Method for screening anti-cancer compounds inhibiting function of TM4SF5 and anti-cancer composition containing chalcone compounds
The present invention relates to a method for screening an anticancer compound and an anticancer compound screened using the method, and more particularly, to a method for screening an anticancer compound, the method comprising: culturing cancer cells expressing the oncogenic protein transmembrane 4 L6 family member 5 (TM4SF5), expressed as the polypeptide of SEQ ID NO: 2, treating the cancer cells with an anticancer candidate, and determining that the anticancer candidate is an anticancer substance when the candidate exhibits antagonistic activity against tumor formation and metastasis based on several events through the molecular mechanism of TM4SF5. The present invention also relates to chalcone compounds screened to have anticancer activity using the method, and an anticancer composition comprising the compound as an effective ingredient.
1. A method for screening an anticancer compound for liver cancer, comprising:
(a) culturing an artificially established hepatocarcinoma cell line expressing the polypeptide of SEQ ID NO: 2 from a vector, and treating the hepatocarcinoma cells with an anticancer candidate;
(b) detecting at least one of the following in the hepatocarcinoma cells treated with the anticancer candidate: (i) phosphorylation of a tyrosine residue at positions 577 (Tyr577) of an amino acid sequence encoding focal adhesion kinase (FAK), (ii) binding of FAK to Rho-GTPase activating protein (RhoGAP) or FAK to GTPase regulator associated with FAK (GRAF), (iii) cytosolic p27 Kip1 expression level and stability, (iv) RhoA activity, and (v) Rac1 activity; and
(c) determining whether the anticancer candidate can be an anticancer substance for liver cancer when the following events are exhibited, compared to when treatment with the anticancer candidate is not conducted: (i) decreased phosphorylation of FAK on Tyr577, (ii) inhibition of binding of FAK to RhoGAP or FAK to GRAF, (iii) reduced cytosolic p27 Kip1 expression level and stability, (iv) increased RhoA activity, or (v) decreased Rac1 activity.
2. The method for screening an anticancer compound according to claim 1 , wherein the cytosolic p27 Kip1 is the phosphorylated p27 Kip1 on Ser 10.
3. The method for screening an anticancer compound according to claim 1 ,
wherein the step (b) further includes detecting at least one selected from the group consisting of cell morphology, expression of a protein involved in cell adhesion formation, cell-cell contact pattern or contact growth, expression of α-smooth muscle actin (α-SMA) or vimentin, expression of E-cadherin, and epithelial-mesenchymal transition (EMT); and
the step (c) further includes the following events: a change in cell morphology from a rod shape into a polygonal shape, reduced expression of proteins involved in cell adhesion formation, maintenance of cell-cell contact, contact inhibition of cell growth, reduced expression of α-SMA or vimentin, increased expression of E-cadherin, or reduced epithelial-mesenchymal transition (EMT).
4. The method for screening an anticancer compound according to claim 3 , wherein the protein involved in cell-cell adhesion formation is selected from the group consisting of E-cadherin, zonula occludens-1 (ZO1), β-catenin and desmoplakin.
5. The method for screening an anticancer compound according to claim 3 , wherein contact inhibition of cell growth is through the reduction of cell number, reduction of cell population in S-phase, or inhibition of multilayer growth.
6. The method for screening an anticancer compound according to claim 5 , wherein the reduction of cell number or reduction of S-phase cell population is mediated through the inactivation of N-linked glycosylation of membrane proteins.
7. The method for screening an anticancer compound according to claim 1 ,
wherein the step (b) further includes detecting at least one selected from the group consisting of cell migration or motility in the presence of an extracellular matrix or serum, the invasion into collagen gels which comprise the extracellular matrix, the invasion into Matrigel which is an extracellular matrix complex, and matrix metalloproteinase (MMP) activity; and
the step (c) further includes the following events: decreased cell migration or motility in the presence of an extracellular matrix or serum, decreased invasion into collagen gels, decreased invasion into Matrigel, or reduction of MMP activity.
8. The method for screening an anticancer compound according to claim 7 , wherein said MMP is MMP-2 or MMP-9.
9. The method for screening an anticancer compound according to claim 3 ,
wherein the step (b) further includes detecting at least one selected from the group consisting of cell migration or motility in the presence of an extracellular matrix or serum, the invasion into collagen gels which comprise the extracellular matrix, the invasion into Matrigel which is an extracellular matrix complex, and matrix metalloproteinase (MMP) activity; and
the step (c) further includes the following events: decreased cell migration or motility in the presence of an extracellular matrix or serum, decreased invasion into collagen gels, decreased invasion into Matrigel, or reduction of MMP activity.
10. The method for screening an anticancer compound according to claim 9 , wherein said MMP is MMP-2 or MMP-9.
11. The method of claim 1 , wherein the artificially established hepatocarcinoma cell line expressing the polypeptide of SEQ ID NO: 2 from a vector is an SNU449 cell line or an SNU398 cell line transfected with a retroviral vector comprising the nucleic acid sequence of SEQ ID NO: 1.