Blockade of neutral sphingomyelinase 2 exerts antitumor effect on metastatic castration resistant prostate cancer cells and promotes tumor regression when combined with Enzalutamide

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related deaths among American men. The development of metastatic castration resistant PCa (mCRPC) is the current clinical challenge. Antiandrogens such as Enzalutamide (ENZ) are commonly used for CRPC treatment. However, patients with androgen receptor (AR)-negative tumors do not respond to ENZ, while AR-positive tumors frequently develop resistance, limiting the long-term efficacy of this therapy. This study investigates the efficacy of neutral sphingomyelinase 2 (n-SMase2) inhibition by DPTIP, both alone and in combination with ENZ, as a therapeutic strategy for mCRPC. In vitro assays were conducted to determine the half-maximal inhibitory concentration (IC₅₀) of DPTIP and ENZ in mCRPC cells. The effect of these treatments on cell proliferation, migration, and colony formation was assessed. The antitumor effect of DPTIP was also evaluated in a preclinical PCa mouse model. Elevated n-SMase2 expression was observed in PCa patients compared to normal subjects at both mRNA and protein levels. In CWR-R1ca and PC-3 cells, DPTIP had IC₅₀ values of 10.31 and 14.57 µM, while ENZ had IC₅₀ values of 33.7 and 81 µM, respectively. Combined treatment significantly suppressed cell proliferation, colony formation, and migration of mCRPC cells. Mechanistically, the ERK1/2 activity and the expression of nSMase2 and NF-kB p65 were inhibited by DPTIP. The in vivo combination of DPTIP and ENZ reduced tumor size and weight more effectively than either drug alone, without significant changes in body weight. This study highlights the therapeutic potential of targeting n-SMase2 for mCRPC. Inhibition of n-SMase2 using DPTIP, both as a standalone treatment and in combination with ENZ, effectively suppressed the growth and migration of mCRPC cells. These findings suggest a promising novel approach to treating mCRPC and warrant further investigation in clinical settings.

Keywords: DPTIP; Enzalutamide; Prostate cancer; mCRPC; mouse model; n-SMase2.

Figure 1

Expression of n-SMase2 in human PCa tissues. A: Expression of n-SMase2 gene SMPD3 transcript retrieved from TCGA data of PCa compared to normal tissues. B: The protein expression of nSMase2 was evaluated by immunohistochemistry in tissue microarray slide comprising 100 cases of PCa tissue cores, 7 BPH, and 11 normal and tumor adjacent tissues cores. The protein expression was represented according to Gleason score (GS). Another slide was incubated only with secondary antibody and kept as a negative control (CTR). C: The immunostaining of nSMase2 was expressed as immunohistochemical score (IHS) which was used to compare PCa with normal tissues, age at diagnosis, Gleason score (GS), pathological stages (T2-4), lymph node metastatic status and distant metastasis (Met). Quantification of immunohistochemical score of n-SMase2 staining. Magnification is 50 µm (solid line) and 20 µm (dashed line). Data considered significant at P<0.01 regarding control groups. N: number; N0/N1: involvement of lymph nodes.

Figure 2

Cytotoxic effect of treatment of mCRPC cells with combined drugs. (A, B) CWR-22RV1 (A) and CWR-R1Ca (B) cells were treated with 0.1 and 0.5 of IC₅₀ of DPTIP (D) or enzalutamide (E), individually and in combination, for 72 hours. Cell viability assay was performed as indicated and the percentage of O.D. change relative to control cells was calculated. (C) Western blot analysis was performed for AR, AR-V7 and n-SMase2 expressions. β-actin was used as loading control. (D) The endogenous expression of n-SMase2 was calculated relative to the house keeping protein and expressed as arbitrary unit compared to the control cells. *, # denote significance at P<0.05 relative to vehicle control cells and individual treatment, respectively.

Figure 3

Effect of treatment of mCRPC cells with DPTIP and Enzalutamide on colony formation. CWR-22RV1 and PC-3 cells were treated with 0.1 and 0.5 IC₅₀ of DPTIP (D) and Enzalutamide (E), and their combinations for 14 days, fixed, stained and dried out for imaging. The number and size of cell colonies were counted and presented as mean + SEM as indicated in bar graphs. *, # depict statistical significance at P<0.05 regarding vehicle control or individual treatment, respectively. Each treatment was conducted in duplicate, and the experiment was independently repeated twice.

Figure 4

Effect of DPTIP and ENZ on mCRPC cell migration. A: About 5 × 10⁴ cells were seeded in upper insert in FBS-depleted medium while complete medium was added to lower wells. Cells were treated with 0.5 IC₅₀ of DPTIP and ENZ, and their combinations for 36 h. The migrated cells were fixed, stained, and imaged. B: The migrated cells were counted and presented as percentage of migratory cells. *, #, ≠ depict significance at P<0.05 compared to vehicle control, ENZ and DPTIP, respectively. NS: Nonsignificant. Magnification was 100×.

Figure 5

Molecular effect of DPTIP and Enzalutamide treatments on PC-3M cells. A: Cells were treated with 0.1 and 0.5 IC₅₀ of DPTIP (D) and Enzalutamide (E), and their combinations for 24 and 48 h. Cell lysates were collected and subjected to Western blot analysis. The membrane-bound proteins were incubated with antibodies against nSMase2, phosphorylated and total ERK1/2, and NF-kB p65. B: The relative expression was calculated as fold change relative to control cells and represented as mean ± SEM as indicated in bar graphs. *, # depict statistical significance at P<0.05 regarding vehicle control or individual treatments, respectively.

Figure 6

RNA-seq and molecular pathways in mCRPC cells treated with DPTIP. A: Visualizing GSEA enrichment results for DPTIP-treated vs. vehicle control PC-3M cells using ggplot2. B: Top five non-redundant enriched terms for the GO class molecular function. C: Quantitative Real-Time PCR analysis was performed as indicated and presented as fold change to cells received vehicle. *, # depict significance at P<0.01 regarding vehicle control cells.

Figure 7

Antitumor effect of DPTIP in preclinical PCa model. (A) A representative experimental design for mice treatment. Mice were injected with CWR-R1ca-luc cells (2 × 10⁶ cells). After 2 weeks, intraperitoneal injection of Enzalutamide (Enz) and DPTIP (10 mg/Kg each) was initiated and continued for another 3 weeks. (B) In Vivo Imaging was acquired at the baseline (week 1) and after 3 weeks of drug treatment. (C, D) Tumor size (C) and total body weight (D) were recorded twice a week for 3 weeks. (E) Tumor mass was excised after mice sacrifice and weighed. * depicts significance at P<0.05. NS: non-significant data.