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Human apoptosis antibody array-membranes studying the apoptotic effect of marine bacterial exopolysaccharides in HepG2 cells

1 Department of Microbial Biotechnology, National Research Centre, Giza, Egypt
2 Department of Hormones, National Research Centre, Giza, Egypt
3 Department of Microbiology, Specialized Hospital, Ain Shams University, Cairo, Egypt
4 Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt

Date of Submission04-Jun-2019
Date of Acceptance26-Nov-2019
Date of Web Publication28-Jul-2020

Correspondence Address:
Salma M Abdelnasser,
Department of Microbial Biotechnology, National Research Centre, Dokki, Giza
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_391_19

 > Abstract 

Background: Hepatocellular carcinoma (HCC) is considered as the third leading cause of cancer-related deaths, in spite of great advances in its treatment. The carbohydrate polymers, exopolysaccharides (EPSs), showed anticancer activity in diverse cancers.
Objective: The purpose of this study is to investigate a panel of 43 apoptotic proteins to assess the possible apoptotic induction effect of bacterial EPSs showing promising cytotoxic effects in HepG2 cells in our previous study, in an attempt to introduce exopolysaccharides as new source for cancer treatment.
Materials and Methods: Apoptosis-related proteins panel were examined through the analysis of Human Apoptosis Antibody Array-Membrane (43 targets).
Results: EPS-6 induces apoptosis through upregulation of different pro-apoptotic proteins as cytochrome C (9.52 fold) and tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R1) (153.49 fold). EPS-RS induces apoptosis through up regulation of second mitochondria-derived activator of caspases (SMAC) (15.75 fold) and the six insulin-like growth factors binding proteins (IGFBP-1 through – 6) (76.81 fold, 7.68 fold, 55.15 fold, 4.9 × 107 fold, 29.69 fold, and 28.92 fold), respectively.
Conclusion: Our results suggested that EPS-6 and EPS-RS could be considered as promising agents in hepatocellular carcinoma treatment.

Keywords: Exopolysaccharides, HepG2, proapoptotic, protein arrays

How to cite this URL:
Abdelnasser SM, Yahya SM, Mohamed WF, Gadallah MA, Shady HM, Mahmoud MG, Asker MM. Human apoptosis antibody array-membranes studying the apoptotic effect of marine bacterial exopolysaccharides in HepG2 cells. J Can Res Ther [Epub ahead of print] [cited 2022 Jun 25]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=291046

 > Introduction Top

According to the Barcelona Clinic Liver Cancer treatment algorithm, portal vein tumor thrombus (PVTT) and hepatocellular carcinoma (HCC) patients could undergo surgical resection only if the remains of the liver function was adequate.[1] Several studies [2],[3] showed that surgical resection in HCC and PVTT patients was a dynamic treatment preference to promote life quality and expand the survival period. However, patients undergoing resection, their 5-year survival rate altered between 50% and 75%, and the recurrence rate, including metastasis and tumor development could reach 50%, besides some cases had impaired liver function and showed bad surgical performance.[4] Following the aforementioned studies and findings,[5] they did not recommend surgical resection for HCC patients associated with tumor metastasis or vascular invasion in their study. Consequently, there is a need to investigate new therapeutic approaches for HCC, as it considered the second dominant cause in cancer deaths.[6]

Regarding the presently available knowledge concerning the molecular divergences that underlie carcinogenesis, several targets for the treatment and prevention of cancer seems to exist. A mutual denominator of many of these targets is the elimination of tumor cells via the inducement of apoptosis while being of no effect on nontransformed cells.[7] In this context, natural biomolecules having the capacity to promote apoptosis in tumor cells could be implied as chemotherapeutic and chemopreventive cancer agents. Due to its diverse structure and function besides its reliable processing cost, bacterial exopolysaccharides (EPSs) are considered attractive sources of biopolymers to overcome cancer as recent treatment strategies show limitations because severe side effects and multidrug resistance occurred in the clinical application.[8]

