Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Kubo Makoto Last modified date:2020.04.03

Associate Professor / Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University / Department of Clinical Medicine / Faculty of Medical Sciences

1. Mori H, Kubo M, Kai M, Velasquez VV, Kurata K, Yamada M, Okido M, Kuroki S, Oda Y, Nakamura M, BRCAness Combined With a Family History of Cancer Is Associated With a Poor Prognosis for Breast Cancer Patients With a High Risk of BRCA Mutations, Clin Breast Cancer., 10.1016/j.clbc.2018.05.008, 18, 5, e1217-e1227, 2018.04.
2. Mori H, Kubo M, Kai M, Kurata K, Yamada M, Nakamura M, Efficacy and Safety of Bi-weekly Pegfilgrastim for Dose-dense Chemotherapy-induced Neutropenia in Breast Cancer Patients, Anticancer Research, 10.21873/anticanres.12740 , 38, 7, 4381-4386, 2018.04, Abstract
The dose-dense doxorubicin and cyclophosphamide (ddAC) for patients with HER-2-negative breast cancer is recommended by the National Comprehensive Cancer Network guideline in US. However, there are little data on serum G-CSF concentrations in patients undergoing bi-weekly dose-dense therapy with pegfilgrastim. The objective of this study was to compare the serum G-CSF concentrations in patients receiving pegfilgrastim in bi- or tri-weekly regimens.
This study included 26 patients who received ddAC or docetaxel and cyclophosphamide (TC) for primary breast cancer. Serum G-CSF concentrations were measured by ELISA.
Serum G-CSF concentrations peaked in the second week of ddAC cases and in the ninth week of TC cases. Neutrophils gradually increased until the sixth week in ddAC cases, while they were slightly decreased during the first three weeks in TC cases. Treatments were completed without febrile neutropenia or treatment delays.
Primary prophylactic pegfilgrastim administrations increased serum G-CSF concentrations, helping to maintain the absolute neutrophil counts that are required to undergo chemotherapy. The treatment of ddAC with 3.6 mg pegfilgrastim is completely safe for female Japanese patients.
3. Nobuhiro Torata, Makoto Kubo, Daisuke Miura, Kenoki Ohuchida, Yusuke Mizuuchi, Yoshinori Fujimura, Eisuke Hayakawa, Masaya Kai, Yoshinao Oda, Kazuhiro Mizumoto, Makoto Hashizume, Masafumi Nakamura, Visualizing Energy Charge in Breast Carcinoma Tissues by MALDI Mass-spectrometry Imaging Profiles of Low-molecular-weight Metabolites, Anticancer Res, doi:10.21873/anticanres.12723, 38, 7, 4267-4272, 2018.04, Abstract
Metabolomics is widely used for biomarker discovery, but conventional mass-spectrometry extraction procedures lose the spatial localization of metabolites. In this study, we directly analyzed breast carcinoma tissues embedded in frozen tissue microarrays (fTMAs) using MALDI mass-spectrometry imaging (MALDI-MSI).
A total of 119 breast tissues (84 carcinoma and 35 normal) were used. MSI data were extracted from each tissue.
Overall, 185 of 1,915 peaks which were commonly detected in 60% of target areas were subjected to further analysis. One hundred and fifty-two peaks of carcinoma showed significantly higher intensity than normal. Comparing metabolite profiles from carcinoma and normal tissues, energy charge (EC) and the sum of adenosine phosphate compound (AXP) indicated significantly higher intensities in cancerous tissues than normal. But comparisons of EC and AXP among lymph node metastasis, tumor size and tumor subtypes indicated no significant differences.
Breast carcinoma tissues had higher EC and AXP values than normal. MALDI-MSI could be a tool for characterizing breast carcinoma.
4. Mori H, Kubo M, Yamaguchi R, Nishimura R, Osako T, Arima N, Okumura Y, Okido M, Yamada M, Kai M, Kishimoto J, Oda Y, Nakamura M, The combination of PD-L1 expression and decreased tumor- infiltrating lymphocytes is associated with a poor prognosis in triple-negative breast cancer, Oncotarget, 8, 9, 15584-15592, 2017.04, This study included patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. Among the 248 TNBCs studied, programmed cell death ligand-1 (PD-L1) expression was detected in 103 (41.5%) tumors, and high levels of tumor-infiltrating lymphocytes (TILs) were present in 118 (47.6%) tumors. PD- L1 expression correlated with high levels of TILs, but was not a prognostic factor. Patients with TILs-high tumors had better overall survival than those with TILs- low tumors (P = 0.016). There was a strong interaction between PD-L1 expression and TILs that was associated with both recurrence-free survival (P = 0.0018) and overall survival (P = 0.015). Multivariate Cox proportional hazards model analysis showed that PD-L1-positive/TILs-low was an independent negative prognostic factor for both recurrence-free survival and overall survival. Our findings suggest that PD-
L1-positive/TILs-low tumors are associated with a poor prognosis in patients with TNBC, and that it is important to focus on the combination of PD-L1 expression on tumor cells and TILs present in the tumor microenvironment. These biomarkers may be useful for stratification of TNBCs and for predicting prognosis and developing novel cancer immunotherapies..
5. Mori H, Kubo M, Nishimura R, Osako T, Arima N, Okumura Y, Okido M, Yamada M, Kai M, Kishimoto J, Miyazaki T, Oda Y, Otsuka T, Nakamura M, BRCAness as a Biomarker for Predicting Prognosis and Response to Anthracycline-Based Adjuvant Chemotherapy for Patients with Triple-Negative Breast Cancer, Plos One, 11, 12, e0167016, 2016.04, BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous tumor that encompasses many different subclasses of the disease. In this study, we assessed BRCAness, defined as the shared characteristics between sporadic and BRCA1-mutated tumors, in a large cohort of TNBC cases. MethodsThe BRCAness of 262 patients with primary TNBCs resected between January 2004 and December 2014 was determined through the isolation of DNA from tumor tissue. Classification of BRCAness was performed using multiple ligation-dependent probe amplification (MLPA). The tumor subtypes were determined immunohistochemically using resected specimens. ResultsOf the 262 TNBCs, the results of the MLPA assays showed that 174 (66.4%) tumors had BRCAness. Patients with BRCAness tumors were younger than patients with non-BRCAness tumors (P = 0.003). There was no significant difference between the two groups regarding their pathological stages. The BRCAness group had a significantly shorter
recurrence-free survival (RFS) compared with the non-BRCAness group (P = 0.04) and had a shorter overall survival (OS) although this did not reach statistical significance. Adjuvant treatments with anthracycline-based regimens provided significantly greater benefits to the BRCAness group (P = 0.003 for RFS, and P = 0.03 for OS). Multivariate Cox proportional hazard model analysis showed that BRCAness was an independent negative prognostic factor, and the anthracycline-based adjuvant chemotherapy was an independent positive prognostic factor for both RFS and OS in TNBC.ConclusionsThe 66.4% patients of TNBCs showed BRCAness. BRCAness is essential as a biomarker in the subclassification of TNBCs and might be of use for predicting their prognosis. Furthermore, this biomarker might be a predictive factor for the effectiveness of anthracycline-based adjuvant chemotherapy for patients with TNBCs..
6. Suyama K, Onishi H, Imaizumi A, Shinkai K, Umebayashi M, Kubo M, Mizuuchi Y, Oda Y, Tanaka M, Nakamura M, Katano, M, CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells, Cancer Letters, 374, 1, 44-53, 2016.04, Hedgehog (Hh) signaling has been found to be activated in breast cancer stem cells (BCSCs). However, the precise role of the BCSCs marker, CD24, remains unclear. Here, we describe a relationship between CD24 and Sonic Hedgehog (SHH), and reveal a role for this relationship in the induction of a malignant phenotype of breast cancer. CD24 siRNA-transfected breast cancer cells (BCCs) demonstrated higher expression of SHH and GLI1, increased anchorage-independent proliferation, and enhanced invasiveness and superior tumorigenicity compared with control. Conversely, CD24 forced-expressing BCCs possessed decreased SHH and GLI1 expression, anchorage-independent proliferation, and invasiveness. Suppression of SHH decreased invasiveness through inhibition of matrix metalloproteinase (MMP)-2 expression, GLI1 expression, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo in CD24 siRNA transfected BCCs. DNA microarray analysis identified STAT1 as a relationship between CD24 and SHH. CD24 siRNA-transfected BCCs with concurrent STAT1 inhibition exhibited decreased SHH expression, invasiveness, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo. These results suggest that CD24 suppresses development of a malignant phenotype by down-regulating SHH transcription through STAT1 inhibition. CD24 gene transfer or STAT1 inhibition may represent new effective therapeutic strategies to target refractory breast cancer.

Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
7. Takizawa K, Yamamoto H, Taguchi K, Ohno S, Tokunaga E, Yamashita N, Kubo M, Nakamura M, Oda Y, Insulin-like growth factor II messenger RNA-binding protein-3 is an indicator of malignant phyllodes tumor of the breast, Hum Pathol, 55, 9, 30-38, 2016.04, Abstract
The aim of this study was to elucidate the clinicopathological and prognostic significance of the expressions of insulin-like growth factor II mRNA-binding protein-3 (IMP3) and epidermal growth factor receptor (EGFR) in phyllodes tumors (PTs). Immunohistochemical staining for IMP3 and EGFR was performed in 130 cases of primary PTs (83 benign, 28 borderline, 19 malignant), 34 recurrent/metastatic PTs, and 26 fibroadenomas (FAs). Among the primary tumors, a high expression of IMP3 was significantly more frequently present in malignant PTs (17/19, 89%) than in the FAs (0/26, 0%), benign PTs (0/83, 0%) and borderline PTs (3/28, 11%). The recurrent and metastatic lesions of malignant PTs also showed high IMP3 expression (3/5 [60%] and 6/6 [100%], respectively). Most malignant PTs showed strong IMP3 expression at the interductal area or more diffusely, whereas weak and focal (low) expression of IMP3 was limited to the periductal area in FAs and benign PTs. EGFR overexpression was significantly correlated with tumor grade and high IMP3 expression. Overexpressions of IMP3 and EGFR were significantly associated with shorter periods of metastasis-free and disease-free survival. The results suggest that high expressions of IMP3 and EGFR with a characteristic staining pattern may be helpful for both identifying malignant PT and predicting the prognosis of these tumors..
8. Jinnouchi M, Yabuuchi H, Kubo M, Tokunaga E, Yamamoto H5, Honda H6, Utility of adaptive control processing for the interpretation of digital mammograms, Acta Radiologica, 1-7, 2015.04, Abstract
Adaptive control processing for mammography (ACM) is a novel program that automatically sets up appropriate image-processing parameters for individual mammograms (MMGs) by analyzing the focal and whole breast histogram.
To investigate whether ACM improves the image contrast of digital MMGs and whether it improves radiologists' diagnostic performance in reading of MMGs.
One hundred normal cases for image quality assessment and another 100 cases (50 normal and 50 cancers) for observer performance assessment were enrolled. All mammograms were examined with and without ACM. Five radiologists assessed the intra- and extra-mammary contrast of 100 normal MMGs, and the mean scores of the intra- and extra-mammary contrast were compared between MMGs with and without ACM in both the dense and non-dense group. They classified 100 MMGs into BI-RADS categories 1-5, and were asked to rate the images on a scale of 0 to 100 for the likelihood of the presence of category 3-5 lesions in each breast. Detectability of breast cancer, reading time, and frequency of window adjustment were compared between MMGs with and without ACM.
ACM improved the intra-mammary contrast in both the dense and non-dense group but degraded extra-mammary contrast in the dense group. There was no significant difference in detectability of breast cancer between MMGs with and without ACM. Frequency of window adjustment without ACM was significantly higher than that with ACM. Reading time without ACM was significantly longer than that with ACM.
ACM improves the image contrast of MMGs and shortens reading time.
© The Foundation Acta Radiologica 2015 Reprints and permissions:
Digital mammography; extra-mammary contrast; image processing; intra-mammary contrast; observer performance.
9. Kamitani T, Matsuo Y, Yabuuchi H, Fujita N, Nagao M, Kawanami S, Yonezawa M, Yamasaki Y, Tokunaga E, Kubo M, Yamamoto H, Honda H, Differentiation between benign phyllodes tumors and fibroadenomas of the breast on MR imaging, Eur J Radiol, 83, 8, 1344-1349, 2014.04, Abstract
The purpose of this study was to determine the factors that contribute to the differentiation between phyllodes tumors (PTs) and fibroadenomas (FAs) on MR imaging.
This retrospective study included 19 PTs and 18 FAs with ≥ 2 cm diameter. The presence or absence of a capsule and internal septum, the extent of lobulation, and the apparent diffusion coefficient (ADC) values were determined. The presence or absence of a cystic component, the time-intensity curve, and the signal intensity on delayed-phase contrast-enhanced T1WI were also evaluated in 31 patients (16 PTs and 17 FAs) who underwent a contrast-enhanced study.
Cystic components were seen in 10 of the 16 PTs (63%) and in 4 of the 17 FAs (24%; P=0.03). The PTs showed strong lobulation more frequently compared to the FAs (14/19 [74%] vs. 7/18 [39%], respectively; P=0.04). Though there was no significant difference, PT tended to be heterogeneous more frequently on the delayed phase of the contrast-enhanced T1WI compared to the FA (11/16 [69%] vs. 7/17 [41%], respectively). No significant difference was found in the other findings.
Although PTs and FAs show similar MR findings, the presence of a cystic component, strong lobulation, and heterogeneity on delayed-phase contrast-enhanced T1WI suggests a PT.
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Breast; Fibroadenoma; MRI; Phyllodes tumor.
10. Nagamatsu I, Onishi H, Matsushita S, Kubo M, Kai M, Imaizumi A, Nakano K, Hattori M, Oda Y, Tanaka M, Katano M, NOTCH4 is a potential therapeutic target for Triple-negative Breast cancer, Anticancer Res, 34, 1, 69-80, 2014.04, BACKGROUND/AIM:

