Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin

Abstract

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC.

Figure 1

Hyper-sialylation associated with ovarian cancer drives the specificity of SNA. (a) Binding of FITC-SNA to the surface of SKOV3 and IOSE-364 was seen through confocal imaging. Scale bar =10 μm (b) Immunohistochemical analysis showing binding of FITC-SNA (green, shown by arrow marks) to ovarian tissue sections. The nuclei were stained with DAPI. Bar=100 μm. (c) Surface binding of SNA was quantitated by flow cytometry in SKOV3 cells. (d) Quantitation of cellular viability was performed using MTT reagent in SKOV3 and WST-1 reagent in IOSE-364 cell lines. (e) BrdU proliferation assay was performed in SKOV3 and IOSE-364 cell lines with different doses of SNA as indicated

Figure 2

SNA induces apoptosis in OC cells. (a) After 24 h of SNA treatment SKOV3 and IOSE-364 cells were analyzed for apoptosis in a FACS flow cytometer. (bandc) Western blot analysis for cleaved caspase-3, -7, -9, Bax and Bcl-2 was done in SKOV3 and OAW-42 cells using SNA-treated whole cell proteins. GAPDH was used as loading control. (d) TUNEL assay was done in SKOV3 cells and then observed by confocal microscopy. Scale bar=10 μm

Figure 3

SNA exposure results in mitochondrial dysfunction. (a) SKOV3 cells stained with MitoTracker Red CMXROS were imaged for mitochondrial structure by confocal microscope. Scale bar represents 10 μm. (b-c) Graphs showing the mitochondrial length and perimeter of SKOV3 cells after indicated time period of SNA exposure. (d) Membrane potential of OAW-42 and IOSE-364 cells stained with JC-1 dye were measured by flow cytometry. (e) ROS production in OAW-42 and SKOV3 cells stained with Mitosox was analyzed by flow cytometry. (f) Western blot depicting cytosolic release of cytochrome-c in OAW-42, IOSE-364 cell lines after 24 h of SNA treatment with GAPDH as loading control. (g) Expression of cytochrome-c after 0, 4 and 8 h of SNA treatment in SKOV3 cells with GAPDH as loading control

Figure 4

A shift in cellular respiration towards. OXPHOS occurs under SNA exposure. OCR was measured by extracellular flux analyzer (Seahorse Bioscience). The basal OCR ATP production, proton leak and reserve respiratory capacity of untreated and SNA administered SKOV3 (a) and IOSE-364 (b) cells were measured by XF24 flux analyzer. The data shown here are mean ±S.E.M. for three experiments performed independently

Figure 5

Mitochondrial fission promoted by mitochondrial translocation of Drp-1 upon SNA exposure, results in decrease of cellular viability. (a) Q-PCR of mitochondrial fission and fusion genes after 4 h of SNA treatment in SKOV3 and IOSE-364 cells. (b) Western blots of Mfn-1 and Drp-1 in SKOV3 cells with GAPDH as loading control and Drp-1 in IOSE-364 cells with tubulin as loading control. (c) Confocal microscopy depicting colocalization of Drp-1(green) with mitochondria (red) in SKOV3 after SNA treatment for 8 h. Nuclei were stained with DAPI. Scale bar=10 μm. (d) Microscopic images of colocalization of Drp-1 (green) with mitochondria (red) in OAW-42 cells treated with SNA for the mentioned time points. ROI indicates merged region of interest. Scale bar=10 μm. (e) Qualitative analysis by PDM imaging. (f) ICA plots generated. (g) Statistics of colocalization study done by ICA. (h) Cell cycle analysis of SKOV3 treated with SNA for longer time periods (24 and 36 h) as observed by flow cytometry. (i) Cells quantitated in each phase of cell-cycle represented as bar diagram

Figure 5

Mitochondrial fission promoted by mitochondrial translocation of Drp-1 upon SNA exposure, results in decrease of cellular viability. (a) Q-PCR of mitochondrial fission and fusion genes after 4 h of SNA treatment in SKOV3 and IOSE-364 cells. (b) Western blots of Mfn-1 and Drp-1 in SKOV3 cells with GAPDH as loading control and Drp-1 in IOSE-364 cells with tubulin as loading control. (c) Confocal microscopy depicting colocalization of Drp-1(green) with mitochondria (red) in SKOV3 after SNA treatment for 8 h. Nuclei were stained with DAPI. Scale bar=10 μm. (d) Microscopic images of colocalization of Drp-1 (green) with mitochondria (red) in OAW-42 cells treated with SNA for the mentioned time points. ROI indicates merged region of interest. Scale bar=10 μm. (e) Qualitative analysis by PDM imaging. (f) ICA plots generated. (g) Statistics of colocalization study done by ICA. (h) Cell cycle analysis of SKOV3 treated with SNA for longer time periods (24 and 36 h) as observed by flow cytometry. (i) Cells quantitated in each phase of cell-cycle represented as bar diagram

Figure 6

SNA induces apoptosis through activation of the AKT-ERK1/2 pathways. Cells were stimulated with 12 μg/ml of SNA for indicated time periods. (a and c) Lysates prepared from the SKOV3 cells were analyzed for p-AKT, p-ERK1, T-AKT, T-ERK1, Bax, Bcl-2 with GAPDH as loading control. (b) Lysates from IOSE-364 were checked for the expression of p-ERK1 after 30-min incubation and T-ERK1 after 24-h incubation with GAPDH as loading control. (d) Apoptotic induction in SKOV3 cells were quantified by flow cytometry after 24 h of SNA treatment in presence or absence of 10 μM AKTi by Flow cytometry. (e) OAW-42 cell lysates were analyzed for p-Drp-1 and Drp-1 after 30-min and 24- h incubation with SNA, respectively. GAPDH was used as loading control. (f) The Q-PCR of Mfn-1, Drp-1 and Fis-1 after 4 h of SNA treatment was observed in IOSE-364 and SKOV3 cells

Figure 7

Schematic representation of SNA-mediated induction of apoptosis in OAW-42 cells. Hyper-sialylation associated with OC drives SNA binding to these cells leading to the activation of AKT and ERK1/2 pathways. Meanwhile in response to SNA administration, mitochondrial membrane permeabilization occurs in association with cytochrome-c release into the cytosol and ROS generation leading to mitochondrial dysfunction. The resulting shift in the cellular bioenergetics promotes cell cycle arrest finally culminating into apoptosis via caspase activation