Introduction
Chemoresistance increases the seriousness of cancer, since, in the absence of effective chemotherapy, other treatment modalities (surgery, radiotherapy) are often doomed to failure, especially when cancer is diagnosed at an advanced stage. For ovarian cancer, which is the fifth most frequent cause of tumour death in women, the 5-year survival rate for stage III and IV disease (representing around 75% of cases) is approximately 20% - 30%. These disappointing results are mainly due to intrinsic chemoresistance or to progressively acquired resistance, despite a good response to surgery and to first-line platinum-based chemotherapy [
1,
2].
When active, chemotherapies lead to the apoptotic death of cancer cells [
3,
4]. Apoptosis is controlled by members of the BCL-2 family, which promote or inhibit this process [
5]. The main anti-apoptotic members are Bcl-2, Mcl-1 and BCL-x
L, whereas the main pro-death members are Bax, Bak, Bad and Bim [
6,
7]. Overexpression of anti-apoptotic Bcl-2 family proteins has been reported as contributing towards cell survival and drug resistance in a variety of human malignancies [
8,
9]. In various cancers, Bcl-x
L and Mcl-1 are frequently overexpressed [
8], suggesting that resistance to apoptosis could imply these two key anti-apoptotic factors. Furthermore, Bcl-x
L and Mcl-1 are frequently associated with chemotherapeutic resistance and relapse [
9,
10]. Therefore, anti-apoptotic strategies, such as concomitant inhibition of Bcl-x
L and Mcl-1 are expected to play a potentially important role in the near future to overcome drug resistance in certain cancers [
9,
11,
12]. Given that Bcl-x
L inhibitors are currently under clinical evaluation (e.g. BH3 mimetic compounds such as antimycin A3, gossypol, HA14-1 [
13] or ABT-737) [
14‐
16]), and that there are few or no available specific inhibitors of Mcl-1 [
17], the discovery of new Mcl-1 inhibitors is of crucial interest.
Citrate is an intermediate of the tricarboxylic acid cycle (TCA), which acts as an “energy gauge” inside the cell since, when in excess, it inhibits glycolysis by allosteric retro-control on phosphofructokinase (PFK). This enzyme catalyses the first irreversible reaction of glycolysis and constitutes the main control checkpoint of this pathway. Because glycolysis furnishes essential intermediates (such as ribose, glycerol, serine …) required for cancer cell proliferation and a significant share of ATP, especially in hypoxic conditions [
18‐
21], its blockage could arrest cancer cell growth [
22‐
25]. When citrate is abundant, PFK activity is virtually switched off, leading to a slowdown in TCA. Interestingly, citrate leads to early down-expression of Mcl-1 [13;28].
ABT-737 is a small chemical molecule that mimics the direct binding of Bad to Bcl-2, Bcl-x
L and Bcl-w, but not to Mcl-1 [
26]. It has demonstrated preclinical activity as a single agent in a wide variety of cancer cells, such as hematopoietic cell lines [
27‐
30] and, to a lesser extent, solid tumour cell lines [32;34–37]. However, ABT-737 is unable to induce cell death as a single agent in other tumour models, such as prostate cancer [38]. This relative resistance has previously been linked to a persistence of Mcl-1 expression [38–40]. Our work confirms the primordial importance of the concomitant inhibition of the two key anti-apoptotic proteins, Mcl-1 and Bcl-x
L. Citrate, which reduces the expression of Mcl-1, dramatically increases cell death in chemoresistant human ovarian carcinoma cell lines exposed to interfering RNA targeting Bcl-x
L or ABT-737.
Discussion
In the absence of effective chemotherapy, other anti-cancer treatment modalities (surgery, radiotherapy) are most often doomed to failure, as demonstrated for ovarian cancers, for which 5-year survival rates are generally poor, as for many other solid cancers. Hypoxic cells are believed to play an important role in chemoresistance [
17‐
19], since these cells may adopt resistance strategies, such as enhanced glycolysis, glutaminolysis, overexpression of anti-apoptotic factors, etc., to survive harsh chemotherapy conditions [
20,
21].