The prognosis for triple-negative breast cancer (TNBC) is poor. In the present study, we evaluated whether NOTCH4 receptor is a potential new therapeutic target for TNBC.


In vitro proliferation and invasiveness were evaluated in TNBC cells with or without small-interfering RNA (siRNA) for NOTCH4, and with or without NOTCH4 plasmid transfection. In vivo, MDA-MB-231 cells with or without NOTCH4 siRNA were subcutaneously implanted into the flank regions of mice. The frequency of nuclear translocation of NOTCH4 was assessed by immunohistochemistry in 21 TNBC samples and 46 non-TNBC samples.


NOTCH4 inhibition in TNBC cells reduced proliferation and invasiveness, and NOTCH4 overexpression in TNBC cells increased proliferation and invasiveness. NOTCH4 inhibition reduced tumour volume and tumourigenicity of mouse xenografts. TNBC cells had a higher frequency of nuclear translocation of NOTCH4 than other cells.


NOTCH4 is a new potential therapeutic target for triple-negative breast cancer.
11. Kubo M, Kanaya N, Petrossian K, Ye J, Warden C, Liu Z, Nishimura R, Osako T, Okido M, Shimada K, Takahashi M, Chu P, Yuan YC, Chen S, Inhibition of the proliferation of acquired aromatase inhibitor-resistant breast cancer cells by histone deacetylase inhibitor LBH589 (panobinostat), Breast Cancer Res Treat, 137, 1, 93-107, 2013.04.
12. Kubo M, Onishi H, Kuroki S, Okido M, Shimada K, Yokohata K, Umeda S, Ogawa T, Tanaka M, Katano M, Short-term and low-dose prednisolone administration reduces aromatase inhibitor-induced arthralgia in patients with breast cancer., Anticancer Res. , 32, 6, 2331-2336, 2012.04, Aromatase inhibitors (AIs) are important therapeutic drugs for postmenopausal women with hormone receptor-positive breast cancer. However, adverse effects of AIs such as arthralgia have been extensively reported. We performed a joint prospective, multi-institutional investigation to find out whether a low-dose and short-term prednisolone is effective against AI-induced arthralgia in 27 patients with breast cancer. Patients were administered 5 mg of oral prednisolone once a day in the morning for only one week. Patients were then asked to answer a questionnaire about joint pain symptoms at one week, one month and two months after the beginning of prednisolone use. Joint pain symptoms improved in 67% of patients immediately after prednisolone use, with 63% still reporting analgesic effect at one month, and 52% at two months after beginning internal use of prednisolone. At one week, one month and two months after the use of prednisolone, 30%, 30% and 26% of patients reported improved daily life, respectively. Our results suggest that prednisolone could substitute non-steroidal anti-inflammatory drugs, acetoaminophen or cyclooxygenase-2 inhibitors in patients with AI-induced arthralgia..
13. Yabuuchi H, Matsuo Y, Sunami S, Kamitani T, Kawanami S, Setoguchi T, Sakai S, Hatakenaka M, Kubo M, Tokunaga E, Yamamoto H, Honda H, Detection of non-palpable breast cancer in asymptomatic women by using unenhanced diffusion-weighted and T2-weighted MR imaging: comparison with mammography and dynamic contrast-enhanced MR imaging, Eur Radiol, 21, 1, 11-17, 2011.04, Abstract
OBJECTIVE: To compare the detectability of non-palpable breast cancer in asymptomatic women by using mammography (MMG), dynamic contrast-enhanced MR imaging (DCE-MRI) and unenhanced MR imaging with combined diffusion-weighted and T2-weighted images (DWI+T2WI).

METHODS: Forty-two lesions in 42 patients with non-palpable breast cancer in asymptomatic women were enrolled. For the reading test, we prepared a control including 13 normal and 8 benign cases. Each imaging set included biplane MMG, DCE-MRI and DWI+T2WI. Five readers were asked to rate the images on a scale of 0 to 100 for the likelihood of the presence of cancer and the BI-RADS category. Confidence level results were used to construct receiver operating characteristic analysis. Sensitivity and specificity were calculated for each technique.

RESULTS: DWI+T2WI showed higher observer performances (area under the curve, AUC, 0.73) and sensitivity (50%) for the detection of non-palpable breast cancer than MMG alone (AUC 0.64; sensitivity 40%) but lower than those of DCE-MRI (AUC 0.93; sensitivity 86%). A combination of MMG and DWI+T2WI exhibited higher sensitivity (69%) compared with that of MMG alone (40%).

CONCLUSION: DWI+T2WI could be useful in screening breast cancer for patients who cannot receive contrast medium and could be used as a new screening technique for breast cancer.

