ognizant Communication Corporation

ONCOLOGY RESEARCH
Featuring PRECLINICAL AND CLINICAL CANCER THERAPEUTICS

ABSTRACTS
VOLUME 17, NUMBERS 11-12

Oncology Research, Vol. 17, pp. 505-578
0965-0407/09 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Chemopreventive Effect of Nonsteroidal Anti-inflammatory Drugs on 9,10-Dimethylbenz[a]anthracene-Induced Lung Carcinogenesis in Mice

R. K. Saini and S. N. Sanyal

Department of Biophysics, Panjab University, Chandigarh-160014, India

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, celecoxib, and etoricoxib were studied as chemopreventive agents in lung cancer in mice induced by 9,10-dimethylbenz[a]anthracene (DMBA). The animals were subjected to a single intratracheal instillation of DMBA by surgical intervention, while they were treated with oral NSAIDs daily at their following anti-inflammatory dose: aspirin 25 mg/kg, celicoxib 6 mg/kg, and etoricoxib 0.6 mg/kg body weight, respectively. The animals were sacrificed after 18 weeks of treatment. Results showed a significant incidence of pulmonary tumors, dysplastic changes in histopathology, and signs of inflammatory occurrence in the DMBA-treated animals, which were grossly reversed by the NSAIDs. A greater number of macrophages, neutrophils, and lymphocytes were seen in the bronchoalveolar lavage (BAL) smear while the inflammatory cell counts decreased in DMBA + NSAIDs groups. A significant increase in the drug-metabolizing enzymes viz. cytochrome p450, cytochrome b5, and glutathione-S-transferase was noted in the DMBA group, which was reverted back in the NSAID-treated mice. Similarly, the subcelluler enzymes were elevated in DMBA, but significantly fell in the NSAID groups. DMBA also caused a higher level of lipid peroxidation as well as the different antioxidant enzyme activity, which were corrected by the NSAIDs. A marked elevation was noticed in the total lipid composition and its individual constituents in the DMBA group, which was reverted back appreciably by the NSAIDs. The results suggest that the DMBA-induced lung tumor development in balb/c mice could be a reliable model to test the chemopreventive potential of the NSAIDs.

Key words: 9,10-Dimethylbenz[a]anthracene; Mice lung tumorigenesis; Nonsteroidal anti-inflammatory drugs

Address correspondence to Dr. S. N. Sanyal, Professor, Department of Biophysics, Panjab University, Chandigarh-160014, India. Tel: 91-172-2534119; Fax: 91-172-657082; E-mail: sn.sanyal@yahoo.com




Oncology Research, Vol. 17, pp. 519-526
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Angiogenic Pathway Inhibition of Corydalis yanhusuo and Berberine in Human Umbilical Vein Endothelial Cells

Jian-Li Gao, Jun-Min Shi, Simon Ming-Yuen Lee, Qing-Wen Zhang, and Yi-Tao Wang

Institute of Chinese Medical Sciences, University of Macau, Macao, China

Corydalis yanhusuo, a well-known traditional Chinese medicine, is widely used in China as an analgesic for patients with terminal cancer. In this study, we want to expose the antiangiogenic effects and the underlying mechanisms of C. yanhusuo and the alkaloids obtained from this plant. The constituents of C. yanhusuo were first investigated for their inhibitory effects on angiogenesis, using several bioassays, including vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion, and tube formation. To determine the active antiangiogenic compounds in C. yanhusuo, we studied the antiproliferative activities of several main constituents of C. yanhusuo, which belong to a group of protoberberine alkaloids, on HUVECs and identified berberine as a powerful angiogenesis inhibitor in C. yanhusuo. Both C. yanhusuo extract and its active compound berberine significantly suppressed the VEGF-induced upregulation of matrix metalloproteinase 2 (MMP2) at both mRNA and protein levels. Their functional effects, including the inhibition of MMP2, were shown to be involved VEGF-triggered ERK1/2 pathways. Our findings provide novel insights into the antiangiogenic effects of C. yanhusuo and berberine, and offer scientific evidence for their traditional clinical application as a cancer treatment.

