DIM induces autophagy in ovarian cancer cells
Autophagy is activated during stress conditions for degradation and recycling of macromolecules and organelles in the cell. We previously reported that DIM induces cellular stress leading to DNA damage in ovarian cancer cells [18
]. Hence, we wanted to determine whether or not DIM induces autophagy in ovarian cancer cells. The autophagy inducing effect of DIM was determined using acridine orange. Acridine orange is a lysomotropic agent that moves freely across biological membranes uncharged. Its protonated form accumulates in acidic compartments during autophagy, where it forms aggregates that fluoresces bright red [19
]. Treatment of SKOV-3, OVCAR-3 or TOV-21G cells with various concentrations of DIM for 24 hours resulted in a concentration dependent increase in the number of autophagic cells (Fig ). Our results showed that DIM-induced autophagy was nearly 3 to 6 fold in SKOV-3, 2 to 5 fold in OVCAR-3 and 2 to 4 fold in TOV-21G cells, when compared with their respective controls (Fig ). For example, 75μM DIM treatment for 24h induced autophagy in approximately 30% in SKOV-3 cells, whereas it was 25% and 15% in OVCAR-3 and TOV-21G cells, respectively (Fig ). Autophagy induction was further confirmed by electron microscopy. Electron microscopy figures clearly shows autophagosome formation as depicted by accumulation of double membrane vesicles in SKOV3 cells treated with DIM (Fig ).
DIM induces autophagy in ovarian cancer cells
Autophagy inducing effects of DIM were further confirmed by western blot analysis. SKOV-3, OVCAR-3 or TOV-21G cells were exposed to various concentrations of DIM for 24 hours. Our results reveal that DIM upregulates LC3B in a concentration dependent manner in all the cell lines tested (Fig ). Our quantitation results showed approximately 2 to 5 fold increase in the expression of LC3B by DIM treatment in different cell lines. DIM induced autophagy was accompanied by increase in the accumulation of Atg12 and p62 (Fig ). Autophagy marker p62 is a protein that is selectively incorporated into the autophagosome by directly binding to LC3B and hence aggregate during autophagy [21
]. On the other hand, Atg12 is instrumental in the autophagic vesicle biogenesis [3
]. DIM treatment failed to exert any effect on Beclin 1 or Atg5 in either of the cell lines tested.
DIM increases the expression of LC3B
DIM increases autophagic flux confirming autophagy induction
LC3B is the hallmark of autophagy, however its expression not always means induction of autophagy. Expression of LC3B may represent either the increased generation of autophagosomes or a block in autophagosomal maturation [22
]. For example, agents that impair lysosomal acidification such as chloroquine and bafilomycin A1 leads to accumulation of LC3B even under normal conditions because turnover of LC3B by basal autophagy is blocked. Hence, one cannot differentiate between induction of autophagy and impairment of autophagolysosomal maturation simply by measuring levels of LC3B. Hence, it is important to determine autophagic flux using LC3 turnover assay in presence and absence of chloroquine or bafilomycin A1. Ideally, when the cells are treated with lysosomotropic agents such as chloroquine or bafilomycin A1, the degradation of LC3B is blocked, resulting in the accumulation of LC3B [23
]. Under these conditions, the difference in LC3B levels in the presence and absence of chloroquine or bafilomycin A1 is more under autophagy induced conditions, indicating that autophagic flux is increased [24
Hence, to measure the autophagic flux induced by DIM, SKOV-3 or OVCAR-3 cells were exposed to chloroquine or bafilomycin for one hour followed by treatment with DIM for 24 hours. As expected, bafilomycin or chloroquine treatment alone increased the accumulation of LC3B indicating the blockade of LC3B degradation (Fig ). The expression of LC3B induced by combination of DIM with bafilomycin or chloroquine was higher compared to the expression of LC3B induced by DIM or lysosomal inhibitor treatments alone indicating that autophagic flux was increased in presence of DIM, yet again confirming that autophagy is induced in our model.