Recent reviews showed that EPS derived from marine bacteria exhibit anticancer and immune-modulatory activities. Priyanka et al. showed that the EPS (650 mg/l) with 7.08% uronic acid-containing sugars and sulfate functional group (2.68%), produced from the Indian Southwest coast bacteria Nitratireductor sp. strain PRIM-31, is a promising drug for brain tumors as it can oppose the Akt/P13K pathway through its anionic charge besides, its binding to the epidermal growth factor secreted by the tumor (the presence of uronic acid, sulfate functional groups, and phosphate to this EPS assigns an overall anionic charge to the polymer).[9] Over sulfated EPS (B100S) from the novel halophilic bacterium Halomonas stenophila strain B100 showed proapoptotic effect upon hematopoietic tumor cells, as Western blotting analysis revealed the appearance of cleavage products of caspase 9 and 8 when cells treated with general and specific caspase inhibitors.[10]

Apoptosis is induced by two signaling pathways, the extrinsic and intrinsic pathways.[11] The death receptor (DR) (extrinsic) pathway attached signals coming from the microenvironment to the intracellular signaling chain that regulates the implementation of programmed cell death.[12] Cell surface DRs, including tumor necrosis factor (TNF) receptors, Fas, and (DR4 and DR5) for TNF-related apoptosis-inducing ligand (TRAIL) are considered the mediators for this pathway.[13] Binding of ligands to their analogous DRs on the cell surface starts the transduction of sequence of signals which promote the formation of a death-inducing signaling complex (DISC) at the plasma Membrane.[14] The last step in this process is the activation of caspase-8, followed by the activation of caspase-3 and caspase-7.[15] Engagement of the mitochondrial (intrinsic) pathway is initiated via proteolytic processing of BID (a pro-apoptotic protein of the BCL-2 family) after cleavage, tBID migrate to mitochondrial Membranes causing its perturbations and the release of cytochrome C (cyto-C) and SMAC which in turn results in the activation of caspase-9. Caspase-3 cleaved and activated to engage the caspase cascade and other nucleases and proteases that end the last events of apoptosis.[16]

The tumor suppressor protein p53 has an essential role in inducing apoptosis.[17] The transcription of the proapoptotic BCL-2 family members, as Bax, DR-5, PUMA, BAK, and NOXA is induced by p53.[18] The cytoplasmic interaction of p53 with the antiapoptotic BCL-2 family proteins in the mitochondria induces the release of cyto-C and SMAC/Diablo (apoptogenic factors), as p53 can interact directly with Bak and/or Bax, stimulates a conformational change in them, favoring their migration to the mitochondria which in turn increase the permeability of its outer Membrane.[15]

Previously, we have illustrated that partially purified EPSs from 12 novel Egyptian marine bacterial strains showed promising cytotoxic activity against HepG2 cells. The EPS-6 and EPS-RS produced by the novel Bacillus megaterium SAmt17 (KP733903) and Bacillus subtilis Subsp. Subtilis SAmt3 (KP733900), respectively, Showed the lowest IC50, (218 and 224 μg/ml), respectively, on HepG2 cells.[19] In continuing our research interest, we established the current study to screen and report the dominant apoptotic mediators during the course of (EPS)-induced cytotoxicity in HCC (HepG2) cells by antibody array analyses.

 > Materials and Methods Top

In vitro cytotoxic assay

Cell propagation and maintenance

HCC HepG2 cells were purchased from American Type Culture Collection and maintained in the proper conditions. The cells were cultured in Dulbecco's Modified Eagle's Medium (Lonza, Belgium) supplemented by 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate at 37°C in a humidified incubator with 5% CO2. The cells harvested after trypsinization (0.025% trypsin and 0.02% EDTA) and washed twice with Dulbecco's phosphate-buffered saline. When the cell density reached approximately 80%, cells were split for further culture. The experiments were made up when the cells were in the logarithmic growth phase. EPSs were subjected to further extensive studies at gene and protein levels.

