RETRACTED: An Extensive Pharmacological Evaluation of New Anti-Cancer Triterpenoid (Nummularic Acid) from Ipomoea batatas through In Vitro, In Silico, and In Vivo Studies

Abstract

Prostate cancer (PCa) is the most common cancer in men, accounting for approximately 10% of all new cases in the United States. Plant-derived bioactive compounds, such as pentacyclic triterpenoids (PTs), have the ability to inhibit PCa cell proliferation. We isolated and characterized nummularic acid (NA), a potent PT, as a major chemical constituent of Ipomoea batatas, a medicinal food plant used in ethnomedicine for centuries. In the current study, in vitro antiproliferative potential against PCa cells (DU145 and PC3) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay; Western blot protein expression analysis; absorption, distribution, metabolism, excretion (ADME); pharmacokinetic prediction studies; and bisphenol A (BPA)-induced prostate inhibition in Sprague Dawley rats were conducted to gauge the anti-cancer ability of NA. Significant (p < 0.05 and p < 0.01) time- and dose-dependent reductions in proliferation of PCa cells, reduced migration, invasion, and increased apoptotic cell population were recorded after NA treatment (3−50 µM). After 72 h of treatment, NA displayed significant IC50 of 21.18 ± 3.43 µM against DU145 and 24.21 ± 3.38 µM against PC3 cells in comparison to the controls cabazitaxel (9.56 ± 1.45 µM and 12.78 ± 2.67 µM) and doxorubicin (10.98 ± 2.71 µM and 15.97 ± 2.77 µM). Further deep mechanistic studies reveal that NA treatment considerably increased the cleavage of caspases and downstream PARP, upregulated BAX and P53, and downregulated BCL-2 and NF-κB, inducing apoptosis in PCa cells. Pharmacokinetic and ADME characterization indicate that NA has a favorable physicochemical nature, with high gastrointestinal absorption, low blood−brain barrier permeability, no hepatotoxicity, and cytochrome inhibition. BPA-induced perturbations of prostate glands in Sprague Dawley rats show a potential increase (0.478 ± 0.28 g) in prostate weight compared to the control (0.385 ± 0.13 g). Multi-dose treatment with NA (10 mg/kg) significantly reduced the prostate size (0.409 ± 0.21 g) in comparison to the control. NA-treated groups exhibited substantial restoration of hematological and histological parameters, reinstatement of serum hormones, and suppression of inflammatory markers. This multifaceted analysis suggests that NA, as a novel small molecule with a strong pharmacokinetic and pharmacological profile, has the potential to induce apoptosis and death in PCa cells.

Keywords: BAX; apoptosis; docking; nummularic acid; p53; prostate cancer.

Figure 1

Chemical structure of NA.

Figure 2

Effect of NA on the viability of prostate cancer cells and IC₅₀ values. Note: MTT assay was used to determine the viability of cancer cells. (A) 24, 48, and 72 h treatment of DU145 cells and (B) 24, 48, and 72 h treatment of PC3 cells. (C) IC₅₀ values of NA against DU145. (D) IC₅₀ values of NA against PC3. (E) IC₉₀ values of NA against DU145. (F) IC₉₀ values of NA against PC3. Data are mean ± SEM of % cell viability (n = 3) at p < 0.05 and p < 0.01.

Figure 3

In vitro scratch assay on NA-treated prostate cancer cells. Note: DU145 cells were plated in 6-well plates and scratched at full confluency. (A) Migration of cells to heal the area of scratch was observed at 0 h, 12 h, and 24 h after treatment. (B) Reduction in area of scratch was photographed using Olympus CKX41 microscope and measured using ImageJ software. Data are mean ± SEM percent area of scratch in triplicate with a marked difference at p < 0.05 and p < 0.01.

Figure 4

Inhibition of colony formation by NA in prostate cancer cell lines. Note: DU145 cells were treated with 10 µM and 20 µM concentrations of NA for 48 h and then allowed to form colonies for 5–7 days. (A) Pictures of colonies for DU145 cell lines were taken using Olympus CKX41 microscope: (1) Control, (2) 1% DMSO, (3) NA (10 µM), and (4) NA (20 µM). (B) Densitometry analysis of colonies was performed in control and treated wells. Data are mean ± SEM (n = 3) of colony intensities measured using ImageJ software.

Figure 5

Western blot analysis of proteins associated with NA induced apoptosis. Note: Prostate cells and DU145 cells were treated with NA at concentrations of 5 µM and 10 µM for 48 h. (A) Data show increased cleavage of caspase 3 and downstream PARP, increased expression and upregulation of pro-apoptotic P53 and BAX, and decreased expression downregulation of anti-apoptotic NF-κB and BCL2. GAPDH was used as a loading control. (B) Fold change in cleaved PARP, cleaved caspase 3, P-53, BAX, BCL-2, and NF-κB expression after treating for 48 h with different concentrations of NA compared to control.

Figure 6

(a) Bioavailability radar chart for NA. The pink zone represents the physicochemical space for oral bioavailability, and the red line represents the oral bioavailability properties. (b) Predicted BOILED-Egg plot from swiss ADME online web tool for NA.