Anti-cancer agent sensitivity-determining marker
An anticancer agent and susceptibility technology, which is applied in the field of anticancer agent susceptibility judgment markers, can solve the problems of patients not being able to obtain the effect and difficulty in continuing treatment, so as to achieve the effect of improving cancer treatment effect, preventing cancer progression, and avoiding side effects
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Embodiment 1
[0096] Exploration of predictive biomarkers for oxaliplatin susceptibility using 2D-DIGE
[0097] (1) Using cells
[0098] SW480, Ls174T, and Lovo classified as highly oxaliplatin-susceptible strains in Test Example 1, and HT29, DLD-1, and WiDr classified as low-oxaliplatin-susceptible strains were used.
[0099] (2) Intracellular protein extraction method
[0100] Remove the medium from the dish, wash with ice-cold PBS for 3 times, in order to avoid the stimulation of the cells and the activation of intracellular proteins, directly add the cell lysate (2M thiourea (thiourea), 7M urea (Urea), 4% CHAPS, 50mM Tris-HCl pH 9) lysed the cells and transferred to a 1.5mL microtube. After sonicating under ice-cooling, it was centrifuged at 13,000×rpm at 4° C. for 10 minutes, and the supernatant was collected. Protein quantification was carried out using a 2D quantification kit (Quant kit) (GE Healthcare), prepared to 5 mg / mL with a cell lysate, dispensed and stored at -80°C until a...
Embodiment 2
[0122] Expression and IC of 6 proteins in colorectal cancer cell lines 50 value correlation
[0123] Regarding the six proteins identified in Example 1, the expression levels in the nine colorectal cancer cell lines used in Test Example 1 and the IC calculated in Test Example 1 were confirmed. 50 value correlation.
[0124] (1) Method
[0125] (a) using cells
[0126] Nine colon cancer cell lines described in Test Example 1 were used.
[0127] (b) Extraction of intracellular proteins
[0128] Remove the culture medium from the dish, wash with ice-cold PBS for 3 times, in order to avoid the stimulation of the cells and the activation of intracellular proteins, directly add the cell lysate (9M urea (Urea), 2% CHAPS, 50mM Tris- Cells were lysed with HCl pH 9) and transferred to 1.5 mL microtubes. After sonicating under ice-cooling, it was centrifuged at 13,000×rpm at 4° C. for 10 minutes, and the supernatant was recovered. Protein quantification was performed using the BCA...
Embodiment 3
[0140] Exploration of predictive biomarkers for oxaliplatin susceptibility using SELDI-TOF MS
[0141] (1) Method
[0142] (a) using cells
[0143] SW480, Ls174T, and Lovo classified as highly oxaliplatin-susceptible strains in Test Example 1, and HT29, DLD-1, and WiDr classified as low-oxaliplatin-susceptible strains were used.
[0144] (b) Extraction of intracellular proteins
[0145] It was carried out by the same method as (1) method (b) extraction of intracellular protein in Example 2.
[0146] (c) Sample preparation for protein expression analysis, fabrication of protein chips, and expression analysis of intracellular proteins
[0147]The cell lysis buffer was adjusted to 0.5 mg / mL with a pH 4 dilution / washing buffer (50 mM sodium acetate buffer) (hereinafter, pH 4 buffer) to obtain a sample, and 100 μL of the obtained sample was applied (apply ) on the spot of a cation-exchange chip array (CM10, Bio-Rad) pretreated with pH 4 buffer, incubated for 30 minutes to react...
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