Mechanism of apoptosis with the involvement of calpain and caspase cascades in human malignant neuroblastoma SH‐SY5Y cells exposed to flavonoids

Abstract

Neuroblastoma is the most common extracranial solid tumor in children causing death at pre‐school age, as no cure has yet been developed. We investigated the proteolytic mechanisms for apoptosis in human malignant (N‐type) neuroblastoma SH‐SY5Y cells following exposure to flavonoids such as apigenin (APG), (−)‐epigallocatechin (EGC), (−)‐epigallocatechin‐3‐gallate (EGCG) and genistein (GST). We found decrease in viability of SH‐SY5Y cells with an increase in dose of APG, EGC, EGCG and GST. Predominantly apoptosis occurred following exposure of SH‐SY5Y cells to 50 μM APG, 50 μM EGC, 50 μM EGCG and 100 μM GST for 24 hr. Apoptosis was associated with increases in intracellular free [Ca²⁺] and Bax:Bcl‐2 ratio, mitochondrial release of cytochrome c and activation of caspase‐9, calpain and caspase‐3. Induction of apoptosis with APG and GST showed activation of caspase‐12 as well. Activation of caspase‐3 could cleave the inhibitor‐of‐caspase‐activated DNase (ICAD) to release and translocate caspase‐3‐activated DNase (CAD) to the nucleus. Activation of caspase‐8 cleaved Bid to truncated Bid (tBid) in cells treated with EGC and EGCG. EGC and EGCG induced apoptosis with caspase‐8 activation and mitochondria‐mediated pathway, whereas APG and GST caused apoptosis via an increase in intracellular free [Ca²⁺] with calpain activation and mitochondria‐mediated pathway. Activation of different proteases for cell death was confirmed using caspase‐8 inhibitor II, calpeptin (calpain inhibitor), caspase‐9 inhibitor I and caspase‐3 inhibitor IV. Thus, plant‐derived flavonoids cause cell death with activation of proteolytic activities of calpain and caspases in SH‐SY5Y cells, and therefore serve as potential therapeutic agents for controlling the growth of neuroblastoma.