The Mechanism And The Relationship Between The Expression Of GRP78/94 And The Resistance To VP-16 In Small Cell Lung Cancer (SCLC)

Objective: The mechanism and the relationship between the expression of glucose-regulated protein 78 (GRP78) and GRP94 and resistance to VP-16 in small cell lung cancer ( SCLC) NCI-H446 cell line was examined with an array of analytical methods including Immuno-fluorescence, RT-PCR and Flow Cytometry Machine (FCM).Methods: The cells were divided and cultivated respectively in three groups, namely, the induced group (induced by the inducer of A23187), the inhibited group (inhibited by the inhibitor of BAPTA-AM) and the control group. Immuno-fluorescence assay was used to examine the expression and distribution of GRP78/94 in the NCI-H446 cell line. RT-PCR was adopted to investigate the expression of GRP78 and GRP94 at the level of mRNA in the cells. The differences of GRP78 and GRP94 among the three groups were compared as well. VP16 of the same concentration was added to the three groups and the apoptosis rates and cell cycles of the three groups cells were tested with FCM. The relationship between the expression of GRP78/94 and resistance to VP-16 in the NCI-H446 cell line was investigated.Results: The assay of Immuno-fluorescence analysis showed that GRP78 and GRP94 were both expressed in NCI-H446 with the mainly distribution in cytoplasm and the periphery of nucleus. Furthermore, the fluorescence intensity was strikingly stronger in the induced group compared with the control group. The analysis with RT-PCR demonstrated that the expression of GRP78 and GRP94 at the level of mRNA was characteristic with the manners of concentration-dependent on the inductors or inhibitors, which means that the expression of them may increased ordecreased with the ascending or descending of concentration of the inducer or inhibitor. The cell cycle analysis illustrated that cells in the induced group primarily distributed in G1 phase with relatively less distribution in phases of S and G2, whereas, cells in inhibited group distributed mainly in phases of S and G2 with relatively less in G1 phase. After the treatment of VP-16, the main chemotherapy drug for SCLC targeted in phases of S and G2, the apoptosis rate of the cells in the induced group markedly decreased while that in the inhibited group increased deeply compared with the control, which suggested that the resistance to VP16 in NCI-H446 could be affected by the distribution of cell cycle under the induction of GRP78 and GRP94.Conclusion: This research indicated that there were expression of GRP78 and GRP94 in cytoplasm and the periphery of nucleus in NCI-H44 cell line. The inductors (A23187) or inhibitors (BAPTA-AM) could increase or decrease the expression of them. Moreover, the degree of expression of GRP78 and GRP94 affected the distribution of cell cycle and was related to the apoptosis induced by VP-16. The resistance to VP-16 induced by GRP78 and GRP94 could be functioned by the impact on the cell cycle in SCLC cells. Therefore, to inhibit the expression of GRP78 and GRP94 could provide an important alternative to reduce the resistance and enhance the sensitivity of chemotherapy in SCLC.