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Methiothepin mesylate causes apoptosis of human prostate cancer cells by mediating oxidative stress and mitochondrial dysfunction
Free Radical Biology and Medicine 150 (2020) 12–22
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Original article
Methiothepin mesylate causes apoptosis of human prostate cancer cells by mediating oxidative stress and mitochondrial dysfunction
T
Changwon Yanga, Gwonhwa Songa,∗, Whasun Limb,∗∗ a
Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea b Department of Food and Nutrition, Kookmin University, Seoul, 02707, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Methiothepin mesylate Prostate cancer Mitochondria Apoptosis Oxidative stress
Prostate cancer is difficult to treat if it metastasizes to other organs. The development of prostate cancer independent of androgen is closely related to the action of neuroendocrine products. Serotonin promotes cell growth in various cancers, and antagonists for serotonin receptors are known to inhibit proliferation and induce cell death in various carcinomas. However, little is known about how antagonists for serotonin receptor function in prostate cancer. We verified apoptotic cell death in prostate cancer cell lines after treatment with methiothepin mesylate (MET), an antagonist for serotonin receptor 5-HT1. MET induced hydrogen peroxide (H2O2) production and mitochondrial Ca2+ overload. Moreover, MET induced changes in the expression of proteins associated with endoplasmic reticulum stress, autophagy, and mitochondrial membrane potential. MET also promoted phosphorylation of JNK, which induced cell death mediated by oxidant production, as evidenced by the JNK inhibitor and oxidant scavenger. Finally, MET has the potential to prevent metastasis by inhibiting the migration of prostate cancer cells. Thus, we show that MET is a potentially novel anticancer agent that can suppress the development of prostate cancer caused by neuroendocrine differentiation.
1. Introduction Prostate cancer is the second most common carcinoma among men worldwide [1]. Prostate cancer cells can metastasize to various organs, such as the lymph nodes, liver, and bones, which makes surgical treatment difficult [2]. Androgen ablation is the first-line therapy, but long-term application leads to androgen-independent tumor cell growth [3]. The growth of androgen-independent prostate cancer is associated with abnormal signaling [4,5]. Natural products derived from plants and compounds that regulate transcriptional activity in the nucleus of cells have been suggested as potential adjuvants to treat androgen-independent prostate cancer [6,7]. For example, the herb extract DT-13 reduces cell migration by inhibiting phosphoinositide 3-kinase (PI3K)/ AKT cascades and matrix metalloproteinase (MMP) 2 and MMP9 in PC3 and DU145 cell lines [8]. In addition, sodium butyrate, a histone deacetylase (HDAC) inhibitor, induces apoptosis by increasing mitochondrial damage and nuclear fragmentation following phosphorylation of mitogen-activated protein kinase (MAPK) [9]. However, there is a lack of information on the therapeutic effects of the regulation of neurosecretory products on prostate cancer, although neuroendocrine
∗
differentiated tumor cells are found in the majority of prostatic carcinomas [10]. Serotonin (5-hydroxytryptamine) is known to function in the central nervous system (CNS) as a neurotransmitter but is now thought to elicit physiological changes throughout the body, such as in the enteric nervous system or vascular system [11,12]. Research on the mechanisms of action of serotonin agonists and antagonists is actively pursued in various cell types. The growth-stimulating effects of serotonin have been reported in various cell types, including pancreatic carcinoid cells and small cell lung carcinoma [13,14]. The effect of serotonin on cancer proliferation is based on the activation of MAPK and PI3K/AKT signaling [15]. Moreover, serotonin is a marker of neuroendocrine differentiation, and inhibitors of serotonin uptake are considered to be a potential treatment for prostate cancer [16]. A variety of chemicals, including serotonergic compounds, serotonin transporter inhibitors, and serotonin receptor agonists, affect the proliferation of prostate cancer cell line, but their physiological mechanism in cells is unclear [17]. Methiothepin mesylate (MET) is a non-selective 5-HT1 receptor antagonist with little known physiological effect in human cells. In broilers, MET was reported to mitigate the pulmonary hypertensive
Corresponding author. Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea. Corresponding author. Department of Food and Nutrition, College of Science and Technology, Kookmin University, Seoul, 02707, Republic of Korea. E-mail addresses: [email protected] (G. Song), [email protected] (W. Lim).
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https://doi.org/10.1016/j.freeradbiomed.2020.01.187 Received 3 December 2019; Received in revised form 30 January 2020; Accepted 31 January 2020 Available online 05 February 2020 0891-5849/ © 2020 Elsevier Inc. All rights reserved.
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Table 1 Primary antibodies used in western blot. Primary antibodies 286
Phosphor-Cyclin D1 (Thr ) Cyclin D1 Beclin-1 Atg5 Phosphor-P62 P62 Phosphor-ULK1 ULK1 TUBA GRP78 IRE1α Phosphor-PERK (Thr981) PERK Phosphor-eIF2α eIF2α Cytochrome c IP3R1 IP3R2 MFN2 VAPB GRP75 PTPIP51 Phosphor-AKT (Ser473) AKT Phosphor-S6 (Ser235/Ser236) S6 Phosphor-JNK (Thr183/Tyr185) JNK Phosphor-c-Jun (Ser73) c-Jun Phosphor-ERK1/2 (Thr202/Tyr204) ERK1/2 Phosphor-P38 (Thr180/Tyr182) P38
Dilution
Supplier
1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:2000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000
Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Santa Cruz Santa Cruz Cell Signaling Santa Cruz Santa Cruz Cell Signaling Cell Signaling Cell Signaling Invitrogen Santa Cruz Cell Signaling Invitrogen Cell Signaling Abcam Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling
Catalog Number Technology Technology Technology Technology Technology Technology Technology Technology
Technology
Technology Technology Technology
Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology
3300 2922 3495 12994 16177 88588 5869 8054 sc-5286 sc-13968 3294 sc-32577 sc-13073 3398 5324 11940 PA1-901 sc-398434 11925 PA5-53023 3593 ab182105 4060 9272 2211 2217 4668 9252 3270 9165 9101 4695 4511 9212
2.4. Spheroid formation
response, but there is little information on its effectiveness in human cancer cells [18]. We examined the effects of MET on cell proliferation and apoptosis after treatment of androgen-independent PC3 and DU145 cells. Moreover, we estimated whether MET can cause oxidative stress and mitochondrial dysfunction in PC3 and DU145 cells. In addition, we examined whether MET is involved in the migration of prostate cancer cells via the regulation of PI3K/AKT and MAPK signaling.
For the hanging drop method, 3000 cells were seeded onto the cover of the culture dish and was treated with 20 μM of MET for 10 days. Changes in spheroid morphology with MET treatment were observed with a DM3000 microscope (Leica). Spheroid area was measured using ImageJ software (http://rsb.info.nih.gov/ij/docs/index.html). 3D plot quantification was performed by using ReViSP software (https:// sourceforge.net/projects/revisp/).
2. Materials and methods 2.5. Immunofluorescence microscopy 2.1. Chemicals To clarify whether MET can regulate the expression of proliferating cell nuclear antigen (PCNA), immunofluorescence method was used as previously described [19]. PC3 and DU145 cells were treated with 20 μM of MET for 24 h. MetaMorph software (Molecular Devices) was used to quantify Alexa 488 green fluorescence.
MET and N-acetyl-L-cysteine (NAC) were purchased from SigmaAldrich, Inc. SP600125 was purchased from Enzo Life Science. Ruthenium red was purchased from Abcam. 2.2. Cell culture
2.6. Annexin V and propidium iodide staining PC3 and DU145 cells were purchased from the American Type Culture Collection. RPMI-1640 medium was used to culture the cells. For the experiments, cells were cultured until about 70% of the plate was filled and then starved for 24 h. Dimethyl sulfoxide (DMSO) was used as the vehicle in each experiment.
To determine whether MET is involved in the apoptosis of cells, Annexin V apoptosis detection kit (BD Biosciences) was used as previously described [19]. PC3 and DU145 cells were treated with increasing doses of MET (0, 5, 10, and 20 μM) for 48 h. 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
2.3. Proliferation assay Cell Proliferation ELISA BrdU kit (Roche) was used to analyze the antiproliferative effects of MET. Experiments were performed as described in our previous study [19]. PC3 and DU145 cells were treated dose-dependently with MET for 48 h. Absorbance was measured at 370 nm and 492 nm, respectively.
