DHA predominantly exerts its anticancer effect by triggering the apoptotic cell death process.1,18
Here, we report for the first time that DHA also induces autophagy and this autophagic process contributes to the cytotoxic activity of DHA against wild-type p53 harbored cancer cells by enhancing apoptosis. As a process of consuming cellular components and generating energy, autophagy engages in a complex interconnection with apoptosis according to the nature of stimulus and cell type. It suppresses apoptosis by eliminating damaged organelles under cellular stress response to cancer therapy, or it sensitizes cells to apoptosis, which is ATP-dependent, by acting as an energy source.29–32
We showed that autophagy and apoptosis occurred simultaneously and acted cooperatively to induce cell death in DHA-treated cancer cells, as DHA treatment increased both LC3-II and cleaved PARP in dose- and time-dependent manners ( and B
, left). In addition, the fact that morphological features of both autophagy and apoptosis were observed in the same cells treated with DHA () confirms the above results.
The finding that DHA induces both autophagy and apoptosis prompted us to closely investigate the interconnection between these two cellular processes. The evidence that further autophagy induction promoted apoptosis in cells treated with DHA ( and S2C
) indicates that autophagy contributes to the apoptosis caused by DHA. In this respect, autophagy seems to facilitate the occurrence of apoptosis. This notion is supported by the observation that autophagy inhibition by MG132, 3-MA or ATG siRNA decreased cell sensitivity to DHA-mediated apoptosis (). Furthermore, the form of cell death initiated by autophagy (autophagic cell death) is caspase independent.33
In our system, we observed significant changes in caspase-3 activity in cells preincubated with autophagy inducer or inhibitor ( and E
) and subsequently treated with DHA, which also implies that autophagy, rather than directly causing cell death, assists DHA-induced apoptotic cell death. Indeed, it has been reported that autophagy can function upstream of apoptosis and participate in the process of membrane blebbing, one of the characteristics of apoptotic cell death, by maintaining cellular ATP levels.32,34,35
Whether this is the role played by autophagy in DHA-induced apoptosis needs to be addressed by further investigation. Additionally, although further autophagy induction by PFTα and rapamycin resulted in a statistically significant enhancement of apoptosis in DHA-treated cells, the increase was modest, which is not unexpected since these autophagic inducers share the same signaling with DHA to trigger autophagy and, compared with DHA, they have less potent effect on autophagic induction. Meanwhile, it is also worth noting that autophagy inhibition and knockdown failed to completely block apoptosis in cancer cells treated with DHA, suggesting that DHA-induced apoptosis is not exclusively based on mTOR-mediated autophgay activation and that other signaling pathways may participate in the apoptotic process caused by DHA treatment. At least the mechanisms involving accumulation of reactive oxygen species (ROS) and β-catenin have been reported.1–3
Another question we addressed in this study is the mechanism underlying DHA-induced autophagy. Prompted by the observation of p53 loss in cancer cells treated with DHA (), we further evaluated the specific involvement of p53 in the autophagic process induced by DHA. Based on the finding that p53 inhibition or knockdown led to autophagy and prevention of p53 degradation decreased DHA-induced autophagy (), we therefore propose that p53 may regulate the onset of autophagy induced by DHA in tumor cells harboring wild-type p53. Although p53 is best known as a transcriptional factor that controls cell cycle and apoptosis, increasing evidence suggests that p53 inactivation also triggers autophagy in normal cells, transformed cells and cancer cells.13,14
Despite obtaining similar results in our experimental systems (SiHa, A549 and MCF-7 cancer cells), the effect of p53 on autophagy is not a general phenomenon, since p53 knockdown fails to enhance autophagy in skeletal muscle cells.14,24
One possible explanation for this is the different genetic background of the cell models. Likewise, considering that the DHA-induced autophagy is mediated by p53 and that some cancer cells are p53 deficient/mutant, it is reasonable to assume that the p53-mediated autophagy induced by DHA is dependent on cell type as well. Indeed, when we treated PC3 (p53 null) and DU145 (p53 mutant) prostate cancer cells23
with DHA, an increase in LC3-II was also detected (Fig. S3A
). In addition, PFTα pretreatment neither influenced the basal level of LC3-II nor the increased LC3-II level induced by DHA in both cell lines (Fig. S3B
). These results indicate that p53 loss-mediated autophagy induced by DHA is relatively important and not essential in some cell types and that besides the p53 pathway, DHA may also trigger autophagy through other mechanisms. However, although p53 is not required for autophagy induction, cancer cells harboring wild-type p53 may have an advantage to undergo autophagy via p53 inactivation after DHA treatment and in p53 deficient/mutant cells, DHA may still be able to induce autophagy by directly targeting downstream signaling events involved in p53-regulated autophagy, such as mTOR. The evidence supporting this hypothesis is that DHA, a highly polyunsaturated fatty acid susceptible to peroxidation, can accumulate ROS in cancer cells1,18
and ROS has been shown to induce autophagy by inhibiting mTOR directly.6,36
It is possible that DHA may induce autophagy through inhibiting mTOR directly by producing ROS in p53 deficient/mutant cells. Studies are currently underway to test this hypothesis.
The signaling by which p53 induces autophagy has been suggested to be a direct action of mTOR through AMPK activation.14,15,37
As a central signal integrator, mTOR receives signals arising from nutrients, growth factors and many cellular kinases including AMPK.26
Phosphorylation of AMPK activates downstream signaling that leads to mTOR inhibition and triggers autophagy,38
which is consistent with the AMPK function of initiating catabolic processes.11
Our data are in line with a recent finding by Tasdemir and colleagues14
that inactivation of p53 inhibits mTOR activity via AMPK activation. Additionally, we showed that treatment with the combination of p53 inhibitor and DHA had a stronger effect on the activation of AMPK/mTOR signaling than treatment with either drug alone, indicating that p53/AMPK/mTOR signaling is involved in DHA-induced autophagy. It is worth noting that, downstream of mTOR, p27 was assessed to indicate mTOR activity, since mTOR phosphorylation does not imply its activation.26
p27 inhibits cyclin-dependent kinase, resulting in cell cycle arrest.39
It has also been shown that p27 is sufficient to induce autophagy.40
Therefore, our observation of a marked increase in p27 in cells treated with DHA suggests that p27 may somewhat correlate with the autophagic activation governed by p53 loss. This is supported by the fact that p27 arrests cell cycle in the G1
and S phase41
and that p53 loss induces autophagy in a cell cycle-dependent fashion, with a preference for the G1
and S phases.42
Since both p27 and ATGs are located downstream of mTOR,26
it is possible that the autophagy and the cell cycle arrest induced by p53 attenuation in cancer cells treated with DHA occur in parallel via mTOR inactivation. Nevertheless, considering that only p27 was used to reflect mTOR activity, we could not exclude the possibility that p27 activation may also account for a substantial portion of autophagy induced by DHA.
Collectively, this study highlights the significance of autophagy in DHA-induced cancer cell death. DHA initially triggers a p53-mediated autophagic process via AMPK/mTOR signaling and the DHA-induced autophagy, alongside other signaling sensitizes tumor cells to apoptosis (). Our finding that DHA-induced autophagy contributes to cancer cell death may have important implications in developing future strategies to use DHA in cancer prevention and therapy.
Figure 6 Proposed mechanism of DHA-induced autophagy and apoptosis in human cancer cells harboring wild-type p53. DHA-induced autophagic activation is mediated by the p53/AMPK/mTOR pathway and contributes to apoptotic cell death. DHA causes the phosphorylation (more ...)