Antibody arrays

Protein array is a powerful technology for the simultaneous determination of the expression levels of a number of proteins. Human Apoptosis Antibody Array-Membrane (43 targets) ab134001 Kit (abcam ® England) were used. The manufacturer protocol was followed. After treatment with IC50 concentrations of EPSs,[19] HepG2 monolayers were washed with cold 1X PBS and lysed in 1X lysis buffer (kit component). Samples were incubated at 4°C for 30 min with gentle shaking. After that, lysates were spin down at 14,000 ×g for 10 min. Protein concentrations in each sample were measured using the Bradford assay.[20] Cell lysates were diluted using 6× solubilization buffer consisting of 375 mM Tris-HCl, 9% SDS, 50% glycerol, 0.03% Bromophenol blue, and 10% (v/v) 2-mercaptoethanol and were then heated (70°C for 10 min). Membranes were blocked with 2 ml 1x blocking buffer (kit component). Equal amounts (500 μg) of total proteins were loaded per each array Membrane, and proteins were separated through incubation of arrays harboring samples with 1 ml of 1x Biotin-conjugated Anti-Cytokines (kit component) overnight at 4°C with gentle shaking. Excess anticytokine reagent was removed by extensive washing with wash buffer I and wash buffer II. Blots were probed with 1.5 ml of 1x Streptavidin-HRP (kit component). Finally, Membranes were washed extensively with wash buffer I and wash buffer II and treated with Detection Buffers C and D (kit components). Light-sensitive films (Fuji, Japan) were used for X-ray film exposure.

Antibody arrays data analysis

The intensity score of each duplicated array spot was measured with the Studio Lite (Version 1.37 v) software (rsbweb.nih.gov/ij). The averaged intensities were calculated by subtracting the averaged background signal. The fold change was obtained by comparing the EPS-6- and EPS-RS-treated HepG2 samples to the untreated cells (Control) (determined as a value of 1). The intensity of positive control signals (biotin-conjugated IgG protein) was used to normalize signal responses for comparison of results across multiple arrays. Normalized values calculated according to manufacturer equation. After normalization to positive control signal intensities, relative expression levels were compared, analyte by analyte

 > Results Top

Antibody arrays

As shown in [Figure 1]b, [Figure 2], and [Figure 3]a, treating HepG2 cells with EPS-6 has the following effects on apoptotic proteins: the proapoptotic proteins Bad, Bax, and BID were increased by 29.9 fold, 7.47 fold, and 8.44 fold, respectively on comparing the EPS-6 treated HepG2 sample spots intensities [Figure 1]b to that of the untreated cells [Figure 1]a. Meanwhile, many other proteins were induced, such as caspase-8 (63.17 fold), cyto-C (9.52 fold), second mitochondria-derived activator of caspases (SMAC) (4.6 fold), the six insulin-like growth factors binding proteins (IGFBP-1–6) (38.44 fold, 5.59 fold, 42.73 fold, 2.5 × 108 fold, 16.86 fold, and 13.82 fold), respectively, and heat shock protein (Hsp60) (1.5 fold). DR6 (member of the TNF receptor superfamily) (1.8 × 108 fold), TNF-alpha (4.3 × 107 fold), the two receptors for TNF-alpha: TNFR1 (8.16 fold) and TNFR2 (33.91 fold), TNF-beta (3.5 × 108 fold), Fas (6.65-fold), FasL (6.71-fold), caspase-3 (7.5 × 107 fold), the tumor-suppressor protein p53 (3.28 fold), p21 (3 fold), p27 (4.96 fold), TRAIL-R1 (101 fold) and TRAIL-R2 (11.88 fold), and high temperature requirement A (HTRA) (a serine protease, associated with the degradation of folded proteins under cellular stress conditions) (1.12 fold).
Figure 1: The effect of tested exopolysaccharide-6 and exopolysaccharide-RS on apoptotic proteins. (a) Control sample (i.e., untreated HepG2 cells). (b and c) alterations of apoptosis-related proteins in exopolysaccharide-6- and exopolysaccharide-RS-treated HepG2 cells, respectively. Protein extract (500 μg) were used for apoptosis array analysis. Array spots were visualized according to the manufacturer's instructions. The intensity of each spot was measured as described in “Materials and Methods”

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Figure 2: Array map detecting 43 apoptosis markers in one experiment

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Figure 3: The relative fold change of proteins with significant difference upon (a) exopolysaccharide 6 and (b) exopolysaccharide RS treatment, respectively, compared to control

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Only the expression of BCL-2 was downregulated (0.92 fold), although the expressions of several inhibitors of apoptosis proteins, including cellular inhibitor of apoptosis proteins (cIAP)-2, XIAP, survivin, BCL-W, CD40 (member of the TNF-receptor superfamily), Hsp27 and (Hsp70), TTRAIL-R3 and TRAIL-R4, and type 1 insulin-like growth factor receptor (IGF-1R) and IGF-I and IGF-II were not affected.

