Emodin‐Induced Necroptosis in Prostate Cancer Cells via the Mitochondrial Fission HSP90/MLKL/PGAM Pathway

Currently, prostate cancer is one of the major malignant tumors in males. Recurrence and metastasis are the main obstacles that prevent the effective treatment of prostate cancer. In the present study, we aimed to evaluate emodin (EG) against human prostate cancer PC3 and DU145 cells. Our study showed that EG significantly decreased the cell viability of PC3 and DU145 cells and strikingly induced non‐apoptotic cell death via necroptosis that was visualized through colony formation assay, Hoechst 33258 staining, and TEM analysis. Furthermore, RNA‐sequencing and KEGG functional enrichment analysis revealed that the necroptosis‐related pathway was activated upon EG treatment in PC3 cells. mRNA and protein expression of necroptosis markers were analyzed by qPCR and immunoblotting, which implied that EG‐induced cell necroptosis via enhancing the expression of MLKL and HSP90AA1 activating PGAM pathway which is considered as a key mediator of mitochondrial fission and leading to ROS generation in PC3 and DU145 cells. Thus, our findings suggested that EG is a new small molecule agonist that induced necroptosis in prostate cancer cells via the mitochondrial fission HSP90/MLKL/PGAM pathway.


Introduction
Nowadays, cancer is the most dangerous disease worldwide, and tens of millions of patients are finally diagnosed with cancer each year. Prostate cancer is a global disease, and its incidence rate and mortality rate rank the second most common cancer-causing death in men worldwide. [1] Chemotherapy is the optimum strategy to augment survival rates and is convenient for treating prostate cancer in the current situation. However, multiple drug resistance (MDR) of prostate cancer is one of the main obstacles to achieving successful treatment and management of chemotherapy. [2] Thus, new chemotherapy agents or lead compounds are deserved to find, which induce new programmed cell death mechanisms against prostate cancer to overcome apoptosis resistance.
Necroptosis is an abnormal form of regulatory necrotic cell death and has been proven to be related to the defense mechanism in organisms against acute pancreatitis, retinal detachment, and cancer immunity. [3,4] Main morphological characteristics of necroptosis are rupture of the moderate chromatin condensation, cytoplasmic swelling, and plasma membrane, biochemical features of which include dropped ATP levels and activated RIP1, RIP3, and MLKL. [5][6][7][8] MLKL is a key mediator of necrosis signaling transduction downstream of the kinase RIP3 and RIP5. [5] Activation of PGAM5, a mitochondrial phosphatase, is another crucial component of necroptosis that can lead to the linear rupture of mitochondria, which is a very important segment in the early stage of cell necrosis. More interestingly, PGAM5 plays a pivotal role in the multiple pathways of necroptosis, such as excessive growth of oxygen free radicals and excessive leakage of calcium ions. [6] In addition, PGAM5 is the anchor of RIP1/RIP3/MLKL necrosome on mitochondria presenting a new pathway to induce necroptosis via the mitochondrial fission pathway that involves recruitment and activation of a GTPase dynamin-related protein 1. [9][10][11] Therefore, researchers urged to develop new chemotherapy agents that utilize necroptosis as a mechanism of action for fighting sensitive and drug-resistant prostate cancer cell lines.
Emodin (EG) is a natural anthraquinone compound from herbs, such as Rheum palmatum, Polygonum cuspidatum, and Polygonum multiflorum, which has been used as TCM for about 2000 years. [12] EG is still used in various food additives and herbal preparations, which have been widely used as dietary supplements to help healthy foods exert the antitumor effect in China. [13][14][15][16] Previous studies confirmed that EG can be developed as an excellent anticancer agent. [12] However, EG has never been explored to assess its anticancer effects on prostate cancer and underlying molecular mechanisms as well as whether they trigger necroptosis in prostate cancer cells.
In the present study, the cytotoxicity of EG from Selaginella trichoclada was evaluated against human prostate cancer DU145 and PC3 cell lines by MTT methods. And we performed an RNA-sequencing measure on EG-treated DU145 and PC3 cells to recognize the mechanisms and predict biomarkers. The RNA-sequencing measure is an RNA analysis method based on next-generation sequencing, enabling us to measure and compare gene expression patterns with unprecedented resolution. [17,18] Then, the functional enrichment analysis was performed based on the differentially expressed genes (DEGs) to predict target genes and determine the regulatory relationship. Therefore, we tried to explore the potential molecular regulatory mechanisms of EG's action in DU145 and PC3 cells.