Overexpression of anti-apoptotic proteins such as Bcl-x
L and Mcl-1 have been reported to greatly contribute to cell survival and drug resistance in various human cancers [
22‐
24]. Concomitant inhibitory strategies targeting these proteins have consequently been shown to overcome drug resistance [11;14].
With this in mind, Bcl-x
L inhibitors, such as ABT-737, are currently under clinical evaluation as BH3 mimetic compounds, and few or no specific inhibitors of Mcl-1 are available [45;46]; the discovery of new Mcl-1 inhibitors is therefore of crucial importance. Since we observed that citrate, an inhibitor of PFK, reduces Mcl-1 expression [
11], we chose to test citrate in association with a specific siRNA targeting Bcl-x
L or ABT-737.
Our results confirm, for the two ovarian cancer cell lines studied, the efficiency of citrate in decreasing Mcl-1 expression, as we have previously described on other cell lines [
13,
25]. In contrast, citrate demonstrated no significant effect on Bcl-x
L expression. The decrease in Mcl-1 protein expression but not Bcl-x
L could be due to the activation of caspase 3 as described in other studies [
32,
33] or by proteasome activity which degrades the Mcl-1 protein after ubiquitylation [
27‐
30]. A recent study reported that the inhibition of glycolysis using 2-deoxy-D-glucose (2DG) induced intracellular ATP depletion which led to specific down regulation of Mcl-1 through the translational control [
34].
Concomitant inhibition of Mcl-1 and Bcl-x
L is a highly effective strategy to destroy chemoresistant ovarian cancer cells, in accordance with previous studies we have conducted on cancer cells from other cancer sites [11;14;33]. In the two human ovarian cell lines studied, all cells were totally destroyed after 72 H of exposure to treatment associating siXL1 and citrate 10 mM, compared to the administration of one product alone (Figures
3 and
4). We chose to expose cells to citrate 24 h after siXL1 transfection, to allow the siXL1 sufficient time to play its role. In contrast, since the blockage of Bcl-x
L activity by ABT-737 is very rapid, occurring within two hours, we chose to administrate citrate before ABT treatment. This association of citrate with ABT-737 seemed more efficient than the combination using siRNA targeting Bcl-x
L, and led to almost complete cell death with nuclear apoptotic fragmentation and complete cleavage of PARP. Furthermore, the number of cells in G1 and G2/M phases, likely to resume the cell cycle, was strongly diminished. The mechanisms of cellular death in response to citrate exposure remain to be further investigated. A number of hypotheses can be put forward: - an increase in N-alpha-acetylation of proteins such as caspases, sensitising these cells to apoptosis, given that citrate is the donor of acetyl in cells through ACLY activity [
35], an activating process that could be initiated at apical caspase 8 and 2 level [
25]; a reduction in ATP synthesis due to glycolysis inhibition by citrate effect, which in turn activates the AMPK pathway, resulting in inhibition of the mTOR pathway [
28,
30,
34]. This pathway could inhibit Mcl-1 expression via two mechanisms: - firstly, Mcl-1 could be phosphorylated by activated GSK3 beta and then degraded by proteasome after ubiquitylation [
27‐
30]; - secondly, blockage of 4E-BP1 resulting in the inhibition of Mcl-1 translation [54;55]. According to our results, the first hypothesis appears the most likely. This molecular aspect remains to be elucidated.
In conclusion, the effect of citrate on Mcl-1 expression could usefully be associated with inhibitors of Bcl-xL, since this strategy demonstrates a strong anti-cancer effect on chemoresistant ovarian cancer cells. Given that few or no specific inhibitors of Mcl-1 are currently available, the use of citrate targeting Mcl-1 could be of major interest for it could contribute towards the destruction of chemoresistant cancer cells.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
HL and PI drafted the manuscript. All authors read and approved the final manuscript.