Introduction
Prostate cancer (PCa) is the most common cancer and remains a leading cause of cancer-related deaths among men worldwide [
1‐
3]. The treatment strategies for localized PCa mainly depend on targeting AR (androgen receptor) signaling as it is predominantly activated during PCa progression [
4]. Several inhibitors targeting this pathway (e.g. enzalutamide/abiraterone/apalutamide/bicalutamide. etc.) have been successfully used to constrain tumor progression. However, the efficacy of these treatments is typically limited, which leads to the emergence of drug resistance and tumor recurrence. Most PCa patients ultimately develop to the form of castration-resistant prostate cancer (CRPC), which only has a short median survival time of about 14 months [
5]. Hence, effective therapeutic options to overcome the drug resistance are urgently desired.
Ferroptosis is a novel type of regulated cell death [
6]. Distinct from apoptosis, necroptosis, pyroptosis, and autophagy, it is characterized by the cellular accumulation of iron and toxic lipid peroxides [
7]. Ferroptosis can be triggered by abnormal expression of transporters, such as SLC7A11 inhibition or transferrin and lactotransferrin activation, as well as antioxidant enzymes like GPX4 modulation. Small molecules, such as erastin, JKE-1674 and GPX4-IN-3, as well as various stress conditions like high temperature, low temperature, hypoxia, and radiation, could also induce ferroptosis [
8]. Extensive studies have demonstrated that ferroptosis induction is a promising therapeutic strategy to inhibit cancer progression and overcome drug resistance in cancer cells [
9,
10]. In the context of prostate cancer, the androgen-repressed gene DECR1 has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to androgen receptor (AR)-targeted treatments through regulating polyunsaturated fatty acids (PUFAs) oxidation. Targeting DECR1 disrupts PUFA oxidation and induces ferroptosis in both PCa and CRPC [
11]. This suggests that inducing ferroptosis could overcome resistance to anti-androgens therapy resistance in PCa. Notably, either inducing ferroptosis alone or synergizing with anti-androgen agents have shown significant efficacy in halting cell progression in PCa and CRPC [
12].
Sanguinarine chloride (S.C) is a natural benzophenanthridine alkaloid extracted from various sources, including the roots of Sanguinaria canadensis, seeds of Argemone Mexicana, and the leaves and fruits of Macleaya cordata [
13]. Numerous studies have reported its pharmacological activity in cancer therapy [
13‐
15]. Recently investigations have highlighted that the anti-cancer activity of sanguinarine mainly depends on inducing the generation of reactive oxygen species and suppressing the JAK/STAT pathway [
14,
16‐
18]. Additionally, sanguinarine displayed anti-metastatic properties and the ability to reverse epithelial-to-mesenchymal transition in estrogen receptor-positive (ER +) breast cancer [
19]. Notably, sanguinarine has displayed effectiveness against multidrug resistance in human cervical and ovarian cancer cells [
13,
20].Evidence suggests that sanguinarine may contribute to ferroptosis by reducing intracellular glutathione content in cisplatin-resistant ovarian cancer cells. As the Xc − /GSH/GPX4 axis serves as an important antioxidant system in ferroptosis by catalyzing the reduction of lipid peroxides, sanguinarine might induce ferroptosis in cancer cells through the reduction of intracellular glutathione levels. In fact, recent study demonstrated that sanguinarine could induce ferroptosis in human cervival cancer cells in an H
2O
2-dependent manner [
13]. Despite these discoveries, the detailed understanding of the pharmacological actions of S.C on prostate cancer and castration-resistant prostate cancer, as well as the intricate molecular signaling mechanisms involved in ferroptosis, remains limited.
In this study, we aim to explore the role of S.C in prostate cancer (PCa) therapy. This study demonstrated that S.C effectively decreased the viability, clonogenicity, and tumorigenicity of PCa cells both in vitro and in vivo. In addition to inducing intrinsic apoptosis, S.C triggered ferroptosis in PCa cells, as evidenced by intracellular iron overload, MDA overexpression, and ROS accumulation. Mechanistically, S.C induced intracellular iron overload by up-regulating HMOX-1, a critical enzyme in heme breakdown, leading to heme degradation and the release of labile Fe
2+, a pivotal initiator of ferroptosis [
21,
22]. Additionally, our study uncovered the significant role of ROS in S.C-induced HMOX-1 expression. In this process, ROS may reduce the stability of BACH1, which binds to the HMOX1 promoter region, consequently suppressing its transcription in a USP47-dependent manner. Overall, our findings shed light on the pharmacological effects of S.C in PCa therapy, especially in the context of castration-resistant prostate cancer (CRPC), and present a promising new approach for treating PCa.