Emodin-induced prostate cancer DU145 and PC3 cells death
Emodin was isolated from the 70 % EtOH extract of Selaginella trichoclada (S. trichoclada). [13] To evaluate the effects of different concentrations of EG on prostate cancer cells, we used the MTT assay for testing cytotoxicity in DU145 and PC3 cells. 5-Fu and DMSO were treated as positive and negative control, respectively. The results showed that the activity of DU145 and PC3 cells in the EG-treated-48 h group was significantly lower than that in a negative control group with an IC 50 value of 18.22 � 0.76 μM, and 68.74 � 5.93 μM, respectively ( Figure 1). Notably, EG did not clearly suppress liver cell proliferation (143.25 � 18.9 μM), which suggested that the natural compound EG is less toxic to normal liver cells.

Emodin-induced PCa cell death was apoptosis-independent
Whether EG induces necroptosis, the morphological changes of DU145 and PC3 cells after EG treatment were observed using colony formation assay, Hoechst 33258 staining, and transmission electron microscopy (TEM) analysis. The morphology of PC3 and DU145 cells was clearly visible and the number of cells was significantly decreased as shown in Figure 2A. Then, the long-term inhibitory effects of EG on the proliferation of DU145 and PC3 cells were investigated through colony formation assay. The results of colony formation showed that EG had a dose-dependent effect on the proliferation and population dependence of DU145 and PC3 cells ( Figure 2B). In addition, staining with DNA-binding fluorochrome Hoechst 33258 revealed that there were non-typical apoptotic nuclear condensation and chromatin fragmentation in both cell lines ( Figure 2C). Besides, according to TEM analysis, the morphology of DU145 and PC3 cells after EG treatment exhibited none of apoptosisrelated cell morphological features, such as chromatin condensation and membrane blebbing. There was critical evidence that the swelling of organelles, progressively translucent cytoplasm, and rupture of the cellular membrane were visualized which are some of the classical features of necroptosis ( Figure 3). Thus, we concluded that there is a possibility of necroptosis execution induced by EG in DU145 and PC3 cells.
There is a close correlation between necroptosis and ROS's cellular content in determining cancer cells' fate. So, we urged to investigate the production of intracellular ROS in DU145 and PC3 and employed fluorescent dichlorofluorescein. The various concentrations of EG significantly induced intracellular ROS generation (green fluorescent) in DU145 and PC3 cells (Figure 4). Thus, we inferred that EG induces prostate cancer cell death via necroptosis related to ROS accumulation.

