Kaempferol mitigates Endoplasmic Reticulum Stress Induced Cell Death by targeting caspase 3/7

Abstract

The Endoplasmic Reticulum (ER) plays a fundamental role in executing multiple cellular processes required for normal cellular function. Accumulation of misfolded/unfolded proteins in the ER triggers ER stress which contributes to progression of multiple diseases including neurodegenerative disorders. Recent reports have shown that ER stress inhibition could provide positive response against neuronal injury, ischemia and obesity in in vivo models. Our search towards finding an ER stress inhibitor has led to the functional discovery of kaempferol, a phytoestrogen possessing ER stress inhibitory activity in cultured mammalian cells. We have shown that kaempferol pre-incubation significantly inhibits the expression of GRP78 (a chaperone) and CHOP (ER stress associated pro-apoptotic transcription factor) under stressed condition. Also, our investigation in the inhibitory specificity of kaempferol has revealed that it inhibits cell death induced by diverse stimuli. Further study on exploring the molecular mechanism implied that kaempferol renders protection by targeting caspases. Both the in silico docking and in vitro assay using recombinant caspase-3 enzyme confirmed the binding of kaempferol to caspases, through an allosteric mode of competitive inhibition. Altogether, we have demonstrated the ability of kaempferol to alleviate ER stress in in vitro model.

Figure 1

Kaempferol rescues cells against BFA induced cell death. (a) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. (b) Confirmation of cytoprotective activity of kaempferol by measuring cellular ATP level using CellTitre Glo reagent. (c) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. (d–f) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. (g) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

Figure 2

Kaempferol inhibits UPR signaling (a) IMR32 cells were cultured with BFA in presence or absence of kaempferol pre-incubation. Q-RTPCR was performed to measure the expression of genes (IRE1α, PERK, ATF6, GRP78 and CHOP) involved in UPR signaling. (b) Immunoblot representing the reduction in the expression of GRP78 in BFA treated conditions upon pretreatment with kaempferol. Densitometry analysis of GRP78 was performed. Data represents the average ± SEM of two independent experiments (n = 2). (c) MDA-MB-468 cells were cultured with BFA in presence or absence of kaempferol pre-incubation. Q-RTPCR was performed to measure the expression of GRP78 and CHOP. Data represented as average ± SEM of two independent experiments performed in duplicate. *Represents the significant increase in expression of BFA treated cells with respect to control cells; ^#represents the reduction in gene expression in kaempferol pre-treated cells when compared to BFA alone treated cells (p ≤ 0.05; one way ANOVA).

Figure 3

Kaempferol regulates ER stress induced apoptotic signaling. (a) IMR32 cells were treated with BFA in presence or absence of kaempferol pre—incubation for 3 h, 6 h and 12 h. Lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using antibodies specific for p-p38-MAPK, p38-MAPK, p-eIF2α. (b) Densitometry analysis of ratio of phospho to non-phosphorylated levels of p38 MAPK and eIF2α. Data represents ratios of the average ± SEM of two independent experiments (n = 2). (c) Regulation of Bim, Bcl2 and DR5 gene expression upon BFA treatment in presence and absence of Kaempferol pre-treatment. Data represented as mean ± SEM of two independent experiments performed in duplicate. ^*Represents the significance with respect to control cells (p ≤ 0.05 (one way ANOVA).

Figure 4

Kaempferol maintains cIAPs and inhibits caspase 3/7 activation. (a,b) Expression of cIAP1 and cIAP2 mRNA level was measured by q-RTPCR. ER stress was induced in IMR32 cells with BFA in the presence and absence of kaempferol pre-treatment. Expression of cIAPs level was compared to caspase 3/7 activation in 12 h and 24 h of ER stress induction. Data represented as average ± SEM of two independent experiments performed in duplicate. ^*Represents the comparison of BFA treated condition to control cells; ^#represents the significant difference between BFA treated cells to BFA + kaempferol treated cells at p ≤ 0.05. (c) Regulation cIAPs mRNA level at increasing treatment hours in IMR32 cells cultured with BFA with/without kaempferol pre-treatment. Data represented as average ± SEM of two independent experiments performed in duplicate. (d,e) Protein levels of cIAP1 and cIAP2 in IMR32 cells after 24 hours of treatment with BFA and kaempferol pre-treated conditions by immunocytochemistry (Scale bar: 67 µm). (f) MTT assay showing the reversal of cell death protection by SMAC mimetic compounds co-treatment with KFL + BFA treated cells after 24 hours of incubation. (g) SMAC mimetic increases the caspase 3/7 activity in IMR32 cells upon treated with BFA alone and in BFA + KFL combination. Data represented as average ±SEM of two independent experiments performed in triplicate. * and ^$ Represents significance at p < 0.05 with respect to BFA treated cells and ^#Represents significance at p < 0.05 with respect to BFA + KFL treated cells (One-way ANOVA).

Figure 5

Kaempferol is a general inhibitor of cell death. MDA-MB-468 cells (a–d) and IMR32 (e–g) were treated with staurosporine (100 nM); doxorubicin (1 µg/ml); TNF-α (1 ng/ml) + cycloheximide (0.5 µg/ml) and cycloheximide (25 µg/ml) alone or after pre-incubation with kaempferol. MTT assay was performed after 24 hours to quantitate percentage of cell death. Data represented as mean ± SEM of at least two independent experiments performed in triplicate. ^*Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

Figure 6

Kaempferol binds to the caspase-7 dimerization site: In silico analysis. (a) LigPlot⁺ analysis (2D representation) of docked complexes of 0TE and kaempferol compared to crystal structure (PDB ID: 1SHJ) of DICA docked pose with caspase 7 enzyme dimerization site. Red circles represents the amino acid residues common to the binding site of 0TE and DICA, obtained by docking studies with kaempferol. Green dotted lines represents H-bonding (kaempferol) in docked complex and violet solid line (DICA) represents the covalent bonding as represented in the crystal structure. (b) Superimposed structures of 0TE and kaempferol docked complexes as represented by UCSF Chimera. (c) 3D representation of docked poses of 0TE, kaempferol and DICA using PYMOL (Cysteine ; Valine ; Methionine ) (d) Solid Surface representation of caspase-7 dimer with 0TE ; kaempferol ; and DICA ; docked poses superimposed at the dimerization site.

Figure 7

Kaempferol inhibits the activity of caspase-3 enzyme. (a and b) Caspase-3 inhibition by kaempferol was measured using recombinant caspase-3 (enzyme) and Z-DEVD-aminoluciferin (substrate) with respect to time (a) and concentration (b). Ac-DEVD-CHO served as a positive control. (c) Dose response curve for kaempferol inhibition on caspase-3 enzyme activity. (d) Analysis of the inhibitory potential of estrogen receptor modulators on caspase-3 enzyme activity. Withanaloide A served as (−) control and Ac-DEVE-CHO as (+) control. Data represented as mean ± SEM of two independent experiments performed in duplicate. (e) Immunocytochemistry for XIAP expression levels in IMR32 cells after 24 hours of incubation in respective conditions (Scale bar: 67 µm).

Figure 8

Graphical abstract. Kaempferol alleviates ER stress by inhibiting the executioner caspase 3/7. Endogenous inhibitor of apoptotic proteins-IAPs level did not get altered upon kaempferol treatment and the SMAC mimetics which are inhibitor of IAPs reverses the kaempferol mediated caspase inhibition and allows cell death to occur.