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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 6  |  Page : 319-327

Leonurine suppresses prostate cancer growth in vitro and in vivo by regulating miR-18a-5p/SLC40A1 axis


Department of Urology, Changzhou Cancer (Fourth People's) Hospital, Changzhou, China

Date of Submission10-Jul-2022
Date of Decision29-Sep-2022
Date of Acceptance08-Oct-2022
Date of Web Publication26-Dec-2022

Correspondence Address:
Dr. Bin Liang
Department of Urology, Changzhou Cancer (Fourth People's) Hospital, No. 68 Honghe Road, Xinbei District, Changzhou, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.365459

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  Abstract 


Prostate cancer is a leading cause of cancer-associated death in males. Leonurine (Leo) is a pleiotropic anti-tumor agent isolated from traditional Chinese herb that was used in gynecologic treatments. However, its pharmacological effect against prostate cancer progression remains unclear. Here, we showed that Leo dose dependently inhibited prostate cancer cell proliferation, promoted cell apoptosis, and induced cell cycle arrest. Moreover, we noticed that miR-18a-5p was downregulated and the solute carrier family 40 member 1 (SLC40A1) is upregulated by Leo treatment. SLC40A1 knockdown by siRNA abrogated the inhibitory effect of Leo on prostate cancer progression. Notably, Leo also significantly inhibited prostate cancer progression in a subcutaneous xenograft tumor mouse model in vivo. This study further unveiled the mechanism by which Leo inhibited prostate cancer progression, which provides a promising potential for its future clinical application.

Keywords: Leonurine, miR-18a-5p, prostate cancer, solute carrier family 40 member 1


How to cite this article:
Liang B, Cui S, Zou S. Leonurine suppresses prostate cancer growth in vitro and in vivo by regulating miR-18a-5p/SLC40A1 axis. Chin J Physiol 2022;65:319-27

How to cite this URL:
Liang B, Cui S, Zou S. Leonurine suppresses prostate cancer growth in vitro and in vivo by regulating miR-18a-5p/SLC40A1 axis. Chin J Physiol [serial online] 2022 [cited 2023 Jan 28];65:319-27. Available from: https://www.cjphysiology.org/text.asp?2022/65/6/319/365459




  Introduction Top


Prostate cancer is one of the most prevalent malignant tumors in males and the fifth leading cause of cancer-related death over the world.[1],[2] In the past few decades, the mortality and morbidity of prostate cancer sharply increase along with the advent of elderly society. The onset of prostate cancer is insidious, but the late-stage symptoms, including nocturia, dysuria, hematuria, and severe pain, severely impair patients' quality of life.[3],[4] Despite the recent advances in prostate cancer therapy, most treatments only have limited therapeutic effects but with severe adverse complications.[5] Therefore, it is urgent to develop novel therapeutic targets for prostate cancer treatment.

Solute carrier family 40 member 1 (SLC40A1), also known as ferroportin, is located at chromosome 2, containing 8 exons and participating in iron transportation across cell membranes.[6] SLC40A1 is highly expressed in endothelial cell, hepatic cell, and macrophage, and a previous study reported that SLC40A1 is associated with the etiology of multiple cancer.[7],[8] MicroRNAs are major non-coding RNA molecules that regulate a wide range of biological processes, including cell migration, proliferation, and DNA repair.[9] miR-18a-5p has been documented to participate in cancer progression, and downregulation of miR-18a-5p by competitively binding with RUNX1 would result in the suppression of prostate cancer progression.[10] In our previous study, we have found that miR-18a-5p was upregulated and SLC40A1 was downregulated in prostate cancer tissues and we have further investigated how miR-18a-5p regulates SLC40A1 expression.[11]

Leonurine (Leo) is the main bioactive component of Leonurus japonicus Houtt., which is a classic traditional Chinese medicine firstly used in gynecologic treatments.[12] With recent advances in pharmacology, the function of Leo has been proven to be pleiotropic. Lin et al. reported that Leo attenuated the resistance of human cervical cancer cell to cisplatin by promoting apoptosis and inhibiting drug-resistant protein expression.[13] Mao et al. demonstrated that Leo could enhance H292 lung cancer cell apoptosis through activating the mitochondria-dependent pathway.[14] Notably, Leo was also found to inhibit chronic myelogenous leukemia malignancy through repressing miR-18a-5p function.[15] However, the pharmacological effect of Leo against prostate cancer progression and whether its function depends on miR-18a-5p/SLC40A1 axis remain to be elucidated.

