| 1. |
狩野 有宏, 藤木 美穂, Establishment of adenine nucleotide translocase deficient cell lines and studies on its role in proliferation and apoptosis（アデニンヌクレオチド交換タンパク質欠損株の作成と増殖およびアポトーシスへの影響）, 第45回日本分子生物学会年会, 2022.12, Adenine nucleotide translocase (ANT) is located in the inner mitochondrial membrane and catalyzes the exchange of mitochondrial ATP for cytosolic ADP, which is a key mechanism for oxidative phosphorylation in eukaryotic organisms. ANT consists of four isoforms in humans and three isoforms in mice (Ant3 is not expressed). The ANT1 gene is predominantly expressed in heart and muscle, and the ANT2 gene is mainly expressed in lymphoid cells and the liver. The ANT3 gene is expressed abundantly but at a low level, and ANT4 is only expressed in brain, liver, and testis. Interestingly, it has been reported that the ANT2 gene is abundantly expressed in cancer cells and aids in the importation of ATP produced by glycolysis from the cytosol to the mitochondria. The glycolytic ATP in mitochondria is hydrolyzed by F1F0-ATP synthase in a reverse reaction that produces a proton motive force, which maintains the mitochondrial membrane potential. However, it has been reported that ATP importation is independent of ANT in HepG2 and A549 cells (JBC. 2017; 292(41): 16969). The role of ANT in apoptosis has been also discussed through the part of the mitochondrial permeability pore. Such as ANT1 acts as a proapoptotic and ANT2 acts as an antiapoptotic protein. However, the mechanisms of ANT1, 2 in apoptosis induction and inhibition are unknown and the significance of them in tumor development and metastasis is not fully understood. For these aims, we have established Ant1 and 2 deficient cancer cells each and attempt to knock out both genes simultaneously by the CRISPR/Cas9 technique. In this meeting, we will report the results of apoptotic induction in these cell lines.. |
| 2. |
狩野有宏，深見契弥，Kamita Moses，岩崎琢磨，岩田隆幸，新藤充, ボンクレキン酸によるがん細胞選択的細胞障害性の解析, 第60回天然有機化合物討論会, 2018.09. |
| 3. |
Jing Gong, Moses Kamita, Mitsuru Shindo, Arihiro Kano, Role of M-CSF expressed from 4T1 tumor cells in immune activation, 2018 IMCE International Symposium, 2018.03. |
| 4. |
公 せい、Kamita Moses、新藤 充、狩野 有宏, M-CSFによる担がんマウス脾細胞のIFN-γ分泌抑制, 2017年 生命科学系学会合同年次大会（ConBio2017）（第40回日本分子生物学会年会、第90回日本生化学会）, 2017.12. |
| 5. |
Arihiro KANO, Moses KAMITA, Takuma IWASAKI, Mitsuru SHINDO, Bongkrekic Acid Facilitates Glycolysis in Cultured Cells Resulting from Mitochondrial Dysfunction, International Symposium on Materials for Chemistry and Engineering (IMCE 2017), 2017.02, Bongkrekic acid (BKA) was originally identified as a cause in the poisoning of coconut-fermented food. In an early study by Welling et al., the authors showed that ingestion of BKA first results in hyperglycemia, with subsequent development of hypoglycemia 1. Interestingly, this study reported that all glycogen disappeared from the liver and heart just before death. Subsequent studies showed that BKA inhibits the adenine nucleotide exchange in mitochondria by binding to adenine nucleotide translocator (ANT) 2. The fundamental role of ANT is the exchange of ATP and ADP across the inner mitochondrial membrane. In addition, it forms the inner membrane channel of the mitochondrial permeability transition pore (mPTP) complex, which becomes the path for releasing of cytochrome c in apoptosis induction. Several papers demonstrated that BKA suppresses apoptosis through inhibiting mPTP opening 3. In addition, we previously showed that the tricarboxylic structure of BKA is essential on the apoptotic inhibition in our structure-activity relationship study 4. However, it is thought that BKA also inhibits ATP production simultaneously, which may result in adverse effects on cell viability. BKA has been well studied with isolated mitochondria, although few studies have examined its effects in cultured cells.
In this study, we aimed to clarify the effect of BKA on culture cells. We show BKA causes loss of viability in several tumor cell lines, but not in normal cell lines, such as NIH3T3. The effect of BKA on the viability of tumor cells is dependent on the seeded number in culture. BKA enhances glycolysis in the cells probably to compensate for the loss of ATP production in the mitochondria. The dependence on the cell number is considered to arise from the difference of glucose consumption in the culture media. As well, the sensitivity to BKA observed in tumor cells is thought to be due to the higher glucose consumption rate in cancer cells. Indeed, the both reduced glucose in the culture media and combined treatment with 2-DG showed enhanced cytotoxicity in 4T1 cells, which further indicate that BKA affects energy production in cultured cells. BKA may be expected to a new type anti-cancer drug that targets cancer metabolism.