14. Kanaya N, Kubo M, Liu Z, Chu P, Wang C, Yuan YC, Chen S, Protective Effects of White Button Mushroom (Agaricus bisporus) against Hepatic Steatosis in Ovariectomized Mice as a Model of Postmenopausal Women, PLoS One, 6, 10, e26654, 2011.04, Abstract
Nonalcoholic fatty liver disease (NAFLD) includes various hepatic pathologies ranging from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Estrogen provides a protective effect on the development of NAFLD in women. Therefore, postmenopausal women have a higher risk of developing NAFLD. Hepatic steatosis is an early stage of fatty liver disease. Steatosis can develop to the aggressive stages (nonalcoholic steatohepatitis, fibrosis and cirrhosis). Currently, there is no specific drug to prevent/treat these liver diseases. In this study, we found that white button mushroom (WBM), Agaricus Bisporus, has protective effects against liver steatosis in ovariectomized (OVX) mice (a model of postmenopausal women). OVX mice were fed a high fat diet supplemented with WBM powder. We found that dietary WBM intake significantly lowered liver weight and hepatic injury markers in OVX mice. Pathological examination of liver tissue showed less fat accumulation in the livers of mice on WBM diet; moreover, these animals had improved glucose clearance ability. Microarray analysis revealed that genes related to the fatty acid biosynthesis pathway, particularly the genes for fatty acid synthetase (Fas) and fatty acid elongase 6 (Elovl6), were down-regulated in the liver of mushroom-fed mice. In vitro mechanistic studies using the HepG2 cell line showed that down-regulation of the expression of FAS and ELOVL6 by WBM extract was through inhibition of Liver X receptor (LXR) signaling and its downstream transcriptional factor SREBP1c. These results suggest that WBM is protective against hepatic steatosis and NAFLD in OVX mice as a model for postmenopausal women.

15. Kai M, Onishi H, Souzaki M, Tanaka Haruo, Kubo M, Tanaka M, Katano M, Semi-quantitative evaluation of CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue using a newly developed fluorescence immunohistochemical staining method
, Cancer Sci. , 102, 12, 2132-2138, 2011.04, Abstract
CD44(+) /CD24(-) tumor cells are reported to contain cancer stem cells in breast cancer. The main purpose of the present study is to develop an immunohistofluorescence method that can quantitatively analyze CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue and help better define the role of CD44(+) /CD24(-) tumor cells in breast cancer. The samples used were from 21 primary breast cancer patients who underwent neoadjuvant chemotherapy and 17 cases with sentinel lymph nodes that had lymph node micrometastasis. CD44(+) /CD24(-) tumor cells were distinguished at a single cell level using improved triple-staining immunohistofluorescence and a simulated laser capture microdissection method. The percentage of CD44(+) /CD24(-) cells significantly increased following neoadjuvant chemotherapy treatment (0.93% and 2.78%, before and after, respectively, P = 0.0043). The percentage of CD44(+) /CD24(-) cells was also significantly high in micrometastatic sentinel lymph nodes (0.49% and 1.91%, primary tumors and lymph nodes, respectively, P = 0.0246). The CD44(+) /CD24(-) tumor cell distribution was heterogeneous in both breast cancer tissue and lymph node metastasis. In a xenograft model using immunodeficient mice, the hedgehog signaling inhibitor cyclopamine repressed the tumorigenicity of CD44(+) /CD24(-) cells. Our results suggest that this semi-quantitative immunohistochemical analysis is valuable for detecting a small population of cells in cancer tissues and that the hedgehog signaling pathway inhibitor cyclopamine is useful for regulating the CD44(+) /CD24(-) tumor cells in breast cancer. (Cancer Sci, doi: 10.1111/j.1349-7006.2011.02063.x, 2011).

16. Sagara Y, Kamada Y, Yamamoto Y, Tanaka M, Kubo M, Yamaguchi R, Nishimura R, Mitsuyama S, Study on the state of implementation of HER2 testing and positiveratios in patients with breast cancer in the Kyushu-Okinawaregion of Japan, Breast Cancer, 19, 4, 315-320, 2011.04, BACKGROUND: The development of molecular-targeted agents has improved the recovery rate for cancer. In Japan, trastuzumab has been approved as an adjuvant treatment for human epidermal growth factor receptor-2 (HER2)-positive breast cancer; therefore, accurate management of HER2 testing has become more important. In addition, proper patient selection is required from the viewpoint of health care economics.

METHODS: The current situation of HER2 testing in patients with infiltrating breast cancer from April 2008 to March 2009 was evaluated in 49 institutions (50 departments) treating breast cancer in the Kyushu-Okinawa region.

RESULTS: In a total of 5286 samples, HER2-positive ratio was 14.2%, which may reasonably reflect the current state of HER2 testing for breast cancer in the Kyushu-Okinawa region. There was a moderate discrepancy in HER2-positive ratio between institutions. Immunohistochemistry (IHC) analysis was outsourced in 15 institutions, and fluorescence in situ hybridization (FISH) analysis was outsourced in 23 institutions. The ratio of retesting by FISH analysis for samples judged as 2+ on IHC was 86.1%. There was no correlation between HER2-positive ratio and the number of HER2 tests at the institution. However, a high percentage of HER2 IHC 0-1+ results and a low percentage of HER2 2+ and 3+ results at the institution were significantly correlated with a high percentage of HER2 FISH-positive results for HER2 IHC 2+ cases.

CONCLUSIONS: There is a moderate discrepancy in HER2-positive ratio between institutions. Institutions with a high percentage of HER2 IHC 0-1+ and a low percentage of HER2 2+ and 3+ may have more false negative cases. These institutions should perform internal accuracy evaluations in order to maintain proper diagnostic judgment.

17. Yamasaki A, Kameda C, Xu R, Tanaka Haruo, Tasaka T, Chikazawa N, Suzuki H, Morisaki T, Kubo M, Onishi H, Tanaka M, Katano M, Nuclear factor kappaB-activated monocytes contribute to pancreatic cancer progression through the production of Shh
, Cancer Immunol Immunother, 59, 5, 675-686, 2010.04, Recently, it was reported that Hh signaling is activated in tumor stromal cells but not in tumor cells themselves and that stromal cells may play a role in the proliferation of cancer cells. This suggests the possibility that stromal cells have an important role in the proliferation of tumor cells that may be mediated through Hh signaling. In this report, we present for the first time that inflammation-stimulated monocytes produce Shh through activation of the NF-kappaB signaling pathway, and that the Shh produced promotes the proliferation of pancreatic cancer cells in a paracrine manner through Hh signaling.