In this study, we explored the pharmaceutical effects of Leo on prostate cancer cell proliferation, apoptosis, and cell cycle transition. The results showed that Leo suppressed prostate cancer progression depending on the miR-18a-5p/SLC40A1 axis. This study provides a promising potential of Leo in the clinical treatment of prostate cancer.


  Materials and Methods Top


Cell culture and reagents

PC-3 and DU145 cell lines were purchased from American Type Culture Collection (Bethesda, MD, USA). Cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (HyClone, USA) supplemented with 1% antibiotic cocktail (C0224, Beyotime, China) and 10% fetal bovine serum (Gibco, USA) in a humidified incubator at 37°C with 5% CO2. For SLC40A1 knockdown, cells were transfected with si-SLC40A1 or si-NC using Lipo3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer's instruction. 48 h after transfection, cells were collected for subsequent experiments. The siRNA sequences used in SLC40A1 knockdown are listed in [Table 1]. Leo was obtained from Selleck Co. Ltd (S3890, Shanghai, China).
Table 1: The siRNA sequences used in solute carrier family 40 member 1 knockdown

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Animals and xenograft nude mouse model

Male nude mice (4 weeks old) were purchased from Shanghai JSJ Co. Ltd (Shanghai, China), and mice were housed in specific pathogen-free condition with free access to food and water. All the animal procedures were agreed and authorized by Changzhou Cancer (Fourth People's) Hospital (Approval No. XK2021-047).[16]

Subcutaneous xenograft model was established following a previously published protocol.[17] 5 × 106 cells resuspended in phosphate-buffered saline (PBS) (100 μL) were subcutaneously injected into mice, and a caliper was used to measure tumor size weekly. Tumor volume was calculated as: tumor volume = (tumor length × tumor width2)/2. One week after modeling, mice were randomly subdivided into control group and Leo group with five mice in each group. Mice in the Leo group received Leo (150 mg/kg) treatment via tail vein injection twice a week, whereas mice in the control group received PBS injection as control. Five weeks after modeling, mice were sacrificed and tumors were harvested for weight measurement, miR-18a-5p detection, SLC40A1 detection, and Ki-67 staining.

Cell counting kit-8 assay

To evaluate cell proliferation, cell counting kit-8 (CCK-8) assay was performed using a commercial assay kit (C0037, Beyotime, Shanghai, China). Briefly, PC3 and DU145 cells were plated in 96-well plates at 2000 cells/well and treated with various concentrations of Leo for 3 days. At each indicated time point, cells were incubated with 10% CCK-8 working solution for 1 h, and then, supernatant from each group was collected for absorbance measurement at 450 nm using a microplate reader (Tecan, USA).

Colony formation assay

Cell colony formation capacity was evaluated following a previously published study.[18] In brief, PC3 and DU145 cells were plated in 6-well plates at 1 × 104 cells/well with indicated treatments. Forty-eight hours after incubation, cells were rinsed with PBS, fixed with 4% paraformaldehyde, and stained with 1% crystal violet solution. The number of colony formation units (CFUs) in each well was quantified.

5-Bromo-2-deoxyuridine immunofluorescent staining assay

BeyoClick™ EdU Cell Proliferation Kit (C0075S, Beyotime, Shanghai, China) was used to detect cell proliferative capacity following the manufacturer's instruction. In brief, PC3 and DU145 cells were plated in confocal dishes at 2 × 105 cells/dish with indicated treatments. Forty-eight hours after incubation, cells were rinsed with PBS, fixed with 4% paraformaldehyde, and stained with BrdU-Alexa Fluor 555. Nucleus was counterstained with DAPI. A laser confocal microscope (SP8, Leica, USA) was used to capture images. The number of BrdU+ cells in each field was quantified.

