Usp9x- and Noxa-mediated Mcl-1 downregulation contributes to pemetrexed-induced apoptosis in human non-small-cell lung cancer cells

Abstract

Pemetrexed, a folate antimetabolite, combined with cisplatin is used as a first-line therapy for malignant pleural mesothelioma (MPM) and locally advanced or metastatic non-small-cell lung cancer (NSCLC). Pemetrexed arrests cell cycle by inhibiting three enzymes in purine and pyrimidine synthesis that are necessary for DNA synthesis. Pemetrexed also promotes apoptosis in target cells, but little is known about its mechanism in cancer cells. We have previously shown that pemetrexed can result in endoplasmic reticulum (ER) stress, and it can lead to downstream apoptosis. In this study, we further elucidate this mechanism. Our data show that pemetrexed increases Noxa expression through activating transcription factor 4 (ATF4) and activating transcription factor 3 (ATF3) upregulation. Furthermore, pemetrexed induces apoptosis by activating the Noxa-Usp9x-Mcl-1 pathway. Inhibition of Noxa by small interfering RNA (siRNA) promotes Usp9x (ubiquitin-specific peptidase 9, X-linked) expression. Moreover, downregulation of the deubiquitinase Usp9x by pemetrexed results in downstream reduction of myeloid cell leukemia 1 (Mcl-1) expression. Mechanistically, Noxa upregulation likely reduces the availability of Usp9x to Mcl-1, thereby promoting its ubiquitination and degradation, leading to the apoptosis of neoplastic cells. Thus, our findings demonstrate that Noxa-Usp9x-Mcl-1 axis may contribute to pemetrexed-induced apoptosis in human lung cancer cells.

Figures

Figure 1

Pemetrexed induces Noxa expression and leads to apoptosis. A549 and H1792 cell lines were treated with pemetrexed at the indicated concentrations for 48 h (a). A549 cell lines were treated with 2.5 μmol/l pemetrexed for various time periods (b). A549 and H1792 cell lines were cultured in six-well plates. On day 2, cells were transfected with control (Ctrl) or Noxa siRNA. On day 3, a single well was divided between two wells. On day 4, 2.5 μmol/l pemetrexed was given to A549 and H1792 cell lines for 48 h (c). Cells were harvested and lysates of whole-cell protein were used for western blot analysis (a–c). A549 cell line was transfected with control and Noxa siRNA using the same protocol. Apoptosis was measured by Annexin V-PE/7-AAD staining and analyzed by flow cytometry (d). For the flow cytometry analysis, the % positive cells in the upper right (Annexin V+/7-AAD+: late apoptotic cells) and lower right quadrants (Annexin V+/7-AAD−: early apoptotic cells) were summed to give the total number of apoptotic cells. The experiment was performed in triplicate and repeated three times. The data are presented as mean±S.D. of three replicates in one experiment (**P<0.01, Student's t-test) (e). Representative pictures from three independent experiments are shown

Figure 2

Downregulation of Mcl-1 accelerates pemetrexed-induced apoptosis. H1792 and A549 cells were treated with pemetrexed for 48 h (a). A549 cells were treated with 2.5 μmol/l pemetrexed for the indicated times (b). Both H1792 and A549 cells were transfected with control (Ctrl) or Mcl-1 siRNA. At 2 days after transfection, cells were treated with 2.5 μmol/l pemetrexed for 48 h (c and d). Whole-cell protein lysates were harvested for western blot analysis (b–d). A549 cells were transfected with control and Mcl-1 siRNA and treated with pemetrexed using the same method. The cells were stained by Annexin V-PE/7-AAD staining and analyzed by flow cytometry (d and e). The experiment was performed in triplicate and repeated three times. The data are presented as mean±S.D. of three replicates in one experiment (**P<0.01) (e). Representative pictures from three independent experiments are shown