18. Kameda C, Nakamura M, Tanaka Haruo, Yamasaki A, Kubo M, Tanaka M, Onishi H, Katano M, Oestrogen receptor-alpha contributes to the regulation of the hedgehog signalling pathway in ERalpha-positive gastric cancer
, Br J Cancer, 102, 4, 738-747, 2010.04, BACKGROUND: Oestrogen receptor-alpha (ERalpha) is highly expressed in diffuse-type gastric cancer and oestrogen increases the proliferation of ERalpha-positive gastric cancer. However, a detailed mechanism by which oestrogen increases the proliferation of these cells is still unclear. METHODS: We used 17-beta-oestradiol (E2) as a stimulator against the ERalpha pathway. Pure anti-oestrogen drug ICI 182 780 (ICI) and small interfering RNA against ERalpha (ERalpha siRNA) were used as inhibitors. Cyclopamine (Cyc) was used as the hedgehog (Hh) pathway inhibitor. Two human ERalpha-positive gastric cancer cells were used as target cells. Effects of the stimulator and inhibitor on E2-induced cell proliferation were also examined. RESULTS: In ERalpha-positive cells, E2 increased not only cell proliferation but also one of the ligands of the Hh pathway, Shh expression. 17-beta-Oestradiol-induced cell proliferation was suppressed by ICI, ERalpha siRNA or Cyc. The increased expression of Shh induced by E2 was suppressed by ICI and ERalpha siRNA but not by Cyc. Furthermore, recombinant Shh activated the Hh pathway and increased cell proliferation, whereas anti-Shh antibody suppressed E2-induced cell proliferation. When a relationship between ERalpha and Shh expressions was analysed using surgically resected gastric cancer specimens, a positive correlation was found, suggesting a linkage between the ERalpha and Hh pathways. CONCLUSION: Our data indicate that activation of the ERalpha pathway promotes cell proliferation by activating the Hh pathway in a ligand-dependent manner through Shh induction of ERalpha-positive gastric cancer.

19. Ikebe M, Kitaura Y, Nakamura M, Tanaka Haruo, Yamasaki A, Nagai S, Wada J, Yanai K, Koga K, Sato N, Kubo M, Tanaka M, Onishi H, Katano M, Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway
, J Surg Oncol. , 100, 8, 725-731, 2009.04, BACKGROUND: Inflammation plays a multifaceted role in cancer progression, and NF-kappaB is one of the key factors connecting inflammation with cancer progression. We have shown that lipopolysaccharide (LPS) promotes NF-kappaB activation in colon cancer cells and pancreatic cancer cells. However, it is unclear why inflammatory stimuli can induce NF-kappaB activation in cancer cells. METHODS: We used two human pancreatic cancer cells, Panc-1 and AsPC-1, as target cells. LPS was used as an inflammatory stimulus. To confirm the participation of TLR4/NF-kappaB signaling pathway, we used three different NF-kappaB inhibitors (PDTC, IkappaBalpha mutant, and NF-kappaB decoy ODN) and siRNAs (against TLR4, MyD88, and MMP-9). Effect of LPS on pancreatic cancer cell invasive ability was determined by Matrigel invasion assay. RESULTS: LPS increased the invasive ability of pancreatic cancer cells, while blockade of NF-kappaB pathway decreased the LPS-dependent increased invasive ability. Blockade of TLR4 or MyD88 by siRNA also decreased the LPS-dependent increased invasive ability. CONCLUSION: These results suggest that TLR/MyD88/NF-kappaB signaling pathway plays a significant role in connecting inflammation and cancer invasion and progression. Copyright 2009 Wiley-Liss, Inc.

20. Kameda C, Tanaka Haruo, Yamasaki A, Nakamura M, Koga K, Sato N, Kubo M, Kuroki S, Tanaka M, Katano M , The Hedgehog pathway is a possible therapeutic target for patients with estrogen receptor-negative breast cancer, Anticancer Res, 29, 3, 871-879, 2009.04, Understanding the expression patterns of estrogen receptor-alpha (ERalpha) is essential for determining therapeutic strategies for patients with breast cancer. The prognosis of patients with ERalpha-negative breast cancer is still poor. We have previously shown that Hedgehog (Hh) signaling is constitutively activated in breast cancer and that Hh signaling could be a new therapeutic target. Therefore, in this study, whether or not Hh signaling could be utilized as a therapeutic target for patients with ERalpha-negative breast cancer was examined. For this purpose, three ERalpha-negative breast cancer cell lines were used in which Hh pathway-related molecules such as the ligand Patched1 and the transcriptional factor Gli1 as target cells are expressed. Cyclopamine, an inhibitor of the Hh pathway, significantly suppressed both the cell proliferation and invasion ability of these cancer cells. In addition, the knockdown of Gli1 by RNA interference in these cells also significantly reduced both cell proliferation and invasion ability. Since our previous data have shown a constitutive activation of the Hh pathway in surgically-resected ERalpha-negative breast cancer specimens, the Hh pathway, especially Gli1, may be a useful therapeutic target for patients with ERalpha-negative breast cancer.

21. Tanaka Haruo, Nakamura M, Kameda C, Kubo M, Sato N, Kuroki S, Tanaka M, Katano M, The Hedgehog signaling pathway plays an essential role in maintaining the CD44+CD24-/low subpopulation and the side population of breast cancer cells.
, Anticancer Res. , 29, 6, 2147-2157, 2009.04, The side population (SP) and the CD44(+)/CD24(-/low) population have been reported in separate studies to include more tumorigenic cells than other populations, and to have the ability to form new tumors and undergo heterogeneous differentiation in breast cancer tissue. However, the relationship between these two populations has not yet been explored in breast cancer cells. Here it is shown that the SP and the CD44(+)/CD24(-/low) populations are overlapping. Both populations were resistant to paclitaxel. Components of the Hedgehog (Hh) signaling pathway were more highly expressed in these cell populations at both the mRNA and protein levels compared with other populations. Furthermore, inhibition of Hh signaling activity suppressed the proliferation of both populations. The significance of Hh signaling activity in the proliferation of both populations was confirmed by the effect of an si-RNA against Gli1, a trans-activator of the Hh signaling pathway, on the proliferation of both populations. These data suggest that the Hh signaling pathway is essential for the proliferation of the tumorigenic population of breast cancer cells, and that this pathway might represent a new candidate for breast cancer therapy targeting cancer stem cells.