In situ Cell Death Detection Kit, TMR red (Roche), was used as previously described to confirm DNA fragmentation changes following MET treatment in the cells [19]. The cells were treated with 20 μM of MET for 48 h. Fluorescence images were taken using a LSM710 confocal 13
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Fig. 1. MET reduces the proliferation of PC3 and DU145 cells. [A] The regulatory effects of MET on cell proliferation are identified. [B] Changes in the spheroid morphology following MET treatment are presented with bright field images and 3D plots. Scale bar represents 100 μm. [C] The ability of MET to regulate the expression of PCNA proteins was analyzed by immunofluorescence using Alexa 488 (green). DAPI (blue) was used to stain the nuclei of PC3 and DU145 cells. The scale bars of the first and third vertical panels are 40 μm, and the scale bars of the second and fourth vertical panels are 20 μm. Asterisks indicate a significant change after treatment with MET (***p < 0.001 and **p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
microscope. MetaMorph software was used to quantify the red fluorescence.
2.11. Determination of intracellular Ca2+ concentration Fluo-4 and Rhod-2 (Invitrogen), respectively, were used to measure intracellular and mitochondrial specific Ca2+ concentrations in response to 24 h MET treatment in the cells. The cells were then harvested using trypsin-EDTA. The harvested cells were stained with 3 μM Fluo-4 AM for 20 min at 37 °C and 3 μM Rhod-2 AM for 30 min at 4 °C.
2.8. Cell cycle analysis Following MET treatment, propidium iodide (PI; BD Bioscience) staining was performed with 100 μg/ml RNase A treatment to analyze cell cycle changes. Cells were divided into subG1, G1, S, and G2/M phases according to the PI fluorescence intensity.
2.12. Analysis of mitochondrial depolarization To investigate the mitochondrial dysfunction in PC3 and DU145 cells, mitochondria staining kit (Sigma-Aldrich) containing JC-1 staining was used as previously described [19]. PC3 and DU145 cells were treated with varying concentrations of MET (0, 5, 10, and 20 μM) for 48 h.
2.9. Western blot analysis Western blot was performed as previously described to confirm protein expression changes in response to MET treatment in PC3 and DU145 cells [19]. Details of all antibodies used in the experiments are shown in Table 1.
2.13. Analysis of cell migration Changes in migration in response to MET were investigated by Transwell migration assay. In the upper chamber of the Transwell inserts, PC3 and DU145 were seeded and incubated for 12 h with 20 μM of MET. To detect the cells that invaded the lower surface, cells in the Transwell membrane were fixed by methanol for 10 min. After staining with hematoxylin for 30 min, the upper part of the Transwell membrane was removed with a cotton swab. The Transwell membrane was covered using a glass slide with a Permount solution.
2.10. Determination of oxidants The cells were treated with dihydroethidium (DHE, Sigma-Aldrich) and Amplex red (Invitrogen) to investigate whether MET can induce superoxide anion (O2−) and hydrogen peroxide (H2O2), respectively. PC3 and DU145 cells were treated for 1 h with varying concentrations of MET (0, 5, 10, and 20 μM). 14
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3.3. Methiothepin mesylate increases H2O2 production and Ca2+ levels in prostate cancer cells
2.14. Quantitative RT-PCR analysis Changes in gene expression in prostate cancer cells following MET treatment were analyzed via quantitative RT-PCR using SYBR® Green (Sigma-Aldrich) as previously described [20]. The following primers were used: VEGFA—sense 5′-TTGTACAAGATCCGCAGACG-3′ and antisense 5′-TCACATCTGCAAGTACGTTCG-3′; MMP2—sense 5′-GTGGAT GATGCCTTTGCTCG-3′ and antisense 5′-CCATCGGCGTTCCCATA CTT-3′; MMP9—sense 5′-TTGACAGCGACAAGAAGTGG-3′ and antisense 5′-ACATTGGCCTTGATCTCAGC-3′); and MMP14—sense 5′-GCA GAAGTTTTACGGCTTGC-3′ and antisense 5′-ACATTGGCCTTGATCTC AGC-3′.
Induction of oxidative stress, leading to oxidant production, is considered an effective mechanism of action in cancer therapeutic approaches. We incubated cells with DHE and measured the degree of red fluorescence as an indicator of O2− production. As a result, MET treatment had no significant effect on the production of O2− (Fig. 3A). Next, we treated the cells with Amplex red to detect the production of H2O2, another representative oxidant that could be produced in cells. MET treatment increased intracellular H2O2 production in a concentration-dependent manner (Fig. 3B). In PC3 and DU145 cells, 20 μM of MET increased intracellular H2O2 production by 1.8-fold (p < 0.001) and 1.5-fold (p < 0.01), respectively. Oxidative stress can increase intracellular Ca2+ levels. We confirmed, using fluo-4 staining, that 20 μM of MET increased intracellular Ca2+ concentration by 1.7-fold (p < 0.05) and 3.3-fold (p < 0.001), respectively, in PC3 and DU145 cells (Fig. 3C). Elevated cytosolic Ca2+ enters the mitochondria to maintain Ca2+ homeostasis in the cytoplasm. We found that 20 μM of MET elevated mitochondrial Ca2+ concentration by 1.5fold (p < 0.05) and 2.5-fold (p < 0.01) in PC3 and DU145 cells, respectively (Fig. 3D). Collectively, MET increases H2O2 and Ca2+ levels in prostate cancer and causes Ca2+ influx into mitochondria.
2.15. Statistical analysis The statistical significance of all experiments that analyzed the changing characteristics in response to the treatment of MET in cells was confirmed based on analysis of variance (ANOVA) using the SAS program (SAS Institute). All experiments were performed at least three times, and a probability value of p < 0.05 was considered statistically significant.
3. Results
3.4. Methiothepin mesylate induces mitochondrial dysfunction and ER stress
3.1. Methiothepin mesylate suppresses the proliferation of prostate cancer cells
Excessive influx of Ca2+ into the mitochondria can lead to mitochondrial dysfunction, as represented by a decrease in mitochondrial membrane potential [21]. MET induced depolarization of mitochondria in PC3 and DU145 cells (Fig. 4A). MET treatment at 20 μM increased the loss of mitochondrial membrane potential by 3.1-fold (p < 0.001) in PC3 and DU145 cells. In PC3 cells, MET (20 μM) significantly increased the expression of Beclin-1 and the phosphorylation of P62 and ULK1, but reduced the expression of Atg5 (Fig. 4B). Increased endoplasmic reticulum (ER) stress causes cell death in cancer cells [22]. MET treatment for 24 h increased the expression of ER stress proteins GRP78 and IRE1α and phosphorylation of PERK (Fig. 4C). However, there was no significant change in eIF2α expression in response to MET in DU145 cells, unlike that in PC3 cells. In addition, MET increased the expression of cytochrome c, implying mitochondria-dependent apoptosis (Fig. 4D). MET did not alter IP3R1 expression, and IP3R2 expression was significantly increased only by 10 μM of MET in PC3 cells. In DU145 cells, the expression of IP3R1 and IP3R2 was significantly reduced by MET. Moreover, MFN2 and VAPB expression decreased while the expression of GRP75 and PTPIP51 increased by MET in PC3 cells. In DU145 cells, MET treatment also inhibited the expression of MFN2 and VAPB and elevated the expression of GRP75. PTPIP51 expression was dose-dependently reduced by MET in DU145 cells. These results suggest that MET could induce mitochondrial dysfunction and ER stress-mediated cell death. However, it still remains unclear whether the activity of MET is directly involved in the ER–mitochondria axis.
After dose-dependent MET treatment of PC3 and DU145 cells for 48 h, BrdU incorporation analysis revealed a reduced proliferation in response to MET (Fig. 1A). At the highest concentration of MET (20 μM), the proliferation of PC3 and DU145 cells was suppressed by 34% (p < 0.001) and 46% (p < 0.001), respectively. Next, we studied the effect of MET in the formation of spheroids. We confirmed that spheroid formation was significantly compromised in cells due to MET (20 μM) treatment for 10 days (Fig. 1B). In response to MET in PC3 cells, the tumor area and volume reduced by 71% (p < 0.001) and 80% (p < 0.001), respectively. In DU145 cells, the tumor area and volume reduced by 77% (p < 0.001) and 89% (p < 0.001), respectively, by MET treatment. PCNA is a protein responsible for cell proliferation and is widely used to verify the inhibitory effects of chemicals on proliferation. We treated PC3 and DU145 with MET for 24 h and then detected PCNA in the nucleus by immunofluorescence analysis. In the DAPI stained nuclei in MET-treated PC3 and DU145 cells, few green fluorescent PCNAs were observed (Fig. 1C). Collectively, MET significantly inhibits the proliferation of human prostate cancer cells.
