Kishu Kitayama

A case of bilateral pheochromocytoma during pregnancy

BackgroundPheochromocytoma is a disease where catecholamines are secreted. If pheochromocytoma occurs during pregnancy, it can be difficult to diagnose because it is similar to pregnancy-induced hypertension. Furthermore, bilateral pheochromocytoma during pregnancy is even rarer than unilateral pheochromocytoma.Case presentationA 32-year-old primigravida, who was 12 weeks’ pregnant, was aware of right abdominal discomfort. Masses in both adrenal glands were observed by abdominal ultrasonography. She was diagnosed with pheochromocytoma. Bilateral adrenalectomy was undertaken at 15 weeks’ gestation and she continued pregnancy. At 39 weeks’ gestation, a healthy male neonate was delivered. She was discharged on the 4th postpartum day.ConclusionsWe present a case of bilateral pheochromocytoma during pregnancy that was diagnosed in the first trimester. Differentiating pheochromocytoma from pregnancy-induced hypertension is important. Early diagnosis and appropriate blood pressure management with medical treatment followed by surgical removal of the tumor results in good maternal and fetal outcomes.

CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells

This corrects the article DOI: 10.1038/bjc.2017.85

Pyruvate kinase isozyme M2 and glutaminase might be promising molecular targets for the treatment of gastric cancer.

The aim of this study was to analyze the significance of glucose metabolism‐related enzymes in the proliferation of gastric cancer under hypoxia. Four hypoxia‐resistant gastric cancer cell lines and four parent cell lines were used. Reverse transcription–PCR was used to evaluate the mRNA expression levels of the following metabolism‐related enzymes: pyruvate kinase isozyme M2 (PKM2), glutaminase (GLS), enolase 1 (ENO1), glucose‐6‐phosphate dehydrogenase (G6PDH), and PKM1. The effects of these enzymes on the proliferation of gastric cancer cells were examined using siRNAs, shikonin as a PKM2 inhibitor, or BPTES as a GLS inhibitor, in vitro and in vivo. Levels of both PKM2 and GLS mRNA were significantly high in all hypoxia‐resistant cell lines, compared with those of their parent cells. Knockdown of PKM2 and GLS significantly decreased the proliferation of all hypoxia‐resistant cells. The combination of siPKM2 and siGLS significantly decreased proliferation compared with treatment by siPKM2 or siGLS alone. The knockdown of ENO1, G6PDH, or PKM1 did not decrease the proliferation of all hypoxia‐resistant cells. Combination treatment using shikonin and BPTES inhibited the proliferation of all hypoxia‐resistant cancer cells more than that by either agent alone. The in vivo study indicated that the tumor size treated by the combination of shikonin and BPTES was significantly smaller than that of vehicle‐treated group. These findings suggested that PKM2 and GLS might play important roles in the proliferation of hypoxic gastric cancer cells. A combination of PKM2 and GLS inhibitors could be therapeutically promising for the treatment of gastric cancer.

Abstract 3791: Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma

It has been reported that the examination of circulating tumor cells (CTCs) is beneficial for predicting tumor stage or treatment response. The epitherial cell adhesion molecule (EpCAM) and cytokeratin has been used as epithelial markers for the identification of CTCs. Since the epithelial-mesenchymal transition is one of malignant phenotype of cancer cells, it is necessary to investigate other markers which might detect mesenchymal CTCs. This study aims to clarify the clinical significance of EpCAM negative and CEA positive CTCs in patients with gastric cancer. Forty patients who underwent operation for gastric cancer were enrolled in this study. A total of 10ml peripheral blood samples were obtained preoperatively. Informed consent to participate in this study was obtained from all of the patients before their surgery. This study was approved by the Human Ethics Review Committee of our University. After collection of mononuclear cell fraction, these cells were stained by PAPI, anti-CD45, anti-EpCAM, anti-cytokeratin, and anti-CEA (CD66e) antibody. We enumerated the number of cells which showed EpCAM, cytokeratin, and CEA positive expression among CD45 negative cells. ROC analyses were performed for investigating predictive value of these cell counts for distant metastasis. We examined association between these cell counts and clinicopathological features. ROC analyses revealed that the AUC for predicting distant metastasis using EpCAM, Cytokeratin, and CEA positive cell counts were 0.583, 0.638, and 0.769, respectively. The median number of EpCAM negative and CEA positive cells was 231 (range: 0-61897). The cell counts of EpCAM negative and CEA positive cells were significantly associated with Tumor depth (p=0.0084), lymph node metastasis (p=0.0107), and tumor size (p=0.023). CEA might be useful marker for detecting high risk of recurrence in patients with gastric cancer. Citation Format: Yuichiro Miki, Masakazu Yashiro, Tomohisa Okuno, Kishu Kitayama, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma [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 3791. doi:10.1158/1538-7445.AM2017-3791