22. Koga K, Nakamura M, Nakashima H, Akiyoshi T, Kubo M, Sato N, Kuroki S, Nomura M, Tanaka M, Katano M, Novel link between estrogen receptor alpha and hedgehog pathway in breast cancer., Anticancer Res. , 28, 2A, 731-740, 2008.04, Ligand-dependent constitutive activation of the hedgehog (Hh) pathway is important in the development of various carcinomas including breast cancer. A link between estrogen receptor alpha (ERalpha) and the Hh pathway in human breast cancer is shown here for the first time. In ERalpha-positive cells, estrogen depletion decreased the expression of sonic hedgehog (Shh), a ligand of the Hh pathway, while estrogen supplementation triggered Shh up-regulation. This estrogen-induced Shh expression activated the Hh pathway in a ligand-dependent manner, and increased cell proliferation. These effects were suppressed by ERalpha inhibitors, including ICI 182,780 (ICI), the dominant negative form of ERalpha and small interfering RNA (siRNA) against ERalpha. Consistent with the in vitro data, a positive correlation between ERalpha and Shh expression was found in breast cancer tissues. These data suggest that ERalpha regulates the Hh pathway through Shh induction, and promotes breast cancer development.

23. Nakamura M, Kubo M, Yanai K, Mikami Y, Ikebe M, Nagai S, Yamaguchi K, Tanaka M, Katano M , Anti-patched-1 antibodies suppress hedgehog signaling pathway and pancreatic cancer proliferation., Anticancer Res., 27, 6A, 3743-3748, 2007.04, BACKGROUND: The hedgehog (Hh) signaling pathway is aberrantly activated in many human carcinomas including pancreatic cancer and regulates tumor cell growth. Overproduction of sonic hedgehog (Shh), a ligand of the Hh signaling pathway, increases the Hh signaling activity through transmitting the signal to patched-1 (Ptch1), the receptor of the Hh signaling pathway. MATERIALS AND METHODS: a-Ptch1 antibodies were raised against an oligo-peptide, designed according to the Ptch1 aminoacid sequence. The specificity of a-Ptch1 was examined by immunoblotting and immuno-fluorescence, and biological effects were detected by RT-PCR and cell proliferation assay using two pancreatic cancer cell lines, Panc1 and SUIT-2. RESULTS: a-Ptch1 recognized a 160 kDa protein as shown by immunoblotting and cell surface staining of pancreatic cancer cells. Incubation with a-Ptch1 suppressed Hh signaling activity and proliferation of pancreatic cancer cells. CONCLUSION: These results provide a new strategy for controling Hh dependent development of pancreatic cancer and other Hh related carcinomas.

PMID: 17970037 [PubMed - in process]
24. Nakashima H, Tasaki A, Kubo M, Kuroki H, Matsumoto K, Tanaka M, Nakamura M, Morisaki T, Katano M, Effects of docetaxel on antigen presentation-related functions of human monocyte-derived dendritic cells.
, Cancer Chemother Pharmacol. , 55, 5, 479-487, 2005.04, PURPOSE: Docetaxel (TXT) is a unique chemotherapeutic agent that has been approved for treating various types of malignancies. TXT stabilizes microtubule assembly in cells and causes various dysfunctions of microtubule-dependent cellular events. Patients with advanced malignancies are beginning to receive TXT in combination with immunotherapy; however, the influence of TXT at clinically achievable serum concentrations (less than 10(-6) M) on antigen presentation-related functions of human monocyte-derived dendritic cells (Mo-DCs) remains unclear. METHODS: Immature Mo-DCs (imMo-DCs) were generated from peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor in vitro. Mature Mo-DCs (mMo-DCs) were induced from imMo-DCs with tumor necrosis factor-alpha and prostaglandin E(2). RESULTS: TXT at concentrations lower than 10(-7) M did not significantly affect cellular viability, phagocytosis, or expression of antigen presentation-related molecules of Mo-DCs. In contrast, TXT at concentrations lower than 10(-9) M significantly suppressed directional motility of imMo-DCs toward MIP-1alpha and of mMo-DCs toward MIP-3beta. However, TXT had no effect on either CCR1 expression by imMo-DCs or CCR7 expression by mMo-DCs. No gross changes in the microtubule skeleton were evident by immunofluorescence microscopy after treatment with TXT at less than 10(-8) M. However, reduced numbers of imMo-DCs with podosomes localized primarily in one cell region were observed. CONCLUSIONS: The present results indicate that different concentrations of TXT influence antigen presentation-related functions differently. In particular, TXT at relatively low therapeutic doses disrupts chemotactic motility of Mo-DCs.
25. Tasaki A, Akiyoshi T, Koga K, Nakashima h, Yamanaka N, Kubo M, Matsumoto K, Kojima M, Tanaka M, Nakamura M, Katano M, Immunohistochemical staining of hedgehog pathway-related proteins in human thymomas, Anticancer research, 25, 6A, 3697-3701, 2005.04, The thymus plays an essential role in the maturing of progenitor cells to functional T cells. Recent studies suggest that the Hedgehog (Hh) signaling pathway contributes to this differentiation process. However, there is limited information concerning the expression of Hh pathway-related proteins (Hh proteins) in the human thymus. The staining of Hh proteins in the thymic epithelium of 26 surgically resected thymoma tissues was examined by immunohistochemistry. The staining of sonic Hh (Shh) correlated relatively well with the World Health Organization histological classification of thymoma. The higher the grade, the fainter the staining. However, no significant difference in Shh staining was found between normal and neoplastic epithelia. Interestingly, Gli1 staining in thymomas was significantly greater than that in normal thymus (p < 0.0001). Thus, some members of the Hh signaling pathway may contribute to the development of thymoma..
26. Kubo M, Morisaki T, Matsumoto K, Tasaki A, Yamanaka N, Nakashima H, Kuroki H, Nakamura K, Nakamura M, Katano M, Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production, Cancer Immunol Immunother, 54, 5, 468-476, 2005.04.
27. Koga K, Matsumoto K, Akiyoshi T, Kubo M, Yamanaka N, Tasaki A, Nakashima H, Nakamura M, Kuroki S, Tanaka M, Katano M, Purification, characterization and biological significance of tumor-derived exosomes., Anticancer Res, 25, 6A, 3703-3707, 2005.04, Abstract

