Exploring NCX4040, an Aspirin Derivative, as a Potential Treatment for Benign Prostatic Hyperplasia

Abstract

Benign Prostatic hyperplasia (BPH) is a leading cause of lower urinary tract symptoms which affects men above 50 years of age. Chronic inflammation and abnormal proliferation of stromal and epithelial cells are implicated in BPH disease onset. The symptoms of BPH include back pain and difficulty in emptying bladder. Finasteride, sildenafil and tamsulosin are some of the drugs used to ease difficulty urinating and relax the muscles of the gland. Transurethral resection of the prostate or laser surgery can be performed to treat severe symptoms. However, these therapies have deleterious effects such as low blood pressure, ejaculatory dysfunction, and lump formation. Hence, there is an unmet need for potential drugs against BPH. Nonsteroidal anti-inflammatory drugs (NSAIDs) have proven to be effective in cancers but their applicability in BPH condition is yet to be fully explored. Aspirin, one of the NDSAIDs, has anti-tumor and anti-inflammatory properties at higher doses. NCX4040, a nitric oxide releasing derivative of aspirin, could prove to be effective against BPH, since it can inhibit abnormal cell proliferation and serve as a vasodilator. We hypothesize that NCX4040 would be an effective drug to treat BPH. BPH-1 epithelial and WPMY-1 stromal cells were used as in vitro models of BPH. MTT assay was performed to check the inhibitory effect of NCX4040 and blocking agents like catalase and N-acetyl-L-cysteine (NAC) were explored on cells after treatment. Clonogenic assay was done to explore the colony formation ability of cells. Spheroid assay was performed to analyze the anti-proliferative effect of NCX4040. Annexin V/PI and cell cycle analysis was performed to check for apoptosis and cell cycle arrest in the cells. Western blot was done to assess the signaling molecules altered by NCX4040 in BPH-1 cells. Confocal immunofluorescence was employed to analyze the dynamics of actin filaments after treatment in cells. Our studies revealed that NCX4040 inhibited the cell viability of BPH-1 and WPMY-1 in a dose dependent manner with IC50 predicted at 5µM and 2.5µM respectively. Of note, catalase and NAC blocked the effect of NCX4040 on prostate cells. Colony formation assay result implied a gradual decrease in the number of colonies of cells treated with NCX4040 with 2.5µM and 5µM doses. Spheroid assay in BPH-1 cells showed inhibitory effects after treatment. Cell cycle analysis by flowcytometry inferred that cell cycle arrest at G2/M phase and annexin V analysis indicated that activation of apoptosis in cells following treatment. Phalloidin staining showed decrease in the actin filament intensity in cells. At the molecular level, NCX4040 downregulated the expression of key markers such as RhoA, p65, COX-2, PCNA, Cyclin D3, and PDE-5 in BPH-1 cells. Taken together, NCX4040 could be used as a potential agent to manage BPH with minimal side effects, which needs further evaluation in animal models.