Key words: Corydalis yanhusuo; Berberine; HUVEC; Angiogenesis

Address correspondence to Prof. Yi-Tao Wang or Dr. Simon Ming-Yuen Lee, Institute of Chinese Medical Sciences, University of Macau, Av. Padre Tomas Pereira S.J., Taipa, Macao, China. Tel: 853 8397 4695; Fax: 853 2884 1358; E-mail: jianligao@gmail.com




Oncology Research, Vol. 17, pp. 527-533
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Effects of a-Adrenoceptor Antagonist Doxazosin on MDR1-Mediated Multidrug Resistance and Transcellular Transport

Kohji Takara,5 Toshiyuki Sakaeda,1 Mikio Kakumoto,2 Yusuke Tanigawara,3 Hironao Kobayashi,4 Katsuhiko Okumura,5 Noriaki Ohnishi,5 and Teruyoshi Yokoyama5

1Frontier Education Center, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
2Department of Hospital Pharmacy, School of Medicine, Kobe University, Kobe 650-0017, Japan
3Department of Pharmacy, Keio University Hospital, Tokyo 160-8582, Japan
4Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553-0002, Japan
5Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji 670-8524, Japan

The purpose of this study is to examine the effects of doxazosin, an P-adrenoceptor antagonist, on Pglycoprotein/MDR1-mediated multidrug resistance (MDR) and the transport of anticancer drugs. The effects of doxazosin, prazosin, and terazosin on MDR1-mediated MDR were assessed in human cervical carcinoma HeLa cells and the MDR1-overexpressing derivative Hvr100-6, established by stepwise increases of the vinblastine concentration in the culture medium. The effects of doxazosin on the transcellular transport and intracellular accumulation of [3H]vinblastine, [3H]daunorubicin, and [3H]digoxin, all MDR1 substrates, were evaluated using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK<sb>1 cells. The sensitivity to vinblastine and paclitaxel of Hvr100-6 cells was increased at 3.4- and 17.5-fold, respectively, by the addition of 1 mM doxazosin, whereas prazosin and terazosin had weaker or no such effects. Prazosin at 1 mM had a reversal effect on the sensitivity to vinblastine, whereas terazosin had no effect. In transport experiments, doxazosin concentration dependently increased the apical-to-basal transport of radiolabeled drugs in LLC-GA5-COL150 cells, but did not show remarkable effects on the basal-to-apical transport. In addition, doxazosin restored the intracellular accumulation in a concentration-dependent manner in LLC-GA5-COL150 cells. Doxazosin may partly reverse MDR by inhibiting MDR1-mediated transport, making it a candidate lead compound in the development of a reversing agent for MDR.

Key words: Doxazosin; MDR1/P-glycoprotein; Multidrug resistance; Reversal; Anticancer drug; Digoxin

Address correspondence to Dr. Kohji Takara, Department of Clinical Pharmaceutics, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji 670-8524, Japan. Tel/Fax: +81-79-223-6831; E-mail: takara@gm.himeji-du.ac.jp




Oncology Research, Vol. 17, pp. 535-547
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Hemin Counteracts the Repression of Bcl-2 and Nrf2 Genes and the Cell Killing Induced by Imatinib in Human Bcr-Abl(+) CML Cells

