bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in muscle size and MHC composition in response to resistance exercise with heavy and light loading intensity
Authors: Holm L. , Reitelseder S. , Pedersen T. G. , Doessing S. , Petersen S. G. , Flyvbjerg A. , Andersen J. L. , Aagaard P. , Kjaer M.
Published: September 2008

Muscle mass accretion is accomplished by heavy-load resistance training. The effect of light-load resistance exercise has been far more sparsely investigated with regard to potential effect on muscle size and contractile strength. We applied a resistance exercise protocol in which the same individual trained one leg at 70% of one-repetition maximum (1RM) (heavy load, HL) while training the other leg at 15.5% 1RM (light load, LL). Eleven sedentary men (age 25 ± 1 yr) trained for 12 wk at three times/week. Before and after the intervention muscle hypertrophy was determined by magnetic resonance imaging, muscle biopsies were obtained bilaterally from vastus lateralis for determination of myosin heavy chain (MHC) composition, and maximal muscle strength was assessed by 1RM testing and in an isokinetic dynamometer at 60°/s. Quadriceps muscle cross-sectional area increased ( P < 0.05) 8 ± 1% and 3 ± 1% in HL and LL legs, respectively, with a greater gain in HL than LL ( P < 0.05). Likewise, 1RM strength increased ( P < 0.001) in both legs (HL: 36 ± 5%, LL: 19 ± 2%), albeit more so with HL ( P < 0.01). Isokinetic 60°/s muscle strength improved by 13 ± 5% ( P < 0.05) in HL but remained unchanged in LL (4 ± 5%, not significant). Finally, MHC IIX protein expression was decreased with HL but not LL, despite identical total workload in HL and LL. Our main finding was that LL resistance training was sufficient to induce a small but significant muscle hypertrophy in healthy young men. However, LL resistance training was inferior to HL training in evoking adaptive changes in muscle size and contractile strength and was insufficient to induce changes in MHC composition.
Eccentric contractions do not induce rhabdomyolysis in malignant hyperthermia susceptible mice
Authors: Corona Benjamin T. , Rouviere Clement , Hamilton Susan L. , Ingalls Christopher P.
Published: September 2008

Recent studies suggest a link between exercise-induced rhabdomyolysis and mutations of the ryanodine receptor (RYR1) associated with malignant hyperthermia (MH). We hypothesized that MH-susceptible mice (RYR1Y522S/wt) would exhibit greater anterior crural muscle [tibialis anterior (TA) and extensor digitorum longus (EDL) muscles] damage and strength deficits following the performance of a single or repeated bouts of eccentric contractions compared with wild-type (WT) mice. After a single injury bout, RYR1Y522S/wtmice produced more isometric torque than WT mice immediately and 3 and 7 days postinjury. Moreover, EDL muscle isometric specific force deficits were fully recovered for RYR1Y522S/wtbut not WT mice 14 days postinjury. The percentage of fibers in TA muscle exhibiting signs of muscle damage 7 and 14 days postinjury were at least three times less in RYR1Y522S/wtthan in WT mice. Uninjured and injured EDL muscle from RYR1Y522S/wtmice also displayed greater S-glutathionylation of RYR1 than that from WT mice. During the weekly injury bouts, torque production by RYR1Y522S/wtmice was fully recovered before the third and fourth injury bouts, whereas torque was still reduced for WT mice. Three days after multiple injury bouts, there were ∼50% fewer fibers exhibiting signs of muscle damage in RYR1Y522S/wtthan in WT TA muscle. These findings indicate that the RYR1Y522S/wtmutation protects skeletal muscle from exercise-induced muscle injury and do not support a direct association between MH susceptibility and contraction-induced rhabdomyolysis when core temperature is maintained at lower physiological temperatures during exercise.
EphA4 promotes cell proliferation and migration through a novel EphA4-FGFR1 signaling pathway in the human glioma U251 cell line
Authors: Fukai Junya , Yokote Hideyuki , Yamanaka Ryuya , Arao Tokuzo , Nishio Kazuto , Itakura Toru
Published: September 2008

