Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

<div><p>Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger <i>Zingiber zerumbet</i>, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.</p> </div

Zerumbone induced apoptosis requires Bax.

<p>(A). SiHa and MCF-7 cells were treated with zerumbone 50 µM for 24 h. Then the cells were fixed permeabilised and immunostained for conformationally active Bax (6A7) or conformationally active Bak as described. Analysis was carried out using FACSAria to determine fluorescence intensity. (B). MCF-7 cells expressing Bax EGFP were stained with 50 nm TMRM and treated with zerumbone 50 µM in 10 nM TMRM containing medium for indicated time points. Imaging for EGFP and TMRM was carried out at 0 h, 6 h and 12 h under live cell incubation on stage. The cells with granular perinuclear green fluorescence indicate Bax oligomerisation at mitochondria and loss of TMRM red fluorescence indicate ΔΨm loss. (C). MCF-7 cells expressing Bax EGFP and vector control transfected cells were treated with zerumbone 50 µM for 12 and 24 h and analysed for chromatin condensation. (D). Colon cancer cell line HCT116 and its Bax deficient derivative Bax KO was treated with zerumbone 50 µM for 24 h and 48 h and analysed for chromatin condensation.</p

Calcium dependent calpain and ROS contribute for Bax activation and mitochondrial permeabilization.

<p>(A). SiHa and MCF-7 cells were treated with zerumbone 25 µM and 50 µM for 24 h. Then the cells were trypsinized and loaded with DCF-DA (10 μM) in serum free OptiMEM medium and immediately analysed by flow cytometry (FACSAria). (B). MCF-7 cells expressing cytochrome c EGFP and Bax EGFP were pretreated with N Acetyl Cysteine (NAC) (3 mM) or DMSO alone for 1 h followed by zerumbone treatment for 24 h. The cells with diffuse cytochrome and Bax punctae were counted and represented as graph. Values are average ± SD from four different experiments. (C). SiHa and MCF-7 cells were treated with zerumbone 50 μM for 24 h. Then the cells were trypsinised and stained with 10 μM t-BOC as described. The fluorescence was analysed using flow cytometry. (D). MCF-7 cells expressing cytochrome c EGFP and Bax EGFP were pretreated with indicated inhibitors alone and inhibitors for 1 h followed by zerumbone for additional 24 h. The cells with diffuse cytochrome and Bax punctae were counted and represented as graph. Values are average + SD from four different experiments. (E). MCF-7 cells expressing calcium probe chameleon targeted at ER (D1ER) was treated with zerumbone for 12 h. The ECFP-EYFP ratio imaging was carried out as described under live cell incubation on stage at an interval of 5 minutes from 12 h onwards. The average ratios from healthy cells and rounded dead cells at 24 h were calculated using NIS element software and plotted. The cells expressing chameleon at cytoplasm was also treated in the same manner to perform ratio imaging. The average ratio is shown as graph (n = 4). (**p ≤001). The contrasts of ECFP and EYFP FRET images are linearly adjusted for visual purpose.</p

Zerumbone induced caspase activation subsequent to mitochondrial transmembrane potential loss.