Ioannis D. Bonovolias and Asterios S. Tsiftsoglou

Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

Imatinib is a targeted selective inhibitor of chimaeric Bcr-Abl tyrosine kinase developed for effective therapy of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) patients. Unfortunately, evidence now exists to indicate that a portion of such patients treated with imatinib acquire resistance and subsequently relapse. To understand the heterogeneous basis of imatinib resistance, we have investigated the possible mechanism(s) via which hemin, a key regulator of hematopoiesis that is converted to heme intracellularly, renders CML cells less susceptible to imatinib. Hemin at 30-90 mM protected a substantial proportion (>40%) of human Bcr-Abl(+) CML cells (K-562 and KU-812) from imatinib-induced cell killing by increasing the imatinib IC50 value, reducing DNA damage, and promoting erythroid differentiation. RT-PCR assessment of RNA transcripts encoded by human GAPDH, Gg-globin, Bcr-Abl, HO-2, Hpr-6, CEBPa, Bcl-2a, Bcl-2b, and Nrf2 genes revealed that hemin selectively counteracted the repression of antiapoptotic Bcl-2a, Bcl-2b, and Nrf2 genes in imatinib-treated cells. These genes are markedly repressed by imatinib alone in human K-562 CML cells. Hemin, however, had no detectable effect on the expression of the Bcr-Abl gene. Moreover, inhibition of de novo heme biosynthesis by succinyl-acetone enhanced the killing effect of imatinib. These data clearly indicate that: (a) cellular heme resulted from de novo biosynthesis and hemin uptake alters the developmental stage of human Bcr-Abl(+) CML cells and their susceptibility to imatinib; (b) cellular heme counteracts the ability of imatinib to repress Bcl-2 and Nrf2 gene expression; and (c) inhibitors of de novo biosynthesis can be developed and combined with imatinib to enhance its antileukemic activity.

Key words: Imatinib mesylate; Bcr-Abl kinase; Hemin; CML cells; Protection

Address correspondence to Prof. Asterios S. Tsiftsoglou, Laboratory of Pharmacology, Department of Pharmaceutical Sciences,Aristotle University of Thessaloniki (A.U.TH.), GR-54124, Thessaloniki, Greece. Tel: +30-2310-997631; Fax: +30-2310-997618; E-mail: tsif@pharm.auth.gr




Oncology Research, Vol. 17, pp. 549-558
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Evaluation of Global Genome Methylation in Gastritis Lesion and its Correlation With Clinicopatological Findings

Rouhallah Najjar Sadeghi,1 Homayon Zojaji,1 Seyed Reza Mohebbi,1 Mohsen Chiani,2 Mohsen Vahedi,1 Dariush Mirsattari,1 Mahsa Molaei,1 Reza Mashayekhi,1 and Mohamad Reza Zali1

1Research Center for Gastroenterology and Liver Diseases, Shaheed Beheshti Medical University, Tehran, Iran
2Pilot Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran

Global genome hypomethylation as an epigenetic phenomenon may induce (pre)neoplastic transformation through inducing chromosomal and genomic instability and activating oncogenes. Global genome hypomethylation has a fundamental role in early stages of tumorigenesis but little is known about this epigenetic event in gastric precancerous lesions such as gastritis. Therefore, we decided to evaluate this issue in gastritis lesion for obtaining new insight toward molecular biology of gastric cancer. Here we used a technique composed of restriction enzyme digestion and pyrosequencing known as luminometric methylation assay to evaluate this issue. DNA obtained from normal and gastritis lesions was digested with HpaII (sensitive to methylation in its cut site) and MspI (insensitive). Overhangs resulting from these enzymes then fill in by polymerase extension assay using pyrosequencing instrument. Nucleotide incorporation during polymerase extension generates light, which expresses as pick in the pyrogram. By comparing the height of picks obtained form both enzymes it can be possible to evaluate and compare global genome methylation level of gastritis and normal tissues. If the target site is fully methylated, the HpaII/MspI (their pick height) will approach zero. If not, this ratio will be around 1. In the other conditions this ratio varies between 0 and 1. Comparing the ratio of normal and gastritis sample, it can be inferred whether or not gastritis is hypomethylated. This study was performed on 83 gastritis and normal adjacent tissues. The patients included 34 male and 49 female and were 15 to 83 years old. According to our study, gastritis tissue was hypomethylated more than the normal tissue (p = 0.028). Global genome methylation has no significant correlation with MSI, pathological findings, age, and gender. We conclude that global genome hypomethylation occurs in the gastritis level. This reduction probably continues in the next steps toward gastric cancer and may induce other epigenetic and/or genetic changes (such as MSI) that promote carcinogenesis.