Materials and methods
Materials
The prostate cancer cell lines LNCaP, VCaP, 22RV1, PC3 and DU145 were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection. Enzalutamide resistant cell including LNCaP-enz and 22RV1-enz were kept in our lab. Sanguinarine chloride (T0129), Enzalutamide (T6002), Docetaxel (T1034), z-VAD-fmk (T7020), Ferrostatin-1 (T6500) Deferoxamine Mesylate (T1637) and all components in ourcompound library were purchased from Targetmol. CCK8 reagent (CYT001) was purchased from Yoche. N-Acetyl-L-cysteine (ST2524), Lipid Peroxidation MDA Assay Kit (S0131S), Total Glutathione Peroxidase Assay Kit with NADPH (S0058) and Annexin V-FITC Apoptosis Detection Kit (C1062L) were purchased from Beyotime. Mito-FerroGreen kit (M489) was purchased from Dojindo. Cycloheximide (HY-12320) was purchased from MedChem Express. The primary antibodies used for IHC and Western blotting were following: GAPDH (ab128915), caspase-9 (ab32539) and BAX (ab32503) were purchased from abcam. BIM (A19702), HMOX1 (A11102), BACH1 (A5393), SLC7A11/xCT (A2413), GPX4 (A1933), KEAP1 (A21724), PERK (A18196), USP47 (A15461), Histone H3 (A2348) and ki67 (A21861) were purchased from Abclonal. HIF1α (AF1009) was purchased from affinity. BCL2 (2872), STAT3 (12640S), PARP (9532), cleaved-caspase3 (9664S) and BAK (12,105) were purchased from Cell Signaling Technology. BCL-XL (66,020-1-Ig), caspase 8 (66,093-1-Ig), caspase 9 (66,169-1-Ig), MCL1 (66,026–1-Ig) and NRF2 (16,396-1-AP) were purchased from proteintech.
Cell culture
LNCaP, VCaP, 22RV1, PC3, DU145,LNCaP-enz and 22RV1-enz cells were cultured in RPMI1640 medium or Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 ℃ with 5% CO2.
Plasmid construction and transfection
All overexpression plasmid (pcDNA3.1-HMOX1 and pcDNA3.1-BACH1) was provided by Tsingke Biotechnology. HMOX1 knockdown vectors (pLKO.1-HMOX1-a/b/c) were synthesized by Synbio-Technologies. The target sequences of sh-HMOX1-a/b/c were as following: sh-HMOX1-a, 5ʹ—cctccctgtaccacatctatg—3ʹ; sh-HMOX1-b, 5ʹ—gctcaacatccagctctttga—3ʹ; sh-HMOX1-c, 5ʹ—acagttgctgtagggctttat—3ʹ. Lentivirus was produced by transfection of PLKO.1 vectors together with the packaging plasmid psPAX.2 and the envelope plasmid pMD2.G into HEK293T cells. Virus-containing supernatant was collected at 48 h after transfection. DU145 and PC3 cells were infected with lentiviral supernatants in the presence of polybrene for 12 h. These infected cells were further selected for two days with puromycin (8 μg/ml).
Wound-healing assay
PC3 and DU145 cells were pre-treated with 0, 0.5, 1.0 µM S.C for 24 h. Cells were then plated into 6-well plate at a density of 5 × 105 cells/well and were cultured till to 90% confluence. A wound in each well was created with pipette tip. Cells were then further cultured with serum-free medium for 0 h and 24 h. Wound-healing process was photographed at each time point.
Cell viability assay
Cells were plated into 96-well plates and treated with indicated compounds for 24 h or 48 h. CCK8 reagent was added into wells and incubated for 2–4 h. Subsequently, the absorbance at 450 nm was measured by a microplate reader.
