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

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.

After the pre-treatment with kaempferol for 90 minutes IMR32, MDA-MB-468 cell lines (c, d) and Neuro2A, Hela cell lines (e,f) were treated with CDDO-Me (1 µM) and Tunicamycin (5 µg/ml) respectively for 24 hours. Data represented as average ±SEM; n=2.* represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p≤0.05 (one way ANOVA).

Supplementary Figure 3. Cyto-protective effect of estrogen receptor modulators.
IMR32 cells pretreated with kaempferol 50 µM in presence and absence of PHTTP 25 µM for 90 minutes. ER stress was induced with 1 µg/ml of Brefeldin A. After 24 hours of treatment, cellular ATP levels and Caspase 3/7 activity were measured using CellTitreGloassay and caspase 3/7 Glo assay respectively. Experiments were performed in triplicate (n=3) and data represented as average of ±SEM (a, b).* represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p≤0.05 (one way ANOVA).
Active caspase inhibition by kaempferol pretreatment compared to other ER modulators in in vitro model using IMR32 cells. ER stress was induced with Brefeldin A (1 µg/ml) for 24 hours. Data represented as average of ±SEM of experiment performed in duplicate (n=2) (c).

Supplementary Figure 4. Cyto-protective effect of kaempferol under apoptotic stimuli.
Viable cell count assessment performed after 24 hours of exposure to cell death inducers using Trypan blue exclusion assay in IMR32 cells. Data represented as average of ±SEM of six counting for each condition, from two independent experiments performed in triplicate (a).
ER stress markers mRNA expression at 24 hours after the induction of stress. *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). (b).
Expression of ER stress marker protein GRP78 in IMR32 cells in absence and in presence of Brefeldin A upon treatment with higher concentration of kaempferol (100 µM) (c, d).

Supplementary Figure 5. Transcriptional regulation of anti-apoptotic proteins XIAP and SURVIVIN.
IMR32 cells pre-treated with kaempferol for 90 minutes and ER stress was induced with BrefeldinA(1 µg/ml) for 12 hours. With the pre-treatment of kaempferol no significant regulation of XIAP (a) and Survivin(b) was seen against Brefeldin A induced ER stress. Data represents the average ±SEM of two independent experiments performed in duplicate.
Supplementary Figure 6. Immunocytochemistry analysis of cIAP1 and cIAP2 protein expression.
Images representing the expression of cIAP1(a) and cIAP2(b) proteins in IMR32 cells upon induction of ER stress with Brefeldin A (1 µg/ml) for 6 hours and 12 hours with/without pretreatment with kaempferol (50 µM). Figure 7. In silico analysis of kaempferol binding to caspase 3 enzyme.

Supplementary
Docking studies on binding of 0TE and Kaempferol to caspase 3 dimer interface using PDB:ID 3DEJ as protein structure. Hydrophobic interactions and hydrogen bond formation were analyzed using LigPlot + 2D interaction diagrams (a). 3D Binding site analysis was performed using UCSF chimera and PYMOL (b&c). Surface diagram showing the binding pocket of 0TE and kaempferol at the same allosteric site in the dimerization region of caspase 3 dimer (d).