Kaempferol with Verapamil impeded panoramic chemoevasion pathways in breast cancer through ROS overproduction and disruption of lysosomal biogenesis

Abstract

Background: Reactive oxygen species (ROS) at low level promotes cell survival through lysosome induced autophagy induction. Glucose stress induced acidosis, hypoxia, ROS, upregulates markers related to cancer stemness and multidrug resistance. Also, lysosomal upregulation is proposed to be one of the important indicators of cell survival under ROS induced stress. Studies supported that, stimulation of Lysosome-TFEB-Ca²⁺ cascade has important role in induction of chemoresistance and survival of cancerous cells.

Purpose: To observe the effect of synergistic drug combination, Kaempferol and Verapamil on markers regulating chemoevasion, tumor stemness & acidosis as well as lysosome upregulation pathways, under low as well as high glucose conditions.

Hypothesis: Based on our earlier observation as well as previous reports, we hypothesized, our drug combination Kaempferol with Verapamil could attenuate markers related to chemoevasion, tumor stemness & acidosis as well as lysosome-TFEB-Ca2+ pathway, all of which have indispensable association and role in chemoresistance.

Methods: RNA and protein expression of candidate genes, along with ROS production and Ca2+ concentrations were measured in ex vivo models in altered glucose conditions upon treatment with KV. Also, computational approaches were utilized to hypothesize the mechanism of action of the drug combination. PCR, IHC, western blotting and molecular docking approaches were used in this study.

Results: The overproduction of ROS by our candidate drugs KV, downregulated the chemoresistance and tumor acidosis markers along with ATP1B1 and resulted in lysosomal disruption with reduction of Ca²⁺ release, diminishing TFEB expression under low glucose condition. An anomalous outcome was observed in high glucose conditions. We also observed KV promoted the overproduction of ROS levels thereby inducing autophagy-mediated cell death through the upregulation of LC3-II and p62 in low glucose conditions. The ex vivo studies also corroborate with in silico study that exhibited the parallel outcome.

Conclusion: Our ex-vivo and in-silico studies revealed that our candidate drug combination KV, could effectively target several pathways regulating chemoresistance, that were not hitherto studied in the same experimental setup and thus may be endorsed for therapeutic purposes.

Keywords: Anti-cancerous; Chemoresistance; Low glucose; Reactive oxygen species; Stem cells; Synergism.