<p>(A). Schematic representation of live cell caspase probe employed to detect caspase activation. The cells express ECFP-DEVD-VENUS fusion protein. DEVD is the preferred amino acid sequence of activated caspase 3 and caspase 7 that is linked in between the donor fluorophore, ECFP and acceptor fluorophore, EYFP. If caspases are not activated, when ECFP is excited energy is transferred to EYFP because of the FRET between the donor and acceptor pairs leading to decrease in ECFP fluorescence and increase in EYFP fluorescence. Upon caspase activation, DEVD is cleaved with loss of FRET that changes the ratio of ECFP/EYFP. (B). Cervical cancer cell line SiHa expressing ECFP-DEVD-EYFP were stained with 50 nm TMRM for 10 min. Then the cells were treated with zerumbone 50 µM in medium containing 10 nm TMRM. The images were taken at 0h, 6 h, 12 h and 24 h using the filter combinations described in the materials and methods under live cell incubation on stage. ECFP channel, EYFP FRET channel, ratio image and TMRM channels are shown. Caspase activation is reflected in ratio change and loss of ΔΨm in TMRM fluorescence intensity by decrease, diffuse or loss. (C). Breast cancer cell line MCF-7 expressing ECFP-DEVD-EYFP were stained with 50 nm TMRM for 10 min. Then the cells were treated with zerumbone 50 µM in medium containing 10 nm TMRM. The images were taken at 0 h, 6 h, 12 h and 24 h using the filter combinations described in the materials and methods under live cell incubation on stage. ECFP channel, EYFP FRET channel, ratio image and TMRM channels are shown. Caspase activation is reflected in ratio change and loss of ΔΨm in TMRM fluorescence by decrease, diffuse or loss. (D). SiHa cells and MCF-7 cells expressing FRET probe were treated with zerumbone 50 µM and ratio imaging was carried out as described. The cells with FRET loss were calculated and represented as graph (n = 4). (E). Ovcar 8- ECFP- DEVD-EYFP cells were stained with Hoechst and TMRM, treated with Zerumbone 50 µM. Imaging for Hoechst, TMRM, ECFP, and EYFP FRET were carried out using a 96 well plate Bio-imager as described at the indicated time points. The contrasts of ECFP and EYFP FRET images are linearly adjusted for visual purpose. (F). MCF-7 cells were transfected with vector alone or pc DNA3 Caspase 3 vector. The caspase 3 expression was analysed by western blot and shown. The stable clones were treated with zerumbone 50 µM. After 24 h chromatin condensation was analysed as described. The right panel represents SiHa cells treated with zerumbone 50 µM alone or after pretreatment with caspase inhibitor z VAD-FMK (50 µM) followed by zerumbone 50 µM for 24 h. The chromatin condensation data from three independent experiments were used for generating the graph.</p

ER targeted Bcl 2 prevents cell death induced by zerumbone than wild type Bcl 2 or Bcl-XL.

<p>A. SiHa cells were transfected with vector alone or Bcl-XL –EGFP, Bcl2- EGFP and ER-Bcl2. The whole cell extract prepared from the cell was probed for Bcl2 and Bcl-XL. Beta actin is served as the loading control. (B). The above panel cell lines were treated with zerumbone 50 µM and 100 µM for 24 h. Then the cells were stained with Hoechst to quantify chromatin condensation. The results shown is average ±SD (n = 4)(**p≤001). (C). The whole cell extract prepared from vector alone or BclXL –EGFP, Bcl2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 µM and 100 µM for 24 h were probed with antibodies against caspase 8, hsp70, Bid, Bax, caspase 3 by western blot technique. Beta actin served as loading control. (D). The whole cell extract prepared from vector alone or Bcl-XL –EGFP, Bcl2 EGFP and ER-Bcl2, untreated, or treated with zerumbone 50 µM and 100 µM for 24 h were probed with antibodies against hsp90, Akt, cyclin D1, XIAP, Survivin, PUMA, by westernblot technique. β-actin and hsc70 served as loading control. (E). SiHa vector alone, BclXL –EGFP, Bcl 2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 µM were stained with Cell ROX Red as described and analysed by flow cytometer. (F). SiHa vector alone, BclXL –EGFP, Bcl 2 EGFP and ERBcl2, untreated, or treated with zerumbone 50 µM were stained with t-BOC as described and analysed by flow cytometer. (G). MCF-10 A, Human Mammary epithelial cells, human smooth Muscle cells and endothelial progenitor cells were treated with zerumbone 50 µM for 24 h. Then the cells were stained with TMRM or DCF-DA as described and analysed by flow cytometer. (H). Endothelial progenitor cells and MCF10A cells were stained with TMRM and Hoechst followed by zerumbone50 μM treatment. The wells were repeatedly imaged at the indicated time points.</p

Zerumbone induced cytotoxicity involved chromatin condensation.

<p>(A). The indicated cells were treated with zerumbone 50 µM for 24 and 48 h. Then the cells were trypsinized and stained with trypan blue to calculate the percentage positive cells with trypan blue uptake by microscopy. The data shown are average ±SD (n = 4). (B). The indicated cells were grown on 96 well plate and treated with zerumbone 50 µM for 24 and 48 h. Then the cells were stained with Hoechst dye as described. The cells with intense, condensed chromatin were counted from three independent wells to calculate the percentage of cells with condensed chromatin. The results shown are average ± SD (n = 4). (C). Representative fluorescent image showing condensed chromatin after zerumbone 50 µM treatment for 48 h in SKBr3 cells (40x).</p