Abstract 37: Effect of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment

Background: Many kinds of solid tumor have heterogeneously a hypoxic environment. Cancer cells acquire characteristics that allow them to survive and proliferate in the hypoxic environment. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear. “Warburg effect” is known as anaerobic glycolysis of cancer cells, contributes tumorigenesis and tumor development. The aim of this study was to analyze the significance of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment. Materials and Methods: Two hypoxia-resistant cancer cell lines, OCUM-12hypo cells and OCUM-2MD3hypo cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used. OCUM-12hypo cells and OCUM-2MD3hypo cells were cloned from OCUM-12 cells and OCUM-2MD3 cells by continuous exposure to 1% oxygen, respectively. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. mRNA expression level of enolase 1 (ENO1), pyruvate kinase isozymes M2 (PKM2), and glutaminase (GLS) were evaluated by RT-PCR. Effects of inhibition of glucose metabolism-related enzymes on the proliferation and apoptosis were examined by CCK assay or FACScan analysis using siRNA (siENO1, siPKM2, and siGLS), or PKM2 inhibitor Shikonin and GLS inhibitor BPTES. Results: Glucose metabolism-related enzymes, ENO1 and PKM2, were significantly increased in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. Expression level of ENO1, PKM2, and GLS were also significantly high in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. The proliferation of OCUM-12hypo cells and OCUM-2MD3hypo cells was significantly inhibited by the knockdown of PKM2 and GLS, but not by the knockdown of ENO1. Cell proliferation was also significantly inhibited by PKM2 inhibitor and/or GLS inhibitor. A synergistic anti-tumor effect for gastric cancer cells in hypoxia was found in combination with PKM2 inhibitor and GLS inhibitor. The combination of PKM2 inhibitor and GLS inhibitor increased apoptosis rates of hypoxic gastric cancer cells. Conclusion: PKM2 and GLS might be important enzymes for the proliferation of gastric cancer in hypoxic microenvironment. The combined treatment with PKM2 inhibitor and GLS inhibitor produced synergistic anti-tumor effects, and might be a therapeutically promising for the treatment of gastric cancer. Citation Format: Kishu Kitayama, Masakazu Yashiro, Tamami Morisaki, Go Masuda, Hiroakai Kasashima, Yuichiro Miki, Haruhito Kinoshita, Tastunari Fukuoka, Tsuyoshi Hasegawa, Masaichi Ohira, Kosei Hirakawa. Effect of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 37.

Abstract 4097: Significance of glypican-1 expression on tumor stromal cells in gastric carcinoma

Background: Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors, and it plays a crucial role in cancer growth. Recently, it has been reported that GPC1 enriched on cancer-derived exosomes, and GPC1-positive exosomes in the circulation was frequently found in the patients with early pancreatic cancer. However role of GPC1-positive exosomes derived from tumor stromal cells remains to be unclear. The aim of this study is to clarify the significance of GPC1 expression on stromal cells in gastric carcinoma. Methods: A total of 597 patients with gastric cancer was enrolled in this study. An anti-GPC1 antibody was used for immunohistochemical staining. GPC1 expression was scored by the intensity of staining and the percentage of positive cells. GPC1 expression on cancer cells or stromal cells was evaluated. The corelation between GPC1 expression and clinicopathological features was statistically analyzed. Results: GPC1 expression on cancer cells was found in 473 (79%) of 597 cases. GPC1 expression on stromal cells was found in 147 (75%) of 597 cases. A Kaplan-Meier survival curve showed that overall survival of patients with GPC1-positive stromal cells was significantly better (log-rank p = 0.0028) than that with GPC1-negative stromal cells (5-year survival rate; 72.5% vs 57.9%, respectively). In contrast, overall survival was not statistically different (log rank p = 0.090) between patients with GPC1-positive tumor cells and GPC1-negative tumor cells (5-year survival rate; 71.8% vs. 64.6%, respectively). GPC1 expression on stromal cells was significantly correlated with T stage, lymph node metastasis, lymphatic vessel invasion, and clinical stage. Conclusions: GPC1 expression on stromal cells was associated with the good prognosis of patients with gastric cancer. GPC1-positive exosomes in stromal cells might be correlated with the progression of gastric cancer. Citation Format: Yuichiro Miki, Masakazu Yashiro, Kishu Kitayama, Hiroaki Kasashima, Go Masuda, Naoshi Kubo, Katsunobu Sakurai, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Significance of glypican-1 expression on tumor stromal cells in gastric carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4097.

