Repurposing HIV-Protease Inhibitor Precursors as Anticancer Agents: The Synthetic Molecule RDD-142 Delays Cell Cycle Progression and Induces Autophagy in HepG2 Cells with Enhanced Efficacy via Liposomal Formulation

Abstract

Hepatocellular carcinoma (HCC) remains a global health issue due to high incidence and mortality, complicated by limited therapeutic options and evolution of de novo resistance to conventional chemotherapy. In this study, we investigated the antiproliferative activity of RDD-142, a synthetic precursor of the HIV-1 protease inhibitor (HIV-PI) Darunavir analog, on the human hepatocellular carcinoma line (HepG2) and healthy hepatocyte line (IHH), both as a free molecule and in liposomal formulation. RDD-142 demonstrated a selective cytostatic effect on HepG2, preserving healthy IHH cells. Mechanistically, RDD-142 delayed cancer cell proliferation by attenuating the ERK1/2 signaling pathway, and concurrently, it activated the autophagic process via p62 up-regulation. These effects were linked to RDD-142 inhibitory activity on the chymotrypsin-like subunit of the proteasome, triggering a UPR-mediated stress response. Notably, the liposomal formulation of RDD-142 significantly enhanced intracellular intake and cytotoxic efficacy. RDD-142 demonstrated promising potential as a therapeutic agent for HCC. Its antitumor activity may be further amplified through liposomal nanoformulation, offering a successful strategy to reduce effective dosage and minimize adverse effects.

Keywords: HIV-protease inhibitor precursor; cell cycle delay; drug repositioning; hepatocellular carcinoma; liposomes; proteasome inhibitors.

Figure 1

Chemical structure of RDD-142.

Figure 2

Effect of RDD-142 treatment on cell proliferation. Representative proliferation profile of HepG2 (A) and IHH (C) under 24 h of treatment with RDD-142 at different concentrations (10–100 µM range), assessed using the xCELLigence system. The cell index (CI) was normalized to the last cell index recorded before RDD-142 addition, measured in real time. Representative IC₅₀ curves of RDD-142 in HepG2 (B) and IHH (D) cells calculated as means of normalized cell index ± SEM (n = 3). The normalized CI values, reported in the dose–response curve of RDD-142, were derived from the final measurements, taken after 24 h of treatment, at each tested inhibitor concentration.

Figure 3

HepG2 accumulation in G2/M phase of the cell cycle after RDD-142 treatment. Cytofluorimetric analysis of the cell cycle of HepG2 (A) and IHH (B) cell lines after treatment with increasing concentrations of RDD-142 (20 µM, 30 µM, and 42 µM). Cell cycle histograms, on the left, show a single representative experiment of the proliferation profile of cells after 24 h of treatment with DMSO and RDD-142 at 42 µM. The bar graphs on the right represent the average of cells in all phases of the cell cycle, expressed as means ± SD of three independent experiments; ** p ≤ 0.01; *** p ≤ 0.001. Immunoblot analysis (C) for the evaluation of Cyclin B1 and CDK 1/2 expression levels, as markers of cell cycle progression, in HepG2 cells treated with increasing concentrations of RDD-142 (20 µM, 30 µM, and 42 µM) for 24 h. Densitometry analysis, on the right, was performed by normalizing with the respective vinculin and the values are expressed as means ± SD of three independent experiments; ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

ERK1/2 attenuation and autophagy activation after RDD-142 treatment. (A) Protein expression level of ERK1/2 and its activation, evaluated by a Western blotting experiment after treatment with increasing concentrations of RDD-142 (20 µM, 30 µM, and 42 µM) for 24 h. Densitometry analysis for Western blotting experiments, below immunoblotting panels, was performed by normalizing with the respective vinculin, and the values are expressed as means ± SD of three independent experiments; ns, not significant; *** p ≤ 0.001. (B) LC3B expression evaluated by confocal microscopy after RDD-142 treatment. The bar graph, at the bottom, represents the percentage of LC3B fluorescence (means ± SD) of three acquisition fields; ** p ≤ 0.01; *** p ≤ 0.001. (C) Immunoblotting analysis for the determination of protein expression levels of autophagy after 24 h of treatment with RDD-142 at increased concentrations. Densitometry analysis for Western blotting experiments, below immunoblotting panels, was performed by normalizing with the respective vinculin, and the values are expressed as means ± SD of three independent experiments; ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

RDD-142 inhibits proteasome activity. (A) Representative kinetics of proteasome subunits β1 (caspase-like), β2 (trypsin-like), and β5 (chymotrypsin-like) activities in HepG2 cell lysates following treatment with RDD-142 (20, 30, and 42 µM), DMSO (CTRL), or Bortezomib (40 nM, positive control). Fluorescence was recorded at 1 min intervals and plotted as arbitrary fluorescence units (AFUs), corresponding to the raw intensity of the AMC signal released during the enzymatic reaction, versus time. (B) Quantification of proteasome activity expressed as the fold change relative to the control. Data represent means ± SEM of three independent experiments. Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

RDD-142 inhibits proteasome activity in vitro. Representative results showing the specific activity of the 20S proteasome after treatment with RDD-142 (42 µM), Bortezomib (40 nM), MG-132 (40 µM), or DMSO (vehicle control). Fluorescence was recorded at 1 min intervals and plotted as arbitrary fluorescence units (AFUs), corresponding to the raw intensity of the AMC signal released during the enzymatic reaction, versus time.

Figure 7

Cryo-TEM micrograph of RDD-142 PEG-liposomes. The image was recorded at a nominal magnification of 25,000×.

Figure 8

Colloidal stability of RDD-142 PEG-liposomes assessed by monitoring the mean diameter (MD), polydispersity index (PI), and zeta potential (ZP) for 90 days at 4 °C. Bars represent the mean values ± standard deviations (n = 4).

Figure 9

Effect of treatment with liposomal formulation of RDD-142 on HepG2 and IHH proliferation. Normalized cell index (CI) curves of HepG2 (A) and IHH (C) under 24 h of treatment with liposomal RDD-142 at different concentrations (5–40 µM range), assessed using the xCELLigence system. CI was normalized to the last cell index recorded before the addition of liposomal RDD-142, measured in real time. Representative IC₅₀ curves of liposomal RDD142 in HepG2 (B) and IHH (D) cells calculated as means of normalized CI ± SEM (n = 3).

Figure 10

The slope of the cell index after treatment with liposomal RDD-142. The rate of proliferation of HepG2 and IHH cells at various concentrations of liposomal RDD-142 (5–40 µM range), as determined by analyzing the slope of cell index over 24 h.

Figure 11

Intracellular intake of RDD-142. HepG2 cells were treated with 42 µM RDD-142, in solution or in PEG-liposomes (L-RDD-142) for 3, 6, 12, and 24 h. The amounts of RDD-142 accumulated in the cytoplasm are presented as areas of the chromatographic peak (means ± SD; n = 3).