[Reversion of multidrug resistance of human gastric cancer SGC7901/DDP cells by E2F-1 gene silencing].
OBJECTIVE
To investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms.


METHODS
Gastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups: the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP cells were transfected with recombinant lentivirus vector (Lv-shRNA-E2F-1), while the negative control with an control lentiviral vector (Lv-shRNA-NC) and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration (IC50) of three chemotherapy drugs including adriamycin, 5-fluorouracil (5-Fu) and cisplatine (DDP) of the three cell groups. Flow cytometry (FCM) was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of multidrug resistance-associated genes (MDR1, MRP) and apoptosis-related genes (c-Myc, Skp2, cyclinD1).


RESULTS
The expression of E2F-1 protein in the experimental group was significantly lower than that in the negative control and blank control groups (0.794 ± 0.033 vs. 1.487 ± 0.082 vs. 1.511 ± 0.084, P < 0.01). The IC50 of the three chemotherapy drugs (adriamycin, 5-Fu and cisplatine) in the experimental group was significantly lower than that of the negative control and blank control groups, respectively (P < 0.01). Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [(0.16 ± 0.01)% vs. (0.37 ± 0.01)% vs. (0.35 ± 0.02)%, P < 0.01]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [(33.82 ± 1.26)% vs. (17.34 ± 0.81)% vs. (13.16 ± 1.06)%, P < 0.01]. The results of RT-PCR and Western blot assays showed that mRNA and protein expressions of five genes (MDR1, MRP, CyclinD1, c-Myc, Skp2) in the experimental group were significantly lower than that in the negative control and blank control groups, respectively (P < 0.01).


CONCLUSIONS
E2F-1 gene silencing enhances the chemosensitivity of gastric cancer SGC7901/DDP cells to the chemotherapeutic drugs, directly or indirectly downregulated the expression of MDR1 and MRP, and finally reverses the multidrug resistance of the gastric cancer cells. The mechanism may be associated with the suppression of cyclinD1, c-Myc and Skp2.
Department of Gastrointestinal and Gland Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China.
Chao
C
Lian
Jie
J
Yang
Xiaotong
X
Wang
Yubo
Y
Xie
Email: xiaoqiang20050@aliyun.com.
Qiang
Q
Xiao
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005472:Fluorouracil / Q000494:pharmacology; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology*; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D002945:Cisplatin; D019938:Cyclin D1; D004317:Doxorubicin; D050687:E2F1 Transcription Factor; D005472:Fluorouracil; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; C493753:E2F1 protein, human