Armenuhi Moghrovyan, Lilya Parseghyan, Gohar Sevoyan, Anna Darbinyan, Naira Sahakyan, Monica Gaboyan, Zaruhi Karabekian, and Armen Voskanyan. Korean J Pain 2022;35:140-51. https://doi.org/10.3344/kjp.2022.35.2.140

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom’s toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude Macrovipera lebetina obtusa (Levantine viper) venom with the actions of the same venom with two key enzymatic components inhibited (specifically, phospholipase A2 (PLA2) and metalloproteinases) in the bioenergetic cellular response, i.e., oxygen uptake and reactive oxygen species generation. In addition to the rate of free-radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data show that viper venom drives an increase in both glycolysis and respiration in Vero cells, while the blockage of PLA2 or/and metalloproteinases affects only the rates of the oxidative phosphorylation. PLA2-blocking in venom also increases cytotoxic activity and the overproduction of reactive oxygen species. These data show that certain components of the venom may have a different effect within the venom cocktail other than the purified enzymes due to the synergy of the venom components.

Cancer cells undergo metabolic reprogramming to sustain proliferation, resist apoptosis, and adapt to oxidative stress. In the present study, we comparatively evaluated the anticancer effects of four medicinal plant extracts ( Inula helenium, Hypericum alpestre, Rumex obtusifolius , and Alchemilla smirnovii ) on HeLa cervical cancer cells, with particular emphasis on oxidative stress, l ‐arginine metabolism, and cellular energy pathways. Cell viability was assessed using the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay, while biochemical colorimetric methods were used to determine nitric oxide production, arginase and nitric oxide synthase activities, malondialdehyde levels, superoxide dismutase and catalase activities, proline content, and key glycolytic metabolites. Nuclear morphology changes consistent with apoptotic features were examined by Hoechst 33258 staining. All tested extracts exhibited cytotoxic effects in HeLa cells in a time‐ and concentration‐dependent manner, which were further enhanced when combined with the metabolic inhibitors l‐NAME (Nω‐nitro‐l‐arginine methyl ester) or nor‐NOHA (Nω‐hydroxy‐nor‐l‐arginine). Inula helenium showed the strongest potentiation in combination treatments. Several extracts significantly decreased arginase activity while increasing nitric oxide synthase activity and nitrite levels, indicating modulation of l‐arginine metabolism. Treatments also increased malondialdehyde levels and stimulated antioxidant enzyme activities, consistent with redox imbalance and oxidative stress–mediated cytotoxicity. In addition, Inula helenium and Alchemilla smirnovii reduced intracellular glucose, pyruvate, and lactate concentrations, suggesting partial modulation of glycolytic metabolism. Fluorescence microscopy revealed chromatin condensation, nuclear fragmentation, and apoptotic body formation, particularly after treatment with Hypericum alpestre and Rumex obtusifolius . These findings demonstrate that selected medicinal plant extracts exert cytotoxic effects by simultaneously modulating oxidative stress, amino acid metabolism, and energy pathways in cervical cancer cells. The investigated plants may represent promising sources of bioactive compounds for the development of complementary metabolic anticancer strategies.

Background/Objectives. Programmed death-ligand 1 (PD-L1) is a critical immune checkpoint protein that enables tumors to evade immune surveillance by suppressing T cell activation. Monoclonal antibodies are currently used to modulate PD-1/PD-L1 interactions. However, several immune-associated adverse effects were ascribed to those treatments. This led to the necessity for small-molecule alternatives like BMS-202. Methods. In this study, we investigated the temporal dynamics of cell-surface PD-L1 in response to the small-molecule dimerizer BMS-202. Treatment with a non-cytotoxic concentration of BMS-202 at 5 µM triggered a transient reduction in surface PD-L1 concentration, reaching its lowest level at 15 min. In the attempt to characterize the fate of PD-L1 following exposure to the BMS-202 dimerizer, we employed a low-pH wash internalization assay. Results. The results demonstrated a transient increase in intracellular PD-L1 within 5–15 min of compound exposure, followed by rapid recovery of surface PD-L1 levels. These findings suggest that BMS-202-induced changes in surface PD-L1 are acute and reversible, with levels returning to baseline within 24 h post-exposure. Conclusions. These findings reveal a dynamic regulatory mechanism where small-molecule-induced dimerization triggers rapid protein trafficking and transient surface depletion. Understanding these temporal dynamics is essential for the development of next-generation small-molecule immune checkpoint inhibitors.