Susumu Ishiguro

Human Umbilical Cord Matrix Mesenchymal Stem Cells Suppress the Growth of Breast Cancer by Expression of Tumor Suppressor Genes

Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Electronic sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II.

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

Naive rat umbilical cord matrix stem cells significantly attenuate mammary tumor growth through modulation of endogenous immune responses.

BACKGROUND AIMS Un-engineered human and rat umbilical cord matrix stem cells (UCMSCs) attenuate growth of several types of tumors in mice and rats. However, the mechanism by which UCMSCs attenuate tumor growth has not been studied rigorously. METHODS The possible mechanisms of tumor growth attenuation by rat UCMSCs were studied using orthotopic Mat B III rat mammary tumor grafts in female F344 rats. Tumor-infiltrating leukocytes were identified and quantified by immunohistochemistry analysis. Potential cytokines involved in lymphocyte infiltration in the tumors were determined by microarray and Western blot analysis. The Boyden chamber migration assay was performed for the functional analysis of identified cytokines. RESULTS Rat UCMSCs markedly attenuated tumor growth; this attenuation was accompanied by considerable lymphocyte infiltration. Immunohistochemistry analysis revealed that most infiltrating lymphocytes in the rat UCMSC-treated tumors were CD3(+) T cells. In addition, treatment with rat UCMSCs significantly increased infiltration of CD8(+) and CD4(+) T cells and natural killer (NK) cells throughout tumor tissue. CD68(+) monocytes/macrophages and Foxp3(+) regulatory T cells were scarcely observed, only in the tumors of the phosphate-buffered saline control group. Microarray analysis of rat UCMSCs demonstrated that monocyte chemotactic protein-1 is involved in rat UCMSC-induced lymphocyte infiltration in the tumor tissues. CONCLUSIONS These results suggest that naïve rat UCMSCs attenuated mammary tumor growth at least in part by enhancing host anti-tumor immune responses. Naïve UCMSCs can be used as powerful therapeutic cells for breast cancer treatment, and monocyte chemotactic protein-1 may be a key molecule to enhance the effect of UCMSCs at the tumor site.

Involvement of angiotensin II type 2 receptor (AT2R) signaling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.

AT2R Gene Delivered by Condensed Polylysine Complexes Attenuates Lewis Lung Carcinoma after Intravenous Injection or Intratracheal Spray

Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Cell-penetrating peptides (CPP) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Here, a synthetic CPP (polylysine, K9 peptide) was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R) and complexes were condensed using calcium chloride. The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro and in vivo. This simple nonviral formulation approach showed negligible cytotoxicity in four different human cell lines (cervix, breast, kidney, and lung cell lines) and one mouse cell line (a lung cancer cell line). In addition, this K9-pDNA-Ca2+ complex demonstrated cancer-targeted gene delivery when administered via intravenous injection or intratracheal spray. The transfection efficiency was evaluated in Lewis lung carcinoma (LLC) cell lines cultured in vitro and in orthotopic cancer grafts in syngeneic mice. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth, offering preclinical proof-of-concept for a novel nonviral gene delivery method exhibiting effective lung tumor gene therapy via either intravenous or intratracheal administration. Mol Cancer Ther; 15(1); 209–18. ©2015 AACR.

Exopolysaccharides extracted from Parachlorella kessleri inhibit colon carcinoma growth in mice via stimulation of host antitumor immune responses

The newly purified extracellular polysaccharides (exopolysaccharides) from Parachlorella kessleri (PCEPS) were evaluated on their antitumor and immunomodulatory effects in cell culture and mouse colon carcinoma peritoneal dissemination model. In two-dimensional cell culture, the PCEPS treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the growth of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with PCEPS. The treatment with PCEPS also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage) and CD8+ cytotoxic T cells. In three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with HL-60 human neutrophilic promyeloblasts and Jurkat cells (human lymphoblasts), but not THP-1 human monocyte/macrophage was significantly attenuated by PCEPS treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of PCEPS (10 mg/kg, twice per week) significantly attenuated the growth of CT26 colon carcinoma in syngeneic mice. The present study suggests that PCEPS inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that exopolysaccharides derived from Parachlorella kessleri contain significant bioactive materials that inhibit colon carcinoma growth.

Co-treatment with a C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine strongly attenuated pancreatic cancer growth in mice through T cell activation

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

Abstract 4606: Involvement of an angiotensin II type 2 receptor (AT2R) signalling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice

