title: Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells.
abstract: OBJECTIVE
  To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).
  
  
  METHODS
  The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor (VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells (GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction.
  
  
  RESULTS
  CD44(+) GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth (GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density (MVD) (GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium- (82.87% ± 6.53%) and high-dose XTSJ groups (77.43% ± 7.34%) was detected at 24 h in the CD44(+) GCSCs group compared with the saline group (95.42% ± 5.76%) and the low-dose XTSJ group (90.74% ± 6.57%) (P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44(-) groups; significant differences were only detected in the high-dose XTSJ group at 48 h (78.57% ± 6.94%) and 72 h (72.12% ± 7.68%) when compared with the other CD44- groups (P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium- (1.76 ± 0.15) and high-dose XTSJ (1.33 ± 0.081) groups compared with the GCSCs control group (2.72 ± 0.25) and the low-dose XTSJ group (2.51 ± 0.25) (P < 0.05). We also detected a remarkable decrease of MVD in the medium- (7.10 ± 0.60) and high-dose XTSJ (5.99 ± 0.47) groups compared with the GCSC control group (8.15 ± 0.42) and the low-dose XTSJ group (8.14 ± 0.46) (P < 0.05). Additionally, CD44 expression was decreased in these groups [medium- (4.43 ± 0.45) and high-dose XTSJ groups (3.56 ± 0.31) vs the GCSC control (5.96 ± 0.46) and low dose XTSJ groups (5.91 ± 0.38)] (P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression.
  
  
  CONCLUSIONS
  Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Yan
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Xiu
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Da-Zhi
authors.initials: DZ
authors.lastname: Sun
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Shi
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Yin-Cheng
authors.initials: YC
authors.lastname: Zhang
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Yong-Jin
authors.initials: YJ
authors.lastname: Li
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Wang
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Zhou
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Shou-Han
authors.initials: SH
authors.lastname: Feng
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Can
authors.initials: C
authors.lastname: Lv
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Pin-Kang
authors.initials: PK
authors.lastname: Wei
authors.affiliation: Bing Yan, Ying Zhao, Li-Juan Xiu, Da-Zhi Sun, Xuan Liu, Ye Lu, Jun Shi, Yin-Cheng Zhang, Yong-Jin Li, Xiao-Wei Wang, Yu-Qi Zhou, Shou-Han Feng, Can Lv, Pin-Kang Wei, Zhi-Feng Qin, Department of Traditional Chinese Medicine, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
authors.forename: Zhi-Feng
authors.initials: ZF
authors.lastname: Qin
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D008297:Male; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic; D017207:Rats, Sprague-Dawley; D051881:Receptor, Notch1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000098:blood supply* / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D000072056:Transcription Factor HES-1; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D020533:Angiogenesis Inhibitors; D000972:Antineoplastic Agents, Phytogenic; D051792:Basic Helix-Loop-Helix Proteins; D004365:Drugs, Chinese Herbal; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; D019394:Ki-67 Antigen; D051881:Receptor, Notch1; D000072056:Transcription Factor HES-1; D042461:Vascular Endothelial Growth Factor A; C497483:CD44 protein, human; C497761:NOTCH1 protein, human; C467484:VEGFA protein, human; C000598250:xiaotan sanjie; C078493:HES1 protein, human
keywords: Gastric cancer stem-like cells; Notch-1; Tumor angiogenesis; Vascular endothelial growth factor; Xiaotan Sanjie decoction