The human prostate carcinoma PC-3 and DU-145 cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. PC-3 cells were cultured in F12 medium (containing 300 mg/l L-glutamine and 1.5 g/l NaHCO₃; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.). DU-145 cells were maintained in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Both cell lines were cultured at 37°C in a humidified incubator with 5% CO₂. The HSPE inhibitor, OGT 2115, MG-132 and cycloheximide (CHX) were purchased from MedChemExpress.

The analysis of TCGA prostate adenocarcinoma data (17) was performed by using the UALCAN (https://ualcan.path.uab.edu/analysis.html) platform according to previously published protocols (18,19). P<0.01 and log2 |Fold Change| >1 were considered as the significant thresholds. In the survival analysis, the high and low expression groups were determined using the median expression level as the cut-off. The Pearson correlation coefficient between HPSE and MCL-1 was calculated using the GEPIA (http://gepia.cancer-pku.cn/index.html) platform.

A total of six prostate cancer tissue samples (including the adjacent normal tissue) were collected from Taizhou Central Hospital (Taizhou, China), and written consent was obtained from all patients for the use of their tissues in the present study. Patients who were diagnosed with advanced prostate cancer from May 2021 to Jan 2023 were included in the present study. The inclusion criteria were as follows: i) Patients with stage IV–V prostate cancer; and ii) patients with a single primary tumor or patients who had only one prior tumor. The exclusion criteria were as follows: i) Patients whose prior cancer was prostate cancer; ii) patients with incomplete follow-up data; iii) patients with only death certificates or autopsy records; and iv) patients whose time of malignancy diagnosis was not known. The baseline patient demographics and clinical characteristics are shown in Table I. Gleason score was determined by following the ‘Gleason Grading of Prostatic Carcinoma: Definition of Grading Patterns and Proposal for a New Grading System’ guidelines (20). The present study was performed in accordance with the ethical standards of The Taizhou Central Hospital Research Committee and The Declaration of Helsinki, or comparable ethical standards. The study was approved by The Medical Ethics Committee of Taizhou Central Hospital (approval no. 2021-SC-076).

Each patient and mouse tissue sample was treated according to the following protocol: The 30-µm free-floating sections were deparaffinized, antigen retrieval was performed, and the endogenous peroxidase activity was removed. Briefly, the samples were rehydrated using xylene and graded concentrations of ethanol (100% ethanol for 5 min three times, 95% ethanol for 5 min once and 80% ethanol for 5 min once), incubated in sodium citrate (10 mmol/l, pH 6.0) at 95°C for 10 min and then cooled down to room temperature, followed with blocking for endogenous peroxidase using 3% hydrogen peroxide (Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Sections were permeabilized with 0.1% Triton and blocked in 10% goat serum (Beyotime Institute of Biotechnology) for 30 min at room temperature. The tissue sections were then incubated with the relevant primary antibody overnight at 4°C. The HPSE antibody (1:200; cat. no. 24529-1-AP) was purchased from Proteintech Group, Inc. and the MCL-1 (1:200; cat. no. 94296) and Ki-67 (1:200; cat. no. 12202) antibodies were purchased from Cell Signaling Technology, Inc. Following the primary incubation, sections were washed using PBS and then incubated with a goat anti-rabbit secondary antibody conjugated with HRP (cat. no. 554021; 1:200; BD Pharmingen; BD Biosciences) for 30 min at room temperature. Then, DAB staining and hematoxylin counter-staining were performed for 2 min at room temperature. Images of the sections were collected using a light microscope (Olympus BX-51).

Cell viability was assessed using the MTT assay. PC-3 and DU-145 cells (3×10³ cells/well) were seeded into 96-well cell culture plates and treated with OGT 2115 at different concentrations (300, 100, 33.33, 11.11, 3.67 and 1.22 µM) for 72 h at 37°C. 0.3% DMSO was set as control solvent. Subsequently, 20 µl MTT reagent (5 mg/ml) was added to each well and incubated at 37°C for 4 h. The MTT crystals were dissolved using DMSO for 10 min at room temperature with gentle shaking, and the absorbance at 492 nm was recorded.

