Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.
BACKGROUND
Patients with glioblastoma multiforme, the most aggressive form of glioma, have a median survival of approximately 12 months. Calcium (Ca(2+)) signaling plays an important role in cell proliferation, and some members of the Ca(2+)-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of many types of cancer cells. In this study, we investigated the role of TRPC6 in cell cycle progression and in the development of human glioma.


METHODS
TRPC6 protein and mRNA expression were assessed in glioma (n = 33) and normal (n = 17) brain tissues from patients and in human glioma cell lines U251, U87, and T98G. Activation of TRPC6 channels was tested by platelet-derived growth factor-induced Ca(2+) imaging. The effect of inhibiting TRPC6 activity or expression using the dominant-negative mutant TRPC6 (DNC6) or RNA interference, respectively, was tested on cell growth, cell cycle progression, radiosensitization of glioma cells, and development of xenografted human gliomas in a mouse model. The green fluorescent protein (GFP) and wild-type TRPC6 (WTC6) were used as controls. Survival of mice bearing xenografted tumors in the GFP, DNC6, and WTC6 groups (n = 13, 15, and 13, respectively) was compared using Kaplan-Meier analysis. All statistical tests were two-sided.


RESULTS
Functional TRPC6 was overexpressed in human glioma cells. Inhibition of TRPC6 activity or expression attenuated the increase in intracellular Ca(2+) by platelet-derived growth factor, suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 activation and cell division cycle 25 homolog C expression regulated the cell cycle arrest. Inhibition of TRPC6 activity also reduced tumor volume in a subcutaneous mouse model of xenografted human tumors (P = .014 vs GFP; P < .001 vs WTC6) and increased mean survival in mice in an intracranial model (P < .001 vs GFP or WTC6).


CONCLUSIONS
In this preclinical model, TRPC6 channels were essential for glioma development via regulation of G2/M phase transition. This study suggests that TRPC6 might be a new target for therapeutic intervention of human glioma.
Ding
Xia
X
Laboratory of Neural Signal Transduction, Institute of Neuroscience, Shanghai Institute for Biological Sciences, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai, China.
He
Zhuohao
Z
Zhou
Kechun
K
Cheng
Ju
J
Yao
Hailan
H
Lu
Dongliang
D
Cai
Rong
R
Jin
Yening
Y
Dong
Bin
B
Xu
Yinghui
Y
Wang
Yizheng
Y
D000256:Adenoviridae; D000818:Animals; D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002455:Cell Division; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004195:Disease Models, Animal; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D016195:G2 Phase; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D016086:Lentivirus; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D014183:Transplantation, Heterologous; D015854:Up-Regulation
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human