premal H. , Yazici Sertac , Nilsson Monique B. , Yokoi Kenji , Tsan Rachel Z. , He Junqin , Kim Sun-Jin , Fidler Isaiah J. , Sood Anil K.
Published: September 2005

Purpose: We determined whether the administration of the tyrosine kinase inhibitor, AEE788, which targets the epidermal growth factor receptor and the vascular endothelial growth factor receptor, alone or in combination with paclitaxel, can inhibit progressive growth of human ovarian carcinoma in the peritoneal cavity of female nude mice.

Experimental Design: Western blot analysis and immunohistochemical analysis identified the optimal dose and schedule of AEE788 therapy. In several different experiments, paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells were injected into the peritoneal cavity of nude mice. Seven days later, treatment with saline (control), AEE788 alone, paclitaxel alone, or a combination of AEE788 and paclitaxel began and continued for 45 days when the mice were necropsied. In independent survival experiments, the mice were necropsied when they became moribund.

Results: Oral administration of AEE788 inhibited phosphorylation of the epidermal growth factor receptor and vascular endothelial growth factor receptor for up to 48 hours. Treatment with AEE788 plus paclitaxel significantly reduced tumor weight and increased survival of mice implanted with paclitaxel-sensitive cell lines compared with control mice or mice treated with AEE788 alone or paclitaxel alone. In mice implanted with paclitaxel-resistant cells, the combination therapy also significantly reduced tumor weight but did not prolong survival. The combination therapy induced apoptosis of both tumor cells and tumor-associated endothelial cells.

Conclusions: The administration of AEE788 and paclitaxel inhibits the progression of human ovarian carcinoma in the peritoneal cavity of female nude mice, in part, by inducing apoptosis of tumor-associated endothelial cells.
Combining a Recombinant Cancer Vaccine with Standard Definitive Radiotherapy in Patients with Localized Prostate Cancer
Authors: Gulley James L. , Arlen Philip M. , Bastian Anne , Morin Steven , Marte Jennifer , Beetham Patricia , Tsang Kwong-Yok , Yokokawa Junko , Hodge James W. , Ménard Cynthia , Camphausen Kevin , Coleman C. Norman , Sullivan Francis , Steinberg Seth M. , Schlom Jeffrey , Dahut William
Published: September 2005

Purpose: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps due to occult metastatic disease. One potential solution is the utilization of a well-tolerated systemic therapy (e.g., vaccine) in concert with local therapy.

Experimental Design: We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapy-only arms. Those patients in the combination arm received a “priming” vaccine with recombinant vaccinia (rV) PSA plus r V containing the T-cell costimulatory molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local granulocyte-macrophage colony-stimulating factor and low-dose systemic interleukin-2. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations.

Results: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases in PSA-specific T cells of at least 3-fold versus no detectable increases in the radiotherapy-only arm (P < 0.0005). There was also evidence of de novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence of immune-mediated tumor killing. The vaccine was well tolerated.

Conclusion: This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the majority of patients generating a PSA-specific cellular immune response to vaccine.
A Phase I and Pharmacologic Trial of Two Schedules of the Proteasome Inhibitor, PS-341 (Bortezomib, Velcade), in Patients with Advanced Cancer
Authors: Dy Grace K. , Thomas James P. , Wilding George , Bruzek Laura , Mandrekar Sumithra , Erlichman Charles , Alberti Dona , Binger Kim , Pitot Henry C. , Alberts Steven R. , Hanson Lorelei J. , Marnocha Rebecca , Tutsch Kendra , Kaufmann Scott H. , Adjei Alex A.
Published: September 2005

Purpose: To define the toxicities, pharmacodynamics, and clinical activity of the proteasome inhibitor, PS-341 (bortezomib), in patients with advanced malignancies.

Patients and Methods: Twenty-eight patients (14 male and 14 female) received PS-341 twice weekly for 4 of 6 weeks (schedule I). Because toxicity necessitated dose omissions on this schedule, 16 additional patients (12 male and 4 female) received PS-341 twice weekly for 2 of every 3 weeks (schedule II). A total of 73 courses of treatment was given (median, 2; range, 1-4). Inhibition of 20S proteasome activity in peripheral blood mononuclear cells (PBMC) and accumulation of proteasome-targeted polypeptides in tumor tissue were evaluated as pharmacodynamic markers of PS-341 activity.