Exosomes are nanovesicles that are released into the extracellular environment during the fusion of multivesicular bodies with the plasma membrane. Exosomes released from dendritic cells, dexosomes, have several biological functions, for example as immunostimulants. Some tumor cells also secrete exosomes (Tu-exosomes). Although experimental data obtained with the use of dexosomes suggest a biological function of Tu-exosomes, this still remains poorly understood. To examine the function of Tu-exosomes, we established a method for collecting highly purified Tu-exosomes, using paramagnetic beads coated with antibodies against tumor-specific proteins such as HER2/neu. With these antibody-coated beads (Ab-beads), it was possible to collect HER2-expressing Tu-exosomes of high purity. Tu-exosomes were also collected from malignant ascites, which contain exosomes secreted from various types of cells such as tumor cells, lymphoid cells and mesothelial cells. The isolation of Tu-exosomes was confirmed by FACS analysis. With regard to their biological functions, Tu-exosomes cultured with a human breast cancer cell line bound to the cell surface and increased tumor cell proliferation. These data indicate that Tu-exosomes may have physiological functions.
28. Matsumoto K, Morisaki T, Kuroki H, Kubo M, Onishi H, Nakamura K, Nakahara C, Kuga H, Baba E, Nakamura M, Hirata K, Tanaka M, Katano M, Exosomes secreted from monocyte-derived dendritic cells support in vitro naive CD4+ T cell survival through NF-(kappa)B activation, Cell Immunol, 231, 1-2, 20-29, 2004.04, Abstract

