Abstract:
Objective To establish oxaliplatin-resistant esophageal squamous cell carcinoma (ESCC) cell line and study the reversal effect and mechanism of palbociclib (an inhibitor of cyclin-dependent kinase 4/6) on this cell line.
Methods High-dose shock and low-dose induction were used to establish the oxaliplatin-resistant ESCC cell line, which was named KYSE150-OXA. CCK-8 assay was used to detect and calculate death rate of the cells treated with oxaliplatin (L-OHP), paclitaxel (PTX), 5-fluorouracil (5-FU) and pabociclib (a CDK4/6 inhibitor), respectively. Flow-cytometry was performed to measure the apoptosis and cell cycle of the cells treated with the above chemotherapeutics. Western blot assay was used to determine the expression of multidrug resistance gene 1 (MDR1) and related cell-cycle genes. CalcuSyn software was used to evaluate the combinative effects of palbociclib and L-OHP on proliferation of ESCC cells.
Results We established a L-OHP-resistant cell line KYSE150-OXA, which showed resistant capacity to both PTX and 5-FU, and the highest drug resistance index was 11.23±0.59. The results showed that MDR1 protein was significantly upregulated by (4.46±0.22) fold in drug-resistant cells. Compared with the control, the number of KYSE150-OXA cells in G1 phase was significantly increased. Expression of CDK4, CDK6 and Cyclin D1 was significantly increased, while Cyclin E expression was significantly decreased. Palboiclib was able to increase the sensitivity of KYSE150-OXA to L-OHP. In low concentration, palbociclib and L-OHP showed antagonism effect, but the synergistic effect was gradually increased with the increasing concentration of palbociclib. In the palbociclib treated group, expression of phospho-retinoblastoma protein decreased by (91±2)% and cyclin A decreased by (89±6)%.
Conclusions Oxaliplatin-resistant cell line which was named KYSE150-OXA, is successfully established. The results suggest that palbociclib can reverse oxaliplatin-resistance by inhibiting the abnormal activation of CyclinD-CDK4/6-pRb signaling pathway.