Results: The most common toxicity was thrombocytopenia, which was dose limiting at 1.7 mg/m2 (schedule I) and 1.6 mg/m2 (schedule II), respectively. Sensory neuropathy was dose-limiting in a patient in schedule I. Grade ≥3 toxicities for schedule I were constipation, fatigue, myalgia, and sensory neuropathy. Grade ≥3 toxicities for schedule II were dehydration resulting from diarrhea, nausea and vomiting, fatigue, hypoglycemia, and hypotension. The maximum tolerated dose was 1.5 mg/m2 for both schedules. Reversible dose-dependent decreases in 20S proteasome activity in PBMCs were observed, with 36% inhibition at 0.5 mg/m2, 52% at 0.9 mg/m2, and 75% at 1.25 mg/m2. Accumulation of proteasome-targeted polypeptides was detected in tumor samples after treatment with PS-341. A patient with multiple myeloma had a partial response.

Conclusion: PS-341 given 1.5 mg/m2 twice weekly for 2 of every 3 weeks is well tolerated and should be further studied.
Disruption of Cooperation Between Ras and MycN in Human Neuroblastoma Cells Promotes Growth Arrest
Authors: Yaari Shira , Jacob-Hirsch Jasmine , Amariglio Ninette , Haklai Ronit , Rechavi Gideon , Kloog Yoel
Published: September 2005

Purpose: Our aim was to examine whether active Ras and MycN cooperation contributes to the malignant phenotype of human neuroblastoma with amplified MycN gene, an aggressive incurable tumor.

Experimental Design: Human neuroblastoma LAN-1 cells, in which the MycN gene is amplified, were used to examine the impact of the Ras inhibitor farnesylthiosalicylic acid on cell growth, on the levels Ras and MycN proteins, and on profiles of gene expression.

Results: We show that LAN-1 cells express relatively large amounts of MycN and active Ras-GTP. Inhibition of active Ras by farnesylthiosalicylic acid led to attenuation of the Raf-MEK-ERK and phosphoinositide 3-kinase-Akt-glycogen synthase-3 (GSK-3) pathways, to reduction in cyclin D1, phospho-retinoblastoma, and E2F, and to increase in the cyclin-dependent kinase inhibitor p27 and in retinoblastoma-binding protein-1, an inhibitor of E2F transcriptional activity. Ras inhibition by farnesylthiosalicylic acid or by a dominant-negative Ras also led to complete disappearance of MycN protein from the nuclei of LAN-1 cells. This was a result of blocking of Akt inactivation of GSK-3, leading to GSK-3-dependent phosphorylation with consequent proteosomal degradation of MycN. Loss of active Ras and of MycN in LAN-1 cells was manifested in profiles of gene expression that could be expected from the loss of MycN transcriptional activity and of Ras signaling. These changes explain the farnesylthiosalicylic acid–induced inhibition of LAN-1 cell growth.

Conclusions: Active Ras is needed to block MycN degradation, promoting cooperative Ras- and MycN-dependent cell cycle progression in LAN-1 cells. Ras inhibitors are therefore likely candidates for the treatment of advanced neuroblastoma characterized by high expression of MycN.
9-cis-Retinoic Acid Treatment Increases Serum Concentrations of α-Tocopherol in Former Smokers
Authors: Han Ji-Youn , Liu Diane D. , Lee J. Jack , Kurie Jonathan , Lotan Reuben , Hong Waun K. , Lee Ho-Young
Published: September 2005

Purpose: Low serum concentrations of antioxidants may be associated with an increased risk of cancer. Based on the accumulated evidence, we hypothesized that retinoids would elevate serum α-tocopherol. This study was designed to determine whether 9-cis-retinoic acid (9-cis-RA), the most common chemopreventive agent, could alter serum α-tocopherol in former smokers. Because hyperlipidemia is a known side effect of retinoids, we also evaluated the association between serum α-tocopherol and lipids in the same population.

Experimental Design: Subjects who had stopped smoking at least 12 months before the study were randomly assigned to receive oral 9-cis-RA or placebo daily for 3 months. Clinical information and blood samples were obtained monthly; serum α-tocopherol concentrations were measured by high-performance liquid chromatography and lipid levels by enzymatic assays before treatment and every month during the treatment.

Results: Of the 149 subjects in the study, 113 completed 3 months of treatment and provided samples for evaluation of serum α-tocopherol. Serum α-tocopherol levels in the 9-cis-RA group (n = 52) were higher after treatment (r = 0.445, P < 0.01) than before. The incidences of grade ≥2 hypertriglyceridemia and hypercholesterolemia were higher in the 9-cis-RA group than in the placebo group (P = 0.0005 and P = 0.01, respectively), but there were no serious complications related to hyperlipidemia.