Quantitative real-time polymerase chain reaction assay

Real-time polymerase chain reaction (RT-PCR) was performed to detect miR-18a-5p expression in prostate cancer cells and xenograft tumor tissues.[19] Briefly, after indicated treatments, total RNA of each group was isolated using Trizol reagent (Invitrogen, USA) following the manufacturer's instruction. Then, cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, USA). RT-PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, USA), and the expression U6 was used as an internal control. The primer sequences used in RT-PCR are listed in [Table 2].
Table 2: The primer sequences used in RT-PCR

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Immunoblotting assay

After indicated treatments, cells were lysed in 1% sodium dodecyl sulfate (SDS) lysis buffer with commercial proteinase/phosphatase inhibitor cocktail (Bimake, China) on ice with stirring. Then, total proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membrane (Millipore), blocked with 5% nonfat milk, and finally incubated with primary antibodies at 4°C overnight. The following day, membranes were rinsed with 1X Tris-buffered saline with Tween-20 (TBST) several times and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, an enhanced chemiluminescence system was used for protein signal detection and the grayscale of the corresponding band was quantified using ImageJ software. The following antibodies were used in immunoblotting: anti-cyclin-dependent kinase 2 (CDK2) (1:1000; ab32147, Abcam, USA), anti-cyclin E (1:1000; ab33911, Abcam, USA), anti-GAPDH (1:1000; ab9485, Abcam, USA), anti-Bax (1:1000; ab32503, Abcam, USA), anti-Bcl-2 (1:1000; ab32124, Abcam, USA), anti-SLC40A1 (1:1000; ab217626, Abcam, USA), and anti-rabbit IgG H and L (1:1000; ab6721, Abcam, USA).

Flow cytometry assay

Cell cycle and cell apoptosis were evaluated using flow cytometry. For assessing cell cycle, Cell Cycle Analysis Kit (C1052, Beyotime, Shanghai, China) was used to detect the cell cycle distribution. After indicated treatments, prostate cancer cells were harvested, rinsed with precold PBS, and fixed using 70% ethanol at −20°C for 6 h. Then, cells were stained with the working solution consisting of PI and RNase for 15 min in the dark. A flow cytometry (CytoFLEX, Beckman Coulter) was used to analyze the cell cycle distribution. For assessing cell apoptosis, Annexin V-PE Apoptosis Detection Kit (C1065, Beyotime, Shanghai, China) was used to describe the cell apoptosis ratio following the manufacturer's instruction. After staining in the dark, a flow cytometry (CytoFLEX, Beckman Coulter) was used to analyze the cell apoptosis ratio. All the flowmetry data analysis was performed using FlowJo software.

Ki-67 staining assay

Immunohistochemical staining of xenograft tumor tissues was performed to detect Ki-67 expression according to a previously published paper.[20] Briefly, the harvested tumor samples were fixed, embedded in paraffin, and sectioned into 5 μm thick slides. Afterward, slides were dewaxed, rehydrated, and incubated with primary Ki-67 antibody (1:500; ab15580, Abcam, USA) and corresponding secondary antibody. A light microscope (Leica, USA) was used to capture images.

Statistical analysis

Data were presented as mean ± standard deviation, and at least three times each assay was repeated. Student's t-test or one-way analysis of variance with post hoc test was used to compare results from different groups. P < 0.05 was considered statistically significant. GraphPad software (GraphPad Prism 8, USA) was used to analyze data.