Bafilomycin or chloroquine block DIM induced autophagy in ovarian cancer cells
Furthermore, acridine orange assay in presence of chloroquine or bafilomycin was performed to ensure the inhibition of autophagosomal maturation. Since, chloroquine or bafilomycin inhibits lysosomal acidification, acridine orange staining induced by DIM was drastically reduced in presence of these inhibitors (Fig ). Our results established that bafilomycin or chloroquine treatment blocked DIM induced acridine orange staining in SKOV-3 and OVCAR-3 cells.
DIM induces ER stress in ovarian cancer cells
Our next step was to investigate the molecular mechanism behind DIM induced autophagy. Recent literature suggests that disturbances in the integrity of ER activate autophagy [6
]. Hence, we questioned ourselves whether DIM causes ER stress in ovarian cancer. To address this issue, SKOV-3, OVCAR-3 or TOV-21G cells treated with varying concentrations of DIM were subjected to western blotting. Our results clearly demonstrate that DIM substantially induces the expression of Grp78 in a concentration dependent manner in all the three cancer cell lines (Fig ). Accumulation of Grp78 is an indicator of ER stress. Our results further show that DIM induced ER stress was accompanied by the accumulation of GADD 153 and IRE1 in SKOV-3, OVCAR-3 and TOV-21G cell lines (Fig ). Both GADD 153 and IRE1 are regulated by Grp78 during ER stress. These observations indicate that DIM induces ER stress in ovarian cancer cells.
DIM induces ER stress in ovarian cancer cells: A) Representative blots showing the concentration dependent effect of DIM on Grp78, IRE1 and GADD153 in i) SKOV-3, ii) OVCAR-3 and iii) TOV-21G cells
Cycloheximide, a protein synthesis inhibitor or Mithramycin, an ER stress inhibitor block DIM mediated ER stress
Because we observed significantly enhanced protein expression of Grp78, GADD 153 and IRE1 by DIM treatment leading to unfolded protein response (UPR), we wanted to determine whether blocking protein induction would inhibit expression of Grp78, GADD 153 or IRE1. To do that, we used cycloheximide (CHX), a protein synthesis inhibitor. SKOV-3 or OVCAR-3 cells were treated with 10μg/ml CHX for 2 hours followed by treatment with 75μM DIM for 24 hours. Our data reveals that the drastic increase in the expression of Grp78, GADD 153 and IRE1 induced by DIM was attenuated in presence of CHX treatment in SKOV-3 and OVCAR-3 cells (Fig ).
CHX is a general protein synthesis inhibitor. To confirm that ER stress is induced in our model, we used mithramycin, a specific ER stress inhibitor. Mithramycin is a gene selective sp1 inhibitor that blocks transcription and protein synthesis of ER chaperones [25
As shown in Figure , mithramycin treatment blocked the induction of Grp 78, IRE1α and GADD 153. These results strongly support that DIM activates ER stress in ovarian cancer cells.
Blocking ER stress inhibits DIM induced autophagy
Literature suggests that disturbances in ER homeostasis lead to autophagy. Since we observed that ovarian cancer cells exposed to DIM undergo both ER stress and autophagy, we hypothesized that DIM activates autophagy in ovarian cancer cells by inducing ER stress. To establish that ER stress induced by DIM leads to formation of autophagic vesicles, we exposed ovarian cancer cells to mithramycin or CHX before treating cells with DIM. Our results show that mithramycin or CHX treatment not only blocked the enormous increase in the expression of LC3B associated with DIM, but also abrogated the significant upregulation of p62 and Atg12 (Fig ). These results support our hypothesis that ER stress was involved in the regulation of DIM mediated autophagy in ovarian cancer cells.