3.2. Methiothepin mesylate causes cell death in prostate cancer We next identified whether MET treatment in prostate cancer cells can cause cell death. Annexin V and PI staining revealed that dosedependent MET treatment of PC3 and DU145 cells for 48 h increased the percentage of cells positive for Annexin V (Fig. 2A). At 20 μM, MET increased apoptosis in PC3 and DU145 cells by 13.3 (p < 0.001) and 3.0 (p < 0.001) times, respectively. We also investigated the extent to which the TUNEL reaction occurred, which quantified the DNA fragmentation in the nucleus of the cells. TUNEL reaction occurred more extensively in cells treated with MET compared to the controls (Fig. 2B). Moreover, cell cycle assays confirmed that MET could increase the number of cells in the SubG1 phase, indicating progression of the apoptotic pathway (Fig. 2C). Western blot analysis confirmed that phosphorylation of cyclin D1, an important protein for cell cycle progression, was reduced by MET, demonstrating that MET can regulate the cell cycle of prostate cancer cells (Fig. 2D). Collectively, in prostate cancer, MET can disrupt cell cycle progression by inducing apoptosis.
3.5. Methiothepin mesylate regulates PI3K/AKT and MAPK pathways in prostate cancer cells We analyzed the phosphorylation of AKT, S6, JNK, c-Jun, ERK1/2, and P38 after MET treatment for 2 h, to determine whether MET could regulate the PI3K/AKT and MAPK pathways (Fig. 5A). In PC3 cells, MET dose-dependently reduced phosphorylation of AKT (Fig. 5B). In DU145 cells, 5 μM of MET increased AKT phosphorylation, while 10 and 20 μM of MET inhibited AKT phosphorylation. S6 is a downstream protein of AKT and is significantly inactivated by MET in both cells (Fig. 5C). Moreover, MET activated JNK in the cells (Fig. 5D). Phosphorylation of c-Jun regulated by JNK also increased with MET treatment. Phosphorylation of ERK1/2 was unchanged in PC3 cells treated 15
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Fig. 2. MET promotes apoptosis in PC3 and DU145 cells. [A] Annexin V and propidium iodide stained cells indicate cell death. The number of cells in the upper right and lower right quadrants was measured to quantify the induction of apoptosis following MET treatment. [B] Staining for the TUNEL response (red) indicates that DNA fragmentation occurred. DAPI (blue) stained the nuclei of cells. [C] Cell cycle distribution was confirmed through propidium iodide staining. The effect of MET on the cell cycle was measured based on the number of cells in the SubG1, G1, S, and G2/M phases. [D] Regulation in phosphorylation of Cyclin D1 protein by MET was investigated by western blot. The intensity of the band for the total protein was used to normalize the intensity of the band for the phosphorylated protein. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05). The scale bars of the first and third vertical panels are 40 μm, and the scale bars of the second and fourth vertical panels are 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
addition of ruthenium red in PC3 cells (Fig. 6E). Collectively, increased H2O2 by MET and activation of JNK protein induce apoptosis, and mitochondrial Ca2+ overload may also induce mitochondrial dysfunction-mediated apoptosis in prostate cancer.
with MET, but ERK1/2 was activated by MET in DU145 cells (Fig. 5E). The expression of P38 protein, unlike other MAPKs, decreased in response to MET (Fig. 5F). Collectively, MET can regulate the PI3K/AKT and MAPK pathways in prostate cancer cells. JNK is a MAPK activated by oxidants. We estimated the mechanism of apoptosis induced by oxidant generation using SP600125, a selective inhibitor of JNK, and NAC, an oxidant scavenger. Co-treatment with MET (20 μM) and SP600125 (10 μM) alleviated the increased production of H2O2 in response to treatment with MET alone in PC3 cells (Fig. 6A). NAC also reduced the increased H2O2 levels by MET to the levels of the control cells. Moreover, the addition of SP600125 and NAC alleviated apoptosis induced by MET, although NAC alone increased apoptosis in PC3 cells (Fig. 6B). Ruthenium red is a chemical that prevents mitochondrial Ca2+ uptake and it significantly reduced the mitochondrial Ca2+ concentration increased by MET in the PC cells (Fig. 6C). In addition, mitochondrial depolarization increased by MET was mitigated by the combination with ruthenium red in PC3 cells (Fig. 6D). The apoptosis induced by MET was also alleviated by the
3.6. Methiothepin mesylate suppress migration in prostate cancer cells We performed a Transwell migration assay to investigate whether MET affects the migration of prostate cancer cells. MET (20 μM) treatment for 12 h significantly inhibited cells from passing through the membrane (Fig. 7A). MET inhibited 69% and 75% of migration in PC3 and DU145 cells, respectively. VEGFA, MMP2, MMP9, and MMP14 are proteins that allow cells to migrate in the extracellular matrix. We found that MET can inhibit the mRNA expression of VEGFA, MMP2, MMP9, and MMP14 (Fig. 7B). Collectively, MET shows therapeutic effects through the inhibition of migration of prostate cancer cells.
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Fig. 3. MET induces increased H2O2 production and Ca2+ concentrations in PC3 and DU145 cells. [A] O2− production was detected by DHE staining. [B] Confirmation of H2O2 generation changes following MET treatment was performed using the Amplex red reagent. [C] Changes in intracellular Ca2+ concentrations by MET were performed by Fluo-4 staining. [D] Changes in mitochondrial Ca2+ concentrations by MET were performed by Rhod-2 staining. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion
high-grade prostate cancer tissues [25]. In addition, their antagonists inhibit the proliferation of prostate cancer cells. 5-HT1A/1B is also highly expressed in high-grade prostate cancer cells [26]. Most serotonin receptors belong to the G-protein coupled receptor (GPCRs) superfamily and differ in downstream signals for each class [27]. 5-HT1 receptors, in general, negatively correlate with the adenylyl cyclase (AC) pathway [28]. The activity of 5-HT1 receptors inhibits cyclic adenosine monophosphate (cAMP) accumulation, implying coupling with Gαi [29]. In addition, 5-HT1A activation inhibits Ca2+ influx in central neurons [30]. However, little is known about the metabotropic effects of 5-HT1 regulation in prostate cancer. In this study, MET promoted intracellular Ca2+ release, but further research is needed regarding the mechanism of Ca2+ regulation of serotonin receptors in prostate cancer cells. Serotonin exerts a mitogenic effect in which 5-HT1A and 5-HT1D receptors
The results of studies showing prostate cancer cells that survive androgen ablation therapy have necessitated further studies of alternative tumor growth mechanisms. Neuroendocrine differentiation in prostate cancer tissues is involved in the mechanism of tumor progression. The products secreted from neuroendocrine cells, including serotonin, are associated with tumor growth independent of androgen [23,24]. Serotonin is a monoamine neurotransmitter with various receptors and different physiological activities in different cell types. Serotonin receptors are generally divided into seven families, of which there are various subtypes. Several serotonin receptors are known to be expressed in prostate cancer tissue. 5-HT2B is expressed in low- and high-grade prostate cancers, and 5-HT4 is predominantly expressed in 17
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Fig. 4. Effects of MET on mitochondrial membrane potentials and expression of proteins related to ER stress, autophagy, and ER-mitochondria axis in PC3 and DU145 cells. [A] MET-induced reduction of mitochondrial membrane potential was analyzed by JC-1 staining. The lower right/upper right values were calculated for quantification. [B] The expression of Beclin-1, Atg5, p-P62, P62, p-ULK1, ULK1, and TUBA were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. [C] The expression of GRP78, IRE1α, p-PERK, t-PERK, p-eIF2α, t-eIF2α, and TUBA were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. [D] The expression of cytochrome c, IP3R1, IP3R2, MFN2, VAPB, GRP75, PTPIP51 were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. Asterisks indicate the significance of the effects of MET (***p < 0.001, **p < 0.01, and *p < 0.05).