Total RNA from cells following treatment (PC-3 cells were treated with OGT 2115 at concentrations of 0, 10, 20 and 40 µM, whereas DU-145 cells were treated with concentrations of 0, 25, 50 and 100 µM, both for 24 h) was extracted using RNAiso reagent (Takara Biotechnology Co., Ltd.) and complementary (c)DNA was synthesized using an RT kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. Subsequently, the cDNA was amplified using SYBR (Takara Biotechnology Co., Ltd.) in a PCR Thermal Cycler Dice Real-Time System according to the manufacturer's protocol. mRNA expression levels were analyzed using the 2^−∆∆Cq method (21) and were normalized to the internal reference gene, GAPDH. The primers used for qPCR were as follows: GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-GCCTTCTCCATGGTGGTGAA-3′; and MCL-1 forward, 5′-GGGCAGGATTGTGACTCTCATT-3′ and reverse, 5′-GATGCAGCTTTCTTGGTTTATGG-3′.

PC-3 cells were treated with OGT 2115 at concentrations of 0, 10, 20 and 40 µM, whereas DU-145 cells were treated with concentrations of 0, 25, 50 and 100 µM, both for 24 h. For the CHX and MG132 assays, CHX or MG132 was added to the culture medium at a final concentration of 35 µM or 10 mM, respectively. The CHX group was pretreated with OGT 2115 at concentrations of 0, 25, 50 and 100 µM for 18 h, and cell lysates were collected 6 h after CHX treatment. The MG-132 group was pretreated with OGT 2115 at concentrations of 0, 25, 50 and 100 µM for 22 h, and cell lysates were collected 2 h after MG-132 treatment. Total protein from cells following treatment was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Equivalent amounts of proteins (50 mg; quantified by BCA kit, Beyotime Institute of Biotechnology) were then separated via SDS-PAGE using a 10% gel and then transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). Subsequently, the membranes were blocked with non-fat dry milk (5%) in TBS with 0.1% Tween-20 for 2 h at room temperature, then probed with primary antibodies against HPSE (1:1,000; cat. no. 24529-1-AP; Proteintech Group, Inc), MCL-1 (1:1,000; cat. no. 94296; Cell Signaling Technology, Inc.) and tubulin (1:5,000; cat. no. sc-32293, Santa Cruz Biotechnology, Inc.) for 12 h at 4°C. Following the primary antibody incubation, the membranes were incubated with an anti-rabbit IgG HRP-conjugated secondary antibody (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) for 2 h at room temperature. The separated proteins were detected using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). Tubulin was used as the loading control. The blots were scanned and semi-quantified using an Image Quant LAS 4000 Min. (GE Healthcare).

A synthetic siRNA targeting MCL-1 (si-MCL-1; 5′-GUGCCUUUGUGGCUAAACATT-3′) was purchased from Shanghai GenePharma Co., Ltd. Scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as a negative control (NC). When the cell density reached 60–70%, PC-3 and DU-145 cells were transfected with siRNA using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol with a final siRNA concentration of 100 nM.

PC-3 cells were treated with OGT 2115 at concentrations of 0, 10, 20 and 40 µM, whereas DU-145 cells were treated with concentrations of 0, 25, 50 and 100 µM. Following treatment with OGT 2115, the cells were cultured for 24 h and apoptosis detected via flow cytometry. For this, PC-3 and DU-145 cells were digested using trypsin for 2 min at 37°C, washed with PBS and resuspended in 100 µl 1X annexin V binding buffer containing 5 µl annexin V-FITC and 10 µl PI (Beyotime Institute of Biotechnology). Cells were then incubated for 15 min at room temperature in the dark. The percentage of PI-positive annexin V-FITC-positive/negative (PI⁺/AV⁻ plus PI⁺/AV⁺) and PI-negative (PI⁻)/AV⁺ cells were quantified using flow cytometry (CytoFlex S; Beckman Coulter, Inc.), and Kaluza Analysis software version 1.2 (Beckman Coulter, Inc.) was used for subsequent analysis.