ER stress regulates DIM induced autophagy
Silencing of Grp78, GADD 153 or LC3B inhibits DIM induced autophagy
Both Mithramycin and CHX treatment blocked induction of several molecules that regulate ER stress and autophagy. However, chemical inhibitors are known to be associated with off-target effects. Therefore, to firmly establish the role of ER stress in DIM mediated autophagy, we sought to determine the effects of DIM in SKOV-3 cells in which Grp78 and GADD 153 were transiently silenced using respective siRNA’s. SKOV-3 ovarian cancer cells were transfected with specific siRNA’s followed by treatment with DIM. Fig shows that silencing Grp78 not only abrogated the induction of its downstream molecules IRE1 and GADD 153, but also blocked the DIM induced LC3B, p62 and Atg12. These results indicate that induction of ER stress not only regulates UPR as indicated by expression of IRE1 and GADD 153, but also controls autophagy as evident by the expression of LC3B, p62 and Atg12.
Because silencing Grp78 blocked DIM induced expression of GADD 153, we wanted to evaluate the effect of DIM on SKOV-3 cells after silencing GADD 153. Similar to our Grp78 data, silencing GADD 153 in ovarian cancer cells blocked DIM induced up regulation of LC3B, p62 and Atg12 (Fig ). These results confirmed that DIM-induced autophagy was mediated by the activation of ER stress in ovarian cancer cells.
To gain further insight into this mechanism, we silenced LC3B using its specific siRNA. Fig shows that silencing LC3B not only blocked DIM induced expression of LC3B, but also p62 and Atg12 which were associated with autophagy. These results indicate that LC3B was required for the induction of p62 and hence autophagy.
DIM regulates autophagy by activation of AMPK
Several recent studies showed that activation of AMPK is important in regulating autophagy. We wanted to know whether this was the case in our model. Our western blotting showed that DIM activated AMPK by phosphorylating it at Thr-172 in SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells (Fig ). To confirm the role of AMPK in DIM induced autophagy, we exposed the cells to compound C, a chemical inhibitor of AMPK before treating with DIM. Our results from Fig shows that treating SKOV-3 or OVCAR-3 cells to compound C inhibited DIM-induced activation of AMPK (Fig6B i-ii). Interestingly, blocking AMPK activation also blocked the expression of LC3B, p62 and Atg 12 in SKOV-3 and OVCAR-3 cells, indicating that AMPK also mediates the effect of DIM on autophagy (Fig ).
AMPK activation by DIM is necessary for DIM induced autophagy
To further confirm the role of AMPK, we silenced AMPK by using specific siRNA against AMPK. Genetically silencing AMPK not only inhibited the activation of AMPK by DIM by blocking its phosphorylation at Thr 172, but also abrogated DIM induced expression of LC3B and p62 (Fig ). Taken together, these results strongly establish that AMPK is a major regulator of DIM induced autophagy in ovarian cancer cells.
DIM elevates cytosolic calcium to regulate AMPK and autophagy
We wanted to gain further insight into how DIM caused activation of AMPK. Many studies suggested that increase in the cytosolic calcium leads to activation of AMPK. It is well known that endoplasmic reticulum is the store house of calcium. Perturbation in the ER results in the release of calcium into cytosol thereby causing deregulation of calcium homeostasis leading to activation of AMPK and autophagy. To test whether DIM treatment leads to the release of calcium in ovarian cancer cells, we loaded cells with Fluo-3AM and continuously monitored calcium levels using Accuri C6 flow cytometer. Baseline reading was taken for a minute, followed by exposure of cells to DIM. Figure clearly demonstrates an increase in the intensity of the baseline after DIM treatment suggesting the release of calcium in different ovarian cancer cells.
Increase in the cytosolic calcium plays a crucial role in DIM induced autophagy
To test whether increase in the cytosolic calcium leads to activation of AMPK, we treated SKOV-3 or OVCAR-3 cells with BAPT-AM, a calcium chelater followed by exposure to DIM for 4 hours. We observed that BAPT-AM significantly blocked the activation of AMPK (Fig ). Furthermore, the expression of LC3B or Atg12 induced by DIM was also significantly suppressed by BAPT-AM treatment (Fig ). Taken together, these results suggest that increase in the cytosolic calcium regulates autophagy in DIM treated ovarian cancer cells.