The Eph receptor tyrosine kinases and their ephrin ligands form a unique cell-cell contact-mediated bidirectional signaling mechanism for regulating cell localization and organization. High expression of Eph receptors in a wide variety of human tumors indicates some roles in tumor progression, which makes these proteins potential targets for anticancer therapy. For this purpose, we did gene expression profiling for 47 surgical specimens of brain tumors including 32 high-grade glioma using a microarray technique. The analysis, focused on the receptor tyrosine kinases, showed that EphA4 mRNA in the tumors was 4-fold higher than in normal brain tissue. To investigate the biological significance of EphA4 overexpression in these tumors, we analyzed EphA4-induced phenotypic changes and the signaling mechanisms using human glioma U251 cells. EphA4 promoted fibroblast growth factor 2-mediated cell proliferation and migration accompanied with enhancement of fibroblast growth factor 2-triggered mitogen-activated protein kinase and Akt phosphorylation. In addition, active forms of Rac1 and Cdc42 increased in the EphA4-overexpressing cells. Furthermore, we found that EphA4 formed a heteroreceptor complex with fibroblast growth factor receptor 1 (FGFR1) in the cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling. Thus, our results indicate that EphA4 plays an important role in malignant phenotypes of glioblastoma by enhancing cell proliferation and migration through accelerating a canonical FGFR signaling pathway. [Mol Cancer Ther 2008;7(9):2768–78]
Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells
Authors: Wilson Catherine , Wilson Timothy , Johnston Patrick G. , Longley Daniel B. , Waugh David J.J.
Published: September 2008

Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c-FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8–induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor– and/or nuclear factor-κB–mediated transcription attenuated IL-8–induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8–induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy. [Mol Cancer Ther 2008;7(9):2649–61]
Soluble EP2 neutralizes prostaglandin E2–induced cell signaling and inhibits osteolytic tumor growth
Authors: Takahashi Tetsuyuki , Uehara Hisanori , Bando Yoshimi , Izumi Keisuke
Published: September 2008

Prostaglandin E2 (PGE2) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE2 exerts its effects by binding and activating E-prostanoid receptor (EP). In this study, we propose a new approach for blocking EP-mediated cell signaling using a soluble chimeric EP2 fragment. Mammalian expression vectors encoding several human EP2 cDNAs were introduced into 293 cells and the culture medium was tested for their function as a decoy receptor for PGE2. PGE2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE2-induced cyclic AMP production, cyclic AMP–responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1β, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-κB ligand mRNA. A stable transfectant expressing FuEP2/Ex2 was established from human prostate cancer PC-3 cells (PC3-FuEP2/Ex2). PC3-FuEP2/Ex2 cells grew at similar rates to vector control cells under normal culture conditions, although PGE2-induced growth stimulation was suppressed. Intraosseous injection of PC3-FuEP2/Ex2 cells into the tibia of athymic nude mice revealed that the degrees of tumor growth and osteolysis were decreased compared with control cell-injected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1β, and IL-6 mRNA levels were reduced in the tumor lesions. These data suggest that FuEP2/Ex2 is useful for treating osteolytic bone metastasis and cancers that depend on EP signaling for their growth and development. [Mol Cancer Ther 2008;7(9):2807–16]
Glucose-regulated protein 78 regulates multiple malignant phenotypes in head and neck cancer and may serve as a molecular target of therapeutic intervention
Authors: Chiu Ching-Chi , Lin Chien-Yu , Lee Li-Yu , Chen Yin-Ju , Kuo Ting-Fang , Chang Joseph Tung-Chieh , Liao Chun-Ta , Wang Hung-Ming , Yen Tzu-Chen , Shen Chia-Rui , Liao Shuen-Kuei , Cheng Ann-Joy
Published: September 2008