Key words: Global genome methylation; Hypomethylation; Gastritis; Gastric cancer; MSI; Pyrosequencing

Address correspondence to Rouhallah Najjar Sadeghi, Research Center for Gastroenterology and Liver Diseases, Taleghani Hospital, Yaman St., Shahid Chamran Highway, Tehran, Iran 1985717413. Tel: +98 21 22432515; Fax: +98 21 22432517; E-mail: najjarsadeghi@yahoo.com




Oncology Research, Vol. 17, pp. 559-564
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Gemcitabine Plus Irinotecan as First-Line Weekly Therapy in Locally Advanced and/or Metastatic Pancreatic Cancer

B. Neri,1 G. Cipriani,1 R. Grifoni,1 E. Molinara,1 P. Pantaleo,1 S. Rangan,1 A. Vannini,1 P. Tonelli,2 A. Valeri,2 D. Pantalone,3 A. Taddei,3 and P. Bechi3

1Department of Oncology, Centre of Experimental and Clinical Oncology, Florence University, AOU Careggi, Florence, Italy
2Unit of General Surgery, AOU Careggi, Florence, Italy
3Department of Critical Medicine and Surgery, Florence University, AOU Careggi, Florence, Italy

Single-agent gemcitabine has been established as standard treatment for advanced pancreatic cancer since clinical studies have shown an improvement in overall survival and significant clinical benefit when compared to the best supportive care despite low overall objective response. Several phase II studies have tested other single agents and different gemcitabine-based regimens in pancreatic cancer, but both response and survival rates have remained low. Irinotecan, a topoisomerase I inhibitor currently approved for the treatment of metastatic colon cancer, has also demonstrated improved response rate in patients with pancreatic cancer. Our purpose was to determine the activity and toxicity of this regimen in patients with unresectable or metastatic pancreatic cancer. Patients with histologically confirmed pancreatic adenocarcinoma received gemcitabine 1000 mg/m2 plus irinotecan 100 mg/m2 IV on days 1, 8, and 15 of a 28-day cycle for 6-8 months. From February 2004 to April 2006, 33 patients were entered into this study, 32 of whom were evaluable for treatment response, toxicity, median time to progression, and median survival. Characteristics included a median age of 63 years (range 41-79), 21 males (64%), and 12 females (36%). One patient discontinued treatment due to adverse effects. The total number of cycles administered was 188 and the median number of cycles for patients was 5.6 (range 2-7). Thirty-two patients were assessable for toxicity and response. Grade 3 hematological toxicity occurred in 9% of patients and was primarily neutropenia. No grade >2 gastrointestinal toxicities or death due to treatment were observed. The most frequent nonhematological adverse event was fatigue. Ten patients responded to treatment with two complete responses (6.3%) and eight partial responses (25.0%), for an overall response rate of 31.3%; 11 patients achieved stable disease (34.3%). The median time to tumor progression and the median survival were 9.2 (95% CI: 6.0-12.4) and 11.8 (95% CI: 7.7-15.9) months, respectively, with a 2-year survival of 22%. On the basis of this trial, the combination of gemcitabine plus irinotecan, administered in a weekly schedule and at this dose, is well tolerated and offers encouraging activity in the treatment of advanced and/or metastatic pancreatic cancer.

Key words: Chemotherapy; Gemcitabine; Irinotecan; Pancreatic cancer

Address correspondence to Prof. Burno Neri, Centro di Oncologia Sperimentale e Clinica, Day Hospital Oncologico, Azienda Ospedaliero Universitaria Careggi, V. le Pieraccini 17, 50139 Firenze, Italia. E-mail: bruno.neri@unifi.it




Oncology Research, Vol. 17, pp. 565-570
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Weekly Administration of Docetaxel and Epirubicin as First-Line Treatment for Hormone-Refractory Prostate Carcinoma

B. Neri,1 E. Molinara,1 P. Pantaleo,1 S. Rangan,1 A. Crisci,2 A. Della Melina,2 A. Raugei,2 D. Villari,2 and G. Nicita2

1Department of Oncology, Center for Experimental and Clinical Oncology, University of Florence, Florence, Italy
2Department of Urology, University of Florence, Florence, Italy