We investigated the effect of exosomes secreted from human monocyte-derived dendritic cells (Mo-DCs), which are generated from PBMCs in response to treatment with GM-CSF and IL-4, on naive CD4+ T cell survival in vitro. Exosomes isolated from culture supernatants of Mo-DCs (>90% purity) were purified with anti-HLA-DP, -DQ, -DR-coated paramagnetic beads. Purified exosomes prolonged the survival of naive CD4+ T cells (>98% purity) in vitro. Treatment with neutralizing mAb against HLA-DR significantly decreased the supportive effect of purified exosomes on CD4+ T cell survival. Exosomes increased nuclear translocation of NF-(kappa)B in naive CD4+ T cells, and NF-(kappa)B activation was significantly suppressed by anti-HLA-DR mAb or NF-(kappa)B inhibitor pyrrolidine dithiocarbamate (PDTC). In addition, PDTC inhibited the effect of exosomes on naive CD4+ T cell survival. Thus, exosomes secreted by Mo-DCs appear to support naive CD4+ T cell survival via NF-(kappa)B activation induced by interaction of HLA-DR and TCRs.
29. Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, Kuroki S, Katano M, Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer
, Cancer Res. , 64, 17, 6071-6074, 2004.04, The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Genetic analysis has demonstrated a critical role for the Hh pathway in mammary gland morphogenesis. Disruption of Patched1, a component of the Hh pathway, results in abnormal growth of mammary duct. Recent studies have shown constitutive activation of the Hh pathway in various types of malignancies. However, it remains unclear whether this pathway is activated in human breast cancer. Here, we determined the expression of the components, including Sonic Hh, Patched1, and Gli1, of the Hh pathway by immunohistochemical staining in a series of 52 human breast carcinomas. All of 52 tumors display staining of high intensity for Gli1 when compared with adjacent normal tissue. The nuclear staining ratio of Gli1 correlates with expression of estrogen receptor and histologic type. Exposure to cyclopamine, a steroidal alkaloid that blocks the Hh pathway, suppresses expression of Gli1 and the growth of the Hh pathway-activated breast carcinoma cells. These data indicate that the Hh pathway is a new candidate for therapeutic target of breast cancer.
30. Yamanaka N, Morisaki T, Nakashima H, Tasaki A, Kubo M, Kuga H, Nakahara C, Nakamura Katsuya, Noshiro H, Yao T, Tsuneyoshi M, Tanaka M, Katano M , Interleukin 1beta enhances invasive ability of gastric carcinoma through nuclear factor-kappaB activation
, Clin Cancer Res. , 10, 5, 1853-1859, 2004.04, PURPOSE: We examined the role of interleukin (IL)-1beta in activation of nuclear factor kappaB (NF-kappaB) and the biological function of activated NF-kappaB in gastric carcinoma cells. EXPERIMENTAL DESIGN: Human gastric carcinoma cell line GCTM-1 was used to examine NF-kappaB activation by immunostaining and electrophoretic mobility shift assay. Matrix metalloproteinase (MMP)-9 expression, which plays an important role in tumor invasion, was assessed by semiquantitative reverse transcription-PCR, Western blotting, and immunostaining. The invasive ability of GCTM-1 cells was measured by Matrigel invasion assay. In vivo expression of IL-1beta and MMP-9 and activation of NF-kappaB in 10 surgically resected gastric carcinoma specimens were examined immunohistochemically. RESULTS: IL-1beta enhanced NF-kappaB activation, MMP-9 expression, and the invasive ability of GCTM-1. A NF-kappaB inhibitor, pyrrolidine dithiocarbamate, suppressed both MMP-9 expression and invasiveness of IL-1beta-treated GCTM-1 cells. IL-1beta did not increase the invasive ability of GCTM-1 cells transfected with MMP-9 antisense oligonucleotide. Concomitant expression of IL-1beta and nuclear NF-kappaB was observed in 3 of 10 gastric carcinoma specimens. Cells producing IL-1beta were tumor-infiltrating macrophages in two specimens and gastric carcinoma cells in one specimen. CONCLUSIONS: One of the molecules that may play a role in NF-kappaB activation in some gastric carcinomas is IL-1beta. The present results suggest that IL-1beta increases the invasive ability of carcinoma cells through activation of NF-kappaB and the resulting MMP-9 expression.
31. Nakamura Katsuya, Nakahara C, Kuga H, Yamanaka N, Tasaki A, Nakashima H, Kubo M, Fujii H, Sueishi K, Tanaka M, Katano M, Novel histoculture drug response assay with a simulated microgravity culture system, Preclinica, 2, 5, 356-361, 2004.04, This study focused on the development of a next generation histoculture drug response assay (HDRA) that can provide information on chemosensitivity of carcinoma cells but not contaminating cells. For HDRA, we used a rotary cell culture system with four disposable vessels that provides a simulated microgravity (SM) condition. This new HDRA was named tentatively SM-HDRA. In SM-HDRA, we examined two types of carcinoma specimens, first, pancreatic carcinoma specimens transplanted into nude mice, and next, human carcinoma specimens resected surgically from 12 patients with colorectal or gastric carcinoma. The in vivo response of pancreatic carcinoma transplanted into nude mice was cultured in medium containing gemcitabine for 4 days. Human carcinoma specimens exposed to 5-fluorouracil (5-FU) or cisplatin (CDDP) were examined. The response was evaluated by hematoxylin-eosin staining, Ki-67 immunohistostaining, TUNEL assay, and secretion of carcinoemb
yonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) into culture supernatants. Eleven of the twelve (91.6%) surgical specimens were successfully evaluated for SM-HDRA. SM-HDRA provided information not only about the chemosensitivity of carcinoma cells without interference from normal cells but also about heterogeneous drug responses in the carcinoma tissue, revealing different chemosensitivities. SM-HDRA is a new drug response assay and will be a useful tool for in-depth analyses of complicated carcinoma tissues and development of therapeutic strategies..
32. Yamanaka N, Sasaki N, Tasaki A, Nakashima H, Kubo M, Morisaki T, Noshiro H, Yao T, Tsuneyoshi M, Tanaka M, Katano M, Nuclear factor-kappaB p65 is a prognostic indicator in gastric carcinoma
, Anticancer Res., 24, 2c, 1071-1076, 2004.04, Background: In common with other investigators, we have reported the constitutive activation of transcription factor nuclear factor-κB (NF-κB) in a variety of carcinomas, but there is no definite information on its clinical significance. Materials and Methods: NF-κB p65 activation was determined by immunohistochemical analysis of surgically resected specimens from 63 gastric carcinomas. The 63 patients were divided into a high NF-κB group (21 patients) and a low NF-κB group (42 patients). Forty-seven of the 63 patients underwent curative resection. The 47 patients consist of 13 high NF-κB patients and 34 low NF-κB patients. Results: The high NF-κB group demonstrated a shorter overall survival rate compared with the low NF-κB group (P = 0.015). In the 47 patients who underwent curative resection, the high NF-κB group also showed a poor survival prognosis (P = 0.032). Multivariate ana
ysis indicated that NF-κB activation is a potential prognostic factor in gastric carcinoma. Conclusions: Constitutive activation of NF-κB p65 may be a new prognostic parameter in gastric carcinoma..
33. Tasaki A, Yamanaka N, Kubo M, Matsumoto K, Kuroki H, Nakamura Katsuya, Nakahara C, Onishi H, Kuga H, Baba E, Tanaka M, Morisaki T, Katano M , Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells, J Immunol Methods, 287, 1-2, 79-90, 2004.04, Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and n
crotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo..
34. Kubo M, Morisaki T, Kuroki H, Tasaki A, Yamanaka N, Matsumoto K, Nakamura Katsuya, Onishi H, Baba E, Katano M , Combination of adoptive immunotherapy with Herceptin for patients with HER2-expressing breast cancer.
, Anticancer Res., 23, 6a, 4443-4449, 2003.04, Clinical use of Herceptin (trastuzumab), which is a humanized monoclonal antibody against HER2, started for patients with HER2-overexpressing breast cancer. To potentiate the efficacy of the Herceptin therapy, this study focused on the combination of Herceptin with activated immune lymphocytes. We used peripheral blood mononuclear cells (PBMCs) as effector cells and used HER2-unexpressing K562 cells, HER2-weakly-expressing breast carcinoma cells (Breast-M), or HER2-strongly-expressing breast carcinoma cells (BT-474) as target cells. Interleukin-2 (IL-2)-activated PBMCs, IL-2/OKT-3-activated PBMCs and a streptococcal preparation OK-432-activated PBMCs were generated and used as effector cells. Cytotoxic activity was determined with 4-hour 51Cr release assay. Both fresh PBMCs and activated PBMCs exhibited Herceptin-dependent cytotoxicity. Importantly, Herceptin-dependent cytotoxicity was found even at a lower effector to target cell ratio (E/T ratio) than that of Herceptin-independent cytotoxicity. In addition, Herceptin-dependent cytotoxicity by these activated PBMCs was observed even in HER2-weakly-expressing Breast-M cells. Since gamma-globulin or anti-CD16 antibody abrogated Herceptin-dependent cytotoxicity, it seems likely that antibody-dependent cellular cytotoxicity (ADCC) plays an important role in the Herceptin-dependent cytotoxicity. We present a recurrent breast cancer patient with malignant pleural effusion, in which HER2-strongly-expressing tumor cells were present, who was undergoing Herceptin therapy. Cluster formation between tumor cells and intrapleural mononuclear cells was induced 24 hours after intravenous injection of Herceptin (4 mg/kg). Mononuclear cells bound specifically to HER2-strongly-expressing tumor cells but not to other cells, such as mesothelial cells, suggested a Herceptin-mediated binding like ADCC in vivo. Taken together, these findings suggest that the combination of Herceptin with various types of activated lymphocytes may be a new therapeutic strategy, not only for HER2-strongly-expressing breast cancer but also for HER2-weakly-expressing cancer.
35. Morisaki T, Matsumoto K, Kuroki H, Kubo M, Baba E, Onishi H, Tasaki A, Nakamura M, Inaba S, Katano M, Combined immunotherapy with intracavital injection of activated lymphocytes, monocyte-derived dendritic cells and low-dose OK-432 in patients with malignant effusion.
, Anticancer Res., 23, 6a, 4459-4465, 2003.04, We have conducted a pilot study with combined immunotherapy using autologous lymphocytes activated ex vivo and monocyte-derived dendritic cells in combination with low-dose OK-432, a streptococcal preparation, in five patients with peritoneal or pleural carcinomatosis who were resistant to standard chemotherapy. All patients were given 3 to 10 courses of the combined immunotherapy. No severe adverse reactions occurred. Effusion production was decreased in all of the patients. Significant decreases in tumor markers of both effusions and sera as well as effusion volume occurred in all of the patients. Cytological examinations revealed a marked decrease or disappearance of cancer cells in those effusions. Three patients showed increase in IFN-gamma levels in the effusions. The overall prognosis of the patients was acceptable and the mean survival time was more than 9 months. The locoregional immunotherapy seems to be encouraging in view of therapeutic modality in patients who are resistant to standard chemotherapy. Our study provides a new protocol for immunotherapy and warrants further phase I/II clinical study for chemo-resistant patients with malignant effusion.
36. Morisaki T, Matsumoto K, Onishi H, Kuroki H, Baba E, Tasaki A, Kubo M, Nakamura M, Inaba S, Yamaguchi K, Tanaka M, Katano M, Dendritic cell-based combined immunotherapy with autologous tumor-pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives, Hum Cell, 16, 4, 175-182, 2003.04, Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to trigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.
37. Inoue S, Tanaka Haruo, Fujita E, Kubo M, Hirakata H, Fukushima Y, Nakafusa Y, Maeda T, Fujishima M, Eosinophilic pleural effusion and peripheral eosinophilia--an uncommon complication of thoracoscopic parathyroidectomy., Nephrol Dial Transplant, 13, 11, 2929-2931, 1998.04.