  Results Top


Leo inhibits prostate cancer cell proliferation

The chemical structure of Leo is presented in [Figure 1]a. Uncontrolled proliferation is one of the key features of malignant tumor development. To determine the pharmaceutical effect of Leo on prostate cancer progression, we first analyzed the proliferation capacity of PC3 and DU145 cells with/without Leo treatment. The proliferation curves revealed that Leo inhibited prostate cancer cell proliferation in a dose-dependent manner [Figure 1]b. A marker for proliferation inhibition is the decrease in CFU and BrdU+ cell ratio.[21] In line with proliferation curves, Leo dose dependently suppressed the colony formation potential of prostate cancer cells along with decreased BrdU+ cell ratio [Figure 1]c and [Figure 1]d. These results imply that Leo could inhibit prostate cancer cell proliferation.
Figure 1: Leo inhibits prostate cancer cell proliferation. (a) Chemical structure of leonurine. (b) CCK-8 assay was used to evaluate cell viability of PC3 and DU145 cells treated with various concentrations of Leo at 0 h, 24 h, 48 h, and 72 h, separately (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (c) Colony formation assay was performed to assess proliferation capacity of PC3 and DU145 cells treated with various concentrations of Leo. The number of colonies was quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (d) Fluorescent staining of BrdU was performed to identify BrdU+ cell in PC3 and DU145 cells treated with various concentrations of Leo. The number of BrdU+ cells was quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. ** indicates P < 0.01, *** indicates P < 0.001 compared with the Leo (0 μM) group. SLC40A1: Solute carrier family 40 member 1, CCK-8: Cell counting kit-8, ANOVA: Analysis of variance, BrdU: 5-Bromo-2-deoxyuridine.

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Leo leads to prostate cancer cell cycle arrest and apoptosis

Cell proliferation depends on proper cell cycle transition. To explore whether Leo inhibits prostate cell proliferation by cell cycle arrest, we analyzed the cell cycle distribution of PC3 and DU145 cells with/without Leo treatment by flow cytometry. The results demonstrated a significant cell cycle arrest after Leo treatment, as indicated by increased cell ratio in G1 phase and decreased cell ratio in S phase [Figure 2]a. CDK2 and cyclin E are the major regulators facilitating the transition from G1 phase to S phase.[22] Consistent with cell cycle analysis, CDK2 and cyclin E expressions were significantly suppressed by various concentrations of Leo in PC3 and DU145 cells, as displayed by immunoblotting [Figure 2]b.
Figure 2: Leo leads to prostate cancer cell cycle arrest. (a) Flow cytometry was performed to evaluate cell cycle in PC3 and DU145 cells treated with various concentrations of Leo. The cell ratios of G1 phase, G2 phase, and S phase were quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (b) Immunoblotting was performed to detect CDK2 and cyclin E protein expression in PC3 and DU145 cells treated with various concentrations of Leo. The grayscale value of the corresponding band was quantitated with ImageJ (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. * indicates P < 0.05 and ** indicates P < 0.01 compared with the Leo (0 μM) group. ANOVA: Analysis of variance.

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Escape from programmed cell death is another key feature of malignant tumor development. Next, we assessed whether Leo induces apoptosis in prostate cancer cells. To test this, we analyzed cell apoptosis ratio of PC3 and DU145 cells with/without Leo treatment using FITC-Annexin/PI staining. The results showed that Leo dose dependently induced prostate cancer cell apoptosis [Figure 3]a. Bax promotes cell apoptosis, whereas Bcl-2 suppressed this process.[23] Notably, a significant increase in Bax expression and a reduction of Bcl-2 expression induced by Leo treatment were confirmed by the immunoblotting [Figure 3]b. Collectively, these results indicate that Leo leads to prostate cancer cell cycle arrest and apoptosis.
Figure 3: Leo leads to prostate cancer cell apoptosis. (a) Flow cytometry was performed to evaluate the cell apoptosis ratio of PC3 and DU145 cells treated with various concentrations of Leo. The cell apoptosis ratio was quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (b) Immunoblotting was performed to detect Bax and Bcl-2 protein expression in PC3 and DU145 cells treated with various concentrations of Leo. The grayscale value of the corresponding band was quantitated with ImageJ (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. * indicates P < 0.05 and ** indicates P < 0.01 compared with the Leo (0 μM) group. ANOVA: Analysis of variance.