Clonogenic assays
The clonogenic assay was performed to assess the clonogenicity capabilities of the indicated cells, following a previously described protocol [
23]. Briefly, cells were treated with varying concentrations of S.C (0, 0.1, 0.5, and 1 μM) and allowed to grow for ten days, with regular changes of fresh medium every 3–4 days. Following removal of the media, the colonies were washed with ice-cold PBS, fixed with 4% paraformaldehyde, stained with crystal violet solution for 15 min at room temperature, and rinsed with distilled water to remove excess dye. The resulting colonies were then counted for each sample.
Hoechst 33,258 staining
PC3 and DU145 cells were seeded into 24-well plates and treated with S.C at concentrations of 0.5 μM and 1.0 μM for either 24 h or 48 h. Subsequently, the cells were fixed with 75% ethanol, stained with Hoechst 33,258 solution, and visualized using a fluorescence microscope.
mRNA expression analysis by real-time PCR
mRNA expression levels were analyzed following a previously described protocol [
23]. Briefly, total RNAs were extracted from the stimulated prostate cancer cell lines using TRIzol reagent (Takara Biotechnology Co., Dalian, China). Subsequently, the extracted RNA was converted into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China). The expression levels of the target genes were quantified using the SYBR Premix Ex Taq kit (Takara, Dalian, China) and the Bio-Rad CFX-96 thermal cycler(Bio-Rad, Hercules, CA). Normalization of the target gene mRNA expression levels was achieved by co-amplification of
β-actin. Primers sequences used for real-time PCR were:
HMOX1 (forward:5ʹ—gctatgtgaagcggctccac—3ʹ; reverse: 5ʹ—cagggctttctgggcaatc—3ʹ);
β-actin (forward: 5ʹ—gcacagagcctcgcctt—3ʹ; reverse:5ʹ—gttgtcgacgacgagcg-3ʹ).
Western blotting assay
Prostate cancer cell lines were treated in vitro as indicated and lysed in the cell lysis buffer (catalog no. P0013B; Beyotime Biotechnology, China) presented with protease /phosphatase inhibitor cocktail (catalog no. 5872; Cell Signaling Technology, Danvers, MA). The lysate were quantified with the bicinchoninic acid (BCA) kit (Beyotime Biotechnology, China). Then, the supernatants were subjected to 10% SDS-PAGE gel and then transferred to PVDF membranes (0.45 μm, Amersham, cat no. 10600023) for detection of NRF2,STAT3, BACH1, PERK, KEAP1, HIF-1a, USP47, H3, HMOX1, SLC7A11, GPX4, PARP, pro-caspase3, caspase3, pro-caspase9, caspase9, caspase8, BCL2, BAK, BIM, BAX and GAPDH proteins.
Malondialdehyde (MDA) and total glutathione peroxidase assay
PC3 and DU145 cells were plated in 100 mm dishes and treated with DMSO or S.C for 48 h. Then, the contents of MDA and total glutathione peroxidase level were quantified using the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime, China) and Total Glutathione Peroxidase Assay Kit (S0058, Beyotime, China) in accordance to the manufacturer’s instructions.
Annexin V-FITC–propium iodide assay
VCaP, PC3 and DU145 cells were plated in 60 mm dishes and treated with DMSO or S.C (1.0 μM)for 48 h. Then, cells were used to determine number of apoptotic cells with the Annexin V-FITC apoptosis kit according to the manufacture’s instruction (C1062L, Beyotime, China). Cells were subjected to flow cytometry (BECKMAN CytoFLEX) to calculate the apoptotic cells.
Reactive oxygen species (ROS) measurement
Intracellular ROS levels were measured using the fluorescent probe DCFH-DA as described previously [
23]. Briefly, the stimulated prostate cancer cell lines were exposed to 25 μM DCFH-DA for 30 min in dark. Then, Cells were subjected to flow cytometry (BECKMAN CytoFLEX) to monitor the fluorescence.