Similarly, as shown in [Figure 1]c, [Figure 2] and [Figure 3]b, treating HepG2 cells with EPS-RS has the following effects on apoptotic proteins: the proapoptotic proteins Bad, Bax, and BID were increased by (130.87 fold, 49.1 fold, and 68.41 fold), respectively on comparing the EPS-RS treated HepG2 sample spots intensities [Figure 1]c to that of the untreated cells [Figure 1]a. Many other proteins were also induced, such as Caspase-3 (5.1 × 108 fold), cyto-C (19.49 fold), SMAC (15.75 fold), the six IGFBP-1–6 (76.81 fold, 7.68 fold, 55.15 fold, 3.2 × 108 fold, 29.69 fold, and 28.92 fold), respectively, and Hsp60 (8.21 fold). DR6 (member of the TNF receptor superfamily) (3.1 × 108 fold), TNF-alpha (7.4 × 107 fold), the two receptors for TNF-alpha: TNFR1 (153.4 fold) and TNFR2 (21.8 fold), Fas (12.24 fold), FasL (12.48 fold), caspase-8 (159.98 fold), the tumor-suppressor protein p53 (12.21 fold), p21 (9.32 fold), p27 (20.80 fold), TRAIL-R1 (153.49 fold) and TRAIL-R2 (21.80 fold), and HTRA (a serine protease, associated with the degradation of folded proteins under cellular stress conditions) (4.50 fold).

None of the apoptosis inhibitor proteins as BCL-2, cIAP-2, XIAP, survivin, BCL-W, CD40 (member of the TNF-receptor superfamily), Hsp27 and Hsp70, TRAIL-R3 and TRAIL-R4, type 1 IGF-1R, and IGF-I and IGF-II showed significant effects.

 > Discussion Top

Tumor cells had the ability to resist apoptosis or programmed cell death.[21] The programmed cell death mechanism maintains proper cell number and eliminates damaged or probable malignant cells.[22] Impairment in the regulation of cell death was involved in different types of malignancies.[23] In the context of targeting different elements of the apoptotic machinery through downregulation of antiapoptotic genes as BCL-2 and upregulation of proapoptotic genes as p53 and members of the TNF superfamily,[24] several studies had reported the induction of apoptosis in different tumor cells as HeLa cells [25] and in human T leukemia cells [10] after treatment with EPSs.

Considering the aforementioned findings, proteins from control and EPS-treated cells were captured on Proteome Profiler™ Arrays as shown in [Figure 1]a, [Figure 1]b, [Figure 1]c for the determination of the relative levels of proteins associated with apoptosis.

The transcription factor p53 was known as the key element regulating the expression of the anti- and proapoptotic genes.[26] Chemo and/or radiation therapy cause the generation of free radicals and or/DNA damage; this, in turn, activates the nuclear P53 which directly triggered mitochondrial death signaling and commencing apoptosis.[27] P53 is a tumor suppressor gene and one of the frequently mutated genes involved in different cancers.[28] The first-known oncogene that prevents cell death rather than stimulating its proliferation was the BCL-2, besides its first detection incorporated in the chromosomal translocation in B-cell lymphoma.[28] The three functional subgroups of the BCL-2 family were: the antiapoptotic (BCL-XL, BCL-2, BFL-1, BCL-W, and MCL-1), the proapoptotic (BOK, BAK, and BAX), and the BH3-only proteins (BID, BIM, NOXA, BAD, BMF, PUMA, HRK, and BIK).[29]

The role of p53 in controlling the expression of genes that mediate cell division cycle arrest and/or apoptosis in response to cellular stresses can be reviewed in Brinkmann et al.[30]

Our result showed similarity with the immune cytochemistry analysis which revealed that apoptosis in HepG2 cells was induced by the bioflavonoid (quercetin) through BCL-2 protein expression reduction and upregulation of the P53 protein, while the absorption spectroscopy showed no significant change in caspase-8 activity.[24] Apoptosis induction in MCF7 cells through upregulation of the P53 and downregulation of BCL-2 after incubation with acetone leaf extract of Ebenaceae was also studied.[31]

Apoptosis induction could be achieved by activating DR signaling pathways. Stimulation of DR4, TNF receptor 1 (TNFR1), and Fas can start multiple signaling pathways resulting in either tumor elimination or promotion in the cancer microenvironment.[32] Signaling pathway is transduced after member of the TNF superfamily cytokines induced receptor trimerization.[33] In our study, results showed upregulation of TNF superfamily cytokines as Fas ligand (FasL) [Figure 3] and TRAIL [Figure 3], such upregulation-induced apoptosis and drove cells to start the tumor elimination signaling pathway. The EPSs in this study had no effect on TNF-alpha gene expression [Figure 3].