involved in tumor development, such as the activation of oncogenes. Therefore, in cancer cells, the level of oxidative stress is considered to be higher than the basal level. In view of these cancer-specific characteristics, oxidant generation within cancer cells is increasingly being considered as a target mechanism for cancer therapy, because this more likely affects the survival of cancer cells than normal cells [36]. 1-(1Naphthyl)piperazine, a serotonergic derivative of quipazine, has anticancer potential through cell cycle regulation and induction of apoptosis based on oxidant generation in melanoma cells [37]. It has already been proven that various types of antidepressants can induce mitochondria-dependent cell death [38]. Paroxetine, which inhibits serotonin reuptake and acts as an antidepressant, increases oxidant production and intracellular Ca2+ levels in human breast cancer cells, leading to a loss in mitochondrial membrane potential [39]. Fluoxetine, another serotonin reuptake inhibitor, also promotes the accumulation of Ca2+ into the mitochondria along with an increase in cytosolic Ca2+ in HeLa cells [40]. MET was also confirmed to promote H2O2 production in prostate cancer cells. Ca2+ is a secondary messenger involved in numerous cellular physiology processes. Disruption of Ca2+ homeostasis induces cell death by apoptosis or autophagy [41]. In particular, Ca2+ overload into mitochondria causes mitochondrial dysfunction, which is represented by a decrease in mitochondrial membrane potential [42]. Ruthenium red inhibits mitochondrial Ca2+ uniporters and inhibits mitochondrial Ca2+ accumulation [43]. In our study, the combination with ruthenium red mitigated mitochondrial depolarization and cell death caused by MET. These findings imply that Ca2+ overload into mitochondria is involved in MET-induced apoptosis of prostate cancer.
play a role in human small cell lung carcinoma [14]. In addition, serotonin promotes proliferation in carcinoid valve disease, which is supposed to be mediated by 5HT1b [31]. In human pancreatic carcinoid cells, serotonin exerts mitogenic effects, which are alleviated by the 5HT1A/1B receptor antagonist [13]. Moreover, 5-HT receptor antagonists and serotonin-uptake inhibitors, including 6-nitroquipazine, zimelidine, and fluoxetine, exert growth inhibitory effects on prostate cancer [16,32]. Histological studies have confirmed high expression of 5-HT1A/ 1B in prostate cancer tissues that metastasize to the lymph nodes and bones [26]. Further, the expression of these receptors was higher in prostate cancer tissues than in benign prostate hyperplasia tissues. PC3 and DU145 cells are hormone-independent prostate cancer cell lines that show higher expression of 5-HT1A/1B receptors than that in LNCaP cells, a hormone-dependent cell line, especially at high growth density [33]. MET is a serotonin receptor antagonist and has a high affinity to 5HT1A/1B [34]. Although some physiological functions have been shown in poultry and gastropods, little is known about the effects of MET in human cells [18,35]. In previous studies using PC3 and DU145 cells, MET dramatically reduced viability at concentrations between 10 and 20 μM [33]. Similarly, in our study, MET significantly reduced cell proliferation at concentrations of 10 and 20 μM in PC3 and 5, 10, and 20 μM in DU145 cells. However, it is still unknown whether the activation or inhibition of 5HT1A/1B is involved in the survival and growth of prostate cancer cells, as suggested in a previous study [33]. Oxidants can regulate the activity and expression of signaling proteins and transcriptional factors involved in cell survival and stress response [36]. Furthermore, oxidants stimulate several mechanisms 18
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Fig. 5. Effects of MET on the regulation of signal transduction in PC3 and DU145 cells. Phosphorylated AKT [A], S6 [B], JNK [C], c-Jun [D], ERK1/2 [E], and P38 [F] were estimated in response to MET (0, 5, 10, and 20 μM). The experiment was conducted at least three times. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05).
induces ER stress-dependent apoptosis [46]. In addition, oxidant generation and JNK phosphorylation are involved in the induction of autophagy [47,48]. MET increases the expression of ER stress proteins in prostate cancer cells. Since DU145 cells are autophagy-deficient cells, the results of western blot analysis performed only on PC3 cells show an increased expression of Beclin-1, p-P62, and p-ULK1 but a decreased expression of Atg5 [49]. The expression of the proteins responsible for the contact between mitochondria and ER was also regulated by MET. The ER-mitochondrial linkage is formed by various proteins covered in
Serotonin activates the PI3K/AKT and MAPK signaling pathways in prostate cancer cells [15]. Tianeptine sodium salt (TSS), a serotonin facilitator, has been shown to have an anti-invasive effect by suppressing the PI3K/AKT signaling and MMP9 in prostate cancer cells [44]. MET suppresses AKT and its downstream protein S6, with a decrease in cell migration. In addition, of the three MAPKs, JNK, ERK1/2 and P38, only JNK is activated by MET. In our previous study JNK activation was also associated with increased oxidant production and mitochondrial dysfunction [45]. Moreover, the activation of JNK in prostate cells 19
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Fig. 6. Effects of co-treatment with MET and inhibitors against JNK (SP600125), oxidants (NAC), and mitochondrial Ca2+ uptake (ruthenium red) in PC3 and DU145 cells. [A] Changes in intracellular H2O2 levels following MET, SP600125, and NAC treatment were confirmed by measuring Amplex red fluorescence. [B] Annexin V and propidium iodide fluorescence were measured to determine the regulation of apoptosis following MET, SP600125, and NAC treatment. [C] Rhod-2 fluorescence was observed to measure mitochondrial specific Ca2+ concentration by MET and ruthenium red treatment. [D] JC-1 fluorescence was measured to identify changes in mitochondrial membrane potential caused by MET and ruthenium red. [E] Annexin V and propidium iodide fluorescence was observed to analyze the effect on the regulation of apoptosis by MET and ruthenium red treatment. Asterisks indicate the significance of the effect of MET (***p < 0.001 and **p < 0.01).
Fig. 7. MET regulates the migrating properties of PC3 and DU145 cells. [A] The effect of MET on migration was confirmed using Transwell inserts. The number of cells migrated through the membrane was measured on images of five non-overlapping locations. [B] The mRNA expression of migration-related genes in response to MET treatment was measured via quantitative RT-PCR. Expression of all genes was confirmed in triplicate. Asterisks indicate the significance of the effect by MET (***p < 0.001). Scale bar represents 100 μm. 20
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Author's contributions GS and WL designed and directed the study. CY, GS, and WL performed experiments, wrote, and prepared the manuscript. CY designed the figures. All authors provided critical feedback and helped to shape the manuscript. Data availability statement The authors confirm that the data supporting the findings of this study are available within the article. Declaration of competing interest The authors have declared no conflict of interest. Acknowledgements This research was supported by a grant of the National Research Foundation of Korea (NRF) grant, funded by the Ministry of Science and ICT (MSIT) [grant number: 2018R1C1B6009048]. References Fig. 8. Schematic diagram describing the effects of MET on prostate cancer cells. MET inhibits AKT signaling pathways while increasing the phosphorylation of JNK proteins. Activated JNK increases intracellular H2O2 production followed by ER stress. Ca2+ released from ER is overloaded into mitochondria and induces mitochondrial dysfunction. The cytochrome c activates the apoptotic pathway, leading to DNA damage in the nucleus. Eventually, the proliferative capacity and migration of prostate cancer cells exposed to MET will decrease.
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this study, such as IP3R, MFN2, VAPB, GRP75, and PTPIP51 [50]. The junction of the ER and mitochondria formed by these proteins is called the mitochondrial-associated ER membrane (MAM), which plays an essential role in Ca2+ homeostasis in the cytoplasm [51]. MAM proteins are also involved in activating mitochondria-mediated apoptosis signals [52,53]. IP3R is a Ca2+ channel located on the ER membrane that regulates the release of Ca2+ from the ER into the cytoplasm [54]. IP3R also facilitates the influx of Ca2+ from the ER to the mitochondria by placing the mitochondria and ER close together by a chaperone protein called GRP75 [55]. Reduced expression of GRP75 prevents cell death mediated by Ca2+ influx into mitochondria [56]. The MFN2 complex and the VAPB-PTPIP51 complex are other tethering proteins responsible for Ca2+ transport between the ER and mitochondria [50]. MFN2 promotes apoptosis through the PI3K/AKT signaling pathway in breast cancer cells [57]. PTPIP51 is known to have higher expression in prostate carcinoma tissue than in benign prostate hyperplastic cells [58]. However, little is known regarding whether MAM proteins responsible for Ca2+ homeostasis are involved in cell death in prostate cancer, and further study is required. In conclusion, this study may be of significance because we have identified specific anticancer mechanisms of serotonin receptor antagonists, which are considered potential therapeutics (see Fig. 8). Serotonin has been shown to contribute to the survival and growth of various types of cancer including prostate cancer. In other carcinomas, therapeutic effects based on the oxidative stress of serotonin receptor antagonists or serotonin transporter inhibitors have been suggested. For the first time, to our knowledge, we found that serotonin antagonists mediate oxidative stress, ER stress, mitochondrial dysfunction, and activation of JNK signaling in prostate cancer cells, leading to apoptosis and reduced cell migration. Considering the significant contribution of neuroendocrine regulation to the proliferation of prostate cancer cells, we believe that various types of modulators of serotonin activity, in addition to MET, may have therapeutic effects.