Glucose-regulated protein 78 (Grp78) is an endoplasmic reticulum chaperone protein and is overexpressed in various cancers. However, it is unclear how significance of this molecule play an active role contributing to the oncogenic effect of head and neck cancer (HNC). To investigate the potential function of Grp78, six HNC cell lines were used. We found that Grp78 is highly expressed in all six cell lines and many of the proteins were localized in the periphery regions, implying other function of this molecule aside from endoplasmic reticulum stress response. Knockdown of Grp78 by small interfering RNA significantly reduced cell growth and colony formation to 53% to 12% compared with that of controls in all six HNC cell lines. Using in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also inhibited to 23% to 2% in all these cell lines tested. In vivo xenograft studies showed that administration of Grp78-small interfering RNA plasmid into HNC xenografts significantly inhibited both tumor growth in situ (>60% inhibition at day 34) and liver metastasis (>90% inhibition at day 20). Our study showed that Grp78 actively regulates multiple malignant phenotypes, including cell growth, migration, and invasion. Because knockdown Grp78 expression succeeds in the reduction of tumor growth and metastatic potential, this molecule may serve as a molecular target of therapeutic intervention for HNC. [Mol Cancer Ther 2008;7(9):2788–97]
Peroxisome proliferator-activated receptor ligand MCC-555 suppresses intestinal polyps in ApcMin/+ mice via extracellular signal-regulated kinase and peroxisome proliferator-activated receptor-dependent pathways
Authors: Yamaguchi Kiyoshi , Cekanova Maria , McEntee Michael F. , Yoon Joo-Heon , Fischer Susan M. , Renes Ingrid B. , Van Seuningen Isabelle , Baek Seung Joon
Published: September 2008

A large body of studies has suggested that peroxisome proliferator-activated receptor γ (PPARγ) ligands, such as thiazolidinedione, are potent candidates for chemopreventive agents. MCC-555 is a PPARγ/α dual agonist and has been shown previously to induce apoptosis in vitro; however, the molecular mechanisms by which MCC-555 affects antitumorigenesis in vivo are poorly understood. In this study, we explored the antitumorigenic effects of MCC-555 both in cell culture and in Apc-deficient mice, an animal model for human familial adenomatous polyposis. MCC-555 increased MUC2 expression in colorectal and lung cancer cells, and treatment with the PPARγ antagonist GW9662 revealed that MUC2 induction by MCC-555 was mediated in a PPARγ-dependent manner. Moreover, MCC-555 increased transcriptional activity of human and mouse MUC2 promoters. Subsequently, treatment with MCC-555 (30 mg/kg/d) for 4 weeks reduced the number of small intestinal polyps to 54.8% of that in control mice. In agreement with in vitro studies, enhanced Muc2 expression was observed in the small intestinal tumors of Min mice treated with MCC-555, suggesting that MUC2 expression may be associated at least in part with the antitumorigenic action of MCC-555. In addition, highly phosphorylated extracellular signal-regulated kinase (ERK) was found in the intestinal tumors of MCC-555-treated Min mice, and inhibition of the ERK pathway by a specific inhibitor markedly suppressed MCC-555-induced Muc2 expression in vitro. Overall, these results indicate that MCC-555 has a potent tumor suppressor activity in intestinal tumorigenesis, likely involving MUC2 up-regulation by ERK and PPARγ pathways. [Mol Cancer Ther 2008;7(9):2779–87]
Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer
Authors: Agarwal Anika , Covic Lidija , Sevigny Leila M. , Kaneider Nicole C. , Lazarides Katherine , Azabdaftari Gissou , Sharifi Sheida , Kuliopulos Athan
Published: September 2008

Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. [Mol Cancer Ther 2008;7(9):2746–57]
1α,25-Dihydroxyvitamin D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model
Authors: Ma Yingyu , Yu Wei-Dong , Hershberger Pamela A. , Flynn Geraldine , Kong Rui-Xian , Trump Donald L. , Johnson Candace S.
Published: September 2008

1α,25-Dihydroxyvitamin D3 (1,25D3) exhibits antitumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 before cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 before cisplatin compared with either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin but increased by 1,25D3 in SCC cells. Knocking down p73 by small interfering RNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin antitumor activity in vitro and in vivo in a SCC model system possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment. [Mol Cancer Ther 2008;7(9):3047–55]
Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model
Authors: Almholt Kasper , Juncker-Jensen Anna , Lærum Ole Didrik , Danø Keld , Johnsen Morten , Lund Leif Røge , Rømer John
Published: September 2008

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm3 in Galardin-treated mice compared with 0.56 mm3 in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application. [Mol Cancer Ther 2008;7(9):2758–67]
Early drug development of inhibitors of the insulin-like growth factor-I receptor pathway: Lessons from the first clinical trials
Authors: Rodon Jordi , DeSantos Victoria , Ferry Robert Jean , Kurzrock Razelle
Published: September 2008