Pancreatic cancer, primarily PDAC, is the most difficult cancer to treat. Therapeutic interventions currently available for PDAC are ineffective. Therefore, obtaining knowledge about developmental mechanisms associated with this cancer could be valuable in the development of early detection and effective treatments for pancreatic cancer. Angiotensin II is the key effecter of the renin-angiotensin system which plays an important role in maintaining blood pressure, body fluid and electrolyte homeostasis, and collagen deposition in the stroma. Expression of the angiotensin II type 1 receptor (AT1R) is shown to be associated with the progression of multiple cancers including PDAC. On the contrary, the expression of AT2R is shown to be involved in the inhibition of the growth of multiple cancers in mice. Accordingly, the objectives of this study were to determine the potential involvement of angiotensin II receptor expression in human PDAC and to evaluate the effect of a novel AT2R agonist on the growth of murine PDAC in syngeneic mouse models. Expression of AT1R and AT2R in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using 28 surgically dissected human PDAC specimens. In this study, though a strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, AT1R expression in the PDAC area was stronger than its expression in the normal area. A moderate AT2R expression was detected in 71% of the PDAC specimens and normal area of the pancreas, and its expression levels in the two areas were similar. Both AT1R and the AT2R mRNA levels were significantly higher in the PDAC area than in the normal pancreas tissue. Cell culture studies clarified that the AT2R agonist significantly attenuated both murine and human PDAC cells with negligible cytotoxicity in normal epithelial cells. Administrations of the AT2R agonist, but not control saline, in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in the syngeneic mice. Immunohistochemical analysis of the PDAC grafts revealed that the agonist treatment induced apoptosis in tumor cells but had no effect on stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development. In addition, the novel AT2R agonist is suggested to be an effective therapeutic for the treatment of PDAC. This work was supported by the Kansas State University (KSU) Johnson Cancer Research Center, NIH RR017686, RR15563, Kansas Bioscience Authority research grant and the Greek Ministry of Education and Research and program ARISTEIA II to AGT Citation Format: Susumu Ishiguro, Kiyoshi Yoshimura, Sonshin Takao, Atsushi Kawabata, Terrahn Wall, Ryouichi Tsunedomi, Masaaki Oka, Makoto Inui, Charalambos Pappas, Andreas G. Tzakos, Masaaki Tamura. Involvement of an angiotensin II type 2 receptor (AT2R) signalling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2014-4606

Abstract 1107: Co-treatment with a C1B5 domain peptide of protein kinase Cγ and a low dose of gemcitabine effectively inhibited pancreatic cancer growth in mouse peritoneal cavity

Although the gemcitabine is an effective chemotherapeutic agent for pancreatic cancer, unacceptable side effects often accompany. Since we have previously discovered that PKCγ C1B domain peptides effectively control tumor growth without any side effect (Kawabata et. al, Cancer Biol Ther, 2012), we sought to examine the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer. Although individual and co-treatment with C1B5 peptide (1µM) and gemcitabine (20 nM) weakly inhibited growth of PAN02 murine pancreatic acinar cell carcinoma in 2D culture, either treatment effectively attenuated spheroid growth on PAN02 cells in 3D culture with 48.2% and 35.8% inhibition, respectively. Combination treatment with the C1B5 peptide and gemcitabine further attenuated the growth of PAN02 cells (69.5% inhibition). In mice bearing peritoneal allograft tumors of PAN02 cells (2.5 x 105 cells/mouse), combination treatment with C1B5 peptide at 20 mg/kg (every other day) and gemcitabine 15 mg/kg (every three days) markedly inhibited tumor growth of PAN02 allografts (94% inhibition) more than individual treatment with gemcitabine (76% inhibition) or C1B5 peptide (39% inhibition). The tumor growth inhibition by the combination treatment was similar to the higher dose (50 mg/kg) of gemcitabine alone treatment. Peritoneal cavity infiltrated neutrophils and granzime B+ lymphocyte numbers were significantly higher in combination treatment group than in control group. In cell culture study, the treatment with C1B5 peptide alone (1µM) significantly increased INF-γ, IL-2, and TNF-α mRNA levels, suggesting that C1B5 peptide directly stimulated Jurkat cell activation. These studies suggest that stimulation of leucocyte migration toward cancer tissues and activation of cytotoxic T cells may play important roles in tumor growth attenuation by the combination treatment of C1B5 peptide and gemcitabine. Taken together, the current study suggests that C1B5 peptide offers an effective combination treatment strategy to reduce side effects associated with gemcitabine without losing tumoricidal effect of this agent. This work is supported in part by Kansas State University Johnson Cancer Research Center, NIH grants P20 GM103418, and Kansas State Bioscience Authority Collaborative Cancer Research grant. Citation Format: Alejandro Zulbaran, Kelsey Monson, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Naomi Ohta, Masaaki Tamura. Co-treatment with a C1B5 domain peptide of protein kinase Cγ and a low dose of gemcitabine effectively inhibited pancreatic cancer growth in mouse peritoneal cavity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1107. doi:10.1158/1538-7445.AM2017-1107

Abstract 3751: Apoptosis inducer gene delivery by polylysine-calcium complexes attenuates mouse lung carcinoma allograft growth after intravenous injection or intratracheal spray

Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Nanoparticle-based gene delivery technique potentially overcomes these concerns and may be applicable to cancer gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Our previous study demonstrated that an apoptosis inducer, angiotensin II type 2 receptor plasmid DNA (pAT2R) encapsulated in a modified HIV-1 TAT peptide (dTAT-pAT2R), significantly attenuated the growth of Lewis lung carcinoma (LLC) allograft in mouse lungs (Kawabata et al., Cancer Res, 2012). Here, we report a newly synthesized polylysine CPP (K9 peptide)-based gene therapy for lung cancer treatment. The pAT2R and K9 peptide (K9-pAT2R) complexes were condensed using calcium chloride (K9-pAT2R-Ca2+). The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro. This simple non-viral formulation approach showed negligible cytotoxicity in several different human and mouse cell lines (human cervix, breast, kidney, and human and mouse lung cell lines). Additionally, this K9-pDNA-Ca2+ complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray into LLC orthotopic allograft-bearing mice. Average lung weights (mg) of the K9-pAT2R-Ca2+ IT (190.6±48.3) and the K9-pAT2R-Ca2+ IV (201.6±67.0) treated groups were significantly smaller than that of the control PBS group (325.7±69.4, P