Determination of intracellular labile iron
For the cellular labile iron assay, indicated cells were plated in 60 mm dish at the density of 5 × 105 cells and treated with S.C or DMSO for 48 h. Then, cells were washed with HBSS three times and stained with Mito-FerroGreen (5.0 μM) in HBSS for 30 min at 37 ℃ in the dark. Cells were subjected to flow cytometry (BECKMAN CytoFLEX) to monitor the fluorescence. Intracellular labile iron level was quantified with the MFI value.
Transmission electron microscopy
PC3 and DU145 cells were treated with S.C or DMSO for 24 h. Cells collected by trypsinization were fixed with 2.5% glutaraldehyde, followed by 1% OsO4. After dehydration, thin sections were stained with uranyl acetate and observed under a transmission electron microscope (JEM-1230, JEOL, Japan).
Immunohistochemistry and histology
The xenograft tumor samples were collected and fixed in paraformaldehyde overnight. Samples were washed with PBS and dehydrated with ethanol, followed by embedding, sectioning and staining. For Primary antibodies used for immunohistochemical staining were as follows: rabbit polyclonal anti-HMOX1 (1:50; Abclonal) and rabbit monoclonal anti-ki67 (1:100; Abclonal, Lebanon, NH). Sections were also stained with hematoxylin–eosin. Images were obtained at 200 ×magnification on an Olympus microscope.
RNA-sequencing (RNA-Seq) and bioinformatics analysis
DU145 or PC3 cells were seeded into 100 mm dishes and were treated with S.C (1 μM) or DMSO for 12 h. Then, total RNA was extracted by trizol and was used for RNA-Sequencing by Genewiz-Azenta (Suzhou, China). 3 biological replicates were prepared for each sample. The differentially expressed genes were defined with a cutoff of fold change (FC) of ≥ 2 and the q value < 0.05.
Xenograft experiments/subcutaneous tumor model
All animal studies and procedures have been approved and performed in accordance with the Animal Care Welfare Committee of Lanzhou University Second Hospital Ethics approval and consent to participate (D2023-136). The CRPC cell line DU145 was used to build the subcutaneous tumor model. DU145 cells were harvested and suspended in PBS at a density of 2 × 107 cells/mL and mixed with matrix gel at a ratio of 1:1. Then, 50 μl suspension was implanted subcutaneously into the flank of 6–8 weeks old NCG (NOD-Prkdcem26Il2rgem26/Gpt) (GemPharmatech™) male mice for 2 weeks. Once these mice developed palpable tumors, mice were randomly divided into three groups: the control group, S.C (2.5 mg/kg) and S.C (5.0 mg/kg) treatment group. Mice weight and tumor size were measured every 2 days. After 7 times drug administration (ip, every two days), mice were sacrificed and tumors were dissected for further analysis, including HE staining, protein immunoblotting etc. Tumor volumes were measured by calipers and calculated as (length × width2)/2.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0(GraphPad Software, Inc., SanDiego, CA, USA). Data are presented as mean ± SD. Significant differences were examined using student’s t test or one-way ANOVA. Differences were considered statistically significant only when p < 0.05.
Discussion
Different candidate treatment options are available for prostate cancer patients, depending on the disease stage. For localized/locally advanced PCa, radical prostatectomy and radiotherapy are the standard treatment approaches. For metastatic androgen-sensitive PCa, androgen deprivation therapy (ADT) is standard treatment. However, drug resistance is inevitable and most patients develop into lethal metastatic castration-resistant prostate cancer (mCRPC). Until now, CRPC remains incurable [
37]. The inactivation of apoptosis is central to the development of CRPC. Several anti-apoptotic members of the
bcl-2 gene family, including BCL2, BCL-X, and MCL-1 showed high expression during the progression of prostate cancers [
38,
39]. ADT treatment also up-regulate the expression of anti-apoptotic proteins. For instance, androgens repress BCL2 expression via negatively modulating the activities of the E2F site in its promoter through activating the CDKI-RB axis [
40]. ADT treatment significantly increases the expression of oncogene
bcl-2. The high expression of these anti-apoptotic proteins could effectively prevent ADT-induced apoptosis in prostate cancer [
39]. Distinct from apoptosis, necroptosis, and pyroptosis, ferroptosis is a type of regulated cell death (RCD) characterized by intracellular iron accumulation and lipid peroxidation [
7]. Extensive studies suggest that ferroptosis plays a pivotal role in tumor suppression and reversing drug resistance [
9,
41].Targeted ferroptosis with its inducer significantly halted the tumor growth of treatment-resistant prostate cancer. The combination of a ferroptosis inducers (FINs) with enzalutamide or abiraterone acetate is highly synergistic for inducing advanced prostate cancer cell death in vitro and in vivo [
42]. Targeted ferroptosis is a promising therapeutic approach to overcome drug resistance in prostate cancer [
43]. Regretfully, there is still no specific ferroptosis inducer currently applied in clinical treatment.