Androgen-independent prostate carcinoma (AICP) is one of the tumors that continue to respond poorly to chemotherapy. Recently, protocols based on the use of docetaxel have significantly improved survival for patients in this disease. In other types of neoplastic disease, combined therapy with taxanes and anthracycline derivatives has been shown to produce additive effects in terms of growth inhibition, and superior tolerability when associated with weekly administration schedules. These findings prompted us to examine the tolerability and efficacy of weekly treatment of AICP with docetaxel (DOX) plus epirubicin (EPI). We enrolled 35 chemotherapy-naive men with AICP (mean age 72 years, range 68-77) and normal hepatic, renal, and cardiac function. The chemotherapy protocol provided for the IV administration of DOX (30 mg/m2) and EPI (30 mg/m2) on days 1, 8, and 15 every 28 days. Treatment was continued for 6 months or until disease progression and/or unacceptable toxicity was observed. Serum levels of prostate-specific antigen (PSA) were monitored in all patients, and reductions from baseline values of >50% were considered indicative of positive responses to treatment. Thirty-four patients were included in the analysis of toxicity, and objective responses to treatment were assessed in the 28 patients with measurable lesions. Nineteen patients (56%) experienced PSA reductions of >50% that persisted for more than 4 weeks. The response to therapy was classified as complete in 1 of the 28 patients (4%) with measurable disease (at the lymph node level). Thirteen others (13/28, 46%) had partial responses, in nine (32%) the disease remained unchanged, and progression was observed in the remaining five (18%); overall response rate was 50% (CR + PR). Of the 27 patients with pain at the time of enrollment, 16 (59%) experienced pain reduction during treatment. The median time to disease progression was 11.7 months (95% CI: 7.7-15.7) while the median survival time was 18.7 months (95% CI: 12.3-25.1). During the study, four patients developed grade 3 anemia and leukopenia, which was reversible in all cases. Lower grades of asthenia, nausea, vomiting, diarrhea, and peripheral edema were also observed. There were no cases of cardiotoxic effects. Alopecia was frequent but reversible in all cases. The results of this preliminary study indicate that the combined administration of DOX and EPI for treatment of AIPC is effective and well tolerated. The weekly administration of the drug combination appears to be a promising approach to the treatment of these tumors.

Key words: Prostate carcinoma; Chemotherapy; Docetaxel; Epirubicin; Weekly regimen

Address correspondence to Prof. Bruno Neri, Centro di Oncologia Sperimentale e Clinica, Day Hospital Oncologico, Azienda Ospedaliero Universitaria Careggi, V. le Pieraccini 17, 50139 Firenze, Italia. E mail: bruno.neri@unifi.it




Oncology Research, Vol. 17, pp. 571-580
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Expression and Mutation Analysis of TIG1 (Tazarotene-Induced Gene 1) in Human Gastric Cancer

Min Soo Son,1 Min-Ju Kang,2 Ho Chul Park,1 Sung-Gil Chi,2 and Yong Ho Kim1

1Department of Surgery, College of Medicine, Kyung Hee University, Seoul, Korea
2School of Life Sciences and Biotechnology, Korea University, Seoul, Korea

Tazarotene-induced gene 1 (TIG1) has been known to function as a cell adhesion molecule, which leads to better cell to cell contact and reduced proliferation. We investigated expression and mutation status of TIG1 in primary gastric tumors and cell lines to explore the candidacy of the gene as a tumor suppressor. A total of 172 gastric tissue specimes, including 80 primary adenocarcinomas, 12 benign tumors, and 80 adjacent normal mucosa, and 15 gastric cancer cell lines were used. TIG1 expression was analyzed by semiquantitative RT-PCR and immunoblot analysis. To screen for the presence of somatic mutations, RT-PCR-SSCP analysis was carried out. The effect of 5-aza-2´-deoxycytidine treatment was examined to elicit whether TIG1 reduction is associated with abnormal DNA hypermethylation. Compared to noncancerous tissues, a substantial reduction of TIG1 expression was observed in 73.3% (11/15) cancer cell lines, and seven of these exhibited nearly undetectable levels of expression. Decreased expression of TIG1 was also found in 62 (77.5%) primary carcinoma tissues compared to adjacent noncancerous tissues, indicating a tumor-specific reduction of TIG1. Expression levels of TIG1 were significantly low in primary carcinomas and cancer cell lines compared to those of normal tissues. Moreover, loss or reduction of TIG1 was significantly high in advanced tumors compared to early tumors and more frequent in poorly differentiated tumors than well or moderately differentiated tumors. TIG1 expression was reactivated or its level was elevated following 5-aza-2´-deoxycytidine treatment, indicating that TIG1 expression is transcriptionally silenced in these cancer cells by abnormal DNA hypermethylation. These data indicate that TIG1 undergoes frequent epigenetic inactivation due to aberrant DNA hypermethylation in gastric cancers, and its altered expression is associated with the malignant progression of tumors.