The insulin-like growth factor-I receptor (IGF-IR) was first cloned in 1986. Since then, intense work has defined classic phosphorelays activated via the IGF-IR, which regulate cell proliferation, apoptosis, motility, and fate. The understanding of the roles of hormones in cancer and the growth hormone–IGF–IGF-binding protein axis specifically has yield to a second wave of development: the design of specific inhibitors that interrupt the signaling associated with this axis. The ability to manipulate these pathways holds not only significant therapeutic implications but also increase the chance of deeper insight about the role of the axis in carcinogenesis and metastasis. Nowadays, >25 molecules with the same goal are at different stages of development. Here, we review the clinical and preclinical experience with the two most-investigated strategies, tyrosine kinase inhibitors and monoclonal antibodies, and the advantages and disadvantages of each strategy, as well as other alternatives and possible drug combinations. We also review the biomarkers explored in the first clinical trials, the strategies that have been explored thus far, and the clinical trials that are going to explore their role in cancer treatment. [Mol Cancer Ther 2008;7(9):2575–88]
Novel opportunities for thymidylate metabolism as a therapeutic target
Authors: Wilson Peter M. , Fazzone William , LaBonte Melissa J. , Deng Jinxia , Neamati Nouri , Ladner Robert D.
Published: September 2008

For over 40 years, the fluoropyrimidine 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens employed in the treatment of colorectal cancer and is frequently combined with the DNA-damaging agents oxaliplatin and irinotecan, increasing response rates and improving overall survival. However, many patients will derive little or no benefit from treatment, highlighting the need to identify novel therapeutic targets to improve the efficacy of current 5-FU-based chemotherapeutic strategies. dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi, providing substrate for thymidylate synthase (TS) and DNA synthesis and repair. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA as uracil is lethal. Importantly, uracil misincorporation represents an important mechanism of cytotoxicity induced by the TS-targeted class of chemotherapeutic agents including 5-FU. A growing body of evidence suggests that dUTPase is an important mediator of response to TS-targeted agents. In this article, we present further evidence showing that elevated expression of dUTPase can protect breast cancer cells from the expansion of the intracellular uracil pool, translating to reduced growth inhibition following treatment with 5-FU. We therefore report the implementation of in silico drug development techniques to identify and develop small-molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU prodrug capecitabine remain central agents in the treatment of a variety of malignancies, the clinical utility of a small-molecule inhibitor to dUTPase represents a viable strategy to improve the clinical efficacy of these mainstay chemotherapeutic agents. [Mol Cancer Ther 2008;7(9):3029–37]
Purified human chorionic gonadotropin induces apoptosis in breast cancer
Authors: Lopez Dayami , Sekharam Madhavi , Coppola Domenico , Carter W. Bradford
Published: September 2008

Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study, the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breast cancer xenografts grown in nude mice were examined. It was shown that intratumoral injection of purified hCG increased the apoptotic index in breast cancer xenografts. These results were supported by the findings that exposure of breast cancer cells to purified hCG decreased cell viability in five different breast cancer cell lines. In some of these cell lines, the effects of hCG in cell viability appear to correlate with activation/expression of the hCG/luteinizing hormone receptor. Preoperative apoptotic induction by factors such as purified hCG may improve local control or work synergistically with neoadjuvant chemotherapy to improve complete pathologic response of locally advanced breast cancer. [Mol Cancer Ther 2008;7(9):2837–8]
Molecular chemotherapy of pancreatic cancer using novel mutant bacterial cytosine deaminase gene
Authors: Kaliberova Lyudmila N. , Della Manna Debbie L. , Krendelchtchikova Valentina , Black Margaret E. , Buchsbaum Donald J. , Kaliberov Sergey A.
Published: September 2008

