Unveiling the anti-cancer mechanisms of calotropin: Insights into cell growth inhibition, cell cycle arrest, and metabolic regulation in human oral squamous carcinoma cells (HSC-3)

Abstract

Background: Calotropin, a cardiac glycoside obtained from the plant Calotropis gigantea, has demonstrated promising potential as an anti-tumorigenesis compound.

Objective: The main objective of this study was to investigate the potential anti-cancer properties of calotropin against HSC-3 oral squamous cancer cells and to elucidate the underlying mechanisms involved in its action.

Material and method: Calotropin were treated in HSC-3 to evaluate cell viability by MTT assay. Flow cytometry analysis divulged that calotropin G0/G1 phase cell cycle arrest and apoptosis in HSC-3 cells. Calotropin displayed inhibitory properties against aerobic glycolysis, a metabolic alteration using glucose uptaken, lactose production and LDHA activity assays. Furthermore, migration and invasion assays help that calotropin has ability to reduce the migratory and invasive of HSC-3 cells, using transwell and Matrigel assay. Validation of mRNA expression through RT-PCR. Molecular docking was implemented to validate the binding association of calotropin with apoptosis and metastatic regulating targets.

Result: The results exemplify that increasing doses of calotropin effectively hold back the HSC-3 cell progression. Migration and invasion assays help that calotropin has ability to reduce the migratory and invasive of HSC-3 cells, indicating its potential to inhibit cancer metastasis. These results imply that calotropin may influence genes linked to metastasis and apoptosis in order to achieve its beneficial effects on cancer. Docking results provided further support, showing a high binding energy between calotropin and metastasis-mediated pathways.

Conclusion: Overall, our findings shed an experimental evidence on how calotropin inhibits the HSC-3 oral squamous cancer cell growth, highlighting the drug's potential as a treatment for oral cancer. Further, investigation on in-vivo experiment is warranted to explore its potential mechanism of action and to develop a novel drug towards clinical trial.

Keywords: Apoptosis; Calotropin; Cell growth; Metastatic property; Oral cancer; Therapeutic targets.

Graphical abstract

Fig. 1

Cytotoxicity of calotropin in HSC-3 cells. (a) HSC-3 administrated with 10–100 μM dose of calotropin for 24 h and 48 h cytotoxicity was analyzed by MTT assay. (b) Cell viability was evaluating by trypan blue assay (right). The software Graph Pad Prism8 was used to determine IC50 values. Data were shown as means ± S.D.

Fig. 2

Drug-protein interaction networking. (a) DPI network constructed by STITCH database. The link of calotropin with aerobic glycolysis, cell cycle, apoptosis, and metastatic associated genes were indicated. (b) Network of interactions between the high nodal strength targets with the calotropin were highlighted using Enrichr database.

Fig. 3

Calotropin inhibits aerobic glycolysis regulation in HSC-3 cells. (a) HSC-3 cells were exposed with 2DG (5 mM, 10 mM) for 24 h and 48 h. Cell viability was measured. (b–d) Glucose uptaken, lactose production, and LDH activity were calculated at 24 h and 48 h treatment of calotropin in HSC-3 cells. Glucose uptake, lactate production, and LDH activity were counted. The t-test was used to evaluate the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig. 4

Calotropin induces cell cycle arrest in HSC-3 cells. Human oral squamous carcinoma cell line, HSC-3 were analyzed for cell cycle distribution by flow cytometry. The t-test was used to investigate the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig. 5

Calotropin induces cell death in HSC-3 cells. OSCC cell line, HSC-3 were analyzed for apoptotic rate using annexin V-FITC by flow cytometry. (LL-Live cells; UL-early apoptosis; UR-Late apoptosis; LR-Necrosis). The t-test was used to test the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig. 6

Calotropin inhibits motility and invasive of oral cancer cells. The treatment of calotropin for 24 h and 48 h in HSC-3 shows inhibition of migration and invasion. Cell invasion was examined using Matrigel-coated Transwell chambers in the HSC-3 cells and treated with calotropin. Quantification of the migration and invasion were displayed as the percentage of migration and invasion representing the number of cells per field compared with the control. The t-test was used to determine the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig. 7

Effect of calotropin in HSC-3 cells. The effect of calotropin on Apoptosis-Related Genes expression (Bax, BCL2, CDH1, CDH2, SNAI2, SNAI1, CLDN1, and VIM) in HSC-3 Cells. As previously mentioned, the expression capacities were measured using qRT-PCR. The relative level of each gene obtained by normalization with β-actin in the untreated group was set subjectively at 1, and Values are reported as the mean ± SEM of three independent experiments, with the level in other groups having been determined in accordance. (*p < 0.05, **p < 0.01; ***p < 0.001) with untreated groups.

Fig. 8

Association of high binding energy in calotropin (a–h) The binding affinity of calotropin in the active docking site of apoptotic and metastatic regulating targets (BAX, BCL2, CDH1, CDH2, SNAI1, SNAI2, CLDN1, and VIM) the binding site is lined by important residues. The hydrogen bond lined with active residues. The molecular docking analysis were performed in Autodock 1.5.4 and visualized using Biovia Discovery tool.

Fig. 9

Diagrammatic illustration of calotropin role in oral cancer cells. Calotropin inhibits HSC-3 cell growth and suppresses metabolic regulations. The treatment of calotropin regulates G0/G1 phase cell cycle arrest and induces apoptosis in HSC-3 oral cancer cells. Calotropin inhibits the metastatic property in oral cancer cells. Molecular docking analysis revealed high binding energy shows strong association of calotropin with apoptotic and metastatic regulating targets.