PC-3 cells (2×10⁶ cells/200 µl) were inoculated subcutaneously into the right side of male BALB/c nude mice (age, 4–5 weeks; Beijing Vital River Laboratory Animal Technology Co., Ltd.). The mice were housed in sterile cages under laminar airflow hoods at 20°C, in a specific pathogen-free environment, under a 12-h light/dark cycle and provided with autoclaved chow and water ad libitum. Animal health and behavior were monitored every day. Nude mice were divided into the following two groups: i) Vehicle control (4% ethanol, 5% PEG 400 and 5% Tween 80) group, n=7; and ii) OGT 2115 treatment group, n=6 (gavage; 40 mg/kg; once daily). When the tumors reached 30–40 mm³ the mice were administered saline or OGT 2115 via gavage daily for 35 days. Vernier calipers were used to measure the length and width of the xenografts twice a week. The tumor volume was calculated using the formula volume=(Width² × length)/2. At the end of the experiment, the mice were anesthetized with isoflurane (4%) and O₂ gas at 300–500 ml/min using the R540 Mice and Rat Animal Anesthesia Machine (RWD Life Science Co., Ltd.) for 10 min prior to sacrifice via cervical dislocation. The humane endpoints followed to determine whether animals should be euthanized before the end of the study were body weight loss of >20% and a tumor volume of >1,500 mm³. In the present study, no animals reached these humane endpoints during the 3-month experiment. Subsequently, the tumors were dissected and weighed. A part of the tumor tissue was fixed with 10% formalin for 24–48 h at room temperature for subsequent experiments, the rest were immediately frozen in liquid nitrogen for storage in the event of further experimentation.

All experimental procedures involving animals were performed in accordance with The National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals in China (22). The present animal study was approved by The Medical Ethics Committee of Taizhou University Medical School (Taizhou, China; approval no. 2021-SX-015).

To determine cell death, a TUNEL assay was conducted. Each sample was fixed using 4% Paraformaldehyde Fix Solution (cat. no. P0099: Beyotime Institute of Biotechnology) for 96 h at room temperature. The 5-µm free-floating sections were deparaffinized, antigen retrieval was performed, and the endogenous peroxidase activity was removed using 3% hydrogen peroxide (Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Briefly, the samples were rehydrated using xylene and graded concentrations of ethanol (100% ethanol for 5 min three times, 95% ethanol for 5 min once and 80% ethanol for 5 min once), then immersed in 50 µl TUNEL reaction solution (cat. no. C1088; Beyotime Institute of Biotechnology), and then the slides were incubated for 60 min at 37°C in a humid darkened chamber. 4′,6-diamidino-2-phenylindole was subsequently applied to the slides for 5 min at room temperature in the dark to stain the nuclei, then mounted with Antifade Mounting Medium (cat no. P0128M; Beyotime Institute of Biotechnology), after which the slides were imaged with a fluorescence microscope, five fields of view observed by microscopy for each slide.

All statistical analyses were performed using GraphPad Prism 8.0 (Dotmatics). Statistical significance was assessed using an unpaired Student's t-test for two groups or a one-way ANOVA followed by Tukey's post hoc test for more than two groups. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To verify the abnormal expression of HPSE and MCL-1 in the development of prostate cancer, immunohistochemistry was performed on adjacent normal and prostate cancer tissues from patients. The results demonstrated that HPSE and MCL-1 were expressed in the cytoplasm and that the protein expression levels in the prostate cancer tissues were markedly higher compared with the adjacent normal tissues (Fig. 1A and B).

The expression profile of HPSE in prostate cancer tissues was also explored using a TCGA dataset (17). As shown in Fig. 1C, statistical tabulation analysis of the dataset demonstrated that HPSE expression was associated with the pathological tumor grade, and expression in Gleason score 7, 8 and 9 tumors was significantly higher than that in Gleason score 6 tumors (P<0.001). Moreover, the 5,000 days overall survival rate of patients with high expression of the HPSE gene was significantly lower than that of patients with Low/Medium expression (P=0.015; Fig. 1D).

A weak correlation between HPSE and MCL-1 expression was also determined (r=0.36, P<0.01; Fig. 1E) using the GEPIA (http://gepia.cancer-pku.cn/index.html). As HPSE expression is known to be associated with tumor progression (23) and MCL-1 is related to apoptosis (24), these results suggested that HPSE and MCL-1 may be involved in cell survival in prostate cancer cells.

To further explore the role of HSPE in the progression of prostate cancer, PC-3 and DU-145 prostate cancer cells were treated with the HSPE inhibitor, OGT 2115 (Fig. 2A). The results demonstrated that, compared with cells treated with the control solvent, OGT 2115 treatment led to significantly decreased cell viability in both cell lines in a dose-dependent manner (Fig. 2B). The IC₅₀ of OGT 2115 in PC-3 cells was 18.4 µM and the IC₅₀ in DU-145 cells was 90.6 µM.