Figure 3

ATF3 and ATF4 are upregulated by pemetrexed, and knockdown of their expression by siRNA protects NSCLC cells from pemetrexed-induced apoptosis. A549 and H1792 cells were treated with the indicated pemetrexed doses for 48 h (a). A549 cells were treated with 2.5 μmol/l pemetrexed for the indicated times (b). A549 and H1792 cells were transfected with control (Ctrl) or ATF3/ATF4 siRNA for 48 h and then treated with 2.5 μmol/l pemetrexed for 48 h (c and d). Cells were harvested and lysates of whole-cell protein were used for western blot analysis (a–c). Representative pictures from three independent experiments are shown

Figure 4

Knockdown of ATF3 or ATF4 expression by siRNA protects NSCLC cells from pemetrexed-induced apoptosis. A549 (a) and H1792 (b) cells were transfected with control (Ctrl) or ATF3/ATF4 siRNA for 48 h and then treated with 2.5 μmol/l pemetrexed for 48 h. Cells were harvested and subjected to Annexin V-PE/7-AAD staining and flow cytometry analysis. The % positive cells in the upper right (Annexin V+/7-AAD+: late apoptotic cells) and lower right quadrants (Annexin V+/7-AAD−: early apoptotic cells) were summed to give the total number of apoptotic cells. The experiment was performed in triplicate and repeated three times. The data are presented as mean±S.D. of three replicates in one experiment (**P<0.01)

Figure 5

Noxa regulates Usp9x and Mcl-1 level after pemetrexed treatment. A549 and H1792 cells were cultured in six-well plates and transfected with control (Ctrl) or Noxa siRNA. Then, 24 h later, single wells were equally divided into two wells. On the second day, cells were treated with 2.5 μmol/l pemetrexed for 48 h (a). The indicated concentrations of pemetrexed were given to H1792 and A549 cells for 48 h (b). A549 cells were treated with 2.5 μmol/l pemetrexed for the indicated time (c). Whole-cell protein of lysates were harvested and used for western blot analysis. Representative pictures from three independent experiments are shown

Figure 6

Knockdown of Usp9x expression attenuates Mcl-1 expression and increases pemetrexed-induced apoptosis in NSCLC cells. H1792 and A549 cells were transfected with control (Ctrl) or Usp9x siRNA for 2 days and then were treated with 2.5 μmol/l pemetrexed for 48 h. Cells were subjected to preparation of the whole-cell protein lysates for western blot analysis (a). A549 cells were prepared to flow cytometry analysis (b). Apoptosis was measured by Annexin V-PE/7-AAD staining. In the flow cytometry analysis, the percent positive cells in the upper right and lower right quadrants were summed to yield the total number of apoptotic cells. The experiment was performed in triplicate and repeated three times. The data are presented as mean±S.D. of three replicates in one experiment (**P<0.01) (c)

Figure 7

Pemetrexed treatment reduces the availability of Usp9x to Mcl-1 and enhances Mcl-1 ubiquitination in lung cancer cells. A549 cells were transfected with plasmids pcDNA3.1-Myc-Mcl-1 or pcDNA3.1 and Ub-HA that could express HA-tagged ubiquitin. After 12 h, the cells were treated with pemetrexed (5 μmol/l) for 8 h, and MG132 (10 μmol/l) was cotreated for another 4 h. For immunoprecipitation, cells were lysed and incubated with Myc antibody for 1 h and then incubated with protein A/G agarose (1 : 1 mix) at 4°C overnight. The beads were washed three times and then boiled in the loading buffer. The Mcl-1 ubiquitination level were analyzed by western blot assay using anti-HA antibody (a). Plasmid pcDNA3.1-Myc-Mcl-1 was transfected into A549 cells. After 12 h, the cells were treated with DMSO or pemetrexed (5 μmmol/l) for 12 h. For immunoprecipitation, cells were lysed and incubated with Myc antibody for 1 h and then incubated with protein A/G agarose (1 : 1 mix) at 4°C overnight. The beads were washed three times and then boiled in the loading buffer. Western blot was performed to examine the levels of proteins binding to Mcl-1 such as Usp9x and Noxa (b). Representative pictures from two independent experiments are shown