CAL-33

Grape Cane Extract Inhibits Cell Viability of HNSCC Cells

Plants are abundant sources of biologically active molecules, with phenolic compounds notably recognized for their antioxidant and anticancer properties. Despite their potential health benefits, grape canes - the by-product of vine pruning - have been underutilized, although they contain valuable phenolic antioxidants. Head and neck squamous cell carcinoma (HNSCC) is a common cancer with resistance to conventional treatments. Polyphenols, known for their anticancer potential and low toxicity, offer promising alternatives. Therefore, Squillaci et al. evaluated the antioxidant and anticancer efficacy of phenolic extracts from "Greco" grape canes on HNSCC cells. First, the antiproliferative activity of "Greco" grape cane extracts on oral Cal-33 and laryngeal JHU-SCC-011 HNSCC cell lines was assessed using an MTT assay. Treatment with extracts prepared at pH 7 for 60 minutes led to a marked dose- and time-dependent inhibition of cell proliferation, achieving an IC50 of 0.25 mg/mL after 72 hours (Fig. 1). Further analysis identified that the polyphenol-rich fraction FR 5 exhibited significant cytotoxicity, with IC50 values of 0.10 mg/mL and 0.12 mg/mL for Cal-33 and JHU-SCC-011 cells, respectively, approximately half the concentration needed by the crude extract for similar effects (Fig. 2). This indicates that polyphenols are key contributors to the antiproliferative effects of the extracts.

Fig. 1. Effect of "Greco" grape cane extracts prepared at different pHs and extraction times on cell viability of Cal-33 and JHU-SCC-011 cell lines (Squillaci G, Vitiello F, et al., 2022).

Fig. 2. Effect of polyphenol-rich fractions on HNSCC cell viability (Squillaci G, Vitiello F, et al., 2022).

Apoptosis Assessment in Cal-33 and JHU-SCC-011 Cell Lines Treated with AdoMet

S-Adenosyl-l-methionine (AdoMet) is a multifunctional biomolecule, involved in many biological processes, especially as a main methyl donor. It has antiproliferative and proapoptotic effects on multiple types of cancers, and recent studies have shown its utility for gastric, prostate and head and neck squamous tumours (HNSC). But the exact molecular basis of AdoMet's activity and its influence on HNSC remains unknown. Mosca et al. tested the apoptotic effect of AdoMet on Cal-33 and JHU-SCC-011 cell lines. Double-labeled with Annexin V-FITC and PI, FACS analysis determined the degree of cell death. The findings indicated that, in Cal-33 cells, at least a quarter of the cells had become dead after 48 hours (Fig. 3a), while after 72 hours of infection, approximately 19% of JHU-SCC-011 cells were dead (Fig. 3b). The AdoMet treatment knocked down full-length caspase 9 (cause of apoptosis), caspase 6, and caspase 7 (upstream caspases) in both cell lines, as well as cleavage of PARP, with an especially dramatic effect at 72 hours (Fig. 4). Both lines of cells had higher levels of proapoptotic Bax and lower levels of antiapoptotic Bcl-2 when treated with AdoMet. This shift in the ratio of Bax to Bcl-2 points towards a caspase-dependent apoptotic response. Finally, AdoMet promotes apoptosis in Cal-33 and JHU-SCC-011 by a caspase-dependent pathway with a higher Bax/Bcl-2 ratio (Fig. 4).

Fig. 3. Representative dot plots of Annexin V-FITC and PI-stained head and neck cancer cells (Mosca L, Pagano M, et al., 2019).

Fig. 4. Effects of AdoMet on the expression levels of relevant proteins regulating apoptotic pathways in head and neck cancer cells (Mosca L, Pagano M, et al., 2019).