The effect of OGT 2115 on prostate cancer cell apoptosis was further determined using Annexin V-FITC/PI flow cytometry. PC-3 cells were treated with OGT 2115 at concentrations of 0, 10, 20 and 40 µM, whereas DU-145 cells were treated with concentrations of 0, 25, 50 and 100 µM. Following treatment with OGT 2115, the cells were cultured for 24 h and apoptosis detected via flow cytometry. In a dose-dependent manner, from the lowest OGT 2115 concentration to the highest, the apoptosis rates of the PC-3 cells were 4.21, 5.51, 8.12 and 9.50%, respectively, whereas the apoptosis rates of the DU-145 cells were 3.15, 11.02, 22.94 and 34.24%, respectively (Q1-LR in Fig. 3A).

In addition, OGT 2115 reduced MCL-1 protein expression levels in PC-3 and DU-145 cells but promoted the protein expression levels of caspase-3 and Bax (Fig. 3B and C).

To explore how the antitumor effect of OGT 2115 was associated with MCL-1 protein expression levels, RNA silencing experiments were conducted. MCL-1 protein expression levels were successfully downregulated using si-MCL-1 in PC-3 and DU-145 cells (Fig. 4A). Flow cytometry demonstrated that the PC-3 cell death rate was 6.25% in the si-NC group and 9.58% in the si-MCL-1 group (Fig. 4B). The cell death rate in the si-NC + OGT 2115 20 µM group was 19.4% and the cell death rate in the si-MCL-1 + OGT 2115 20 µM group was 22.64%. In DU-145 cells, the cell death rate was 7.24% in the si-NC group and 12.8% in the si-MCL-1 group (Fig. 4B). As shown in Fig. 4C, the cell death rate in the si-NC + OGT 2115 100 µM group was 22.68%, whereas the cell death rate of the si-MCL-1 + OGT 2115 100 µM group was 24.63%. These results support the suggestion that OGT 2115 inhibits prostate cancer cell viability.

To further validate the role of OGT 2115 in prostate cancer, in vivo experiments were conducted. PC-3 cells were injected subcutaneously into the right side of nude mice. When the tumors reached 30–40 mm³ the mice were administered saline or OGT 2115 via gavage daily for 35 days. The results demonstrated that tumor growth was significantly inhibited in the OGT 2115 group compared with the control group at days 28 and 35 (Fig. 5A). The average tumor volume of the control group was 399.2 mm³ and the largest was 738.1 mm³, whereas the average tumor volume of the OGT 2115 group was 201.7 mm³ and the largest was 365.5 mm³. Furthermore, Ki67 immunohistochemical analysis was conducted to assess the proliferative ability of OGT 2115-treated prostate cancer cells. The results demonstrated a significant decrease in Ki67⁺ cells in the OGT 2115 group compared with the control group (Fig. 5B). To further explore the effect of OGT 2115 on apoptosis in nude mice xenografts, TUNEL staining was performed. The results demonstrated that OGT 2115 treatment significantly increased the percentage of TUNEL⁺ apoptotic cells in nude mice xenografts compared with the control (Fig. 5B). Furthermore, the HPSE and MCL-1 protein expression levels in the OGT 2115 group were markedly lower compared with the control group (Fig. 5C).

As OGT 2115 had been determined to reduce MCL-1 protein expression levels in prostate cancer cells and xenografts, the underlying mechanism of OGT 2115-regulated MCL-1 expression was further investigated. RT-qPCR and western blotting were conducted to determine the MCL-1 mRNA and protein expression levels. In PC-3 cells, OGT 2115 treatment was administered at 0, 5, 10 and 20 µM for 24 h. In DU-145 cells, OGT 2115 treatment was administered at 0, 25, 50 and 100 µM for 24 h. The results of the RT-qPCR analysis demonstrated that OGT 2115 significantly reduced the MCL-1 mRNA expression levels in PC-3 and DU-145 cells compared with the 0 µM group, in a dose-dependent manner (Fig. 6A). Furthermore, the results of the western blotting analysis demonstrated that OGT 2115 markedly reduced the MCL-1 protein expression levels in prostate cancer cells in a dose-dependent manner (Fig. 6B). The protein synthesis inhibitor, CHX, was also administered to the OGT 2115 group for 6 h before detecting changes in MCL-1 protein expression. The results demonstrated that CHX did not affect the MCL-1 protein expression levels (Fig. 6B). Furthermore, the OGT 2115 group was treated with MG132, an inhibitor of proteasomal degradation. The results demonstrated that MG-132 markedly inhibited the decrease in MCL-1 protein expression levels caused by OGT 2115 treatment (Fig. 6B). It can therefore be concluded that OGT 2115 facilitates MCL-1 protein degradation instead of protein production in prostate cancer cells.