EPS-RS and EPS-6 upregulate FasL, while EPS-RS only showed TRAIL upregulation. Similar apoptosis induction through activation of FASL occurred in AGS human gastric cancer cells after treatment with Poncirin (flavanone glycoside) as illustrated by western blotting.[34] Although our results showed induction of apoptosis via the upregulation of TRAIL in HepG2 cells, previous studies documented resistance to TRAIL-induced apoptosis in some cancer cells including human prostate cell lines as LNCaP and DU145.[35] Considering the aforementioned context and TRAIL strong antitumor activity through apoptosis induction in tumor cells and imperceptible toxicity on normal cells, combinatorial strategies depending on TRAIL and natural therapeutic agents were established.[36] Combination of TRAIL and subtoxic doses of ursolic acid (i.e., form of glycosides) induce significant apoptosis in LNCaP and DU145 prostate cancer cells resisting Trail cytotoxic effect.[37] Combined treatment of TRAIL- and goniothalamin-induced apoptosis via TRAIL-mediated pathway via upregulation of death receptor DR5 and downregulation of cellular FLICE-like inhibitory protein (cFLIP) (antiapoptotic regulator) in TRAIL-refractory colorectal cancer; LoVo cells.[37]

Only few studies concerned about studying the disruption in different cellular pathways at posttranslational level upon treating cells with the targeted compounds.[38] In this study, we followed this context to determine the apoptotic proteins whose activities were altered once exposed to the carbohydrate polymers under study. We screened Human Apoptosis Antibody Array-Membrane (43 targets) for EPS-6- and EPS-RS-treated HepG2 cells for determination of the relative levels of expression of a panel of proteins associated with apoptosis in an attempt to confirm the proapoptotic action of these EPSs. As shown in [Figure 3]a and [Figure 3]b, all the proapoptotic proteins showed upregulation expression levels with different expression folds; however, contentious results for the antiapoptotic proteins were observed.

From the apoptotic proteins that target mitochondria was the SMAC. SMAC bind to proteins that prevent apoptosis (IAPs) causing their deactivation and prevent them from stopping the apoptotic process to proceed. SMAC and other proapoptotic proteins upregulation are one of our goals for considering EPS-6 and EPS-RS as promising therapeutic agents.

IGFBPs mediate the induction of apoptosis, besides regulating the insulin-like. IGF-I and IGF-II had antiapoptotic action as IGF-I-induced Bcl protein expression and downregulated Bax expression leading to an increase in the BCL/Bax heterodimer which results in apoptosis suppression. IGF-IR mediated the antiapoptotic action of IGFs. According to these evidence IGF-I, IGF-II, and IGF-IR were expected to be downregulated, but in our study, they are upregulated.

Besides, the known p53-dependent p21 expression activation that induces antiapoptotic effect leading to cell growth arrest in cells showing DNA damage, induction of apoptosis, and cell proliferation inhibition through the upregulation of p21 expression independently of the cellular p53 status (i.e., mutant or not) was explained through the results of Yang et al.[39] Their novel anticancer compounds induced apoptosis in breast cancer cells through p21 upregulation independently of the p53.[39]

P21 could show proapoptotic or antiapoptotic activity, depending on subcellular localization, cellular conditions, and cell type. In the nucleus, cyclin-dependent kinase inhibitor P21 and p27 interfered the growth arrest in G1 and G2 phases through binding and inhibiting the complex cyclin-dependent kinase/cyclin formation, such inhibition implemented DNA repair and assigned a tumor suppressor role for p21.[40] In the cytoplasm, P21 considered oncogen as apoptosis was inhibited through its interaction with the proapoptotic proteins as apoptosis signal-regulating kinase 1 and stress-activated protein kinase, which in turn prevents cell death.[41]