Key words: Tazarotene-induced gene 1 (TIG1); Hypermethylation; Gastric cancer

Address correspondence to Yong Ho Kim, M.D., Department of Surgery, College of Medicine, Kyung Hee University, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Republic of Korea. Tel: +82-2-958-8246; Fax: +82-2-966-9366; E-mail: kyjho@khmc.or.kr or Sung-Gil Chi, Ph.D., School of Life Sciences and Biotechnology, Korea University, 136-701 Seoul, Republic of Korea. Tel: +82-2-3290-3443; Fax: +82-2-927-5458; E-mail: chi6302@korea.ac.kr




Oncology Research, Vol. 17, pp. 581-591
0965-0407/09 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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A Bone Metastasis Model With Osteolytic and Osteoblastic Properties of Human Lung Cancer ACC-LC-319/bone2 in Natural Killer Cell-Depleted Severe Combined Immunodeficient Mice

Shinsaku Otsuka,1 Masaki Hanibuchi,1 Kenji Ikuta,1 Seiji Yano,2 Hisatsugu Goto,1 Hirokazu Ogino,1 Tadaaki Yamada,2 Soji Kakiuchi,3 Yasuhiko Nishioka,1 Takashi Takahashi,4 and Saburo Sone13

1Department of Respiratory Medicine and Rheumatology, Institute of Health Biosciences, University of Tokushima Gradate School, Tokushima, 770-8503, Japan
2Division of Medical Oncology, Cancer Research Institute, Molecular Carcinogenesis, Kanazawa University, Kanazawa, 920-0934, Japan
3Department of Medical Oncology, Institute of Health Biosciences, University of Tokushima Gradate School, Tokushima, 770-8503, Japan
4Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 464-8601, Japan

Lung cancer is commonly associated with multiple-organ metastasis, and bone is a frequent metastatic site for lung cancer. Lung cancer frequently develops osteolytic, and less frequently osteoblastic, metastasis to bone. Osteolytic metastasis models of lung cancer have been reported, but no osteoblastic metastasis model is available for lung cancer. In the present study, we established a reproducible model of human lung cancer with both osteolytic and osteoblastic changes in natural killer cell-depleted severe combined immunodeficient mice. Intravenous inoculation of ACC-LC-319/bone2 cells resulted in the development of metastatic colonies in the lung, liver, and bone of the mice. As assessed sequentially by X-ray photographs, osteolytic bone lesions were observed by day 28, and then osteoblastic lesions were detected by day 35. Histological examination revealed the presence of bony spurs, a hallmark of osteoblastic bone metastasis, where osteoclasts were hardly observed. Treatment with an anti-human vascular endothelial growth factor antibody, bevacizumab, as well as zoledronate, inhibited the number of experimental bone metastases, including osteoblastic changes produced by ACC-LC-319/bone2 cells. These results indicate that our bone metastasis model by ACC-LC319/bone2 might be useful to understand the molecular pathogenesis of osteolytic and osteoblastic metastasis, and to identify molecular targets to control bone metastasis of lung cancer.

