2020 Fiscal Year Research-status Report
Modulation of circadian clock and its therapeutic implications in invasive breast carcinoma
Project/Area Number |
20K17585
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Research Institution | Kagawa University |
Principal Investigator |
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Project Period (FY) |
2020-04-01 – 2023-03-31
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Keywords | breast carcinoma / metastasis / circadian clock |
Outline of Annual Research Achievements |
In fiscal year 2020, in vitro studies were conducted in both mouse and human breast cancer cells. First, we measured the mRNA expression of core clock genes in mouse breast cancer cells (4T1) and mouse healthy mammary tissue. Quantitative real time PCR data revealed that the mRNA expression of Per1 and Per2 was significantly lower in breast carcinoma cells compared to the normal mammary tissue. Since glucose metabolism has a significant impact on clock genes, before going to the knockdown or over-expression studies, we conducted pharmacological studies with rare sugar, D-allose. Accumulating evidences have indicated that d-allose reduces glucose uptake through down-regulation of glut-1. Therefore, we have checked the cell proliferation in mouse (4T1) and human (MDA-MB-231) breast carcinoma cells and found that d-allose reduced the cell proliferation in a dose- and time-dependent manner. Following intervention with d-allose (50 mM) for 48 hrs, quantitative real time PCR was performed to check the mRNA expression of core clock genes. The gene expression data revealed that Per1, Per2 and Cry2 expression were increased after treatment with D-allose compared to the vehicle or D-glucose treatment which might be associated with reduced cell proliferation in breast cancer cells. Therefore, these data are in line with our hypothesis that modulation of circadian clock might be a potential therapeutic approach for invasive breast carcinoma.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We are conducting experiment step by step. After completing the in vitro studies, we will conduct in vivo experiment.
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Strategy for Future Research Activity |
a) Clock genes-manipulated breast cancer cells (1x106) will be injected in the mammary fat pad of 6-weeks-old female immunodeficient nude (MDA-MB-231) and/or Balb/C mice (4T1 cell line). Metastatic potential (migrated cells foci) of clock genes-manipulated breast cancer cells will be analyzed in the lungs of xenograft mice following fixation in Bouin’s solution in accordance with our standardized protocol. b) Identified genes from in vitro microarray assay will be examined in the lung tissues (containing metastatic foci) by quantitative reverse transcription PCR (qRT-PCR). Moreover, the corresponding proteins of the identified genes will be investigated by western blotting and/or immunohistochemistry.
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