Considering the above evidence, our results showed upregulation for the p21, p27, and p53 proteins expression levels which correspond to that of Nguyen et al.[42] Induction of apoptosis after triple-negative breast cancer cells quercetin treatment through FasL, p21, and p51 increased expression level.[42] Also in human hepatocellular cancer cells lacking p53, p21 induced the proapoptotic protein Bax to a level suitable to inhibit the antiapoptotic BCL-2. Breast cancer MCF-7 cells treated with the acetone leaf extract of Diospyros lycioides-induced apoptosis via upregulation of P53 and downregulation of BCL-2 as antiapoptotic protein.[31] P27 was induced by cell-cell contact and transforming growth factor-β1.[43]

Hsp27 and Hsp70 prohibited apoptosis by inhibiting caspase-3 and inhibiting the signaling pathway mediated through TNF-α or Fas receptors. Hence, downregulation for HSP27 and HSP70 had been required for apoptosis induction, and this was clear with the results of Choghaei et al.[44] Results revealed apoptosis induction in MCF-7 breast cancer cells treated with microRNA-29a inhibitor through downregulation of the molecular chaperones HSP27 and HSP70 and upregulation of HSP60.[44] Knocking down of HSP27 by (siRNA HSP27) increased apoptosis in RD rhabdosarcoma cells treated with actinomycin D.[45]

Hsp60 showed proapoptotic activity through caspase-3 activation or an antiapoptotic one through Bax complexes segregation. In human cancers in vivo, the molecular chaperone Hsp60 was noticeably upregulated and coordinated a cytoprotective pathway focused on stabilization of survivin levels and control of p53 action. On the contrary, removal of the Hsp60 lead to the loss of the mitochondrial survivin, which prevent apoptosis, parallel exceeded expression of p53, and stimulation of p53-dependent apoptosis in tumor cells. This Hsp60 cytoprotective action was particularly active only in tumors in vivo, where it was upregulated, when compared to normal cells, and loss of Hsp60 in normal cells did not lead to cell death or loss of mitochondrial function.[46] Transfection of MCF-7 or WT HCT116 cells with siRNA to knockdown Hsp60 resulted in loss of mitochondrial Membrane potential, release of the mitochondrial cyto-C in the cytosol, and proteolytic processing of effector caspase-3.[46]

Following the above-reviewed results, apoptosis could be induced through upregulation or downregulation of HSP60 but only through downregulation of HSP27 and HSP70. In the study, upregulation for HSP27, HSP70, and HSP60 was illustrated.

Apoptosis induced via downregulation of two members of proteins that inhibit apoptosis (IAPs), livin, and survivin. Livin bound SMAC (IAP antagonist) and caspase-3, caspase-7, and caspase-9 resulting in inhibition of apoptosis.[47] Survivin could inhibit apoptosis through binding the effector caspases-3 and -7 which in turn prevent caspase activity or through binding to pro-caspase-9, preventing cyto-C release and hence inhibited apoptosis via the mitochondrial-mediated pathway. Induction of apoptosis via downregulation of livin and survivin after treating the pancreatic cancer cells PANC-1 with Oxymatrine (extract from Chinese herb Sophora Flavescens Ait) was illustrated in the reverse transcription polymerase chain reaction results of Ling et al.[48]

X-linked inhibitor of apoptosis protein (XIAP) or inhibitor of apoptosis protein 3 (IAP3) prohibit apoptotic cell death.[49] Apoptosis was induced in human pancreatic cancer cells (BxPC-3 and PanC-1) resistant to AZD5582 (a synthetic novel small-molecule IAP inhibitor) through downregulation of the antiapoptotic protein Mcl-1 which is achieved via silencing of XIAP (i.e., transfecting cells with siRNA targeting XIAP).[50]

As unexpected observation, this study array results illustrated upregulation for survivin, livin, and XIAP.

TRAIL induced apoptosis only if bound to R-1 or R-2 although it could bound to four transmembrane receptors (TRAIL-R1 to TRAIL-R4) as R-1 and R-2 were the only receptors among the four receptors containing a full death domain (DD) required for induction of apoptosis, R-3 and R-4 play the role of regulatory receptors but could not mediate apoptosis upon TRAIL binding. TRAIL-R3 and TRAIL-R4 inhibited apoptosis; R-3 did not have an intracellular domain as it formed of a glycosylphosphatidylinositol protein and R-4 had coexpression with R-2 and hence could form complex that inhibit ligand binding. In review of these results, upregulation for TRAIL-R1 and TRAIL-R2 had been illustrated in our study. TRAIL binding to its receptor 1 or 2 led to the DISC formation, and this complex mediated caspase-8 self-cleavage which in turn induced apoptosis, DISC, a complex of Fas-associated DD (FADD), cFLIP, caspases-8 or -10, and receptor oligomers.[51] Induction of apoptosis and higher rates of cell death (80%) were reported to happen in myeloid leukemia cell lines as Kasumi-1 (ACC 220) and MM6 (ACC 124) through downregulation of TRAIL-R3 using TRAIL-R2 inducing targeted antibodies.[52] On contrary to our expections, TRAIL-R3 and TRAIL-R4 protein expression level showed upregulation in the study array.