The combination of molecular chemotherapy with radiation therapy has the potential to become a powerful approach for treatment of pancreatic cancer. We have developed an adenoviral vector (AdbCD-D314A) encoding a mutant bacterial cytosine deaminase (bCD) gene, which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil. The aim of this study was to investigate AdbCD-D314A/5-FC-mediated cytotoxicity in vitro and therapeutic efficacy in vivo alone and in combination with radiation against human pancreatic cancer cells and xenografts. AdbCD-D314A/5-FC-mediated cytotoxicity alone and in combination with radiation was analyzed using crystal violet inclusion and clonogenic survival assays. CD enzyme activity was determined by measuring conversion of [3H]5-FC to [3H]5-fluorouracil after adenoviral infection of pancreatic cancer cells in vitro and pancreatic tumor xenografts by TLC. S.c. pancreatic tumor xenografts were used to evaluate the therapeutic efficacy of AdbCD-D314A/5-FC molecular chemotherapy in combination with radiation therapy. AdbCD-D314A infection resulted in increased 5-FC-mediated pancreatic cancer cell killing that correlated with significantly enhanced CD enzyme activity compared with AdbCDwt encoding wild-type of bCD. Animal studies showed significant inhibition of growth of human pancreatic tumors treated with AdbCD-D314A/5-FC in comparison with AdbCDwt/5-FC. Also, a significantly greater inhibition of growth of Panc2.03 and MIA PaCA-2 tumor xenografts was produced by the combination of AdbCD-D314A/5-FC with radiation compared with either agent alone. The results indicate that the combination of AdbCD-D314A/5-FC molecular chemotherapy with radiation therapy significantly enhanced cytotoxicity of pancreatic cancer cells in vitro and increased therapeutic efficacy against human pancreatic tumor xenografts. [Mol Cancer Ther 2008;7(9):2845–54]
Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95
Authors: Park Margaret A. , Zhang Guo , Mitchell Clint , Rahmani Mohamed , Hamed Hossein , Hagan Michael P. , Yacoub Adly , Curiel David T. , Fisher Paul B. , Grant Steven , Dent Paul
Published: September 2008

Prior studies have noted that inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors [PD184352; AZD6244 (ARRY-142886)] interacted in a synergistic manner with geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin] to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of extracellular signal-regulated kinase 1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was reactive oxygen species dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase-8 or overexpression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase-8 with CD95. Inhibition of p38 MAPK or knockdown of BID, FAS-associated death domain, or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data show that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the apoptotic signal from CD95 to promote cell death. [Mol Cancer Ther 2008;7(9):2633–48]
Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells
Authors: Bataller Marc , Méndez Carmen , Salas José A. , Portugal José
Published: September 2008

During a normal cell cycle, polyploidy and aneuploidy can be prevented by several checkpoints, which are mainly p53 dependent. Here, we show that treatment of HCT-116 (p53+/+) colon carcinoma cells with the novel antitumor antibiotic mithramycin SK (MSK) results in polyploidization and mitotic catastrophe, which occurs after a transient halt in G1 phase followed by the overtaking of the G2-M checkpoint when treated cells are incubated in a fresh drug-free medium. Cells reentering aberrant mitosis mainly died by necrosis, although active caspase-3 was observed. Our results indicate that a decrease in p53 RNA and protein levels, together with concomitant changes in the expression of other proteins such as p21WAF1, were involved in MSK-induced polyploidy. Furthermore, the effects of MSK on HCT-116 (p53+/+) cells cannot be attributed exclusively to the down-regulation of p53 by MSK, because these effects differed from those observed in MSK-treated HCT-116 (p53−/−) cells. The p53−/− cells died mainly from G2-M through early p53-independent apoptosis, which appeared to be mediated by caspase-2, although secondary necrosis was also observed. [Mol Cancer Ther 2008;7(9):2988–97]
A role for macroautophagy in protection against 4-hydroxytamoxifen–induced cell death and the development of antiestrogen resistance
Authors: Samaddar Julia S. , Gaddy Virgil T. , Duplantier Jennifer , Thandavan Sudharsan Periyasamy , Shah Manish , Smith Marlena J. , Browning Darren , Rawson Jim , Smith Sylvia B. , Barrett John T. , Schoenlein Patricia V.
Published: September 2008