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Leo suppresses prostate cancer cell growth depending on the upregulation of solute carrier family 40 member 1

SLC40A1 is documented to be a major cancer-suppressing protein.[24] Our previous study found that miR-18a-5p regulated downstream SLC40A1 expression to modulate cancer cell proliferation and apoptosis.[11] In line with these findings, we also noticed a dose-related reduction in miR-18a-5p expression and an increase in SLC40A1 protein expression after Leo treatment in PC3 and DU145 cells [Figure 4]a and [Figure 4]b. To further investigate whether SLC40A1 is essential for the protective effect of Leo against prostate cancer progression, we transfected PC3 and DU145 cells with si-SLC40A1 or si-NC to knockdown SLC40A1 protein expression [Supplementary Figure 1]a. Notably, SLC40A1 knockdown was proven to suppress prostate cancer cell proliferation [Supplementary Figure 1]b. In the subsequent experiments, we choose the siRNA sequence with best knockdown efficiency for validation. As anticipated, SLC40A1 knockdown obviously abrogated Leo-mediated prostate cancer cell proliferation inhibition, as displayed in colony formation assay [Figure 4]c. Furthermore, Leo-induced apoptosis and cell cycle arrest were also reversed by SLC40A1 knockdown [Figure 4]d and [Figure 4]e. These data reveal that Leo suppresses prostate cancer cell growth depending on the upregulation of SLC40A1.
Figure 4: Leo suppresses prostate cancer cell growth depending on the upregulation of SLC40A1. (a) RT-PCR was performed to detect miR-18a-5p expression in PC3 and DU145 cells treated with various concentrations of Leo (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (b) Immunoblotting was performed to detect SLC40A1 protein expression in PC3 and DU145 cells treated with various concentrations of Leo. The grayscale value of the corresponding band was quantitated with ImageJ (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. ** indicates P < 0.01 compared with the Leo (0 μM) group. (c) Colony formation assay was performed to assess the proliferation capacity of PC3 and DU145 cells transfected with si-NC or si-SLC40A1 under Leo (800 μM) treatment. The number of colonies was quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups.(d) Flow cytometry was performed to evaluate the cell apoptosis ratio of PC3 and DU145 cells transfected with si-NC or si-SLC40A1 under Leo (800 μM) treatment. The cell apoptosis ratio was quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. (e) Flow cytometry was performed to evaluate cell cycle in PC3 and DU145 cells transfected with si-NC or si-SLC40A1 under Leo (800 μM) treatment. The cell ratios of G1 phase, G2 phase, and S phase were quantified (n = 3). One-way ANOVA with post hoc test was used to compare results from different groups. ** indicates P < 0.01 compared with the control group. ##indicates P < 0.01 compared with the Leo (800 μM) + si-NC group. ANOVA: Analysis of variance.

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Leo suppresses prostate cancer growth in vivo

To confirm the effects of Leo on prostate cancer progression in vivo, we established a subcutaneous xenograft model in nude mice and treated them with Leo or PBS. Notably, Leo treatment significantly attenuated xenograft prostate cancer progression in nude mice, as indicated by the decreased tumor volume [Figure 5]a. After 5 weeks' treatment, tumors derived from Leo-treated mice were much smaller and lighter than those from control mice. Meanwhile, no significant weight loss was detected in Leo-treated mice, indicating that the toxicity of Leo was limited [Figure 5]b. Notably, the miR-18a-5p expression was also attenuated by Leo treatment, whereas the downstream SLC40A1 protein expression was promoted [Figure 5]c and [Figure 5]d. Ki-67 is a biomarker labeling cells in highly active proliferation period.[20] As expected, Leo treatment obviously diminished Ki-67 expression in xenograft tumor tissue in vivo [Figure 5]e. Thus, we conclude that Leo could suppress prostate cancer growth in vivo.
Figure 5: Leo suppresses prostate cancer growth in vivo. A xenograft tumor model was established by subcutaneously injecting PC3 cells, and nude mice bearing tumor were treated with PBS as control or Leo (150 mg/kg) intraperitoneally twice a week for 5 weeks. (a) Tumor size was measured every week starting from the time of injection. After 5 weeks, the mice were sacrificed, and tumors were imaged and weighted (n = 6). Student's t-test or one-way ANOVA with post hoc test was used to compare results from different groups. (b) Body weight of mice at sacrifice (n = 6). Student's t-test was used to compare results from different groups. (c) RT-PCR was performed to detect miR-18a-5p expression in the excised tumor masses (n = 6). Student's t-test was used to compare results from different groups. (d) Immunoblotting was performed to detect SLC40A1 protein expression in the excised tumor masses. The grayscale value of the corresponding band was quantitated with ImageJ (n = 6). Student's t-test was used to compare results from different groups. (e) Immunohistochemical staining of Ki-67 was used to identify the Ki-67+ cell in the excised tumor masses. The cell ratio positive for Ki-67 staining was quantified with ImageJ (n = 6). Student's t-test was used to compare results from different groups. **indicates P < 0.01 compared with the control group, SLC40A1: Solute carrier family 40 member 1, ANOVA: Analysis of variance.