Key words: Bone metastasis model; Osteoblastic bone metastasis; Osteolytic bone metastasis; Lung adenocarcinoma

Address correspondence to Saburo Sone, Department of Respiratory Medicine and Rheumatology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho Tokushima, 770-8503, Japan. Tel: +81-88-633-7127; Fax: +81-88-633-2134; E-mail: ssone@clin.med.tokushima-u.ac.jp




Oncology Research, Vol. 17, pp. 593-599
0965-0407/09 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Cloning of a Novel Splicing Variant of RIN1 and its Expression in Gastric and Colon Cancer

Masako Fujioka, Takanori Goi, Yasuo Hirono, Kanji Katayama, and Akio Yamaguchi

First Department of Surgery, University of Fukui, Fukui, Japan

The regular RIN1 gene is a molecule located on chromosome 11q13.2, and contains a coding region of 2352 bp with a 3´ domain that binds to H-Ras protein, suggesting that it is an important molecule in the intracellular signaling pathway. In this study, we confirmed the existence of a novel form of the RIN1 gene with a different splicing pattern, successfully cloned it, and examined its expression in gastric and colon cancer cell lines. A 612-bp band (the RIN1 variant mRNA) was identified in the RT-PCR product from the colon cancer cell line Colo320D. (A 2352-bp band representing the regular RIN1 gene in HT29 cell line.) The 612-bp band was sequenced and compared with that of the regular RIN1 gene. As a result, the 612-bp product was found to contain a tyrosine phosphorylation site on the 5´ side and Ras and 14-3-3 binding domains on the 3´ side, indicating that it is a product with a different splicing pattern. The expression of the RIN1 variant mRNA was observed in two of six gastric cancer cell lines and four of five colon cancer cell lines. We identified a novel RIN1 gene with a splicing pattern different from that of the regular RIN1 gene. Comparison of both genes revealed that the novel RIN1 products had a structure conserving the Ras and 14-3-3 binding domains, but lacking two tyrosine phosphorylation sites. Novel RIN1 variant protein was expressed primarily in the cytoplasm and no expression in the cell membrane, and RIN1 variant protein was bound to 14-3-3 protein. In addition, the novel RIN1 mRNA was found to be expressed in gastric and colon cancer cell lines, suggesting that it is an important gene for the function of cancer cells.

Key words: Colorectal cancer; Splicing variant; RIN1 gene

Address correspondence to Takanori Goi, First Department of Surgery, University of Fukui, 23-3, Eiheiji-cho, Yoshida-gun, Fukui, Japan. Tel: 81-776-61-3111, ext. 2343; Fax: 81-776-61-8113; E-mail: tgoi@u-fukui.ac.jp




Oncology Research, Vol. 17, pp. 601-612
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E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Comparative Proteomic Analysis of Mouse Melanoma Cell Line B16, a Metastatic Descendant B16F10, and B16 Overexpressing the Metastasis-Associated Tyrosine Phosphatase PRL-3

Sang Hee Kim,1* Yongmo Kim,1* Moonil Kim,2 Dae Shick Kim,3 Sang Chul Lee,1 Seung-Wook Chi,1 Do Hee Lee,1 Sung Goo Park,1 Byoung Chul Park,1 Kwang-Hee Bae,1 and Sunghyun Kang1

1Medical Proteomics Research Center, KRIBB, Daejeon, 305-806, Korea
2BioNanotechnology Research Center, KRIBB, Daejeon 305-806, Korea
3Department of Pathology, Samsung Medical Center and Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea

Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of B16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the B16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase b subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.

Key words: B16 Differential in-gel electrophoresis (DIGE); Melanoma; Metastasis; Proteomics

Address correspondence to Sunghyun Kang, Translational Research Center, KRIBB, 52 Eoeun-dong, Yuseong, Daejeon 305-806, Korea. Tel: +82-42-860-4243; Fax: +82-42-860-4593; E-mail: skang@kribb.re.kr or Kwang-Hee Bae, Translational Research Center, KRIBB, 52 Eoeun-dong, Yuseong, Daejeon 305-806, Korea. Tel: +82-42-860-4268; Fax: +82-42-860-4593; E-mail: khbae@kribb.re.kr

*Both authors equally contributed to this work.