This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor–positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT–induced cell death, but not 4-OHT–induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT–resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT–resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer. [Mol Cancer Ther 2008;7(9):2977–87]
Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility
Authors: Paccione Rachel J. , Miyazaki Hiroshi , Patel Vyomesh , Waseem Ahmad , Gutkind J. Silvio , Zehner Zendra E. , Yeudall W. Andrew
Published: September 2008

At later stages of tumor progression, epithelial carcinogenesis is associated with transition to a mesenchymal phenotype, which may contribute to the more aggressive properties of cancer cells and may be stimulated by growth factors such as epidermal growth factor and transforming growth factor-β. Previously, we found that cells derived from a nodal metastatic squamous cell carcinoma are highly proliferative and motile in vitro and tumorigenic in vivo. In the current study, we have investigated the role of vimentin in proliferation and motility. Cells derived from nodal metastasis express high levels of vimentin, which is undetectable in tumor cells derived from a synchronous primary lesion of tongue. Vimentin expression was enhanced by epidermal growth factor and transforming growth factor-β both independently and in combination. Use of RNA interference resulted in the generation of stable cell lines that express constitutively low levels of vimentin. RNA interference-mediated vimentin knockdown reduced cellular proliferation, migration, and invasion through a basement membrane substitute by 3-fold compared with nontargeting controls. In addition, cells with reduced vimentin reexpressed differentiation-specific keratins K13, K14, and K15 as a result of increased gene transcription as judged by quantitative PCR and promoter-reporter assays. Furthermore, cells in which vimentin expression was reduced showed a greatly decreased tumorigenic potential, as tumors developing from these cells were 70% smaller than those from control cells. The data suggest that reversal of the mesenchymal phenotype by inhibiting vimentin expression results in reexpression of epithelial characteristics and reduced tumor aggressiveness. [Mol Cancer Ther 2008;7(9):2894–903]
Penta-1,2,3,4,6-O-galloyl-β-d-glucose induces p53 and inhibits STAT3 in prostate cancer cellsin vitroand suppresses prostate xenograft tumor growthin vivo
Authors: Hu Hongbo , Lee Hyo-Jeong , Jiang Cheng , Zhang Jinhui , Wang Lei , Zhao Yan , Xiang Qiu , Lee Eun-Ok , Kim Sung-Hoon , Lü Junxuan
Published: September 2008

Penta-1,2,3,4,6-O-galloyl-β-d-glucose (PGG) is a naturally occurring gallotannin from some Oriental herbs. Several cell culture studies suggested a potential for PGG as a novel agent for the chemoprevention and treatment of cancer. Here, we investigated the cell death signaling mechanisms induced by PGG in human prostate cancer cells of different p53 functional status. We observed the induction of G1- and S-phase arrests and caspase-mediated apoptosis in the androgen-dependent human LNCaP cells, which express wild-type p53, and in the androgen-independent, p53-mutant DU145 cells. In LNCaP cells, caspase-mediated apoptosis induction by PGG was associated with and mediated in major part by activation of p53 as established through small interfering RNA knockdown and dominant-negative mutant approaches. Intracellular reactive oxygen species production by PGG was found to be crucial for these molecular and cellular actions. In DU145 cells, which harbor constitutively active signal transducer and activator of transcription 3 (STAT3), caspase-mediated apoptosis induction by PGG was associated with an inhibition of STAT3 Tyr705 phosphorylation and the down-regulation of STAT3 transcriptional targets Bcl-XL and Mcl-1. Overexpression of Bcl-XL or knockdown of its binding partner Bak attenuated apoptosis induction. Furthermore, we provide, for the first time, in vivo data that PGG significantly inhibited DU145 xenograft growth in an athymic nude mouse model in association with an inhibition of pSTAT3. Our data support PGG as a multitargeting agent for chemoprevention and therapy of prostate cancer by activating the p53 tumor suppressor pathway and by inhibiting STAT3 oncogenic signaling. [Mol Cancer Ther 2008;7(9):2681–91]
N-(4-Hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing
Authors: Wang Hongtao , Maurer Barry J. , Liu Yong-Yu , Wang Elaine , Allegood Jeremy C. , Kelly Samuel , Symolon Holly , Liu Ying , Merrill, Alfred H. , Gouazé-Andersson Valérie , Yu Jing Yuan , Giuliano Armando E. , Cabot Myles C.
Published: September 2008

