Biotin-Linked Ursolic Acid Conjugates as Selective Anticancer Agents and Target-Identification Tools for Cancer Therapy

Abstract

Ursolic acid (UA), a naturally occurring pentacyclic triterpenoid, exhibits potent anticancer properties; however, its poor solubility and bioavailability limit its therapeutic application. To overcome these challenges and facilitate molecular target identification, a set of biotin-conjugated UA derivatives (5a-d) was synthesized through selective C-28 alkylation and biotinylation. The use of microwave-assisted synthesis significantly enhanced both reaction efficiency and product purity. Among the derivatives, compound 5c exhibited increased cytotoxicity and selectivity against bladder cancer cell lines, surpassing UA in its ability to induce apoptosis, generate reactive oxygen species (ROS), and halt cell cycle progression at the G1 phase. Proteomic profiling revealed that 5c interacts with proteins involved in ER stress, RNA processing, cytoskeletal remodeling, and metabolic regulation. These findings underscore the potential of biotinylated UA derivatives as multifunctional chemical probes for mechanistic studies in the development of targeted therapies for cancer.

Keywords: Ursolic acid; anticancer agents; bioconjugate; biotin; in vitro assays; microwave-assisted synthesis; natural product; proteomics; selectivity index.

Figure 1

Molecular targets of UA.

Scheme 1

Synthesis of UA-biotin hybrid conjugates via alkyl linkers at C-28.

Figure 2

The percent cell viability of tumorigenic human (5637, T24, HT-1376), mouse (MB49), and non-tumorigenic bladder epithelial (UROtsa) cell lines after treatment with a selected dose of UA and 5c. The percentage cell viability was calculated relative to the untreated control cells. MTT reduction assay was performed after 72 h of respective treatment. Results are expressed as the mean of percent cell viability ± standard deviation (SD) (n = 4), and one-way ANOVA was performed to determine the significance.

Figure 3

Estimation of oxidative stress: Treatment with UA or 5c increased ROS levels in various bladder cancer cells. ROS levels were estimated at 24 h using DCFDA. The relative fluorescence unit (RFU) was normalized to that observed in untreated cells and expressed as a percent increase. Data are Means ± SD, (n = 4). Relative significance, expressed as p-value are calculated based on one-way ANOVA.

Figure 4

Estimating apoptotic cell death: Representative quadrant analysis images of bladder cancer cells 5637 and MB49 treated for 24 h with UA or 5c (10 & 15 μM each), or Staurosporine (Sta, 0.2 μM; a positive inducer of apoptosis). The intensity of Annexin V-FITC (bound to externalized phosphatidyl serine) and PI (DNA) in a single cell suspension was measured using the NovoCyte Quanteon flow-cytometer. The intensity of green and red fluorescence were recorded, and quadrant analysis was performed using NovoExpress software (version 1.6.1).

Figure 5

Quantitative representation of apoptotic cell death: The absolute quantification of early and late apoptosis in 5637 & MB49 cells is presented as bar graphs. Data shown are mean ± SD of three experiments (n = 3), and one-way ANOVA was performed to determine the significance of the differences between untreated control and treated cell samples.

Figure 6

The representation of cell cycle phase distribution after treatment of UA and 5c in the different bladder cancer cell lines (5637, HT-1376, T24, MB49). The flow cytometric images shown here are from the 3 independent experiments of data acquisition using NovoCyte Quanteon flow cytometer and NovoExpress software (version 1.6.1).

Figure 7

UA & 5c induced cytotoxicity reveals cell cycle arrest in G1 phase: Bar graph representing the different phases of the cell cycle. The phases of the cell cycle resolution were analyzed using NovoCyte Quanteon flow cytometry in the 5637, HT-1376, T24, and MB49 cells after 24 h of treatment with UA and 5c. The data were analyzed using NovoExpress software (version 1.6.1). The data shown here are the mean ± SD of n = 3, and a one-way ANOVA was performed to assess significance (* p > 0.05; *** p > 0.001; ns = Statistically non-significant).