Oncology Research, Vol. 17, pp. 613-620
0965-0407/09 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
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Knockdown of Bmi-1 Impairs Growth and Invasiveness of Human Gastric Carcinoma Cells

Jun Xiao and Changsheng Deng

Department of Gastroenterology and Hubei Provincial Center of Clinical Study for Digestive Diseases, Zhongnan Hospital, the Key Laboratory of Allergy and Immune-related Diseases, Wuhan University School of Medicine, Wuhan, PR China

The effect of B cell-specific MLV integration site-1 (Bmi-1) RNA interference (RNAi)-mediated inhibition of Bmi-1 expression on the proliferation, apoptosis, and invasiveness of human gastric carcinoma AGS cells was investigated. RT-PCR and Western blot analyses demonstrated that Bmi-1 expression and the Bmi-1 protein level were significantly decreased in Bmi-1 shRNA transfected AGS cells compared to untransfected and nonspecific shRNA transfected AGS cells. Bmi-1 RNAi-mediated inhibition of Bmi-1 expression significantly affected cell growth and invasiveness, and resulted in increased AGS cell apoptosis. This was not observed in untransfected and nonspecific shRNA transfected AGS cells. Inhibition of Bmi-1 expression in human gastric carcinoma cells affects cell proliferation and invasiveness.

Key words: Apoptosis; Bmi-1; Gastric carcinoma; Proliferation; RNA interference (RNAi)

Address correspondence to Prof. Changsheng Deng, M.D., Ph.D., Department of Gastroenterology, Wuhan University Zhongnan Hospital, Donghu Road 169, Wuhan 430071, Hubei Province, P.R. of China. Tel: 00 86 27 67813072; Fax: 0086 27 67813061; Email: xiaojunzhongnan@163.com




Oncology Research, Vol. 17, pp. 621-630
0965-0407/09 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Antitumor Effects of Targeting hTERT Lentivirus-Mediated RNA Interference Against KB Cell Lines

Dan Chen,12 Hongzhang Huang,1 Chaobin Pan,3 Jianguang Wang,3 Bin Zhang,3 and Anxun Wang2

1Department of Oral & Maxillofacial Surgery, Guanghua College of Stomatology, Sun Yat-Sen University, Guangzhou, Guangdong Province, 510055, PR China
2Department of Oral & Maxillofacial Surgery, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong Province, 510080, PR China
3Department of Oral & Maxillofacial Surgery, Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong Province, 510120, PR China

Squamous cell carcinomas are the leading frequent malignant tumors in the oral and maxillofacial region. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a sequence-specific RNA degradation process. In this study, we screened and identified an in vitro-transcribed 21-bp shRNA targeting human telomerase reverse transcriptase (hTERT) from three candidates and generated a lentivirus vector. Subsequent experiments indicated that this lentiviral transgenic system could effectively transfer into target KB cells, above 80% gene transfer efficiency at MOI of 2.5, and significantly and specifically inhibited hTERT expression both in mRNA (73.42%) and protein (74.67-82.91%) levels. To further evaluate the role of hTERT-targeted RNAi, we found that hTERT inhibition consequently induced suppression of cyclin D1 (54.67%), upregulation of caspase-3 (100.10%), and caspase-9 (42.67%) of KB cells. Therefore, the apoptosis rates of KB cells were increased by 206.33%. In conclusion, these data indicated the potential of lentivirus vectors in cancer gene therapy, especially after development of more efficient vector production methods, and higher virus titers demonstrated that targeting hTERT RNAi may result in telomere uncapping, which triggers cell cycle arrest and apoptosis signal and leads to tumor suppression.

Key words: RNA interference (RNAi); Gene therapy; Oral squamous cell carcinoma; Human telomerase reverse transcriptase; Lentiviral vector

Address correspondence to Hongzhang Huang, Department of Oral & Maxillofacial Surgery, Guanghua college of Stomatology, Sun Yat-Sen University, No. 56, Linyuanxi Road, Guangzhou City, Guangdong Province, 510055, People's Republic of China. Tel: +86 20 83820121; Fax: +86 20 83822807; E-mail: Huanghongzhang.sysu@yahoo.com.cn