Emodin-mediated PCa cell death via necroptosis
Afterward, we performed transcriptome analysis data of prostate cancer cells treated with EG based on RNA-sequencing. This high-throughput approach allows us to discover genes and pathways that have been altered after EG treatment. The volcano plots of DEGs were shown in Figure 5A, the red and green colors representing up-expression and down-expression, respectively. A total of downregulated and upregulated genes (using 2-fold as the cut-off value) were identified according to RNA-sequencing data ( Figure 5B). KEGG analysis was applied to explore the biological functions of EG in prostate cancer, which suggested that highly enriched DEGs resulting after EG treatment in PC3 cells were associated with the necroptosis cell death pathway when compared with untreated cells (Figure 5D). KEGG enrichment analysis results showed that DEGs such as CAMK2 A, CAMK2B, H2AFY2, STAT6, PYGM, XIAP, TLR4, PARR4, IFNAR2, CASP8, and RIRK1 genes were all significantly downregulated by EG induction ( Figure 5C). Besides, BIRC3, PLA2G4A, TNFRSF10A, IL-1β, TICAM1, SQSTM1, HSP90AB1, PARP2, HIST1H2AE, MLKL, PPID, TNFAIP3, HSP90AA1, H2AFZ, PPIA, CHMP4A, FAF1, HIST2H2AC, RBCK1, SLC25A4, PGAM5, FTL, SLC25A5, VDAC2 and FTH1 genes were significantly upregulated following treatment with EG ( Figure 5C). Noted that MLKL and PGAM5 play a pivotal role in inducing necroptosis via the mitochondrial fission pathway. [19] A previous large-scale study documented that the molecular chaperone heat shock protein 90 (HSP90AA1) is highly expressed in tumor tissues in most cancer types except prostate cancer, which is a potential diagnostic and prognostic factor for cancer. [20] There is a great strategy that increases the expressions of HSP90AA1 and PGAM5 to inhibit cell proliferation in prostate cancer. Our findings provide a sight that EG could be a small molecule agonist to upregulate necroptosis in PC3 prostate cancer cells via the mitochondrial fission pathway.
We evaluated the expression of HSP90 and PGAM5 genes in DU145 and PC3 cells by referring to the results of cell morphology analysis and RNA-seq measure using RT-qPCR analysis. Figure 6A is showing the heatmap of DE-mRNAs in the EG group and control group, blue represents the up-regulated PGAM5 and HSP90AA1 mRNA levels. Then, for further exploration of the cellular signaling mechanism underlying EG against DU145 and PC3 cells, we analyzed the level of PGAM5 and HSP90AA1 genes. EG significantly up-regulated PGAM5 and  HSP90AA1 gene expression, stipulating further evidence that EG could be considered a strong agonist featuring necroptosis as a cell-killing mechanism against prostate cancer cells (Figure 6B and 6C). Interestingly, PGAM5 and HSP90AA1 mRNA expression were dose-dependent in PC3 cells ( Figure 6B). These results indicate that the mitochondrial fission HSP90/MLKL/ PGAM signaling is critical for EG-induced necroptosis in PC3 cells.
To further confirm the results, we investigated the expression of PGAM5 protein by immunofluorescence staining. As shown in Figure 7, fluorescence imaging revealed that EG treatment induced the expression level of PGAM5 protein in DU145 and PC3 cells (the red colors represent PGAM5 protein). Interestingly enough, PGAM5 protein appeared in the nucleus of DU145 and PC3 cells after EG treatment. PGAM5 is responsible for the execution of the early stage of necroptosis, which consists of the activation of mitochondrial fission, Ca 2 + overload, and excessive ROS generation leading to mitochondrial fragmentation and disruption of mitochondrial movement which eventually evoke cell death. Subsequently, Western blotting further analyzed the protein level of HSP90 and PGAM5. EG significantly increased the expressions of HSP90 and PGAM5 proteins in DU145s and PC3 cells compared with the control group ( Figure 8). Finally, those findings provide strong evidence that EG induces necroptosis in prostate cancer via the mitochondrial fission HSP90/MLKL/PGAM pathway.
For nearly four decades, natural products from herb medicines or plants remain a major source of lead compounds and candidate drug discovery. Natural products have been confirmed to reduce infection rates and the mortality rate of different animal models. Emodin is an active ingredient of TCM as a naturally occurring anthraquinone derivative. Emodin inhibits cell growth in various types of cancer cells and regulates genes and proteins related to the control of cell apoptosis, cell invasion, metastasis, and cell cycle arrest. [8,21] Moreover, the synergistic enhancement of apoptosis and combination of chemotherapy drugs is very important for cancer treatment, which has attracted attention as a promising therapeutic approach. [22] However, prostate cancer cells are resistant to chemotherapy or defective in apoptosis induction. Therefore, the development of new drugs to enhance different forms of non-apoptotic cell death is expected to provide a promising therapeutic strategy for cancer patients.
In the present article, we noted that Emodin-induced death in DU145 and PC3 prostate cancer cells are independent of apoptosis. There is a close correlation between necroptosis and ROS's cellular content in determining cancer cells' fate. [11] Moreover, we observed the accumulation of intracellular ROS production in DU145 and PC3 cells, as well as the rupture of the cellular membrane, progressively translucent cytoplasm, and swelling of organelles, which are typical features of the necroptosis process. Necroptosis is an abnormal form of necrotic cell death, which plays a vital role in cancer treatment. Necroptosis is related to the defense mechanism in organisms against acute pancreatitis, retinal detachment, and cancer immunity. [23] Afterward, we performed RNA-sequencing-based transcriptome analysis data on prostate cancer cells after being treated with EG. Transcriptome analysis showed that BIRC3, PLA2G4A, TNFRSF10A, IL-1β, TICAM1, SQSTM1, HSP90AB1, PARP2, HIST1H2AE, MLKL, PPID, TNFAIP3, HSP90AA1, H2AFZ, PPIA, CHMP4A, FAF1, HIST2H2AC, RBCK1, SLC25A4, PGAM5, FTL, SLC25A5, VDAC2, and FTH1 genes were upregulated which are the regulatory genes of necroptosis cell death and could be considered as potential targets of EG in prostate cancer treatment. Inhibition of HSP90 blocks membrane translocation of activated MLKL, which implicates HSP90 as a modulator of necroptosis at the level of MLKL. [24] In human tumor cells, knockout of PGAM5 alleviates necroptosis induced by TNF-α, a calcium ionophore, and ROS, which plays a key role in oxidative stress-induced necroptosis. [6] PGAM5 increases the phosphorylation level of Cyclophilin D to open mitochondrial permeability transition pores (mPTPs) and increase ROS production, evoking endothelial necroptosis. [11] The mPTPs opening is mainly caused by the increase of the matrix Ca 2 + concentration and oxidative stress. [25,26] Above mentioned studies also confirm our findings that EG enhances the expression of the HSP90AA1 gene and activates the PGAM pathway leading to intracellular ROS generation. We determined that mitochondrial fission mediated by PGAM takes place in response to EG which ultimately results in necroptosis. The sketch map of the underlying molecular mechanisms associated with the anticancer effect of EG in PC3 and DU145 cells is illustrated in Figure 9.

