Note: The Notch inhibitor cowanin accelerates nicastrin degradation

Note for: The Notch inhibitor cowanin accelerates nicastrin degradation

(doi: 10.1038/s41598-018-23698-4)

Notch alteration contributes to carcinogenesis. Therefore, it could be used as the target to treat the cancer. Using cell-based system to screen compound from Garcinia speciosa. 1/12 was found to inhibit notch signaling pw. HES1 and HES5 are the targets. Cowanin decreases HES1 and HES5 protein level. Cytotoxic to leukemic HPB-ALL.

Notch signaling - many fundamental process;

1.proliferation

2.stem cell maintenance

3.differentiation

(well just like wnt signaling pw)

It is important for neuronal diff. It has been regulated through hairy and enhancer of split 1 (HES1), HES5 and HES related (HESR/HEY) family genes.

Aberration in Notch signaling participates in both multiple hematologic and solid malignancies.

Notch signaling is activated by interaction

between the ligand-expressing cell and the signal-receiving cell.

Various Notch inhibitors;

1. γ-secretase inhibitors

2. DAPT (N-[N-(3,5-difluorop henacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

3. RO4929097

4.  MK-0752

5. F0384014

6. SHAM1

7. MAML1-derived peptide that inhibits the binding of full-length MAML1 to NICD1-RBP-J

Natural product;

1.curcumin

2.genistein

Role in this study; searching for the new Notch signaling inhibitors from natural resources by using cell-based reporter system -- the approach of using cell-based system is limited for natural compound screening.

they develop cell-based reporter assay with the T-Rex system to evaluate Notch signaling inhibitory activity.

Platform;

1.using luciferase as a readout for inhibitor action but using fluorometric microculture cytotoxicity assay as a readout for cell viability -- therefore, if extract giving low luciferase activity but high cell viability --> good candidate

2.cell line LS174T (colon cancer - this cell has notch signaling activity)

3.using crude extract first (MeOH fraction - Garcinia speciosa)

4.using 100 ug/ml crude extract to treat the cell.

5.seperate more with different solvent systems from the positive result -- elucidate the chemical constituents

Solvent system; non-polar to highly polar (hexane, EtOAc, nBuOH, and water)

6.combine fractions between Hexane and EtOAC --> analyse;

α-mangostin (1), cowanin (2), cowanol (3), norcowanin (4), rubraxanthone (5), cowaxanthone (6)β-mangostin (7), 6-methoxy-γ-mangostin (8), 9-hydroxycalabaxanthone (9), cowaxanthone B (10), fuscaxanthone A (11)and a new compound (12).

7.using pure compound now to test on the inhibitory activity by using their own cell-based system (now the unit change by weight to the same particle -- molar).

8.criteria to determine the potency; high IC50<10uM, moderate IC50 -- 10-20 uM

9.They explain by using SAR (structure-acivity relationship)

10.investigate more at the molecular level; using WB and looking specific proteins that are part of the notch signaling.

11.using cancer cell to confirm the inhibition from cell-based assay --> T-ALL and HPB-ALL, using normal human embryonic kidney 293 cells as normal cell line.

12.also they compare the cowanin with the available inhibitor on the market

Cowanin (2) was shown to inhibit γ-secretase activity by reducing nicastrin levels, which was the result of the acceleration of nicastrin degradation.

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