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [3H]palmitic acid showed that 4-HPR elicited a dose-responsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor d-erythro-N,N-dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR–induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther 2008;7(9):2967–76]
Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes
Authors: Stahnke Bettina , Thepen Theo , Stöcker Michael , Rosinke Reinhard , Jost Edgar , Fischer Rainer , Tur Mehmet K. , Barth Stefan
Published: September 2008

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcγRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8+ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64+ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC50) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64+ AML cells, whereas CD64− AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924–32]
Genes Differentially Expressed in Prolactinomas Responsive and Resistant to Dopamine Agonists
Authors: Passos Vanessa Q. , Fortes Maria Angela H.Z. , Giannella-Neto Daniel , Bronstein Marcello D.
Published: September 2008

Background/Aims: Prolactin (PRL) secretion and its gene expression are inhibited by dopamine. Prolactinomas are the most common secreting pituitary adenomas, and dopamine agonists (DA) are the first choice for their treatment. However, a subset of patients is resistant to DA. As the mechanisms involved in DA resistance are not fully understood, the aim of this study was to obtain new insights regarding the molecular differences between the prolactinomas that are responsive to DA and those that are resistant. Methods: Tumor tissue samples were collected from 17 patients who harbored prolactinomas, which were classified as responsive or resistant according to their clinical and laboratorial reaction to DA. The expression of 6 genes was evaluated by real-time polymerase chain reaction: dopamine receptor type 2 (DRD2), nerve growth factor-β (NGFB) and its receptor (NGFR), estrogen receptor-α (ERA), estrogen receptor-β (ERB) and the pituitary tumor transforming gene (PTTG). Results: Median DRD2 and NGFR expression in responsive patients was significantly higher than in resistant ones (p = 0.029 and p = 0.020, respectively). Moreover, the expressions of DRD2 and NGFR were positively correlated with PRL decrease during treatment (r = 0.66, p = 0.005 and r = 0.57, p = 0.044, respectively). Furthermore, ERB expression was positively correlated to PTTG expression (r = 0.68, p = 0.032) and negatively correlated to NGFB expression (r = –0.75, p = 0.02). Conclusions: DRD2 and NGFR expressions are related to the responsiveness of prolactinoma to DA. However, PTTG, ERB and ERA expressions are not. Also ERB, ERA and PTTG expressions did not present a clear correlation to tumor aggressiveness. Furthermore, the response of prolactinomas to DA should be viewed as a spectrum ranging from the most responsive to the most resistant ones.
The Role of Cellular Adaptation to Mechanical Forces in Atherosclerosis
Authors: Hahn Cornelia , Schwartz Martin A.
Published: September 2008

Atherosclerosis is a chronic inflammatory disease that originates at regions of arteries exposed to disturbances in fluid flow and results in progressive plaque formation in those areas. Recent work on cellular responses to flow has identified potential mechanosensors and pathways that may influence disease progression. These results led us to hypothesize that the same mechanisms that mediate adaptive responses in the vasculature become maladaptive at sites of disturbed flow. Subsequent changes in gene expression and matrix remodeling help to entrain these inflammatory pathways. These events synergize with systemic risk factors such as hyperlipidemia, smoking, and diabetes, leading to disease progression.
Critical Role of Macrophages in Glucocorticoid Driven Vascular Calcification in a Mouse-Model of Atherosclerosis
Authors: Preusch Michael R. , Rattazzi Marcello , Albrecht Claudia , Merle Uta , Tuckermann Jan , Schütz Günther , Blessing Erwin , Zoppellaro Giacomo , Pauletto Paolo , Krempien Robert , Rosenfeld Michael E. , Katus Hugo A. , Bea Florian
Published: September 2008

Objective—

Macrophage-derived products are known to play a crucial role during atherogenesis and vascular calcification. Glucocorticoids (GC) are important modulators of immune cell functions, but their specific effects on macrophages behavior during plaque formation are not defined. The present study was therefore designed to investigate the effects of macrophage-specific deletion of the glucocorticoid receptor (GR
LysMCre
) on atherogenesis and vascular calcification in a hyperlipidemic mouse-model.

