Takashi Tatara1,2, Toru Mukohara3,4,5, Rina Tanaka6, Yohei Shimono1,7, Yohei Funakoshi1, Yoshinori Imamura1, Masanori Toyoda1, Naomi Kiyota1,4, Midori Hirai6, Yoshihiro Kakeji2, Hironobu Minami1,4. 1. Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan. 2. Division of Gastrointestinal Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan. 3. Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan tmukohar@east.ncc.go.jp. 4. Cancer Center, Kobe University Hospital, Kobe, Japan. 5. Division of Breast and Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan. 6. Division of Pharmacokinetics and Pharmaceutics, Kobe University Graduate School of Medicine, Kobe, Japan. 7. Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
Abstract
BACKGROUND: Our hypothesis was that three-dimensional (3D) culture better represents differential in vivo responses to trastuzumab between PIK3CA-wild-type (wt) and mutant (mt) cell lines than does two-dimensional (2D) culture. MATERIALS AND METHODS: Apoptosis and cell signaling proteins were evaluated in response to trastuzumab with and without BKM120, a pan-phosphatidylinositol 3-kinase (PI3K) inhibitor, using western blot analysis of four breast cancer cell lines with human epidermal growth factor receptor 2 (HER2) amplification. RESULTS: Increased expression of cleaved poly (ADP-ribose) polymerase (PARP) was observed only in 3D-cultured PIK3CA-wt lines in response to trastuzumab, but not in 2D-cultured PIK3CA-wt or PIK3CA-mt lines. Decrease in the ratio of phosphorylated (p-)AKT to AKT in response to trastuzumab was more profound in PIK3CA-wt cells than in PIK3CA-mt cells in 3D culture, while the difference between PIK3CA genotypes was less apparent in 2D culture. Treatment with BKM120 and trastuzumab resulted in a stronger increase in cleaved PARP than either treatment alone. CONCLUSION: 3D Culture appears to better represent trastuzumab-induced apoptosis and resistance to trastuzumab associated with PIK3CA mutation. Copyright
BACKGROUND: Our hypothesis was that three-dimensional (3D) culture better represents differential in vivo responses to trastuzumab between PIK3CA-wild-type (wt) and mutant (mt) cell lines than does two-dimensional (2D) culture. MATERIALS AND METHODS: Apoptosis and cell signaling proteins were evaluated in response to trastuzumab with and without BKM120, a pan-phosphatidylinositol 3-kinase (PI3K) inhibitor, using western blot analysis of four breast cancer cell lines with humanepidermal growth factor receptor 2 (HER2) amplification. RESULTS: Increased expression of cleaved poly (ADP-ribose) polymerase (PARP) was observed only in 3D-cultured PIK3CA-wt lines in response to trastuzumab, but not in 2D-cultured PIK3CA-wt or PIK3CA-mt lines. Decrease in the ratio of phosphorylated (p-)AKT to AKT in response to trastuzumab was more profound in PIK3CA-wt cells than in PIK3CA-mt cells in 3D culture, while the difference between PIK3CA genotypes was less apparent in 2D culture. Treatment with BKM120 and trastuzumab resulted in a stronger increase in cleaved PARP than either treatment alone. CONCLUSION: 3D Culture appears to better represent trastuzumab-induced apoptosis and resistance to trastuzumab associated with PIK3CA mutation. Copyright