Lysicamine Reduces Protein Kinase B (AKT) Activation and Promotes Necrosis in Anaplastic Thyroid Cancer

Abstract

Anaplastic thyroid cancer (ATC) is an aggressive form of thyroid cancer (TC), accounting for 50% of total TC-related deaths. Although therapeutic approaches against TC have improved in recent years, the survival rate remains low, and severe adverse effects are commonly reported. However, unexplored alternatives based on natural compounds, such as lysicamine, an alkaloid found in plants with established cytotoxicity against breast and liver cancers, offer promise. Therefore, this study aimed to explore the antineoplastic effects of lysicamine in papillary TC (BCPAP) and ATC (HTH83 and KTC-2) cells. Lysicamine treatment reduced cell viability, motility, colony formation, and AKT activation while increasing the percentage of necrotic cells. The absence of caspase activity confirmed apoptosis-independent cell death. Necrostatin-1 (NEC-1)-mediated necrosome inhibition reduced lysicamine-induced necrosis in KTC-2, suggesting necroptosis induction via a reactive oxygen species (ROS)-independent mechanism. Additionally, in silico analysis predicted lysicamine target proteins, particularly those related to MAPK and TGF-β signaling. Our study demonstrated lysicamine's potential as an antineoplastic compound in ATC cells with a proposed mechanism related to inhibiting AKT activation and inducing cell death.

Keywords: akt phosphorylation; anaplastic thyroid cancer; lysicamine; natural compounds; necrosis; thyroid cancer.

Figure 1

Lysicamine induces cell toxicity and reduces migration in thyroid tumor cells. (a) Molecular structure of lysicamine, synthesized via benzyne chemistry [27]. (b) A reduction in cell viability was observed in KTC-2, HTH83, BCPAP (TC cell lines), and Nthy-ORI (thyroid non-tumoral cell line) 72 h after treatment with three concentrations of lysicamine. Cell viability was measured via PrestoBlue assay and expressed as a percentage of viability compared to non-treated cells. (c) Representative images of colony assay across all TC cell lines 72 h after lysicamine IC₅₀ treatment vs. no treatment control (DMSO) and subsequent 10 days of culture without treatment. (d) Representative images of wound closure progression 12 h after lysicamine treatment of KTC-2 at IC₅₀ concentration. For (b), data were plotted as mean ± SEM of at least three biological replicates in triplicate; significance was determined by two-way ANOVA multiple comparisons followed by Bonferroni test (post-test); ** p < 0.001 *** p < 0.0001.

Figure 2

Lysicamine induces cell toxicity and reduces thyroid cancer cell spheroids area. (a,b) A reduction in cell viability was observed in spheroids of KTC-2 (a) on day 14 and in HTH83 (b) on days 10 and 14 after 2× IC₅₀ lysicamine treatment. Spheroid viability was measured via PrestoBlue assay and expressed as a percentage compared to untreated control. (c,e) Reduction in KTC-2 (c) and HTH83 (e) spheroids area after 2× IC₅₀ lysicamine treatment. (d,f) Representative images of KTC2 (d) and HTH83 (f) spheroids at days 0, 3, and 10 after treatment with 2× IC₅₀ lysicamine vs. DMSO. The area of the spheroids from the images was quantified by ImageJ, Version 1.53t (imagej.nih.gov) and expressed as a percentage of untreated control. Data were plotted as mean ± SEM of at least three biological replicates in triplicate; significance was determined by two-way ANOVA multiple comparisons followed by Bonferroni test (post-test); * p < 0.05; *** p < 0.0001.

Figure 3

Lysicamine induces necroptosis in TC cells in a ROS-independent Manner. (a) Live cells (AV−/7-AAD−) were decreased in all TC cell lines by Lysicamine; (b) the same treatment resulted in increased necrotic cells (AV−/7-AAD+) in all TC cell lines; and (c) had no effect on Caspase 3/7 activation in all TC cell lines. (d) Lysicamine increased ROS production only in HTH83 (e) in a concentration-dependent manner. (f) In HTH83, ROS inhibition by NAC did not increase live cells (AV−/7-AAD−) or reduce necrotic cells (AV−/7-AAD+). (g) Necrosome inhibition via NEC1 treatment reduced necrotic cells (AV−/7-AAD+) triggered by lysicamine treatment. In all experiments, cells were treated with lysicamine IC₅₀ or DMSO (control) for 48 h, stained, and analyzed via flow cytometry. Results were expressed as percentages compared to untreated controls. Data were plotted as mean ± SEM of at least three biological replicates in triplicate; significance was determined by one-way or two-way ANOVA multiple comparisons followed by Bonferroni test (post-test); * p < 0.05, ** p < 0.001, and *** p < 0.0001.

Figure 4

Lysicamine modulates AKT phosphorylation in thyroid cancer cells. (a) p-AKT was reduced in KTC-2 in a dose-dependent manner after lysicamine treatment. (b) p-AKT was reduced in HTH83 cells with 1.5× IC₅₀ lysicamine treatment. No ERK phosphorylation modulation was observed in both cell lines. Cells were cultured without fetal bovine serum (FBS), and after 24 h, FBS was added alone or combined with DMSO or lysicamine 0.5×, 1×, or 1.5× IC₅₀0. Protein extraction was performed 30 min treatment after. Representative images of two or more biological replicates conducted per cell line are presented.

Figure 5

Lysicamine activity was characterized via in silico analysis. (a) PASS analysis resulted in a list of pharmacological activities. (b) PASS analysis also predicted 36 protein interactions (confidence value > 0.4). (c) SEA analysis predicted 46 direct protein interactions. (d) Among both lists, 7 proteins were shared. Go Biological Process analysis modeled (e) molecular functions and (f) biological processes for proteins predicted by PASS and (g) biological processes by SEA. STRING database was used to generate a protein–protein interaction network for proteins predicted (h) by PASS and (i) by SEA.