Conclusions
In summary, we presented a natural anthraquinone derivate EG that could potentially be a small molecule agonist in prostate cancer treatment inducing necroptosis via the mitochondrial fission HSP90/MLKL/PGAM pathway.

Reagents
Emodin (EG) was separated from S. trichoclada and was prepared into a 1 ml stock solution dissolved by DMSO and stored at À 20°C for later use. The centrifuge tube was purchased from NEST Biotechnology Co. Ltd

Plant material
The whole herbs of Selaginella trichoclada were collected from Shimen (Hunan province, China) in August 2015, which was identified by Prof. Zhenji Li (Xiamen University, Xiamen, China). A voucher specimen (20150801) was deposited in the Xiangya School of Pharmaceutical Sciences, Central South University. [27]

Extraction and isolation
The air-dried whole herbs of S. trichoclada (10.0 kg) were extracted with 70 % EtOH (80 L × 2, 2 h/each) under reflux conditions. The solvent was concentrated under reduced pressure to yield a dried residue (418.0 g), which was suspended in H 2 O, then successively partitioned with petroleum ether, AcOEt, and BuOH. The AcOEt fractions (54.8 g) were preliminarily separated on silica gel CC with

Cell cytotoxicity assay
Emodin was evaluated to observe the effects on human prostate cancer DU145 and PC3 cell lines using the MTT assay. DU145 and PC3 cells were seeded in 96-well cell culture plates (density: 1 × 10 5 cells/well) in RPMI-1640 containing 10 % FBS (MRC, Jiangsu, China) and 1 % penicillin/streptomycin and incubated at 37°C and 5 % CO 2 . On the following day, the cells were treated with various  concentrations of EG for 48 h. The cells were then treated with 10 μl of the MTT solution at 5 mg/mL for 4 h followed by the addition of 150 μl of DMSO to each well. Finally, the measurement of absorbance was carried out at 490 nm using an ELISA reader.

RNA extraction and sequencing
Total RNA was extracted using the mirVanaTM miRNA isolation Kit (Ambion) according to the manufacturer's protocol. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evaluate the quality and concentration of total RNA. Samples with RNA integrity number (RIN) � 7 were analyzed subsequently. According to the manufacturer's instructions, the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) was used to construct the cDNA library. Then, these cDNA libraries were sequenced on the Illumina sequencing platform (Illumina HiSeqTM 2500), and 125bp/150bp paired terminal readings were generated

Bioinformatics analysis
Use Trimmatic to process raw data (raw readings). Remove readings containing ploy-N and low-quality readings to obtain clean readings. Then use hisat2 to map the clean readings to the reference genome. Use cufflinks to calculate the FPKM value of each gene, and obtain the reading of each gene by counting the htseqcount. [28][29][30] DEG uses the DESeq R package function to estimate SizeFactors and nbinomTest for identification. P value < 0.05 and folding change > 2 or < 0.5 were set as the threshold of significant differential expression. The hierarchical cluster analysis of DEG was carried out to explore gene expression patterns. [31] The GO enrichment and KEGG pathway enrichment analysis of DEG were performed using R based on hypergeometric distribution, respectively. [32,33] Then, by using cuff comparison software to compare reference genomes and known annotation genes, gene structure expansion and identification of new transcripts are carried out.

Colony formation assay
DU145 and PC3 cells (1 × 10 6 cells) were seeded into 6-well plates (NEST Biotechnology Co. Ltd) and treated with EG for 48 h and then cultured for 14 days (the medium refreshed every 3 days). On the 14th day, the supernatant was discarded and cells were fixed with 4 % paraformaldehyde for 20 min. After PBS washing was repeated twice. Cells were incubated at room temperature for 20 min after staining with 0.1 % crystal violet. After, cells were washed with water and allowed to dry overnight. After counting colonies containing more than 50 cells, a diagram was drawn by software.

