Gastric cancer is one of the most frequent cancers, and it ranks the third most common cancer in China [Wu et al., 2002]. Although surgical techniques and chemoradiotherapy have effectively promoted advancements in its treatment, its overall survival rate is still low [Yang et al., 2012]. Due to the importance of chemotherapy in treating gastric cancer, the development of multidrug resistance (MDR) becomes a serious obstacle to effective chemotherapy. Mechanisms involved in MDR include decreased drug accumulation in tumor cells, altered intracellular drug distribution, increased detoxification, and uncoupled pathways linking cellular damage with apoptosis [Shi et al., 2008]. Although the molecular mechanisms of the MDR have not yet been demonstrated in gastric cancer cells, some studies have reported that the mechanisms of MDR were associated with the over-expression of P-glycoprotein (P-gp) encoded by MDR1 gene in tumor cells [Han et al., 2006; Song et al., 2012; Wang et al., 2012], thus, the inhibition of P-gp expression in tumor cells could be one of the most effective ways to reverse MDR and make tumor cells resensitize to chemotherapy [Chen et al., 2012]. In addition, Apoptosis was a common pathway that finally mediated the killing functions of anticancer drugs, which was an important cause of MDR [Hong et al., 2005].
E2F is a family of transcription factors implicated in cell cycle control. To date, 8 E2F subunits have been identified. Thus far, E2F-1 is the best characterized member. It promotes cell cycle progression by regulating the expression of critical regulator genes involved in DNA replication and G1/S transition [Han et al., 2006]. Most importantly, it plays a most important role in malignant phenotypes of some cancers. Some previous studies reported that E2F-1 could affect the cell proliferation and apoptosis and it may be involved in regulating MDR in some cancers [Han et al., 2003; Lu et al., 2012]. E2F-1 could affect the cell cycle and apoptosis. Furthermore, apoptosis is just one of the most important mechanisms of reversal MDR. Consequently, E2F-1 may play a crucial role in the control of reversal MDR. We assumed that E2F-1 may influence the MDR of gastric cancer cells through regulating the expression of the MDR1 gene and/or with other genes associated with apoptosis. The aim of this study was to explore the relationship between E2F-1 and MDR of gastric cancer cells.
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The development of MDR to cancer chemotherapy is a major obstacle to the effective treatment of gastric cancer [Fan et al., 2000]. However, the mechanism of MDR remains obscure. P-gp was the first molecule identified as a modulator of MDR. After that, various other molecules were shown to be involved, including transporters that eject anticancer drugs from cells, such as MDR-associated protein (MRP) [Chuman et al., 1996], genes regulating apoptosis, such as p53 [Matsuhashi et al., 2005], PKC [Han et al., 2002], and Bcl-2 family [Xiao et al., 1998]. Recently, the distribution of drugs in cancer cells was also considered to play a part in MDR [Minchinton and Tannock, 2006], but there may be other mechanisms that control MDR of gastric cancer cells [Fan et al., 2000].
Transcription factor E2F-1 has been largely studied as a promoter of S phase transition in the cell cycle and as a regulator of apoptosis, E2F-1 has been shown to eliminate latent neoplastic cells through apoptosis in vitro [Li et al., 2009]. Downregulation of E2F-1 significantly inhibits the growth of prostate cancer cell lines and leiomyosarcoma [Nguyen et al., 2005], and it is linked to low proliferation and longer survival of different types of carcinoma cells in vivo and in vitro [Evangelou et al., 2007]. Our previous study reported that E2F-1 plays diverse roles in cells ranging from regulating cell cycle progression to promoting cell death by apoptosis in gastric cancer cells in vitro and in vivo [Xiao et al., 2007; Xie et al., 2009, 2009; Wang et al., 2011]. The present study is believed to be the first to correlate E2F-1 with MDR of gastric cancer cells, and we found that expression of E2F-1 regulated drug efflux pumping, the cell cycle, and apoptosis. The multiple changes conferred by E2F-1 on gastric cancer cells are not surprising, given the involvement of E2F-1 in a wide range of biochemical reactions, and E2F-1 is a transcription factor that contributes to reversing MDR.
Our study indicated that E2F-1 shRNA led to downregulation of E2F-1 mRNA and protein expression in SGC7901/DDP cells, caused cell cycle arrest in the G0/G1 phase, and induced cell apoptosis. Furthermore, downregulation E2F-1 in SGC7901/DDP cells enhanced the sensitivity of SGC7901/DDP cells to cisplatin, doxorubicin, and fluorouracil. The ability to pump doxorubicin was reduced significantly, moreover, a strong anti-tumor effect of E2F-1 shRNA in vivo was observed, as tumor growth was suppressed and tumor apoptosis was increased in nude mice when E2F-1 mRNA and protein was downregulation by E2F-1 shRNA. These findings suggest that E2F-1 shRNA reversed MDR of human gastric cancer cells.
MDR is mainly due to the overexpression of P-gp (which is encoded by MDR1) and MRP, they function as a drug efflux pump which actively transports drugs from the inside to the outside of cancer cells and prevents the intracellular accumulation of anticancer drugs inside cancer cells necessary for cytotoxic activity, it should be noted that P-gp-mediated drug efflux was not the only mechanism involved in drug resistance. Previous studies have shown that the effect of P-gp on drug resistance is closely related to cell cycle distribution and apoptosis [Kornmann et al., 1999; Sicari et al., 2012; Tsubaki et al., 2012]. S phase kinase-associated protein 2 (skp2) is a key regulator of cell cycle control, c-Myc is required for Skp2 to trigger S phase entry, Skp2-Myc connection is important for S phase progression, skp2 recently has been shown to be a direct downstream target gene of c-myc [Kornmann et al., 1999], in addition, suppression of cyclin D1 (a regulatory kinase of cell cycle distribution) levels has been shown to potentiate the response of human pancreatic cancer cells to cisplatinum [Kornmann et al., 1998]. Besides regulation of cell cycle distribution, apoptosis is a common pathway that finally mediates the killing effects of anticancer drugs, which is an important cause of MDR. Bcl-2 family, which includes anti- and pro-apoptotic members such as Bcl-2 and Bax [Igney and Krammer, 2002; Tait and Green, 2010; Sicari et al., 2012], Bcl-2 binds to the mitochondrial membrane, competitive binding with Bax and forming Bcl-2/Bax heterodimer, which leads to closing mitochondrial permeability transition pore and preventing the release of cytochrome C, thereby inhibiting apoptosis via the Bcl-2/Bax signal pathway, moreover, Survivin induces mitochondrial fragmentation, and reduces mitochondrial respiration [Hagenbuchner et al., 2012], it is closely related to apoptosis. Therefore, in the present study, inhibition of E2F-1 expression may have decreased MDR1, MRP, Bcl-2/Bax, c-Myc, Skp2, Survivin, and Cyclin D1 expression directly or indirectly, which was responsible for reversal of MDR in human gastric cancer cells in vitro and in vivo. Further studies are needed to confirm our results.
The term MDR was originally coined to define a condition enabling a disease-causing organism or cancer cells to resist distinct drugs or chemicals with a wide variety of structure and function, targeted at eradicating the organism/cancer cell. Much routine chemotherapy cannot achieve good therapeutic effects because of MDR [Lu et al., 2012]. In this study, we showed that E2F-1 plays a critical role in reversing MDR. Downregulation of E2F-1 using RNAi reversed the progression of MDR in gastric cancer SGC7901/DDP cells in vitro and in vivo. In conclusion, this study lays the foundation for treatment of MDR in gastric cancer through manipulation of E2F-1 expression.