Introduction
EHMT2, also known as G9a, is an important enzyme for histone H3 dimethylation at lysine-9 [
1,
2]. EHMT2 is overexpressed in breast, prostate, colon, bladder, ovarian, melanoma, lung, and liver cancers [
3-
5]. Recent research shows that enhanced expression of EHMT2 is involved in the proliferation of cancer cells [
6]. BIX-01294, a diazepin-quinazolinamine derivative, is a specific inhibitor of EHMT2 by reducing the H3K9Me2 level [
7]. Furthermore, BIX-01294 is the first small compound that can replace Oct4 to induce neural stem cells to be reprogrammed into iPSCs [
8]. Moreover, BIX-01294 can induce apoptosis in human neuroblastoma cells by raising CASP8 and CASP3 activity [
9]. BIX-01294 also can decrease the proliferating activity of bladder cancer cells [
6]. It was reported that knockdown of
EHMT2 by siRNA [
3,
4,
10,
11] or treatment with BIX-01294 [
10,
12,
13] inhibited growth of bladder, lung, prostate, and breast cancer cells.
A variety of detrimental stimuli, such as ultraviolet light, hypoxia, oxidative stress, can affect the function of ER (endoplasmic reticulum), leading to accumulation of unfolded or misfolded protein in ER and activation of ER stress response [
14-
18]. A large number of unfolded or misfolded proteins can bind to HSPA5, and then activate three pathways through PERK, ERN1 and ATF6. The long-term, severe ER stress will ultimately induce cell apoptosis [
19]. DDIT3 is a transcriptional factor and the downstream member of ER stress pathways [
20,
21]. Recent studies have shown that numerous drugs, such as lonafarnib, MG132, CDDO-Me and pemetrexed up-regulate DDIT3 expression via p-EIF2S1-ATF4 axis and accordingly enhance apoptosis through induction of DR5 or Bim etc. [
22-
24].
Mitochondrion is at the center of intrinsic apoptotic pathway. Under the regulation of BCL-2 family, Cyt c, a component of mitochondrial electron transport chain, can be released into cytoplasm through a hole related to the oligomerization of BAX/BAK and VDAC (voltage-dependent anion channel). The Cyt c in cytoplasm can combine with APAF1 and induce cell apoptosis [
25]. MCL1 drives its pro-survival function by combining with BAX/BAK and inhibiting the oligomerization. PMAIP1 belongs to the pro-apoptotic BH3-only family and has been known to suppress pro-survival MCL1 and A1 through binding to them [
26]. Several BH3-only family proteins, including PMAIP1, can be activated by DDIT3 [
27]. Notably, PMAIP1 can control the stability of MCL1 through regulating its polyubiquitination which E3 ligase HUWE1 or FBW7 are involved in [
28]. Ubiquitin specific peptidase 9x (USP9X), a deubiquitinase belonging to USP family, can stabilize MCL1 through removing the polyubiquitin chains linked by Lys-48 which is the essential sign in proteasomal degradation process, and it is overexpressed in several kinds of cancer cells [
29]. Moreover, over-expression of PMAIP1 may trigger a decrease in the USP9X/MCL1 interaction [
28]. Recent studies also show that high level of PMAIP1 likely reduces the availability of USP9X to MCL1, thereby promoting the ubiquitination and degradation of MCL1, leading to the apoptosis of neoplastic cells [
30].
However, the specific mechanism of apoptosis mediated by BIX-01294 in bladder cancer cells remains unknown. The goal of our research was to investigate which pathway BIX-01294 follows to induce apoptosis in human bladder cancer cells and which proteins play important roles in this process. In our research, BIX-01294 was capable of leading to ER stress. Furthermore, DDIT3, as a downstream transcriptional factor in ER stress, promotes the expression of PMAIP1 which can significantly lower MCL1 levels. Moreover, BIX-01294 also reduces USP9X and then affects the stability of MCL1. According to our present study, we demonstrate that BIX-01294 causes apoptosis in bladder cancer cells through ER stress pathway, resulting in PMAIP1 up-regulation and MCL1 down-regulation.
Materials and methods
Reagents
The powder of BIX-01294 (B9311) trihydrochloride and EHMT2 (G6919) antibody were purchased from Sigma-Aldrich (City of Saint Louis, Missouri, US). CASP3 (IMG-145) antibody was purchased from Imgenex. USP9X (5751), DDIT3 (2895), CASP8 (9746), CASP9 (9502), PARP1 (9542), HSPA5 (3183) and ERN1 (3294) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts, US). ATF4 (SC-200) and MCL1 (SC-12756) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, US). PMAIP1 (OP180) antibody was purchased from Calbiochem (San Diego, California, US).
Cell lines and cell culture
The human bladder cell lines T24 and 5637 were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). T24 and 5637 were grown in monolayer culture at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. The T24 cell line was cultured in McCoy's 5A Modified Media (Sigma-Aldrich) supplemented with 5% (v/v) FBS (SAFC®Global) and the 5637 cell line was cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 5% (v/v) FBS.
Western blot analysis
Preparation of whole cell protein lysates and the procedures for the Western blot analysis were already described [
31].
siRNA transfections
All the siRNAs were synthesized by GenePharma. The small interfering RNA (siRNA) duplexes for control,
DDIT3 siRNA duplexes target the sequence 5'-GCCTGGTATGAG GACCTGC-3' [
32].
