ERK1/2 act a part in GCDA-induced survival of human liver carcinoma cells.
The ERK1/2 cascade is best known for its role in proliferation, differentiation and tumorigenesis (13). Firstly, we measured the endogenous protein levels of ERK1/2 in five HCC cell lines (HepG2, Bel-7402, Bel-7404, QGY-7703 and SMMC-7721). The result of western blot showed that ERK1/2 were extensively expressed in all the liver cancer cells we detected (Figure.1A). Next, to test whether GCDA promoted HCC cell proliferation, we treated QGY-7703 cell line with 100µM GCDA for 0h, 24h, 48h, and 72h, and then checked the viable cells by CCK8. Results indicated that viable cells significantly increased after treatment with GCDA until 72h (Fig. 1B).
To determine whether ERK1/2 affected the GCDA-induced survival of HCC cells, we designed siRNAs targeting ERK1 (225, 888 and 933) and ERK2 (355, 513 and 714). All the siRNAs were transfected into QGY-7703 cells. Then immunoblot was done to determine the interference efficiency. As shown in Fig. 1C, ERK1 and ERK2 proteins expression were inhibited by siRNA888 and siRNA513 mostly, respectively. After siRNA888 targeting ERK1 and siRNA513 targeting ERK2 were transfected into QGY-7703 cell line together, GCDA was used to treat cells for 24h. Apoptotic cells were analyzed using annexin V binding on FASC. Flow cytometry results demonstrated that GCDA could repress apoptosis. But after ERK1/2 were silenced, the apoptotic cells were increased (Fig. 1D&E). In other words, specific depletion of ERK1/2 blocked GCDA-stimulated cell survival. These results indicated that ERK1 and ERK2 molecules might have functions in survival of hepatoma cells mediated by GCDA.
GCDA induces ERK1/2 phosphorylation, which may be involved in prolonged survival of human liver cancer cells.
Next, we investigated potential mechanisms involved in the GCDA-induced HCC cell survival. QGY-7703 cells were treated with 100µM GCDA for 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h. Results demonstrated that the activated ERK1/2 increased obviously after GCDA treatment in QGY-7703 cells, while the expression of endogenous ERK1/2 changed little (Fig. 2A).
Irinotecan has been known as a topoisomerase I inhibitor, which was effective in a variety of cancers (21). The antimetabolite 5-fluorouracil (5-FU), which could inhibit thymidylate synthase, was a widely used antitumor agent (22). In order to check the effect of GCDA-induced ERK1/2 activation on cell survival, QGY-7703 cells were treated with antitumor drug (10µM Irinotecan or 5µg/mL 5-FU) or GCDA (100µM) + antitumor drug (10µM Irinotecan or 5µg/mL 5-FU) for 72h. We observed that Irinotecan or 5-FU induced > 80% of cells undergoing death and GCDA significantly prolonged cell survival following treatment with Irinotecan or 5-FU (Fig. 2B). Therefore, we speculated that phosphorylation of ERK1/2 might participate in chemoresistance induced by GCDA.
The MAPK/ERK1/2 inhibitor PD98059 decreases GCDA-stimulated cell proliferation.
To further verify the role of activated ERK1/2 in HCC cells, the MAPK/ERK1/2 inhibitor PD98059, which could inhibit phosphorylation of ERK1/2, was used (23). We treated QGY-7703 cells with GCDA (100µM) or GCDA (100µM) + PD98059 (10µM) for 24h, 48h and 72h. Then, CCK8 was done to test the viability of QGY-7703 cells. CCK8 experiments showed that suppression of ERK1/2 activation by PD98059 would decrease proliferation of liver cancer cells (Fig. 3A). Next, QGY-7703 cells were treated with or without PD98059 (10µM) for 0.5 h, followed by treatment with GCDA (100µM) or GCDA (100µM) + antitumor drug (10µM Irinotecan or 5µg/mL 5-FU) for 72h. Results of CCK8 showed that PD98059 significantly attenuated the chemoresistance induced by GCDA, which could prolong cell survival following treatment with Irinotecan or 5-FU (Fig. 3B). In conclusion, these findings implied that phosphorylation (or activation) of ERK1/2, which could be attenuated by PD98059, might be important for GCDA-mediated HCC cells’ survival and chemoresistance.
PD98059 suppresses GCDA-induced nuclear aggregation of ERK1/2 and p-ERK1/2.
In unstimulated cells, ERK1/2 molecules are usually located in the cytoplasm (15). Under stimulation, lots of ERK1/2 molecules were translocated to the nucleus (15). ERK1/2 localization plays a significant role in determining the strength of this pathway. Therefore, we examined the localization of ERK1/2 and p-ERK1/2 following GCDA (100µM) or GCDA (100µM) + PD98059 (10µM) treatment. The results of immunofluorescence staining showed that ERK1/2 proteins were distributed in both cytoplasm and nucleus and more p-ERK1/2 proteins accumulated in the nuclear as small spots in resting HCC cells (Fig. 4A&B). Following GCDA treatment, most ERK1/2 proteins gathered in the nucleus, while more p-ERK1/2 proteins accumulated in the nucleus as bigger speckles. However, after PD98059 treatment, the aggregation of ERK1/2 and p-ERK1/2 proteins in nucleus significantly decreased (Fig. 4A&B). Collectively, the above data suggested that nuclear accumulation of ERK1/2 and p-ERK1/2 induced by GCDA could be impaired by PD98059.
PD98059 restrains GCDA-induced increase of Mcl-1 and decrease of Bim.
ERK1/2 signalling has been verified to regulate some members of Bcl-2 family, which can contribute to tumour cell survival via increasing anti-apoptotic factors and decreasing pro-apoptotic members of Bcl-2 family(16). Hence, we inspected the level of some Bcl-2 family members following GCDA (which can activate ERK1/2 pathway) or PD98059 (which can repress ERK1/2 pathway) treatment. Firstly, 100µM GCDA was used to treat QGY-7703 cells for 0h, 0.5h, 1h, 2h, 4h and 8h. Immunoblot had been done to check the levels of Bcl-2, Mcl-1, Bim and Bak. We observed that GCDA could promote expression of Bcl-2 and Mcl-1, both of which are anti-apoptotic Bcl-2 family members, and decrease expression of Bim and Bak, both of which are pro-apoptotic Bcl-2 family members (Fig. 5A). Next, in order to determine if suppression of ERK1/2 signalling regulated Bcl-2 family members’ expression, GCDA (100µM) or GCDA (100µM) + PD98059 (10µM) were used to treat QGY-7703 cells for 8h. Results showed that inhibition of ERK1/2 by PD98059 could block GCDA-induced increase of Mcl-1 and decrease of Bim. However, Bcl-2 and Bak did not change significantly (Fig. 5B). Our data supported the notion that GCDA might facilitate cell survival via regulation proteins of Bcl-2 family, some of which could be inhibited by PD98059. Such results indicated that activation of ERK1/2 pathway induced by GCDA could mediate certain members of Bcl-2 family.