δ‐Tocotrienol induces apoptosis, involving endoplasmic reticulum stress and autophagy, and paraptosis in prostate cancer cells

Abstract Objectives Prostate cancer, after the phase of androgen dependence, may progress to the castration‐resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E‐derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti‐cancer properties of δ‐TT in CRPC cells (PC3 and DU145) and the related mechanisms of action. Materials and methods MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy. Results We demonstrated that δ‐TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ‐TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ‐TT; in autophagy‐defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ‐TT; (c) in both CRPC cell lines, δ‐TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ‐TT. Conclusions These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti‐cancer activity of δ‐TT in CRPC cells.


| INTRODUC TI ON
Prostate cancer (PCa) represents the third cause of cancer-related deaths among men in the Western countries. 1 Androgen-deprivation therapy (GnRH agonists, either alone or in combination with an antiandrogen) represents the treatment of choice for this pathology. 2,3 However, the tumour frequently progresses towards a condition of castration resistance (castration-resistant prostate cancer, CRPC) for which the therapeutic options are limited. 5,6 Several natural compounds were reported to possess anti-cancer properties 7 by inducing apoptosis, through modulation of intracellular pathways including endoplasmic reticulum (ER) stress and autophagy. 8,9 Cells react to severe stress conditions by accumulating misfolded proteins in the ER, leading to dissociation from the chaperone BiP (immunoglobulin heavy-chain-binding protein, GRP78) and activation of three stress sensors: PERK (double-stranded RNA-dependent protein kinase PKR-like ER kinase), ATF6 (activating transcription factor 6) and IRE1α (inositol-requiring enzyme 1α). Each of these proteins couples to a specific intracellular pathway converging to apoptosis. 14,15 The transcription factor CHOP (C/EBP homologous protein, also known as GADD153) plays a key role in the ER stress-related apoptosis pathway.
Autophagy consists of the degradation/recycling of damaged cytoplasmic proteins and organelles that are sequestered in autophagosomes and then degraded in autophagolysosomes. Several markers of the autophagic phases, such as LC3-II (the phosphatidylethanolamine-conjugated form of LC3 microtubule-associated protein 1AB-light chain), and sequestosome 1/p62 (SQSTM1/ p62) 16 were identified. Autophagy is involved both in suppression of cancer growth and in development of drug resistance in cancer cells. 17 There is now evidence connecting the ER stress-autophagy axis with apoptosis, representing a molecular target of cancer treatments. 8 Non-canonical cell death mechanisms are also involved in natural compounds anti-cancer activity. 18,19 Paraptosis is a programmed cell death characterized by cytoplasmic vacuolation resulting from mitochondrial and/or ER swelling and requiring protein synthesis. 21 Vitamin E is composed of two groups of compounds, tocopherols (TPs) and tocotrienols (TTs); each group consists of four isomers: α, β, γ and δ. TTs (but not TPs) have attracted interest for their healthpromoting properties, such as anti-cancer activity. δ-and γ-TT were reported to exert anti-cancer activities through the modulation of different intracellular pathways. 22,23 The bioavailability/safety of these compounds was demonstrated in healthy subjects 25,26 and in cancer patients. 27 Here, we dissected the antitumour effects of δ-TT in CRPC cells by exploring the involvement of the ER stress-autophagy axis in this activity; we also investigated the possible involvement of non-canonical programmed cell deaths (ie paraptosis) in its effects. Z-VAD-FMK (the pan-caspase inhibitor; FMK001) was from R&D System Inc (Minneapolis, MN). The ER stress inhibitors salubrinal (S) and 4-PBA (4-phenylbutyrate), the autophagy inhibitors CQ (chloroquine) and Baf (bafilomycin), the translation inhibitor cycloheximide, and analytical grade solvents were from Sigma-Aldrich; 3-MA (3-methyladenine) was from Selleckchem (Munich, Germany).