Abstract 1730: Cancer stem cells might sustain stemness by the induction of autophagy in starvation stress

Background: Cancer stem cells (CSCs), which have capacity for self-renewal and cancer cell supply, have been thought to be responsible for tumor initiation, distant metastasis, and chemoresistance. Side population (SP) cells extracted by the fluorescence-activated cell sorting technique using Hoechst 33342 are thought to be enriched for CSCs. Autophagy is an intracellular degradation system that is induced under stress, such as starvation. Although autophagy has been reported to be associated with tumor progression under various stresses, the significance of autophagy in CSCs has still remained to be unknown. The aim of this study is to clarify the role of autophagy of CSCs under starvation stress. Materials and Methods: Two gastric cancer cell lines, OCUM-2MD3 and OCUM-12 were used. Two side population (SP) cell lines, OCUM-2MD3/SP and OCUM-12/SP were sorted by flow cytometry as CSC-rich cells from the parent OCUM-2MD3 and OCUM-12 cells, respectively. The effects of starvation stress, including low glucose, low amino acid, or low serum, on the stemness of CSCs were examined. The expression level of autophagy associated proteins, LC3A and LC3B, was evaluated by RT-PCR and western blotting. The effects of autophagy on the stemness and the proliferation of CSCs were examined by SP fraction and MTT assay. Chloroquine was used as an autophagy inhibitor. Results: The percentages of SP cells were significantly higher in the OCUM-12/SP cells (10.2%) and OCUM-2MD3/SP cells (12.3%) than in their parent OCUM-12 cells (2.1%) and OCUM-2MD3 cells (1.2%). The expression level of LC3A and LC3B was high in both OCUM-2MD3/SP cells and OCUM-12/SP cells, in comparison to that in OCUM-2MD3 cells and OCUM-12 cells. Starvation stress increased the autophagy status of OCUM-2MD3/SP and OCUM-12/SP cells. Chloroquine significantly decreased the SP fraction and proliferation activity of OCUM-2MD3/SP cells and OCUM-12/SP cells. Conclusion: Autophagy acts as a survival pathway in CSCs under starvation stress. CSCs might sustain stemness by the induction of autophagy in sarvation status. The autophagy inhibitor might be a promising therapy for CSCs. Citation Format: Go Masuda, Masakazu Yashiro, Kishu Kitayama, Yuichiro Miki, Hiroaki Kasashima, Tamami Morisaki, Tatsunari Fukuoka, Tsuyoshi Hasegawa, Masaichi Ohira, Kosei Hirakawa. Cancer stem cells might sustain stemness by the induction of autophagy in starvation stress. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1730.

Abstract 1281: Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ

Background & Aims: The antitumor activity of prostaglandin (PG) D2 has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD2 is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD2 synthase (PGDS), on gastric cancer cells. This is the first report showing that PG synthase is a promising agent for the treatment of gastric carcinomas that express peroxisome proliferator-activated receptor γ (PPARγ). Methods: The effects of PGD2 and PGDS on the proliferation of gastric cancer were examined in vivo and in vitro for five human gastric cancer cell lines. The expression levels of PGD2 receptors and PPARγ were evaluated by RT-PCR. The effects of PGD2 and PGDS on the expression of c-myc and cyclin D1 were examined by Western blotting in the presence or absence of a PPARγ antagonist. Results: PPARγ was expressed in gastric cancer cell lines, but PGD2 receptors were not. PGD2 and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD2 production of gastric cancer cells. A PPARγ antagonist significantly suppressed the growth-inhibitory effects of PGD2 and PGDS. Expression of c-myc and cyclin D1 was significantly decreased by PGD2; this inhibitory effect was suppressed by the PPARγ antagonist. Both PGD2 and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than after PGD2 treatment. PGD2 decreased the proliferation of gastric cancer cells through PPARγ signaling. Conclusion: PGDS and PGD2 decreased the proliferation of gastric cancer via the PPARγ pathway. PGDS is a promising therapeutic agent for gastric cancer and PPARγ might be a predictive biomarker for PGDS treatment in gastric cancer. Citation Format: Masakazu Yashiro, Tatsunari Fukuoka, Haruhito Kinoshita, Go Masuda, Kishu Kitayama, Yuichiro Miki, Tamami Morisaki, Tsuyoshi Hasegawa, Kosei Hirakawa. Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1281.