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Original article
Methiothepin mesylate causes apoptosis of human prostate cancer cells by mediating oxidative stress and mitochondrial dysfunction
T
Changwon Yanga, Gwonhwa Songa,∗, Whasun Limb,∗∗ a
Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea b Department of Food and Nutrition, Kookmin University, Seoul, 02707, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Methiothepin mesylate Prostate cancer Mitochondria Apoptosis Oxidative stress
Prostate cancer is difficult to treat if it metastasizes to other organs. The development of prostate cancer independent of androgen is closely related to the action of neuroendocrine products. Serotonin promotes cell growth in various cancers, and antagonists for serotonin receptors are known to inhibit proliferation and induce cell death in various carcinomas. However, little is known about how antagonists for serotonin receptor function in prostate cancer. We verified apoptotic cell death in prostate cancer cell lines after treatment with methiothepin mesylate (MET), an antagonist for serotonin receptor 5-HT1. MET induced hydrogen peroxide (H2O2) production and mitochondrial Ca2+ overload. Moreover, MET induced changes in the expression of proteins associated with endoplasmic reticulum stress, autophagy, and mitochondrial membrane potential. MET also promoted phosphorylation of JNK, which induced cell death mediated by oxidant production, as evidenced by the JNK inhibitor and oxidant scavenger. Finally, MET has the potential to prevent metastasis by inhibiting the migration of prostate cancer cells. Thus, we show that MET is a potentially novel anticancer agent that can suppress the development of prostate cancer caused by neuroendocrine differentiation.
1. Introduction Prostate cancer is the second most common carcinoma among men worldwide [1]. Prostate cancer cells can metastasize to various organs, such as the lymph nodes, liver, and bones, which makes surgical treatment difficult [2]. Androgen ablation is the first-line therapy, but long-term application leads to androgen-independent tumor cell growth [3]. The growth of androgen-independent prostate cancer is associated with abnormal signaling [4,5]. Natural products derived from plants and compounds that regulate transcriptional activity in the nucleus of cells have been suggested as potential adjuvants to treat androgen-independent prostate cancer [6,7]. For example, the herb extract DT-13 reduces cell migration by inhibiting phosphoinositide 3-kinase (PI3K)/ AKT cascades and matrix metalloproteinase (MMP) 2 and MMP9 in PC3 and DU145 cell lines [8]. In addition, sodium butyrate, a histone deacetylase (HDAC) inhibitor, induces apoptosis by increasing mitochondrial damage and nuclear fragmentation following phosphorylation of mitogen-activated protein kinase (MAPK) [9]. However, there is a lack of information on the therapeutic effects of the regulation of neurosecretory products on prostate cancer, although neuroendocrine
∗
differentiated tumor cells are found in the majority of prostatic carcinomas [10]. Serotonin (5-hydroxytryptamine) is known to function in the central nervous system (CNS) as a neurotransmitter but is now thought to elicit physiological changes throughout the body, such as in the enteric nervous system or vascular system [11,12]. Research on the mechanisms of action of serotonin agonists and antagonists is actively pursued in various cell types. The growth-stimulating effects of serotonin have been reported in various cell types, including pancreatic carcinoid cells and small cell lung carcinoma [13,14]. The effect of serotonin on cancer proliferation is based on the activation of MAPK and PI3K/AKT signaling [15]. Moreover, serotonin is a marker of neuroendocrine differentiation, and inhibitors of serotonin uptake are considered to be a potential treatment for prostate cancer [16]. A variety of chemicals, including serotonergic compounds, serotonin transporter inhibitors, and serotonin receptor agonists, affect the proliferation of prostate cancer cell line, but their physiological mechanism in cells is unclear [17]. Methiothepin mesylate (MET) is a non-selective 5-HT1 receptor antagonist with little known physiological effect in human cells. In broilers, MET was reported to mitigate the pulmonary hypertensive
Corresponding author. Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea. Corresponding author. Department of Food and Nutrition, College of Science and Technology, Kookmin University, Seoul, 02707, Republic of Korea. E-mail addresses: [email protected] (G. Song), [email protected] (W. Lim).
∗∗
https://doi.org/10.1016/j.freeradbiomed.2020.01.187 Received 3 December 2019; Received in revised form 30 January 2020; Accepted 31 January 2020 Available online 05 February 2020 0891-5849/ © 2020 Elsevier Inc. All rights reserved.
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Table 1 Primary antibodies used in western blot. Primary antibodies 286
Phosphor-Cyclin D1 (Thr ) Cyclin D1 Beclin-1 Atg5 Phosphor-P62 P62 Phosphor-ULK1 ULK1 TUBA GRP78 IRE1α Phosphor-PERK (Thr981) PERK Phosphor-eIF2α eIF2α Cytochrome c IP3R1 IP3R2 MFN2 VAPB GRP75 PTPIP51 Phosphor-AKT (Ser473) AKT Phosphor-S6 (Ser235/Ser236) S6 Phosphor-JNK (Thr183/Tyr185) JNK Phosphor-c-Jun (Ser73) c-Jun Phosphor-ERK1/2 (Thr202/Tyr204) ERK1/2 Phosphor-P38 (Thr180/Tyr182) P38
Dilution
Supplier
1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:2000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000
Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Santa Cruz Santa Cruz Cell Signaling Santa Cruz Santa Cruz Cell Signaling Cell Signaling Cell Signaling Invitrogen Santa Cruz Cell Signaling Invitrogen Cell Signaling Abcam Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling
Catalog Number Technology Technology Technology Technology Technology Technology Technology Technology
Technology
Technology Technology Technology
Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology Technology
3300 2922 3495 12994 16177 88588 5869 8054 sc-5286 sc-13968 3294 sc-32577 sc-13073 3398 5324 11940 PA1-901 sc-398434 11925 PA5-53023 3593 ab182105 4060 9272 2211 2217 4668 9252 3270 9165 9101 4695 4511 9212
2.4. Spheroid formation
response, but there is little information on its effectiveness in human cancer cells [18]. We examined the effects of MET on cell proliferation and apoptosis after treatment of androgen-independent PC3 and DU145 cells. Moreover, we estimated whether MET can cause oxidative stress and mitochondrial dysfunction in PC3 and DU145 cells. In addition, we examined whether MET is involved in the migration of prostate cancer cells via the regulation of PI3K/AKT and MAPK signaling.
For the hanging drop method, 3000 cells were seeded onto the cover of the culture dish and was treated with 20 μM of MET for 10 days. Changes in spheroid morphology with MET treatment were observed with a DM3000 microscope (Leica). Spheroid area was measured using ImageJ software (http://rsb.info.nih.gov/ij/docs/index.html). 3D plot quantification was performed by using ReViSP software (https:// sourceforge.net/projects/revisp/).
2. Materials and methods 2.5. Immunofluorescence microscopy 2.1. Chemicals To clarify whether MET can regulate the expression of proliferating cell nuclear antigen (PCNA), immunofluorescence method was used as previously described [19]. PC3 and DU145 cells were treated with 20 μM of MET for 24 h. MetaMorph software (Molecular Devices) was used to quantify Alexa 488 green fluorescence.
MET and N-acetyl-L-cysteine (NAC) were purchased from SigmaAldrich, Inc. SP600125 was purchased from Enzo Life Science. Ruthenium red was purchased from Abcam. 2.2. Cell culture
2.6. Annexin V and propidium iodide staining PC3 and DU145 cells were purchased from the American Type Culture Collection. RPMI-1640 medium was used to culture the cells. For the experiments, cells were cultured until about 70% of the plate was filled and then starved for 24 h. Dimethyl sulfoxide (DMSO) was used as the vehicle in each experiment.
To determine whether MET is involved in the apoptosis of cells, Annexin V apoptosis detection kit (BD Biosciences) was used as previously described [19]. PC3 and DU145 cells were treated with increasing doses of MET (0, 5, 10, and 20 μM) for 48 h. 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
2.3. Proliferation assay Cell Proliferation ELISA BrdU kit (Roche) was used to analyze the antiproliferative effects of MET. Experiments were performed as described in our previous study [19]. PC3 and DU145 cells were treated dose-dependently with MET for 48 h. Absorbance was measured at 370 nm and 492 nm, respectively.