EHMT2 siRNA duplexes target the sequence 5′-GCATTTCCGCATGAGTGAT-3′.
PMAIP1 siRNA duplexes target the sequence 5′-GGAAGUCGAGUGUGCUACU-3′,
USP9X siRNA duplexes target the sequence 5′-CAATCAAGTTCAATGATTA-3′, The transfection of siRNA was conducted with X-tremeGENE siRNA Transfection Reagent (Roche) following the instructions.
Cell survival assay
On the first day, cells were seeded in 96 well plates. Twenty-four hours later, BIX-01294 was added at the indicated concentrations in each well. After treatment by BIX-01294 for another 24 h, we used 10% TCA to immobilize the cells and then evaluated the living cell numbers using the sulforhodamine B assay as previously described [
33].
Apoptosis assays
Apoptosis was detected by the Annexin V-FITC Apoptosis Detection Kit (BIOBOX) following the manufacturer’s protocol. Activation of the caspase family was evaluated by western blot.
Construction of the plasmid
MCL1 gene was amplified from A549 cells cDNA with PCR technique using the primers described as below: 5′-CGGATCCGCCGCCACCATGTGTTTGGCCTCAAAAGAAACG-3′ (sense); 5′-CGGGCCCCTATCTTATTAGATATGCCAAAC-3′ (antisense). This resultant construct was named pcDNA3.1-MCL1 after
MCL1 gene was cloned into pcDNA3.1 (+) vector by using
Bam HI and
Apa I restriction sites [
30].
Plasmid transient transfections
On the first day, T24 and 5637 cells were seeded into 6-well plates. On the second day, cells were transfected with pcDNA3.1 and pcDNA3.1-MCL1 using X-tremeGENE HP DNA Transfection Reagent (Roche). On the third day, experimental groups were treated by 10 μM BIX-01294 for 24 hours. On the fourth day, the cells were harvested for western blot.
Discussion
To date, over-expression of EHMT2 may be a prognostic marker for patients with melanoma and esophageal cancer [
38,
39]. In addition, EHMT2 inhibitors can prevent the proliferation and self-renewal of AML via reducing HoxA9-dependent transcription and also can increase the intracellular ROS [
7,
40]. BIX-01294 has been demonstrated to induce autophagy in breast and colon cancers [
7]. Earlier reports showed BIX-01294 led to apoptosis by activating CASP8 and CASP3 in human neuroblastoma cells and reduce the proliferation of human bladder cancer cells. In our study, with the increase of BIX-01294 concentration, the cell survival rate of bladder cancer cells T24 and 5637 reduced clearly. Moreover, by treating with BIX-01294, the expression level of some apoptosis-related proteins such as CASP8, CASP9, CASP3 and PARP1 were activated simultaneously. This means that BIX-01294 indeed induces the apoptosis in human bladder cancer cells. However, the mechanisms underlying how BIX-01294 triggers apoptosis in human bladder cancer cells remain unclear.
Cellular apoptosis can be induced by a long-term, severe ER stress. Some earlier studies have found that DDIT3, as a downstream component of ER stress, can be activated by ATF4. We also discovered that ER stress component, like HSPA5, ERN1, ATF4 and DDIT3, increased when the human bladder cancer cells were treated with BIX-01294. Moreover, the cleavage of PARP1 were suppressed in cells transfected with DDIT3 siRNA. This implies the BIX-01294 induces caspase-dependent apoptosis through activating ER stress. Notably, the expression of PMAIP1 was reduced, while MCL1 was rescued when the cells were transfected with DDIT3 siRNA. These results indicate that DDIT3 induces the expression of PMAIP1 which is required for BIX-01294-induced apoptosis.
In terms of mitochondrial apoptotic pathway, PMAIP1 belongs to the BH3-only pro-apoptotic protein family while MCL1 belongs to pro-survival BCL2 protein family. MCL1 can be suppressed by PMAIP1 or stabilized by the deubiquitinase USP9X [
41]. We found that BIX-01294 up-regulated the expression of PMAIP1 and down-regulated the level of MCL1 and USP9X. In addition, in the cells transfected with
PMAIP1 siRNA, the levels of MCL1 and USP9X were augmented, while the cleaved caspases and PARP1 were decreased. These results suggest that BIX-01294 degrades the MCL1 through up-regulation of PMAIP1 and reduction of USP9X, which is consistent to our previous study that PMAIP1 upregulation reduces the availability of USP9X to MCL1, thereby promoting its ubiquitination and degradation, leading to the apoptosis of neoplastic cells [
35]. Since DDIT3 also modulates other apoptotic proteins such as death receptors in the extrinsic pathway, we cannot exclude the role of extrinsic pathway in the apoptosis induction by BIX-01294 in bladder cancer cells. Therefore, further investigation is required to characterize as much as details involved in the BIX-01294-induced apoptosis.
In summary, our data demonstrate that BIX-01294 induces ER stress pathway, resulting in up-regulation of PMAIP1 and down-regulation of USP9X and MCL1, leading to apoptosis in bladder cancer cells. These findings may offer important insight into the molecular mechanism of BIX-01294, thereby accelerating its use in clinical scenarios which may help improve bladder cancer treatments.
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Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
XL and LS conceived the project. JC and XL designed the experiments. JC and WS performed the experiments. XH, MW, XS and YZ help carry out the Western Blot assay. JC and XL analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.