Conclusions: Treatment of former smokers with 9-cis-RA significantly increased their serum α-tocopherol levels, and this could be a benefit. In addition, serum α-tocopherol could serve as a biomarker for 9-cis-RA treatment.
Predictors of the Response to Gefitinib in Refractory Non–Small Cell Lung Cancer
Authors: Kim Kyu-Sik , Jeong Ju-Yeon , Kim Young-Chul , Na Kook-Joo , Kim Yun-Hyeon , Ahn Sung-Ja , Baek Sun-Mi , Park Chang-Soo , Park Chang-Min , Kim Yu-Il , Lim Sung-Chul , Park Kyung-Ok
Published: September 2005

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has a response rate of 10% to 20% in refractory non–small cell lung carcinoma. Although female gender, adenocarcinoma, and never having smoked are possible markers of a favorable response, mutations of the EGFR gene have also been reported to be highly significant predictors of response. Seventy patients with relapsed non–small cell lung carcinoma were enrolled in the Expanded Access Program. After the drug became available commercially, 28 more patients were treated with gefitinib. Response evaluations were feasible in 80 patients. Twenty-seven tumor specimens (8 responders and 19 nonresponders) were available for the sequence analysis of the EGFR gene. The response rate was 25% (20/80) and the disease control rate (remission + stable disease) was 47.5% (38/80). The response rate was significantly higher for adenocarcinoma (41.0%) versus non-adenocarcinoma (9.8%, P = 0.001), in those who never smoked (58.8%) versus smokers (15.9%, P < 0.001), and in females (42.1%) versus males (19.7%, P = 0.049). A deletion or mutation of the EGFR gene was found in six of eight responders. Remission was noted in all patients with a mutation, whereas the response rate was 9.5% (2/21) in patients without a mutation (P < 0.001). The predictors of response showed significant correlations with survival and time to progression. In a multivariate logistic analysis, the independent predictors of response were smoking history and adenocarcinoma. Given that 9.5% of smokers and 6.7% of those with non-adenocarcinoma showed a mutation of the EGFR gene, the genetic profile may replace those variables as an independent predictor of a response.
p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer
Authors: Figueiredo Marxa L. , Kim Yong , St. John Maie A.R. , Wong David T.W.
Published: September 2005

Purpose: To test the potential of p12CDK2-AP1 (p12), a cell cycle regulator and cyclin-dependent kinase-2-associating protein commonly down-regulated in head and neck squamous cell carcinoma (∼70%), as a gene therapy in inhibiting head and neck squamous cell carcinoma growth in vivo.

Experimental Design: We addressed the effect of p12 expression on tumor growth by using a well-established squamous cell carcinoma VII/SF floor of mouth xenograft mouse model. The effect of therapy on tumor growth was determined for: (a) no treatment, (b) PBS, (c) vehicle (1,2-dioleoyloxy-3-trimethylammonium propane:cholesterol liposomes / 5% dextrose), (d) empty vector controls, and (e) p12-encoding vector experimental groups.

Results: p12 gene therapy significantly induced antitumor effects as compared with controls, including (a) size and weight of p12-treated tumors decreased by 51% to 72% compared with all controls (P < 0.02), (b) tumor growth rate post-therapy was inhibited by 55% to 64% compared with empty vector controls (P < 0.0001), and (c) p12 expression was higher in p12-treated than controls (P < 0.002) by two-tailed t test analyses. Mechanistically, p12 treatment affected cell turnover kinetics as assessed by apoptotic and cell proliferation indices. p12 therapy significantly increased terminal nucleotidyl transferase–mediated nick end labeling (P < 0.05) and morphology-based apoptotic indices (P < 0.05) as well as significantly decreased Ki-67 cell proliferation indices (P < 0.001) compared with controls, resulting in a net cell turnover reduction in p12-treated tumors.