In situ Cell Death Detection Kit, TMR red (Roche), was used as previously described to confirm DNA fragmentation changes following MET treatment in the cells [19]. The cells were treated with 20 μM of MET for 48 h. Fluorescence images were taken using a LSM710 confocal 13
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Fig. 1. MET reduces the proliferation of PC3 and DU145 cells. [A] The regulatory effects of MET on cell proliferation are identified. [B] Changes in the spheroid morphology following MET treatment are presented with bright field images and 3D plots. Scale bar represents 100 μm. [C] The ability of MET to regulate the expression of PCNA proteins was analyzed by immunofluorescence using Alexa 488 (green). DAPI (blue) was used to stain the nuclei of PC3 and DU145 cells. The scale bars of the first and third vertical panels are 40 μm, and the scale bars of the second and fourth vertical panels are 20 μm. Asterisks indicate a significant change after treatment with MET (***p < 0.001 and **p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
microscope. MetaMorph software was used to quantify the red fluorescence.
2.11. Determination of intracellular Ca2+ concentration Fluo-4 and Rhod-2 (Invitrogen), respectively, were used to measure intracellular and mitochondrial specific Ca2+ concentrations in response to 24 h MET treatment in the cells. The cells were then harvested using trypsin-EDTA. The harvested cells were stained with 3 μM Fluo-4 AM for 20 min at 37 °C and 3 μM Rhod-2 AM for 30 min at 4 °C.
2.8. Cell cycle analysis Following MET treatment, propidium iodide (PI; BD Bioscience) staining was performed with 100 μg/ml RNase A treatment to analyze cell cycle changes. Cells were divided into subG1, G1, S, and G2/M phases according to the PI fluorescence intensity.
2.12. Analysis of mitochondrial depolarization To investigate the mitochondrial dysfunction in PC3 and DU145 cells, mitochondria staining kit (Sigma-Aldrich) containing JC-1 staining was used as previously described [19]. PC3 and DU145 cells were treated with varying concentrations of MET (0, 5, 10, and 20 μM) for 48 h.
2.9. Western blot analysis Western blot was performed as previously described to confirm protein expression changes in response to MET treatment in PC3 and DU145 cells [19]. Details of all antibodies used in the experiments are shown in Table 1.
2.13. Analysis of cell migration Changes in migration in response to MET were investigated by Transwell migration assay. In the upper chamber of the Transwell inserts, PC3 and DU145 were seeded and incubated for 12 h with 20 μM of MET. To detect the cells that invaded the lower surface, cells in the Transwell membrane were fixed by methanol for 10 min. After staining with hematoxylin for 30 min, the upper part of the Transwell membrane was removed with a cotton swab. The Transwell membrane was covered using a glass slide with a Permount solution.
2.10. Determination of oxidants The cells were treated with dihydroethidium (DHE, Sigma-Aldrich) and Amplex red (Invitrogen) to investigate whether MET can induce superoxide anion (O2−) and hydrogen peroxide (H2O2), respectively. PC3 and DU145 cells were treated for 1 h with varying concentrations of MET (0, 5, 10, and 20 μM). 14
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3.3. Methiothepin mesylate increases H2O2 production and Ca2+ levels in prostate cancer cells
2.14. Quantitative RT-PCR analysis Changes in gene expression in prostate cancer cells following MET treatment were analyzed via quantitative RT-PCR using SYBR® Green (Sigma-Aldrich) as previously described [20]. The following primers were used: VEGFA—sense 5′-TTGTACAAGATCCGCAGACG-3′ and antisense 5′-TCACATCTGCAAGTACGTTCG-3′; MMP2—sense 5′-GTGGAT GATGCCTTTGCTCG-3′ and antisense 5′-CCATCGGCGTTCCCATA CTT-3′; MMP9—sense 5′-TTGACAGCGACAAGAAGTGG-3′ and antisense 5′-ACATTGGCCTTGATCTCAGC-3′); and MMP14—sense 5′-GCA GAAGTTTTACGGCTTGC-3′ and antisense 5′-ACATTGGCCTTGATCTC AGC-3′.
Induction of oxidative stress, leading to oxidant production, is considered an effective mechanism of action in cancer therapeutic approaches. We incubated cells with DHE and measured the degree of red fluorescence as an indicator of O2− production. As a result, MET treatment had no significant effect on the production of O2− (Fig. 3A). Next, we treated the cells with Amplex red to detect the production of H2O2, another representative oxidant that could be produced in cells. MET treatment increased intracellular H2O2 production in a concentration-dependent manner (Fig. 3B). In PC3 and DU145 cells, 20 μM of MET increased intracellular H2O2 production by 1.8-fold (p < 0.001) and 1.5-fold (p < 0.01), respectively. Oxidative stress can increase intracellular Ca2+ levels. We confirmed, using fluo-4 staining, that 20 μM of MET increased intracellular Ca2+ concentration by 1.7-fold (p < 0.05) and 3.3-fold (p < 0.001), respectively, in PC3 and DU145 cells (Fig. 3C). Elevated cytosolic Ca2+ enters the mitochondria to maintain Ca2+ homeostasis in the cytoplasm. We found that 20 μM of MET elevated mitochondrial Ca2+ concentration by 1.5fold (p < 0.05) and 2.5-fold (p < 0.01) in PC3 and DU145 cells, respectively (Fig. 3D). Collectively, MET increases H2O2 and Ca2+ levels in prostate cancer and causes Ca2+ influx into mitochondria.
2.15. Statistical analysis The statistical significance of all experiments that analyzed the changing characteristics in response to the treatment of MET in cells was confirmed based on analysis of variance (ANOVA) using the SAS program (SAS Institute). All experiments were performed at least three times, and a probability value of p < 0.05 was considered statistically significant.
3. Results
3.4. Methiothepin mesylate induces mitochondrial dysfunction and ER stress
3.1. Methiothepin mesylate suppresses the proliferation of prostate cancer cells
Excessive influx of Ca2+ into the mitochondria can lead to mitochondrial dysfunction, as represented by a decrease in mitochondrial membrane potential [21]. MET induced depolarization of mitochondria in PC3 and DU145 cells (Fig. 4A). MET treatment at 20 μM increased the loss of mitochondrial membrane potential by 3.1-fold (p < 0.001) in PC3 and DU145 cells. In PC3 cells, MET (20 μM) significantly increased the expression of Beclin-1 and the phosphorylation of P62 and ULK1, but reduced the expression of Atg5 (Fig. 4B). Increased endoplasmic reticulum (ER) stress causes cell death in cancer cells [22]. MET treatment for 24 h increased the expression of ER stress proteins GRP78 and IRE1α and phosphorylation of PERK (Fig. 4C). However, there was no significant change in eIF2α expression in response to MET in DU145 cells, unlike that in PC3 cells. In addition, MET increased the expression of cytochrome c, implying mitochondria-dependent apoptosis (Fig. 4D). MET did not alter IP3R1 expression, and IP3R2 expression was significantly increased only by 10 μM of MET in PC3 cells. In DU145 cells, the expression of IP3R1 and IP3R2 was significantly reduced by MET. Moreover, MFN2 and VAPB expression decreased while the expression of GRP75 and PTPIP51 increased by MET in PC3 cells. In DU145 cells, MET treatment also inhibited the expression of MFN2 and VAPB and elevated the expression of GRP75. PTPIP51 expression was dose-dependently reduced by MET in DU145 cells. These results suggest that MET could induce mitochondrial dysfunction and ER stress-mediated cell death. However, it still remains unclear whether the activity of MET is directly involved in the ER–mitochondria axis.
After dose-dependent MET treatment of PC3 and DU145 cells for 48 h, BrdU incorporation analysis revealed a reduced proliferation in response to MET (Fig. 1A). At the highest concentration of MET (20 μM), the proliferation of PC3 and DU145 cells was suppressed by 34% (p < 0.001) and 46% (p < 0.001), respectively. Next, we studied the effect of MET in the formation of spheroids. We confirmed that spheroid formation was significantly compromised in cells due to MET (20 μM) treatment for 10 days (Fig. 1B). In response to MET in PC3 cells, the tumor area and volume reduced by 71% (p < 0.001) and 80% (p < 0.001), respectively. In DU145 cells, the tumor area and volume reduced by 77% (p < 0.001) and 89% (p < 0.001), respectively, by MET treatment. PCNA is a protein responsible for cell proliferation and is widely used to verify the inhibitory effects of chemicals on proliferation. We treated PC3 and DU145 with MET for 24 h and then detected PCNA in the nucleus by immunofluorescence analysis. In the DAPI stained nuclei in MET-treated PC3 and DU145 cells, few green fluorescent PCNAs were observed (Fig. 1C). Collectively, MET significantly inhibits the proliferation of human prostate cancer cells.
