| Objective: To explore the relationship between the reversal ofTetramethylpyrazine on HCC cell line BEL-7402/ADM ROS-MAPKsignaling pathway.Methods: Human hepatocellular carcinoma cell line BEL-7402andmulti-drug resistant (MDR) hepatocellular carcinoma cell line (HCC)BEL-7402/ADM were divided into ten groups, BEL-7402、BEL-7402/ADM、BEL-7402/ADM+TMP、BEL-7402/ADM+VRP、BEL-7402/ADM+3-AT；BEL-7402+ADM、BEL-7402/ADM+ADM、BEL-7402/ADM+TMP+ADM、BEL-7402/ADM+VRP+ADM and BEL-7402/ADM+3-AT+ADM. Humanmultidrug resistant hepatocellular carcinoma cell line BEL-7402/ADM weretreated with tetramethylpyrazine (TMP). The comparison of combinationwith/without3-AT (inducer of ROS), UO126(inhibitor of ERK), SP600125(inhibitor of JNK) and SB203580(Inhibitor of p38) were conducted bydetecting and calculating IC50value, resistant fold and reversal fold by usingCCK-8. The fluorescent density of intracellular ROS were determined andanalyzed by Confocal laser scanning microscope (CLSM) and Flow cytometry (FCM) in each group. The accumulation of doxorubicin (or adriamycin, ADM)in cells were determined by FCM assay and high performance liquidchromatography (HPLC). Cell apoptosis of every group was measured byFCM.Results:(1) CCK-8assay showed that the IC50value for the groups ofresistant cells were significantly higher than the parental cells, indicating thatthe groups of resistant cells were resistant to ADM compared with the parentalcells. Among which the resistant index of BEL-7402/ADM cells treated withtetramethylpyrazine (TMP) decreased compared with those without drugs,suggesting TMP reversed MDR HCC like VRP and3-AT. When TMPcombined with UO126(ERK blocker) or SP600125(JNK blocker), it wasfound that resistance index was reduced and reversal fold wasimproved.Furthermore, IC50value was lower than that of parental cells whenTMP conbined with SP600125. However, when TMP combined withSB203580(p38blocker) was treated in resistant cells, the results showed thatresistant index was increased and reversal fold was reduced, enabling thatresistant cells were less sensitive to chemotherapeutic drugs than before.(2)The average intensity of the fluorescence of intracellular ROS of each groupwas collected by FCM analysis. Compared with the BEL-7402/ADM group,intracellular ROS level was increased when the resistant cells were added withreversal agents and ADM indicating that the MDR to chemotherapeutic drugsmay be related to ROS level in resistant cells.(3) The intracellular accumulation of doxorubicin was detected by FCM. The results showed thatVRP,3-AT and TMP increased the intracellular level of ADM in MDR cellsto induce cell deaths.(4) Intracellular concentration of ADM in MDR cellswere determined by HPLC. The result showed that TMP could increasedintracellular concentration of ADM, which was consistent with that of FCM.(5) The result by dual calibration of Annexin V-FITC showed that TMP couldpotentiate the sensitivity of resistant cells to chemotherapeutical agents.Conclusion: MDR HCC cells BEL-7402/ADM had a lower level ofintracellular ROS. Moreover, cells resistance to chemotherapeutical agentsmay be positively related with intracellular level of ERK and JNK, butnegatively with p38. However, increased ROS in cells could reverse MDR byenabling cells sensitivity to chemotherapy. TMP may reverse MDR byincreasing intracellular ROS. Furthermore, TMP combined with either ERK orJNK inhibitors could enhance the reversal effects of TMP. The cell signalingshould be further studied. |
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