Tumor growth suppression by DIM is associated with ER stress and autophagy
We previously demonstrated that DIM inhibits the growth of various ovarian cancer cells in vitro
]. Whether or not, DIM suppresses the growth of ovarian tumors in vivo was not clear. Hence, we used ovarian tumor xenograft model to determine the growth suppressive effects of DIM in vivo. We injected 5 x 106
SKOV-3 cells subcutaneously on both right and left flanks of female athymic nude mice. Mice were randomized into two groups of 10 mice each. Treated group received 2mg/day DIM by oral gavage, whereas, control group received PBS. Tumor volume was recorded thrice a week using vernier calipers and weight of mice was recorded twice a week. Our results show that DIM significantly suppressed the growth of ovarian tumors. For example, at day 42, average tumor volume in the group of mice that received DIM was approximately 100 mm3
which was remarkably lesser as compared to the average tumor volume of 250 mm3
in control group (Fig ). Interestingly, weight of mice from both groups did not differ significantly suggesting that DIM was not toxic to the mice (Fig ). To test whether DIM mediated tumor growth suppression was associated with ER stress and autophagy, tumors from control and DIM treated mice were lysed and subjected to western blotting. As expected, our results show that DIM activates ER stress and autophagy in vivo. We observed an increase in several ER stress markers such as Grp78, GADD 153 and IRE1 in the tumors of DIM treated mice as compared to controls (Fig ). Moreover, we observed an increase in the phosphorylation of AMPK and expression of other autophagy markers such as LC3B and p62 in DIM treated tumors (Fig ). These results suggest that DIM suppressed the growth of ovarian tumors in vivo by inducing ER stress mediated autophagy.
DIM suppresses the growth of ovarian tumors in nude mice by inducing ER stress modulated autophagy
Our study for the first time demonstrates that DIM treatment induces autophagy in ovarian tumor cells in vitro
and in vivo
by enhancing ER stress and activating AMPK. Autophagy is a catabolic process during which damaged organelles and proteins are engulfed and degraded to provide metabolic needs. Autophagy is activated in response to various kinds of stress. Our published studies showed that DIM induces cellular stress in ovarian cancer cells [18
]. Our current results suggest that DIM induces autophagy in SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells in a concentration dependent manner as analyzed by flowcytometry and electron microscopy.
LC3B is accumulated and localized to vesicular structures during autophagy and hence it is considered as a hallmark of autophagy. Activation of autophagy by DIM in our model was confirmed by enhanced expression of LC3B. Our results also showed that autophagy induction was associated with an increase in the expression of p62 and Atg12. Autophagy marker p62 is a protein that is selectively incorporated into autophagosome by directly binding to LC3B and hence aggregate during autophagy. Atg12 is instrumental in the autophagic vesicle biogenesis [3
]. Our studies are in agreement with previous studies, which show that agents such as resveratrol induce autophagy by increasing the expression of LC3B [21
]. Beclin 1 and Atg5 are genes that are responsible for initiation of autophagy. However, to our surprise, Beclin1 or Atg5 expression by DIM treatment was not significantly altered. It is interesting to note that several studies have reported that induction of autophagy can be independent of Beclin 1 or Atg5 [26
DIM treatment in presence of bafilomycin or chloroquine lead to increased expression of LC3B when compared to the cells treated either with DIM or inhibitors alone, suggesting increased autophagic flux. Both bafilomycin and chloroquine inhibit the fusion between autophagosomes and lysosomes, thus prevent the execution step of autophagy. Nonetheless, our results from flowcytometry demonstrated that chloroquine or bafilomycin completely blocked DIM-induced autophagy. Induction of autophagy in ovarian cancer cells was confirmed by genetic silencing of LC3B. Knocking out LC3B inhibited DIM induced expression of LC3B as well as Atg12 and p62. Our observations are in agreement with several studies demonstrating the role of LC3B and p62 in autophagy [21
Our study established that autophagy induced by DIM treatment was mediated by activation of ER stress or AMPK activation. Activation of ER stress in our model was associated with an increase in the expression of Grp78, IRE1 and GADD153. Grp78 is a chaperone that folds protein into its proper confirmation. Grp78 acts as a sensor in ER and it binds to IRE1 under normal conditions [9
]. However, during ER stress, it releases IRE1, which further leads to expression of a transcriptional factor GADD 153 that continues the response of unfolded proteins [5
]. Both IRE1 and GADD 153 were significantly up regulated by DIM treatment in all the three ovarian cancer cells. Genetically silencing Grp78 attenuated DIM induced expression of IRE1 and GADD 153, suggesting the importance of Grp78 in regulating DIM induced ER stress.