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| 6. |
Moses K. Kamita, 狩野 有宏, 新藤 充, Tumor-derived macrophage colony stimulating factor mediates tumor immunosuppression, 2n International Exchange and Innovation Conference on Engineering & Sciences, 2016.10, The mechanisms through which tumors evade immune surveillance remain incompletely understood. In our previous study, immunosuppressive soluble factor(s) secreted by murine mammary tumor (4T1) cells was shown to be a protein between 10-100 kDa. In the current study, identification of the protein in the active fractions revealed the presence of macrophage colony stimulating factor (M-CSF). M-CSF is a cytokine that plays an essential role in the regulating survival, proliferation and differentiation of macrophages and monocytes. Although the involvement of M-CSF in enhancing tumor metastasis has been reported, its involvement in tumor growth is not well reported. Here we show that genetic ablation of M-CSF in 4T1 cells reduces the rate of tumor growth in mice compared with the wild-type 4T1. Knock-out cells also resulted in reduced rate of splenomegaly which has been shown to be high in 4T1 tumor-bearing mice. These data argue that, M-CSF is a key player in suppressing immune response against tumor and may be explored to boost immune based tumor therapy. . |
| 7. |
Moses Kamita, 新藤 充, 狩野 有宏, Immunosuppression by Murine Mammary Carcinoma Cells, mediated by Tumor-Secreted Soluble Factors, 第38回日本分子生物学会年会、第88回日本生化学会大会の合同大会, 2015.12, Current tumor immunotherapies are hindered from effectively fighting cancer due to the ability of tumor cells to create a tolerant microenvironment, secrete immunosuppressive factors and generate suppressive cells such as MDSC. Secretion of factors like IL-10, TGF-β, and VEGF among others has been shown to enhance cancer growth by suppressing T cell activity against tumors. 4T1 tumor cells were derived from originally established spontaneously emerged murine mammary tumor. Recently, leukemoid reaction and massive splenomegaly caused by the inoculation of 4T1 cells was reported1. In this study, to investigate tumor-induced immunosuppression, we first evaluated IFN-γ expression in the splenocytes culture, isolated from 4T1 tumor-bearing mice2. The elevated production of IFN-γ was observed at a week after inoculation, and then reduced to under detection levels in 3 to 4 weeks. Production of IFN-γ was further suppressed in a splenocyte culture in the presence of 4T1-conditioned media. This suppression indicated the presence of immunosuppressive soluble factors in conditioned media secreted by 4T1 cells. Purification of 4T1-conditioned media through chromatography systems and further analysis by MALDI TOF-MS indicated the presence of M-CSF and G-CSF in the active fractions. While the involvement of G-CSF in this activity is well characterized3, M-CSF activity is not well reported. Further studies are underway to evaluate the activity of M-CSF and G-CSF in tumor-associated immune suppression and the underlying mechanisms.. |
| 8. |
Arihiro Kano, Murine mammary carcinoma 4T1 cells secrete soluble factor(s) for specific immunosuppression, Japanese Nano-Macro Materials, Devices and System Research Alliance, 2014.11. |
| 9. |
Studies of tumor models using syngeneic transplantation have advanced our understanding of
tumor immunity, including both immune surveillance and evasion. Murine mammary carcinoma 4T1
cells secrete immunosuppressive soluble factors as demonstrated in splenocyte culture. Cultured
primary splenocytes secrete IFN-γ, which was strikingly elevated when the cells were isolated from
4T1 tumor-bearing mice. The secretion of IFN-γ peaked at a week after 4T1 inoculation and then
declined. This reduction may be due to the relatively decreased lymphocytes and increased
granulocytes in a spleen accompanied by splenomegaly with the 4T1 engraftment. Additionally, IFN-
γ production was further suppressed with the addition of the conditioned media from 4T1 cells to
the splenocyte culture. This suppressive effect was more evident in the splenocytes isolated from
mice that had 4T1 tumors for a longer period of time and was not observed in the conditioned
medium either from CT26 cells or with splenocytes isolated from CT26 tumor-bearing mice. These
results suggest that the IFN-γ suppression is 4T1 tumor-specific. The soluble factor(s) in the 4T1-
conditoned media was a protein between 10 to 100 kDa. The cytokine tip assay demonstrated
several known cytokines that negatively regulate immune responses and may be candidates for this
immunosuppression activity.. |
| 10. |
Arihiro Kano, Formulation of Photofrin® with cationic graft copolymer and tumor delivery
, Technologies for Medical Diagnosis and Therapy Symposium, 2013.10, We have shown that polyethyleneglycol-grafted poly-L-lysine (PLL-g-PEG) exerts
long lifetime in blood circulation and effective accumulation in a tumor [1]. It is
thought that PLL-g-PEG is useful as a drug delivery carrier for tumor treatment. In
this study, we examined the interaction of Photofrin® [2], a photosensitizer used for
photodynamic therapy to treat tumors, with PLL-g-PEG, and in addition, evaluated
the photo-induced cytotoxicity, tumor accumulation, photosensitive reaction in skin,
a major side effect. For this study, PLL-g-PEG, 40 kDa of PLL, 5 kDa of PEG, and 5
to 40-mol% of the PEG grafting ratio, was prepared and used. Photofrin consists of 2
to 6 hematoporphyrins, consequently, it is negatively charged due to 4 to 8 carboxyl
groups. Therefore this carboxylic multimer seemed to form a complex with PLL-g-
PEG by ionic interaction. The result of gel retardation assay showed that 1 mol/L
of NaCl did not affect this interaction suggesting that bonding other than static is
involved. Intravenously injected Photofrin with PLL-g-PEG showed two-fold higher
accumulation in tumors in mice, and interestingly, photosensitive reaction in skin and
tails was completely suppressed [3].. |
| 11. |
Discrimination of single nucleotide polymorphisms by strand exchange assay using partially double-stranded probes. |
| 12. |
Nucleation-Synchronized Strand Exchange Reaction for DNA Mismatches Discrimination. |
| 13. |
Nucleation-synchronized strand displacement for highly sensitive DNA analysis. |
| 14. |
Characterization of IFN-gamma-induced apoptosis in SV40-T-immortalized hepatocytes.
Arihiro Kano, Serdar Gursoy, Toshihiro Akaike, Atsushi Maruyama
The Japanese Biochemical Society. |