Conclusions: We show that this novel therapeutic modality can significantly induce antitumor responses in vivo. These results support a role for p12 as a novel tumor growth suppressor gene therapy and suggest that optimization and/or combination with current therapies may hold considerable promise in preparation for clinical trials.
Serum Cystatin C is a Better Marker of Topotecan Clearance than Serum Creatinine
Authors: Hoppe Antje , Séronie-Vivien Sophie , Thomas Fabienne , Delord Jean-Pierre , Malard Laurence , Canal Pierre , Chatelut Etienne
Published: September 2005

Purpose: To evaluate plasma cystatin level as a covariate to predict topotecan pharmacokinetics. Cystatin C, a member of the cystatin superfamily of cysteine proteinase inhibitors, has been recently proposed as an alternative endogenous marker of glomerular filtration. Renal function is known as a key factor of topotecan clearance.

Experimental Design: Data were obtained from 59 patients who underwent drug monitoring for individual dosing of topotecan. Topotecan plasma concentrations versus time were analyzed using a nonlinear mixed effect model according to a two-compartment pharmacokinetic model and a first-order conditional estimation method. A proportional error model was used for residual and interpatient variabilities. Data-splitting was done randomly to create a model-building data set (44 patients) and a model validation data set (15 patients).

Results: Using the building data set, four covariates significantly decreased the objective function value and interindividual variability on topotecan clearance (CL) when tested individually: ideal body weight (IBW), serum creatinine, age, and cystatin C level. The best model was: CL (L/hour) = 20.2 [cystatin C (mg/L) / 1.06]−0.60 [IBW (kg) / 57]0.95. Prospective evaluation using the validation data set confirmed that the model based on cystatin C had a better predictive value than the models based on serum creatinine or body surface area.

Conclusion: Cystatin C is a marker of drug elimination which is superior to serum creatinine for topotecan. It deserves to be further explored as a promising covariate for drug dosing as well as selection criteria for clinical studies of drugs eliminated mainly or partially by the kidney.
Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity
Authors: Munshi Anupama , Kurland John F. , Nishikawa Takashi , Tanaka Toshimitsu , Hobbs Marvette L. , Tucker Susan L. , Ismail Sheikh , Stevens Craig , Meyn Raymond E.
Published: September 2005

Purpose: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro.

Experimental Design: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays.

Results: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined γ-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, γ-H2AX foci persisted long after ionizing exposure in the NaB-treated cells.

Conclusions: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that γ-H2AX phosphorylation can be used as a predictive marker of radioresponse.
A Molecular Signature in Superficial Bladder Carcinoma Predicts Clinical Outcome
Authors: Dyrskjøt Lars , Zieger Karsten , Kruhøffer Mogens , Thykjaer Thomas , Jensen Jens L. , Primdahl Hanne , Aziz Natasha , Marcussen Niels , Møller Klaus , Ørntoft Torben F.
Published: September 2005

Purpose: Cancer of the urinary bladder is a common malignant disease in the western countries. The majority of patients presents with superficial tumors with a high recurrence frequency, a minor fraction of these patients experience disease progression to a muscle invasive stage. No clinical useful molecular markers exist to identify patients showing later disease progression. The purpose of this study was to identify markers of disease progression using full-genome expression analysis.

Experimental Design: We did a full-genome expression analysis (59,619 genes and expressed sequence tags) of superficial bladder tumors from 29 bladder cancer patients (13 without later disease progression and 16 with later disease progression) using high-density oligonucleotide microarrays. We used supervised learning for identification of the optimal genes for predicting disease progression. The identified genes were validated on an independent test set (74 superficial tumor samples) using in house-fabricated 60-mer oligonucleotide microarrays.

Results: We identified a 45-gene signature of disease progression. By monitoring this progression signature in an independent test set, we found a significant correlation between our classifications and the clinical outcome (P < 0.03). The genes identified as differentially expressed were involved in regulating apoptosis, cell differentiation, and cell cycle and hence may represent potential therapeutic targets.

Conclusions: Our results indicate that it may be possible to identify patients with a high risk of disease progression at an early stage using a molecular signature present already in the superficial tumors. In this way, better treatment and follow-up regimens could be assigned to patients suffering from superficial bladder cancer.
An Innovative Microarray Strategy Identities Informative Molecular Markers for the Detection of Micrometastatic Breast Cancer
Authors: Mikhitarian Kaidi , Gillanders William E. , Almeida Jonas S. , Hebert Martin Renee , Varela Juan C. , Metcalf John S. , Cole David J. , Mitas Michael
Published: September 2005