In this study, we constructed a small molecular library targeting DNA damage/repair, angiogenesis, chromatin/epigenetic regulation, cytoskeletal signaling, JAK/STAT signaling, and other pathways. Based on screening of these small molecules, we found that S.C inhibited the activity of prostate cancer cells, including enzalutamide-resistant prostate cancer cells. Additionally, it enhances docetaxel’s cytotoxic effect on prostate cancer cells. Sanguinarine is a natural product extracted from the roots of Sanguinaria canadensis. Studies have reported that sanguinarine promoted cellular glutathione depletion [
16,
44]. This suggests that sanguinarine may induce cell death through ferroptosis. Recently, two groups reported that sanguinarine facilitates ferroptosis in non-small cell lung cancer and cervical cancer [
13,
45]. However, the mechanism by which sanguinarine triggers ferroptosis remains unclear. In vitro experiments also showed that S.C repressed colony formation and migration in prostate cancer cells. S.C treatment activated intrinsic apoptosis pathway in prostate cancer cells. However, pan-caspase inhibitor z-VAD-fmk only partially prevented S.C-induced cell death. Further studies demonstrated that ferrostatin-1 (fer-1) and deferoxamine (DFO) also partially decreased the cytotoxic activity of S.C in prostate cancer cells. S.C reduced the level of GSH and the expression of GPX4 and SLC7A11. MDA and Fe
2+ levels increased in S.C-treated prostate cancer cells. All results indicated ferroptosis was involved in S.C-induced cell death. Consequently, RNA-seq analysis of S.C treated cell identified HMOX1 as a possible target of S.C. HMOX1 is a stress-induced enzyme that metabolizes heme into carbon monoxide, iron, and biliverdin. Studies showed that HMOX1 mediated ferroptosis by promoting ROS production and iron accumulation [
46,
47]. We indeed found that S.C induced a high expression of HMOX1 in mRNA and protein levels, and HMOX1 knockdown decreased ROS, MDA and iron generation in S.C-treated cells. Consistent with previous research, we found the expression of SLC7A11 and GPX4 did not rely on HMOX1 (Fig.
4k–j). This suggests that S.C may facilitate ferroptosis by attenuating GSH generation and enhancing Fe
2+ overload in prostate cancer cells. Further analysis of transcription factors expression, revealed thatHMOX1 may contribute to S.C-induced ferroptosis. BACH1 down-regulation appears to be involved in S.C-induced ferroptosis as S.C treatment decreased BACH1 expression in nucleus and cytoplasm. BACH1 overexpression abolished S.C-induced HMOX1 expression. Further studies showed that the S.C-reduced BACH1 protein levels relied on decreasing its stability. BACH1 stability was mediated by USP47 via enhancing the deubiquitylation of BACH1 [
36]. Herein, we also found that S.C attenuated USP47 expression in prostate cancer cells. This implies that S.C may increase HMOX1 expression by decreasing BACH1 stability in a USP47 dependent manner. Extensive evidence has identified that ROS induced ferroptosis [
48‐
50]. Our study demonstrated that S.C-induced ferroptosis also depended on ROS accumulation. ROS scavenger NAC significantly decreased iron, MDA levels and enhanced BACH1-mediated HMOX1 transcriptional inhibition effects in S.C treated cells. Finally, in vivo experiments showed that S.C delayed tumor progression. Immunohistochemistry analysis confirmed that the tumor suppressive effect of S.C may indeed rely on activating ROS/USP47/BACH1/HMOX1 axis.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.