3.2. Methiothepin mesylate causes cell death in prostate cancer We next identified whether MET treatment in prostate cancer cells can cause cell death. Annexin V and PI staining revealed that dosedependent MET treatment of PC3 and DU145 cells for 48 h increased the percentage of cells positive for Annexin V (Fig. 2A). At 20 μM, MET increased apoptosis in PC3 and DU145 cells by 13.3 (p < 0.001) and 3.0 (p < 0.001) times, respectively. We also investigated the extent to which the TUNEL reaction occurred, which quantified the DNA fragmentation in the nucleus of the cells. TUNEL reaction occurred more extensively in cells treated with MET compared to the controls (Fig. 2B). Moreover, cell cycle assays confirmed that MET could increase the number of cells in the SubG1 phase, indicating progression of the apoptotic pathway (Fig. 2C). Western blot analysis confirmed that phosphorylation of cyclin D1, an important protein for cell cycle progression, was reduced by MET, demonstrating that MET can regulate the cell cycle of prostate cancer cells (Fig. 2D). Collectively, in prostate cancer, MET can disrupt cell cycle progression by inducing apoptosis.
3.5. Methiothepin mesylate regulates PI3K/AKT and MAPK pathways in prostate cancer cells We analyzed the phosphorylation of AKT, S6, JNK, c-Jun, ERK1/2, and P38 after MET treatment for 2 h, to determine whether MET could regulate the PI3K/AKT and MAPK pathways (Fig. 5A). In PC3 cells, MET dose-dependently reduced phosphorylation of AKT (Fig. 5B). In DU145 cells, 5 μM of MET increased AKT phosphorylation, while 10 and 20 μM of MET inhibited AKT phosphorylation. S6 is a downstream protein of AKT and is significantly inactivated by MET in both cells (Fig. 5C). Moreover, MET activated JNK in the cells (Fig. 5D). Phosphorylation of c-Jun regulated by JNK also increased with MET treatment. Phosphorylation of ERK1/2 was unchanged in PC3 cells treated 15
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Fig. 2. MET promotes apoptosis in PC3 and DU145 cells. [A] Annexin V and propidium iodide stained cells indicate cell death. The number of cells in the upper right and lower right quadrants was measured to quantify the induction of apoptosis following MET treatment. [B] Staining for the TUNEL response (red) indicates that DNA fragmentation occurred. DAPI (blue) stained the nuclei of cells. [C] Cell cycle distribution was confirmed through propidium iodide staining. The effect of MET on the cell cycle was measured based on the number of cells in the SubG1, G1, S, and G2/M phases. [D] Regulation in phosphorylation of Cyclin D1 protein by MET was investigated by western blot. The intensity of the band for the total protein was used to normalize the intensity of the band for the phosphorylated protein. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05). The scale bars of the first and third vertical panels are 40 μm, and the scale bars of the second and fourth vertical panels are 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
addition of ruthenium red in PC3 cells (Fig. 6E). Collectively, increased H2O2 by MET and activation of JNK protein induce apoptosis, and mitochondrial Ca2+ overload may also induce mitochondrial dysfunction-mediated apoptosis in prostate cancer.
with MET, but ERK1/2 was activated by MET in DU145 cells (Fig. 5E). The expression of P38 protein, unlike other MAPKs, decreased in response to MET (Fig. 5F). Collectively, MET can regulate the PI3K/AKT and MAPK pathways in prostate cancer cells. JNK is a MAPK activated by oxidants. We estimated the mechanism of apoptosis induced by oxidant generation using SP600125, a selective inhibitor of JNK, and NAC, an oxidant scavenger. Co-treatment with MET (20 μM) and SP600125 (10 μM) alleviated the increased production of H2O2 in response to treatment with MET alone in PC3 cells (Fig. 6A). NAC also reduced the increased H2O2 levels by MET to the levels of the control cells. Moreover, the addition of SP600125 and NAC alleviated apoptosis induced by MET, although NAC alone increased apoptosis in PC3 cells (Fig. 6B). Ruthenium red is a chemical that prevents mitochondrial Ca2+ uptake and it significantly reduced the mitochondrial Ca2+ concentration increased by MET in the PC cells (Fig. 6C). In addition, mitochondrial depolarization increased by MET was mitigated by the combination with ruthenium red in PC3 cells (Fig. 6D). The apoptosis induced by MET was also alleviated by the
3.6. Methiothepin mesylate suppress migration in prostate cancer cells We performed a Transwell migration assay to investigate whether MET affects the migration of prostate cancer cells. MET (20 μM) treatment for 12 h significantly inhibited cells from passing through the membrane (Fig. 7A). MET inhibited 69% and 75% of migration in PC3 and DU145 cells, respectively. VEGFA, MMP2, MMP9, and MMP14 are proteins that allow cells to migrate in the extracellular matrix. We found that MET can inhibit the mRNA expression of VEGFA, MMP2, MMP9, and MMP14 (Fig. 7B). Collectively, MET shows therapeutic effects through the inhibition of migration of prostate cancer cells.
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Fig. 3. MET induces increased H2O2 production and Ca2+ concentrations in PC3 and DU145 cells. [A] O2− production was detected by DHE staining. [B] Confirmation of H2O2 generation changes following MET treatment was performed using the Amplex red reagent. [C] Changes in intracellular Ca2+ concentrations by MET were performed by Fluo-4 staining. [D] Changes in mitochondrial Ca2+ concentrations by MET were performed by Rhod-2 staining. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion
high-grade prostate cancer tissues [25]. In addition, their antagonists inhibit the proliferation of prostate cancer cells. 5-HT1A/1B is also highly expressed in high-grade prostate cancer cells [26]. Most serotonin receptors belong to the G-protein coupled receptor (GPCRs) superfamily and differ in downstream signals for each class [27]. 5-HT1 receptors, in general, negatively correlate with the adenylyl cyclase (AC) pathway [28]. The activity of 5-HT1 receptors inhibits cyclic adenosine monophosphate (cAMP) accumulation, implying coupling with Gαi [29]. In addition, 5-HT1A activation inhibits Ca2+ influx in central neurons [30]. However, little is known about the metabotropic effects of 5-HT1 regulation in prostate cancer. In this study, MET promoted intracellular Ca2+ release, but further research is needed regarding the mechanism of Ca2+ regulation of serotonin receptors in prostate cancer cells. Serotonin exerts a mitogenic effect in which 5-HT1A and 5-HT1D receptors
The results of studies showing prostate cancer cells that survive androgen ablation therapy have necessitated further studies of alternative tumor growth mechanisms. Neuroendocrine differentiation in prostate cancer tissues is involved in the mechanism of tumor progression. The products secreted from neuroendocrine cells, including serotonin, are associated with tumor growth independent of androgen [23,24]. Serotonin is a monoamine neurotransmitter with various receptors and different physiological activities in different cell types. Serotonin receptors are generally divided into seven families, of which there are various subtypes. Several serotonin receptors are known to be expressed in prostate cancer tissue. 5-HT2B is expressed in low- and high-grade prostate cancers, and 5-HT4 is predominantly expressed in 17
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Fig. 4. Effects of MET on mitochondrial membrane potentials and expression of proteins related to ER stress, autophagy, and ER-mitochondria axis in PC3 and DU145 cells. [A] MET-induced reduction of mitochondrial membrane potential was analyzed by JC-1 staining. The lower right/upper right values were calculated for quantification. [B] The expression of Beclin-1, Atg5, p-P62, P62, p-ULK1, ULK1, and TUBA were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. [C] The expression of GRP78, IRE1α, p-PERK, t-PERK, p-eIF2α, t-eIF2α, and TUBA were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. [D] The expression of cytochrome c, IP3R1, IP3R2, MFN2, VAPB, GRP75, PTPIP51 were analyzed in response to MET (0, 5, 10, and 20 μM) treatment for 24 h. Asterisks indicate the significance of the effects of MET (***p < 0.001, **p < 0.01, and *p < 0.05).
involved in tumor development, such as the activation of oncogenes. Therefore, in cancer cells, the level of oxidative stress is considered to be higher than the basal level. In view of these cancer-specific characteristics, oxidant generation within cancer cells is increasingly being considered as a target mechanism for cancer therapy, because this more likely affects the survival of cancer cells than normal cells [36]. 1-(1Naphthyl)piperazine, a serotonergic derivative of quipazine, has anticancer potential through cell cycle regulation and induction of apoptosis based on oxidant generation in melanoma cells [37]. It has already been proven that various types of antidepressants can induce mitochondria-dependent cell death [38]. Paroxetine, which inhibits serotonin reuptake and acts as an antidepressant, increases oxidant production and intracellular Ca2+ levels in human breast cancer cells, leading to a loss in mitochondrial membrane potential [39]. Fluoxetine, another serotonin reuptake inhibitor, also promotes the accumulation of Ca2+ into the mitochondria along with an increase in cytosolic Ca2+ in HeLa cells [40]. MET was also confirmed to promote H2O2 production in prostate cancer cells. Ca2+ is a secondary messenger involved in numerous cellular physiology processes. Disruption of Ca2+ homeostasis induces cell death by apoptosis or autophagy [41]. In particular, Ca2+ overload into mitochondria causes mitochondrial dysfunction, which is represented by a decrease in mitochondrial membrane potential [42]. Ruthenium red inhibits mitochondrial Ca2+ uniporters and inhibits mitochondrial Ca2+ accumulation [43]. In our study, the combination with ruthenium red mitigated mitochondrial depolarization and cell death caused by MET. These findings imply that Ca2+ overload into mitochondria is involved in MET-induced apoptosis of prostate cancer.