There is increasing evidence that molecular detection of micrometastatic breast cancer in the axillary lymph nodes (ALN) of breast cancer patients can improve staging. Molecular analyses of samples obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (n = 489 patients) indicate that whereas the majority of molecular markers are informative for the detection of metastatic breast cancer (significant disease burden), only a few are sensitive for the detection of micrometastatic disease (limited disease burden). Frequency distribution and linear regression analyses reveal that relative levels of gene expression are highly correlated with apparent sensitivity for the detection of micrometastic breast cancer (P < 0.05). These data provides statistical validation of the concept that the most informative markers for detection of micrometastatic disease are those that are most highly expressed in metastatic disease. To test this hypothesis, we developed an innovative microarray strategy. RNA from a metastatic breast cancer ALN was diluted into RNA from a normal lymph node and analyzed using Affymetrix microarrays. Expression analysis indicated that only two genes [mammaglobin (mam) and trefoil factor 1 (TFF1)] were significantly overexpressed at a dilution of 1:50. Real-time reverse transcription-PCR analysis of pathology-negative ALN (n = 72) confirm that of all the markers tested, mam and TFF1 have the highest apparent sensitivity for detection of micrometastatic breast cancer. We conclude that a dilutional microarray approach is a simple and reliable method for the identification of informative molecular markers for the detection of micrometastatic cancer.
High Mutagen Sensitivity in Peripheral Blood Lymphocytes Predicts Poor Overall and Disease-Specific Survival in Patients with Stage III Non–Small Cell Lung Cancer Treated with Radiotherapy and Chemotherapy
Authors: Chang Joe Y. , Komaki Ritsuko , Sasaki Ryohei , Liao Zhongxing , Stevens Craig W. , Lu Charles , Fossella Frank V. , Allen Pamela K. , Cox James D. , Spitz Margaret R. , Wu Xifeng
Published: September 2005

Purpose: To investigate whether the bleomycin sensitivity assay, an in vitro peripheral blood lymphocyte assay, can predict outcome in patients with inoperable stage III non–small-cell lung cancer (NSCLC) treated with definitive radiotherapy and chemotherapy.

Experimental Design: We identified 102 patients with inoperable stage III NSCLC cell lung cancer treated with definitive radiotherapy and chemotherapy. The patients' pretreatment peripheral blood lymphocyte cultures were treated with the radiomimetic mutagen bleomycin. An index of bleomycin sensitivity was determined by counting the number of chromatid breaks in 50 metaphases. The correlation between bleomycin sensitivity (expressed as mean breaks per cell) and clinical outcome was analyzed.

Results: High bleomycin sensitivity (defined as a mean of >1.02 chromatid breaks/cell, representing the third quartile of bleomycin sensitivity) predicted poor disease-specific survival and overall survival. The 6-year disease-specific survival was 27% in patients with high bleomycin sensitivity compared with 46% in patients without such sensitivity (P = 0.0094). The association remained statistically significant when adjusted for smoking status, age, and radiation dose. The 6-year overall survival was 19% for patients with high bleomycin sensitivity and 29% for those without (P = 0.0193). There was a trend toward worse local regional control and worse disease-free survival among patients with high bleomycin sensitivity. There was no difference between the two groups in distant metastasis-free survival or radiation treatment-related complications.

Conclusions: High bleomycin sensitivity correlated with poor overall survival and disease-specific survival in these patients with stage III NSCLC treated with radiotherapy and chemotherapy. Bleomycin sensitivity may function as a biomarker for poor clinical outcome for this group of patients.
Characterization of a New Antibody Raised against the NH2 Terminus of P-Glycoprotein
Authors: Rao Prema S. , Govindarajan Rajgopal , Mallya Kavita B. , West William , Rao U. Subrahmanyeswara
Published: September 2005

Purpose: Cancers exposed to chemotherapy develop multidrug resistance, a major cause for chemotherapy failure. One mechanism of multidrug resistance development is due to overexpression of P-glycoprotein (Pgp) in these cancer cells. Thus, a prechemotherapy evaluation of Pgp in cancer cells aids in the design of alternative regimens that can circumvent such failure. As few Pgp-specific antibodies are available in detecting low levels of Pgp, there is a need for preparing an antibody that allows the detection of Pgp by various immunologic methods.

Experimental Design: We selected the amino acid stretch 11 to 34 in the cytoplasmically located NH2 terminus of Pgp as antigen, which was chemically synthesized and used to raise an antibody in a rabbit, termed NH211 antibody. We compared the properties of NH211 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry.

Results: Immunoblotting analysis suggested that NH211 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH211 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH211 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections.