| Cell lines and cell culture
Normal prostate epithelial RWPE-1 (provided by Dr N. Zaffaroni; IRCCS, National Institute of Cancer, Milano, Italy) and cancer (DU145 and PC3) cell lines were from American Type Culture Collection (ATCC, Manassas, VA, USA). RWPE-1 cells were cultured in keratinocyte-SFM medium supplemented with Bovine Pituitary Extracts and EGF (2.5 μM) (Thermo Fisher Scientific), DU145 and PC3 cells in RPMI medium supplemented with FBS (7.5% and 5% respectively), F I G U R E 1 δ-TT decreases cell viability and exerts a cytotoxic effect on DU145 and PC3 prostate cancer cells. A, RWPE-1 normal epithelial prostate cells and DU145 and PC3 prostate cancer cells were treated with δ-TT (5-20 μg/mL) for 24 h. Cell viability was then evaluated by MTT assay. The IC 50 values were 2.91 × 10 −5 M and 3.22 × 10 −5 M for DU145 and PC3 cells, respectively. B, Prostate cancer cells were treated with δ-TT (5-20 μg/mL) for 24 h. Total, live and dead cells were evaluated by Trypan blue exclusion assay. C, DU145 and PC3 cancer cells were treated with δ-TT (15 μg/mL) for 36-72 h. Cell viability was then evaluated by MTT assay. D, Prostate cancer cells were treated with δ-TT (15 μg/mL) for 48 h and then, after withdrawal of the treatment, were left to grow for 11-12 d, dependently on the cell line-specific proliferation rate. A colony formation assay was performed to evaluate the ability of the cells to form proliferating colonies (dimensions of colonies) and the survival of colony-forming cells (number of colonies). Each experiment was repeated three times. Data in (A-C) represent mean values ± SEM and were analysed by Dunnet's test after one-way analysis of variance. * P < 0.05 vs 0, controls (vehicle) F I G U R E 2 δ-TT triggers apoptosis in DU145 and PC3 prostate cancer cells. A, DU145 and PC3 cells were treated with δ-TT (5-20 μg/ mL) for 24 h (left panels) or with δ-TT (15 μg/mL) for 6-24 h (right panels). Western blot analysis was carried out to analyse the expression levels of cleaved caspase 3 (ie active) and PARP. Tubulin expression was evaluated as a loading control. 0 and C, controls (vehicle). One representative of three different experiments, for each of the analyses performed, is shown. B, DU145 and PC3 cells were treated with δ-TT (15 μg/mL) for 18 h; the intracellular localization of cytochrome c was then evaluated by immunofluorescence analysis. One representative of three experiments performed is shown. Scale bars are 20 μm. The arrow indicates the cytochrome c-mitochondrial colocalization in controls cells. C, To confirm the involvement of apoptosis in the antitumour activity of δ-TT, DU145 and PC3 cells were treated with the pan-caspase inhibitor Z-VAD-FMK (50 μM) for 4 h before the tocotrienol (15 μg/mL for 24 h). Cell viability was then evaluated by MTT assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analysed by Bonferroni's test after one-way analysis of variance. * P < 0.05 vs controls (vehicle). ♦ P < 0.05 vs δ-TT-treated cells glutamine and antibiotics. Cells were cultured in humidified atmosphere of 5% CO 2 /95% air at 37°C.

| Colony formation assay
Cells were seeded (100-250 cells/well, depending on the cell type) in 6-well plates. After each treatment, a colony formation assay was performed to assess dimensions and numbers of colonies.
Colonies were fixed with 70% methanol and stained with Crystal Violet 0.15%. Images of stained colonies were captured by a Nikon photo camera.

| Western blot assay
Cells were seeded at 5 × 10 5 cells/dish in 10-cm dishes. After each treatment, cells were lysed in RIPA buffer; protein preparations (15-40 μg) were resolved on SDS-PAGE and transferred to nitrocellulose (or PVDF for the Western blot of LC3) membranes.
Membranes were incubated with the specific primary antibodies.
After each treatment, cells were fixed and stained with the specific

| Statistical analysis
Statistical analysis was performed with a statistic package (GraphPad Prism5, GraphPad Software San Diego, CA, USA). Data are represented as the mean ± SEM of three-four independent experiments. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Dunnet's or Bonferroni's test. A P value <0.05 was considered statistically significant.  Figure 1D).  respectively. Figure 3C confirms that δ-TT induces the expression of CHOP and ATF4 (as in Figure 3A); pre-treatment with both ER stress inhibitors significantly reduced the expression of both proteins. These results support that δ-TT triggers ER stress in CRPC cells.

| ER stress mediates the antitumour activity of δ-TT in prostate cancer cells
Data from the literature support that ER stress is involved in the antitumour activity of δ-TT in cancer cells. 29,30 To confirm this hypothesis in prostate cancer cells, DU145 and PC3 cells were treated with two ER

| δ-TT triggers autophagy in PC3 prostate cancer cells
To assess whether δ-TT might trigger the autophagy pathway in  Figure 5B). These results are in agreement with data reporting that DU145 cells are autophagydefective due to an alternative splicing of ATG5 transcript and lack of a full-length ATG5 protein. 31 Thus, further studies investigating the involvement of autophagy in δ-TT anti-cancer activity were performed in PC3 cells.

| δ-TT triggers ER stress-related autophagy in PC3 prostate cancer cells
To confirm the activation of an autophagic flux in PC3 cells, we investigated the presence of autophagosomes in δ-TT-treated (15 μg/ mL, 18 hours) cells by TEM. Figure 6A shows

hours). We showed that both ER stress inhibitors counteracted
the tocotrienol-triggered increase of the LC3-II/LC3-I ratio as well as that of SQSTM1/p62 expression ( Figure 6D,E).
In conclusion, in PC3 cells (but not in autophagy-defective DU145 cells), δ-TT-induced autophagy is related to the upstream activation of the ER stress pathways (ER stress-autophagy axis).