Morphological analysis by Hoechst 33258 staining assay
In order to investigate apoptosis, morphological analysis was performed by Hoechst 33258 staining. Fix cells with methanol acetic acid for 10 min, then use 100 % in the dark at room temperature 100 μl Hoechst 33258 stain for 10 min. Then the cells were washed twice with PBS for examination and immediately photographed under the fluorescence microscope with an excitation wavelength of 330-380 nm. The definition of apoptotic cells is based on changes in nuclear morphology, such as chromatin condensation and fragmentation.

Cell morphological assessment
The DU145 and PC3 cells were seeded into a 6-well plate and treated with EG for 48 h. After drug intervention, the images were observed with an inverted microscope (Leica, Germany). At the same time, the cells were also cultured in a 6 cm culture dish and treated with different concentrations of EG for 48 h. After that, the cells were harvested by trypsinization and mixed with the supernatant. Then, the cells were centrifuged at 2500 rpm at 4°C for 5min. The cells were fixed in 2.5 % glutaraldehyde for 2 h, washed with PBS three times, and then fixed in 1 % osmium tetroxide buffer, dehydrated in graded series ethanol, and embedded in butyl resin. Thin section (50 to 60 nm) and stained with a saturated solution of uranyl acetate bound with lead citrate (HITACHI, Japan).

Reactive oxygen species (ROS) measurement
ROS production was measured in DU145 and PC3 cells after EG treatment using a cellular ROS assay kit. The DCFH-DA is easily oxidized to fluorescent DCF for the detection of intracellular ROS in PC3 and DU145 cells after EG treatment and DMSO (solvent control) as previously described, respectively. DCF was imaged by fluorescence microscopy (Carl Zeiss, Germany).

RT-PCR analysis
Prostate cancer cell lines PC3 and DU145 were cultured in RPMI-1640 medium containing 10 % heat-inactivated FBS and 1 % penicillin/streptomycin. All cells were cultured in an incubator at 37°C and 5 % CO 2 . Real-time PCR was used to detect gene expression. Extraction of total RNA from cells using RNA isolators (Novel Cell and Molecular Biotechnology, No. M050, Suzhou, China). cDNA is synthesized from total RNA by reverse transcription kit. The RT-PCR reactions were performed using ChamQ SYBR qPCR Master Mix. The reaction primers are as follows:

Immunofluorescence staining
DU145 and PC3 cells were seeded into 24 wells plates and EG treatment after 24 h. After washing with PBS for 48 h, the cells were fixed with 4 % paraformaldehyde for 30 min and permeabilized with 0.1 % Triton X-100 into PBS for 30 min. The cells were washed with PBS and blocked with 5 % BSA in PBS for 2 h, and incubated with PGAM5 polyclonal antibody (Proteintech Group, Inc, 28445-1-AP, Wuhan, China) in 5 % BSA at 4°C overnight. After cells were washed with PBS, Cy3-conjugated secondary antibody (Proteintech Group, Inc, SA00009-2, Wuhan, China) was applied for 2 h in dark, and then PBS-washed cells were subjected to staining with DAPI for 3 min. Finally, DU145 and PC3 cells were imaged by a fluorescence microscope (Zeiss, Germany).

Western blotting
The harvested cells were washed with PBS and lysed with protein lysis buffer. Protein concentrations of the proteins were determined by using a BCA Protein Assay Kit (Multisciences, Hangzhou, China). The sample protein was separated by SDS polyacrylamide gel electrophoresis, transferred to the PVDF membrane (Merck Millipore, Billerica, MA), and mixed with the designated PGAM5 polyclonal antibody, HSP90 antibody, and α-microtubulin antibodies were incubated overnight. After washing with TBST three times and incubating with a secondary antibody at room temperature for 2 h, the protein expression was detected with ECL Prime Western blotting kit (NCM Biotech, P10100, Suzhou, China). SPSS 18.0 was used for statistical analysis. Statistical data are expressed as mean � standard deviation. Student t-test was conducted to compare the differences between the two groups. P values less than 0.05 were considered statistically significant.

Author Contributions
Z. X.: performed the experiments and analyzed the data; F. Y. K.: edited; Y. X.: designed the experiments, analyzed the data, and wrote the manuscript. P. Y.: revised the manuscript.