Conclusion: As the NH211 antibody detects Pgp present in cells and tissues, we conclude that the amino acid sequence to which this antibody was raised is highly antigenic and the antibody is useful in the detection of Pgp by a variety of immunologic methods.
CD44 Expression Is Associated with Increased Survival in Node-Negative Invasive Breast Carcinoma
Authors: Diaz Leslie K. , Zhou Xiao , Wright Edward T. , Cristofanilli Massimo , Smith Terry , Yang Ying , Sneige Nour , Sahin Aysegul , Gilcrease Michael Z.
Published: September 2005

Purpose: CD44 is a multifunctional cell surface receptor with many known splice variants, some of which have been reported to play a role in tumor progression. The purpose of this study was to evaluate the prognostic significance of CD44 isoforms in early-stage, lymph node–negative invasive breast carcinoma.

Experimental Design: Immunohistochemical staining for CD44 isoforms was done on archival paraffin tissue sections of invasive breast carcinoma from a cohort of lymph node–negative patients who received no adjuvant tamoxifen or chemotherapy and who had a mean clinical follow-up period of 15 years. Immunohistochemical staining was done with antibodies to CD44s, the standard isoform of CD44, and to isoforms containing variant exon 6 (CD44v6); levels of staining were correlated with clinical outcome data.

Results: There was a trend towards increased disease-free survival for patients whose tumors had high anti-CD44s positivity (P = 0.05), and a significant association was observed between anti-CD44s positivity and disease-related survival (P = 0.04). Expression of CD44v6 isoforms did not correlate with clinical outcome.

Conclusion: CD44 expression, as assessed by immunohistochemical staining with anti-CD44s, may be a favorable prognostic factor in patients with node-negative invasive breast carcinoma.
Development of β-Lapachone Prodrugs for Therapy Against Human Cancer Cells with Elevated NAD(P)H:Quinone Oxidoreductase 1 Levels
Authors: Reinicke Kathryn E. , Bey Erik A. , Bentle Melissa S. , Pink John J. , Ingalls Stephen T. , Hoppel Charles L. , Misico Rosana I. , Arzac Gisela M. , Burton Gerardo , Bornmann William G. , Sutton Damon , Gao Jinming , Boothman David A.
Published: September 2005

β-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces β-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to β-lapachone. Thus, β-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non–small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of β-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to β-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to β-lapachone. Once converted, β-lapachone derivatives caused NQO1-dependent, μ-calpain-mediated cell death in human cancer cells identical to that caused by β-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect β-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of β-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to β-lapachone. The use of β-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.
Systemic Treatment with Tetra-O-Methyl Nordihydroguaiaretic Acid Suppresses the Growth of Human Xenograft Tumors
Authors: Park Richard , Chang Chih-Chuan , Liang Yu-Chuan , Chung Yousun , Henry Ryan A. , Lin Elaine , Mold David E. , Huang Ru Chih C.
Published: September 2005

Purpose: We have previously shown that the transcriptional inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) induces growth arrest in tumor cells and exhibits tumoricidal activity when injected intratumorally into tumor cell explants in mice. The experiments reported here were designed to determine whether M4N can be given systemically and inhibit the growth of five different human xenograft tumors.

Experimental Design: Nude (nu/nu) mice bearing xenografts of each of five human tumor types (i.e., hepatocellular carcinoma, Hep 3B; prostate carcinoma, LNCaP; colorectal carcinoma, HT-29; breast carcinoma, MCF7; and erythroleukemia, K-562) were treated with M4N given i.v. or i.p. in a Cremophor EL–based solvent system or orally in a corn oil based diet. Tumors from the treated animals were measured weekly and analyzed for the expression of the Cdc2 and survivin genes, both previously shown to be down-regulated by M4N.

Results: Systemic M4N treatment suppressed the in vivo growth of xenografts in each of the five human tumor types. Four of the five tumor models were particularly sensitive to M4N with tumor growth inhibitions (T/C values) of ≤42%, whereas the fifth, HT-29, responded to a lesser extent (48.3%). Growth arrest and apoptosis in both the xenograft tumors and in the tumor cells grown in culture were accompanied by reductions in both Cdc2 and tumor-specific survivin gene expression. Pharmacokinetic analysis following oral and i.v. administration to ICR mice indicated an absolute bioavailability for oral M4N of ∼88%. Minimal drug-related toxicity was observed.

Conclusion: These preclinical studies establish that when given systemically, M4N can safely and effectively inhibit the growth of human tumors in nude mice.