Blocking the mediated signaling pathway involving the TNF superfamily transmembrane receptor, CD40 was suggested as mean of therapy for follicular lymphomas (FL) in B-cells. Inhibition of the NF-kB activity by I kB-α phosphorylation inhibitor BAY 117085 caused upregulation for the antiapoptotic proteins Bcl-xL and c-FLIP which inhibited the activation of caspase-8 and the attachment of CD40 to its ligand, such attachment prevent the TRAIL-mediated apoptotic pathway in FL B-cells.[53] In review of these findings, downregulation for CD40 was considered for induction of apoptosis. Neither EPS-6 nor EPS-RS showed downregulation for CD40 protein expression level.

Downregulation for the cIAPs was required also for induction of apoptosis, as they induced the activation of NF-kB pathway through linking K63 polyubiquitination of receptor interacting protein 1downstream of TNF-alpha receptor 1 (TNFR1).[54] Reversing the aforementioned results, EpS-6 and EPS-RS showed upregulation for cIAP-2.

Apoptotic extrinsic pathway was initiated after FasL and TNF-alpha (TNF-alpha: TNFR1 and TNFR2, FasL: CD95/Apo-1) bound to their receptors. Binding occurred between the protein motif of the TNF receptor-associated DD (TRADD) and FADD adaptor proteins with their corresponding intracellular DD. Death effector domain of pro-caspase-8 interacted with that of FADD and TRADD forming the DISC which in turn activated pro-caspase 8 to caspase 8 that activated other effector caspases leading to nucleus disruption and cell death.[55]

Previous study demonstrated induction of apoptosis through upregulation of DR6 (TNF receptor superfamily member 21) protein expression level.[56] Following the aforementioned findings, upregulation for TNF-alpha, TNF-beta, FasL, TNFR1, TNFR2, and DR6 were required for induction of apoptosis. According to the apoptotic array results in the current study, EPS-6 and EPS-RS showed upregulation for the protein expression level of the aforementioned proteins which reflects their promising proapoptotic activity.

HTRA1, HTRA2, HTRA3, and HTRA4 were members of the human HTRA proteases family. They are formed of a protease domain, one PDZ (postsynaptic density of 95 kDa, Discs large and Zonula occludens 1) domain and different N termini. Only HTRA1and HTRA2, were involved in control of cell proliferation and tumor suppression.[57] Absence of HTRA1 decreased the susceptibility to anticancer drugs,[58] and its overexpression prevented tumor growth in vivo and in vitro proliferation.[59]

The processed form of HTRA2/Omi was released into the cytoplasm during apoptosis to activate caspase directly or indirectly through IAPs cleavage as XIAP.[59]

In accordance to these mentioned observations, HTRA1 and HTRA2 were suggested to have a proapoptotic activity. In this study, the apoptosis array spotted HTRA1 and HTRA2 as a total protein (HTRA) which was upregulated significantly after cells treatment with EPS-6 and EPS-RS.

 > Conclusion Top

A significant apoptotic activity was illustrated for the bacterial carbohydrate polymers EPS-6 and EPS-RS using Human Apoptosis Antibody Array-Membrane (43 targets) ab134001 Kit (abcam ® England). Induction of apoptosis occurred via extrinsic- and/or intrinsic-mediated pathways. The overall findings of this study introduced new EPSs from marine bacteria as promising targeted therapy for HCC acting through promoting apoptosis. Further studies on different hepatoma cell lines and in vivo studies are also required to verify the use of these EPSs as a drug.

Financial support and sponsorship

The authors acknowledge the financial support of the National Research Centre, Cairo, Egypt (Grant no: 1159).

Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3]


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