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  Discussion Top


Recent studies have identified a series of traditional Chinese medicine monomers with excellent anti-tumor potential. However, the molecular mechanism underlying the pharmaceutical effects of these drugs has been barely elucidated. Here, we highlighted Leo as an anti-tumor agent by inhibiting cell proliferation and promoting cell apoptosis and cell cycle arrest in prostate cancer PC3 and DU145 cells. Furthermore, downregulation of SLC40A1 caused abrogation of the anti-tumor effect induced by Leo, which indicated that miR-18a-5p/SLC40A1 axis is indispensable for the action of Leo. Thus, targeting SLC40A1 by Leo provides a promising therapeutic strategy to combat prostate cancer progression.

Leo has exhibited an anti-tumor effect in various solid tumors. According to systemic studies, unlimited replicative potential and evading apoptosis are proven to be hallmarks of cancer.[25] Replicative immortality enables cell to escape from cell death, causing uncontrolled growth and metastasis. During cell proliferation, CDK2 and cyclin contribute to cell cycle transition, and Bax and Bcl-2 regulate apoptosis balance. Here, Leo inhibited these cycle-related protein expressions with upregulated Bax and downregulated Bcl-2 expression. In line with our findings, a previous study on leukemia showed that Leo markedly attenuated xenograft tumor growth by inhibiting proliferation.[15],[26] Therefore, we conclude that Leo might exert an anti-tumor effect by not only disrupting cell replicative potential but promoting cell apoptosis.

SLC40A1 and its anti-tumor role in prostate cancer have only been described recently,[11] and this study is the first to report the regulation of SLC40A1 under Leo treatment. SLC40A1 was previously reported to participate in the maintenance of intracellular iron homeostasis by transporting iron ions between cell membrane.[27] Recent studies have found that ferroptosis induced by iron overload is associated with tumorigenesis, and ablation of SLC40A1 leads to escape from ferroptosis, which in turn exacerbated tumor progression.[28],[29] Thus, upregulation of SLC40A1 by drugs provides a feasible way to sensitize tumor cell to ferroptosis. miR-18a-5p is a miRNA modulating gene expression in tumor progression. Previous studies reported that miR-18a-5p could modulate epithelial-mesenchymal transition in breast cancer by downregulating cancer-suppressing gene expression.[30] In this study, we found that miR-18a-5p expression was abnormally upregulated, and its downstream SLC40A1 expression was inhibited. However, Leo treatment significantly reversed this trend by disrupting the inhibition of SCL40A1 expression due to miR-18a-5p both in vitro and in vivo. Furthermore, SLC40A1 knockdown by siRNA transfection counteracted the pharmaceutical effects of Leo on prostate cancer cell, indicating that SLC40A1 might be indispensable for Leo action. Considering the essential role of SLC40A1 in iron metabolism, we plan to investigate the relevance of Leo action and ferroptosis in the future study.

We noticed several limitations in this study. On the one hand, even though xenograft tumor model was used here, patient-derived xenograft (PDX) models would further contribute to the validity of this study. The microenvironment of PDX xenograft closely resembles the original tumor, facilitating drug discovery and translational application of our finding in laboratory.[31] On the other hand, Leo has a broad range of targets; thus, it is difficult to use a single pathway or protein target to explain Leo's pharmaceutical effect. The direct targets and binding domains need to be clarified in our future studies.


  Conclusion Top


This study reveals that Leo inhibits proliferation and induces apoptosis of prostate cells via regulating miR-18a-5p/SLC40A1 signaling. Therefore, Leo might provide a novel and safe therapeutic approach for prostate cancer treatment in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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