play a role in human small cell lung carcinoma [14]. In addition, serotonin promotes proliferation in carcinoid valve disease, which is supposed to be mediated by 5HT1b [31]. In human pancreatic carcinoid cells, serotonin exerts mitogenic effects, which are alleviated by the 5HT1A/1B receptor antagonist [13]. Moreover, 5-HT receptor antagonists and serotonin-uptake inhibitors, including 6-nitroquipazine, zimelidine, and fluoxetine, exert growth inhibitory effects on prostate cancer [16,32]. Histological studies have confirmed high expression of 5-HT1A/ 1B in prostate cancer tissues that metastasize to the lymph nodes and bones [26]. Further, the expression of these receptors was higher in prostate cancer tissues than in benign prostate hyperplasia tissues. PC3 and DU145 cells are hormone-independent prostate cancer cell lines that show higher expression of 5-HT1A/1B receptors than that in LNCaP cells, a hormone-dependent cell line, especially at high growth density [33]. MET is a serotonin receptor antagonist and has a high affinity to 5HT1A/1B [34]. Although some physiological functions have been shown in poultry and gastropods, little is known about the effects of MET in human cells [18,35]. In previous studies using PC3 and DU145 cells, MET dramatically reduced viability at concentrations between 10 and 20 μM [33]. Similarly, in our study, MET significantly reduced cell proliferation at concentrations of 10 and 20 μM in PC3 and 5, 10, and 20 μM in DU145 cells. However, it is still unknown whether the activation or inhibition of 5HT1A/1B is involved in the survival and growth of prostate cancer cells, as suggested in a previous study [33]. Oxidants can regulate the activity and expression of signaling proteins and transcriptional factors involved in cell survival and stress response [36]. Furthermore, oxidants stimulate several mechanisms 18
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Fig. 5. Effects of MET on the regulation of signal transduction in PC3 and DU145 cells. Phosphorylated AKT [A], S6 [B], JNK [C], c-Jun [D], ERK1/2 [E], and P38 [F] were estimated in response to MET (0, 5, 10, and 20 μM). The experiment was conducted at least three times. Asterisks indicate the significance of the effect of MET (***p < 0.001, **p < 0.01, and *p < 0.05).
induces ER stress-dependent apoptosis [46]. In addition, oxidant generation and JNK phosphorylation are involved in the induction of autophagy [47,48]. MET increases the expression of ER stress proteins in prostate cancer cells. Since DU145 cells are autophagy-deficient cells, the results of western blot analysis performed only on PC3 cells show an increased expression of Beclin-1, p-P62, and p-ULK1 but a decreased expression of Atg5 [49]. The expression of the proteins responsible for the contact between mitochondria and ER was also regulated by MET. The ER-mitochondrial linkage is formed by various proteins covered in
Serotonin activates the PI3K/AKT and MAPK signaling pathways in prostate cancer cells [15]. Tianeptine sodium salt (TSS), a serotonin facilitator, has been shown to have an anti-invasive effect by suppressing the PI3K/AKT signaling and MMP9 in prostate cancer cells [44]. MET suppresses AKT and its downstream protein S6, with a decrease in cell migration. In addition, of the three MAPKs, JNK, ERK1/2 and P38, only JNK is activated by MET. In our previous study JNK activation was also associated with increased oxidant production and mitochondrial dysfunction [45]. Moreover, the activation of JNK in prostate cells 19
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Fig. 6. Effects of co-treatment with MET and inhibitors against JNK (SP600125), oxidants (NAC), and mitochondrial Ca2+ uptake (ruthenium red) in PC3 and DU145 cells. [A] Changes in intracellular H2O2 levels following MET, SP600125, and NAC treatment were confirmed by measuring Amplex red fluorescence. [B] Annexin V and propidium iodide fluorescence were measured to determine the regulation of apoptosis following MET, SP600125, and NAC treatment. [C] Rhod-2 fluorescence was observed to measure mitochondrial specific Ca2+ concentration by MET and ruthenium red treatment. [D] JC-1 fluorescence was measured to identify changes in mitochondrial membrane potential caused by MET and ruthenium red. [E] Annexin V and propidium iodide fluorescence was observed to analyze the effect on the regulation of apoptosis by MET and ruthenium red treatment. Asterisks indicate the significance of the effect of MET (***p < 0.001 and **p < 0.01).
Fig. 7. MET regulates the migrating properties of PC3 and DU145 cells. [A] The effect of MET on migration was confirmed using Transwell inserts. The number of cells migrated through the membrane was measured on images of five non-overlapping locations. [B] The mRNA expression of migration-related genes in response to MET treatment was measured via quantitative RT-PCR. Expression of all genes was confirmed in triplicate. Asterisks indicate the significance of the effect by MET (***p < 0.001). Scale bar represents 100 μm. 20
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Author's contributions GS and WL designed and directed the study. CY, GS, and WL performed experiments, wrote, and prepared the manuscript. CY designed the figures. All authors provided critical feedback and helped to shape the manuscript. Data availability statement The authors confirm that the data supporting the findings of this study are available within the article. Declaration of competing interest The authors have declared no conflict of interest. Acknowledgements This research was supported by a grant of the National Research Foundation of Korea (NRF) grant, funded by the Ministry of Science and ICT (MSIT) [grant number: 2018R1C1B6009048]. References Fig. 8. Schematic diagram describing the effects of MET on prostate cancer cells. MET inhibits AKT signaling pathways while increasing the phosphorylation of JNK proteins. Activated JNK increases intracellular H2O2 production followed by ER stress. Ca2+ released from ER is overloaded into mitochondria and induces mitochondrial dysfunction. The cytochrome c activates the apoptotic pathway, leading to DNA damage in the nucleus. Eventually, the proliferative capacity and migration of prostate cancer cells exposed to MET will decrease.
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this study, such as IP3R, MFN2, VAPB, GRP75, and PTPIP51 [50]. The junction of the ER and mitochondria formed by these proteins is called the mitochondrial-associated ER membrane (MAM), which plays an essential role in Ca2+ homeostasis in the cytoplasm [51]. MAM proteins are also involved in activating mitochondria-mediated apoptosis signals [52,53]. IP3R is a Ca2+ channel located on the ER membrane that regulates the release of Ca2+ from the ER into the cytoplasm [54]. IP3R also facilitates the influx of Ca2+ from the ER to the mitochondria by placing the mitochondria and ER close together by a chaperone protein called GRP75 [55]. Reduced expression of GRP75 prevents cell death mediated by Ca2+ influx into mitochondria [56]. The MFN2 complex and the VAPB-PTPIP51 complex are other tethering proteins responsible for Ca2+ transport between the ER and mitochondria [50]. MFN2 promotes apoptosis through the PI3K/AKT signaling pathway in breast cancer cells [57]. PTPIP51 is known to have higher expression in prostate carcinoma tissue than in benign prostate hyperplastic cells [58]. However, little is known regarding whether MAM proteins responsible for Ca2+ homeostasis are involved in cell death in prostate cancer, and further study is required. In conclusion, this study may be of significance because we have identified specific anticancer mechanisms of serotonin receptor antagonists, which are considered potential therapeutics (see Fig. 8). Serotonin has been shown to contribute to the survival and growth of various types of cancer including prostate cancer. In other carcinomas, therapeutic effects based on the oxidative stress of serotonin receptor antagonists or serotonin transporter inhibitors have been suggested. For the first time, to our knowledge, we found that serotonin antagonists mediate oxidative stress, ER stress, mitochondrial dysfunction, and activation of JNK signaling in prostate cancer cells, leading to apoptosis and reduced cell migration. Considering the significant contribution of neuroendocrine regulation to the proliferation of prostate cancer cells, we believe that various types of modulators of serotonin activity, in addition to MET, may have therapeutic effects.
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