Furthermore, role of ER stress by DIM induced autophagy was established in our model. Pharmacologically inhibiting ER stress using chemical inhibitors such as cycloheximide or mithramycin not only blocked the induction of Grp78, IRE1 or GADD 153, but also inhibited DIM induced expression of Atg12, LC3B and p62 in SKOV-3 and OVCAR-3 ovarian cancer cells. ER stress is connected to autophagy in our model by IRE-1 and GADD153. IRE1 activates transcriptional factors such as Hac1 which is capable of inducing several Atg genes leading to the induction of autophagy [5
]. Accordingly, our results showed that blocking the expression of IRE1 inhibited DIM induced autophagy. During ER stress, Grp78 releases IRE1 leading to the expression of GADD 153. Several studies have demonstrated the involvement of GADD 153 in autophagy [30
]. Knocking out Grp78 or GADD 153 blocked the expression of LC3B, Atg12 and p62 induced by DIM. Interestingly knock down of Grp78 also abrogated DIM induced expression of IRE1 and GADD 153, confirming that IRE-1 and GADD 153 are major players of ER stress and regulate autophagy in our model. Similar to our observations, ER stress induced autophagy was observed by capsaicin and atorvastatin in other malignancies [31
ER stress not only activates UPR, but also leads to the release of calcium from ER into cytosol. Our studies demonstrated that DIM treatment elevates cytosolic calcium levels in ovarian cancer cells. Cytosolic calcium levels were reduced in the presence of EGTA, a calcium chelater. Release of calcium into cytosol leads to the activation of various kinases including AMPK which is known to regulate autophagy. AMPK is also an energy sensor and is activated when there is increase in AMP/ATP ratio, which is usually the scenario during cellular stress, the same reason for which autophagy is activated. In agreement with these facts, DIM treatment activated AMPK in all the three ovarian cancer cell lines. BAPT-AM, a calcium chelater not only suppressed DIM induced phosphorylation of AMPK but also attenuated the expression of LC3B and Atg12. Role of AMPK in DIM induced autophagy was established by silencing AMPK. These studies showed that absence of AMPK inhibits the expression of p62 or LC3B, the classic markers of autophagy, hence confirming that AMPK regulates autophagy in our model. All these observations are in agreement with several studies showing that activation of AMPK leads to autophagy [16
Oral administration of 2mg DIM everyday substantially suppressed the growth of ovarian tumors. The tumors from DIM treated mice clearly demonstrated the significant induction of ER stress and autophagy similar to our in vitro
observations. Interestingly, mice that received DIM did not show any significant change in the body weight when compared with the weight of control mice. DIM is an indole compound present in cruciferous vegetables [33
]. Recent clinical trials suggested that DIM is well tolerated in humans [34
]. Interestingly, administration of 2mg/kg/day DIM through oral route suppressed Cervical Intraepithelial Neoplasia with 1-2 grades [34
]. Several pharmacokinetic studies on DIM stated that up to 300mg single dose of DIM can be well tolerated in humans [35
]. Our dose of DIM falls within the accepted and tolerated dose in humans. Nevertheless, further clinical studies are needed to show that DIM can reduce the ovarian tumor growth in humans.
In conclusion, DIM promotes autophagy in ovarian cancer through activation of ER stress and AMPK. To our knowledge, this is the first report showing the regulation of autophagy by DIM in ovarian cancer cells.