Hesperetin Inhibits Bladder Cancer Cell Proliferation and Promotes Apoptosis and Cycle Arrest by PI3K/AKT/FoxO3a and ER Stress-mitochondria
Pathways
Authors: Su Yao , Chen Lin , Yang Jin 
Published: February 2024

Background and Objectives:
Hesperetin (HSE) is a natural flavonoid derived from the
hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs, such as Chenpi
and Hovenia caryophyllus. HSE displays anti-inflammatory and antioxidant activities. However,
its potential mechanism of action on bladder cancer (BLCA) remains unknown. The aim of this
study was to investigate the potential mechanism of action of HSE on BLCA cells.

background:
Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs such as Chenpi and Hovenia caryophyllus. It has been shown that HSE displays anti-inflammatory, antioxidant activity. However, the potential mechanism of action on bladder cancer (BLCA) remains unknown.

Methods:
Network pharmacology analysis was used to construct a composite target network, combined
with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity
evaluation was determined by CCK-8 assay. A clone formation assay was performed to assess
cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration
and invasion ability. Hoechst 33342 staining was visualized to observe morphological features
of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and
mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western
blot analysis was performed to analyze the expression of key proteins associated with cell proliferation,
apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial
pathways.

objective:
The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells.

Results:
Network pharmacology analysis was performed to identify 155 potential candidate targets
of HSE-BLCA, and further topological analysis was performed to obtain 34 hub-gene. Enrichment
analysis yielded patterns of death and key pathways, revealing that the anti-BLCA effect of
HSE may be related to the positive regulation of PI3K/AKT/FoxO3a and ER stress-mitochondrial
pathways. in vitro results showed that HSE blocked cell proliferation, migration, and invasion in a
concentration-dependent manner and triggered apoptosis, G0/G1 phase blockade, ROS production,
and MMP depolarization. In addition, Western blot results showed that HSE downregulated
phosphorylated (p)-3-phosphoinositide-dependent kinase-1 (PI3K), phosphorylated (p)-AKT
serine/threonine kinase 1 (AKT), phosphorylated (p)-Forkhead box O 3a (FoxO3a), anti-apoptotic
proteins, proliferation-associated proteins, and cell cycle promoters, whereas the levels of proteins
related to the expression of cell cycle regulators, pro-apoptotic proteins, and ER stress-mitochondrial
pathway were up-regulated in BLCA cells by the intervention of HSE. PI3K agonist (YS-49)
and ER stress inhibitor (4-PBA) partially or completely reversed HSE-mediated proliferation,
apoptosis, and cycle blockade in BLCA cells.

method:
Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. Clone formation assay to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways.

Conclusion:
The anticancer effects of HSE in BLCA may be attributed to its coordination of actions,
inhibiting cell proliferation, migration, and invasion, inducing apoptosis, G0/G1 phase arrest,
generating reactive oxygen species, causing MMP loss, and engaging the caspase protein family.
These actions are likely mediated through the PI3K/AKT/FoxO3a and ER stress-mitochondrial
pathways. Thus, our findings suggest that HSE is a promising novel therapeutic candidate for
the prevention and treatment of BLCA.