Methods and Results—

Bone marrow was isolated from GR
LysMCre
mice and wild-type controls (GR
flox
) and subsequently transplanted into lethally irradiated LDL-receptor–deficient mice. Animals were fed a Western-type diet for 15 or 24 weeks, and atherosclerotic lesions within the aortic sinus were evaluated. At both time points, no significant difference in serum lipid and corticosterone concentrations, atherosclerotic lesion size and macrophage-content within the lesions could be observed. However, GR
LysMCre
mice showed less calcification as well as a significant reduction of RANKL, BMP2, and Msx2 expression within the vasculature. In vitro studies using conditioned media from macrophages which had been stimulated with dexamethasone demonstrated a dose-dependent increase in calcium deposition by vascular smooth muscle cells.

Conclusion—

This study demonstrates that macrophage-specific glucocorticoid receptor inactivation reduces vascular calcification without affecting atherosclerotic lesion size in LDL receptor–deficient mice.
Expansion of T-Cell Receptor ζ
dim
Effector T Cells in Acute Coronary Syndromes
Authors: Ammirati Enrico , Vermi Anna-Chiara , Cianflone Domenico , Banfi Michela , Foglieni Chiara , Godino Cosmo , Airoldi Flavio , Ferri Luca A. , Gorman Claire L. , Manfredi Angelo A. , Maseri Attilio , Cope Andrew P. , Monaco Claudia
Published: September 2008

Objective—

The T-cell receptor zeta (TCRζ)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCRζ-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCRζ
dim
T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCRζ
dim
T cells in coronary artery disease.

Methods and Results—

We examined the expression of TCRζ-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4
+
TCRζ
dim
T cells were found in patients with acute coronary syndromes (ACS, n=66; median 5.3%, interquartile 2.6 to 9.1% of total CD4
+
T cells;
P
<0.0001) compared to chronic stable angina (CSA, n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein, and it persisted after the acute event. Moreover, TCRζ
dim
cells were also more represented within CD8
+
T cell, NK, and CD4
+
CD28
null
T cell subsets in ACS compared to CSA and controls. Finally, CD4
+
and CD8
+
TCRζ
dim
T cells isolated from ACS displayed an enhanced transendothelial migratory capacity.

Conclusions—

TCRζ
dim
T cells, an effector T-cell subset with transendothelial migratory ability, are increased in ACS, and may be implicated in coronary instability.
Effect of Macrophage Overexpression of Murine
Liver X Receptor
-α (
LXR
-α) on Atherosclerosis in LDL-Receptor Deficient Mice
Authors: Teupser Daniel , Kretzschmar Daniel , Tennert Carsten , Burkhardt Ralph , Wilfert Wolfgang , Fengler Dörte , Naumann Ronald , Sippel Albrecht E. , Thiery Joachim
Published: September 2008

Background—

The nuclear liver X receptor-α (LXR-α) has been implicated in the regulation of intracellular cholesterol homeostasis, inflammatory response, and atherosclerosis susceptibility. The aim of the present study was to test whether transgenic expression of LXR-α might affect these mechanisms and result in a reduction of atherosclerosis.

Methods and Results—

We generated mice with macrophage overexpression of mouse
LXR
-α, evidenced by significantly elevated expression levels of LXR-target genes (
ABCA1
,
ABCG1
) in these cells. For atherosclerosis studies, mice were crossed onto the LDL-receptor deficient background. Plasma lipids and lipoproteins as well as liver triglycerides were not significantly different between transgenic animals and nontransgenic controls. However, lesion area at the brachiocephalic artery (BCA) was significantly reduced (−83%,
P
=0.02) in male
LXR
-α transgenic mice. This was associated with a significantly increased cholesterol efflux to acceptor-free media (+24%,
P
=0.002) and ApoA1 containing media (+20%,
P
<0.0001) as well as reduced lipopolysaccharide (LPS)-induced NO-release from macrophages of transgenic animals, providing a potential mechanism for the reduction of atherosclerosis.

Conclusion—

Our data show for the first time that transgenic overexpression of
LXR
-α in macrophages has significant antiatherogenic properties. We conclude that overexpression of
LXR
-α in macrophages might be useful as a therapeutic principle for the prevention of atherosclerosis.