| Autophagy mediates the antitumour activity of δ-TT in PC3 prostate cancer cells
To assess whether autophagy might mediate the proapoptotic activity of δ-TT in PC3 cells, cells were pretreated with 3-MA  Figure 6F shows that δ-TT increased the expression of cleaved caspase 3 and PARP, as previously observed. 3-MA, given alone, did not affect the levels of these proteins; on the other hand, 3-MA significantly counteracted the effects of δ-TT on the expression levels of cleaved caspase 3 and PARP ( Figure 6F).
As expected, cell viability was significantly suppressed by δ-TT, while it was not affected by 3-MA; however, the autophagy inhibitor significantly reverted (although not completely) the cytotoxic effect of δ-TT ( Figure 6G).

| δ-TT triggers paraptosis in prostate cancer cells
Data reported above suggest that δ-TT may exert its activity by triggering non-canonical pro-death mechanisms in addition to apoptosis (see Figure 2C). Paraptosis represents an alternative cell death mechanism characterized by extensive vacuolation related to ER stress/mitochondria swelling. 18,32,33 Paraptosis was also reported to be dependent on protein synthesis. 34,35 We found that δ-TT ( These data support that paraptosis is involved in the anti-cancer activity of δ-TT in prostate cancer cells.

| D ISCUSS I ON
It is accepted that TTs are associated with significant anti-cancer properties. 24,36 Although most of the studies so far reported were performed with γ-TT, δ-TT was shown to be the most effective vitamin E isoform in triggering cell death in prostate cancer cells 37,38 ; however, the molecular mechanisms/targets of this activity are poorly clarified.
Here, we dissected the molecular mechanisms underlying the antitumour/proapoptotic activity of δ-TT in CRPC cells (DU145 and PC3); the possible involvement of paraptosis in its activity was also investigated.
We confirmed that δ-TT exerts a significant antitumour/cyto- results were reported for γ-TT. 24,41,42 In line with these data, the anti-cancer activity of TTs (specifically γ-and δ-TT) was reported in a wide range of tumours. 24,45,46 To get further insights into the mechanisms and targets of the δ-TT antitumour activity in CRPC cells, we concentrated our studies on the ER stress and autophagy pathways. We observed that, in both DU145 and PC3 cells, δ-TT induces the expression of BiP, p-eIF2α and IRE1α. δ-TT also induced the expression/activation of the transcription factors ATF4 and CHOP (pointing out their cytoplasmic-to-nuclear localization). It is known that the p-eIF2α/ATF4 pathway activates CHOP, a transcription factor that is also activated by IRE1α. 14,51 These results demonstrate that, in CRPC cells, δ-TT triggers the main ER stress branches, leading to the activation of CHOP, deeply involved in the ER stress-related apoptosis. To confirm the involvement of the ER stress pathway in the activity of δ-TT, These data demonstrate that, in CRPC cells possessing an efficient autophagy pathway, δ-TT induces apoptosis by triggering the ER stress-related pro-death autophagy pathway. On the other hand, only the ER stress pathway is involved in the activity of δ-TT in autophagy-defective cells.
To our knowledge, this is the first report describing the involvement of the ER stress-autophagy in the anti-cancer activity of δ-TT in prostate cancer cells. γ-TT was shown to concurrently trigger ER stress and autophagy in inducing apoptosis in breast cancer cells. 52,53 TTs (specifically γ-and δ-TT) were shown to induce apoptosis by triggering the ER stress branches in cervical cancer, 30 breast cancer 54 and melanoma cells. 29 In line with these observations, both the ER stress and the autophagy pathways were reported to mediate the anti-cancer activity of several natural compounds. 55,56 Here, we also observed that abrogation of apoptosis by the pancaspase inhibitor Z-VAD-FMK significantly, but not completely, reverted the cytotoxic effect of δ-TT on CRPC cells. Thus, an additional programmed cell death modality might be involved in the activity of the tocotrienol. Paraptosis, necroptosis, mitotic catastrophe, anoikis were reported to be typical of apoptosis-resistant tumour cells and to mediate the cytotoxic effects of anti-cancer compounds. 18,20,33 This makes these types of cell death a promising target for novel therapeutic strategies. 33 Among them, paraptosis is characterized by: intense cytoplasmic vacuolation, correlated with ER stress and mitochondrial swelling/dilatation; de novo protein synthesis; involvement of JNK and p38 kinases. 18,21,33 We observed that δ-TT induces morphological changes, with an intense cytoplasmic vacuolation in both CRPC cells. In δ-TT-treated cells, we pointed out: by TEM, a significant swelling of mitochondria and dilatation of the ER cisternae; by light microscopy, a cytoplasmic vacuolation that was markedly inhibited in the presence of salubrinal or cycloheximide; by Western blot, increased expression of the active forms of JNK and p38. These data support that, in addition to apoptosis, the non-canonical cell death paraptosis is involved in the antitumour activity of δ-TT in CRPC cells.
So far, the role of paraptosis-like cell death in the cytotoxic activity of δ-TT has been reported only in colon carcinoma cells. 62,63 On the other hand, different natural compounds (such as taxol) were shown to trigger this cell death in different cancer cells. 18,64,65 These data demonstrate that, in CRPC cells, δ-TT exerts an anticancer activity by triggering both apoptosis, involving the ER stressautophagy axis, and paraptosis, providing novel mechanistic insights into this activity.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest related to this article.