Synergistic suppression of a disintegrin acurhagin-C in combination with AZD4547 and reparixin on terminating development for human osteosarcoma MG-63 cell

Current therapies available for the treatment of human osteosarcoma, an aggressive bone tumor, are insufficient. To examine an alternative approach of integrin-based anti-osteosacoma strategy, acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin, was isolated and evaluated for its application in combination with two potent inhibitors of basic fibroblast growth factor (bFGF) and interleukin-8 (IL-8). The investigation of human osteosarcoma MG-63 cells pre-incubated with a FGF receptor-1 (FGFR-1) blocker AZD4547, a CXC-chemokine receptor-1/-2 (CXCR1/2) antagonist reparixin, and acurhagin-C via two given modes of separation and combination was executed. Detected by flow cytometry, integrins-2/-5/-v/-β1, FGFR-1, CXCR1 and CXCR2 constitutively express on the resting membrane. However, bFGF/IL-8-activated
MG-63 cells only statistically enhanced the surface exposure of integrins-5/-1, FGFR-1 and CXCR2. In activated MG-63 cells, acurhagin-C targeting integrin-5 not only might potentiate the inhibitory effect of AZD4547 and reparixin on the surface expression of integrin-5, FGFR-1 and CXCR2, but also acurhagin-C used alone remained effectively to diminish the surface exposure of those targeted receptors.
Hence, a complicated crosstalk mechanism should be involved in the membrane interactions. Furthermore, co-administration of acurhagin-C with AZD4547 and reparixin also showed to have the synergistic suppression toward cell proliferation and the gene expression of matrix metalloproteinase-2. Also, the administration of three-in-one mode could nearly abrogate the cellular attachment onto collagen-IV- and fibronectin-coated wells, as well as penetration into Matrigel-barrier. These data supported an ECD-disintegrin acurhagin-C targeting integrin-5 upon combined used with AZD4547 and reparixin may become a promising therapeutic approach for attenuating osteosarcoma development.

Osteosarcoma, most prevalent in childhood and adolescence, is an aggressive bone tumor with creating osteoid nature [1,2] and thus may lead to osteosarcoma growth. Bone metastasis would confer worse prognosis for osteosarcoma development [2]. Unfortunately, current therapies only minor effective for metastatic osteosarcoma. The real pathogenesis of osteosarcoma is still unclear. Therefore, the extensive investigations for the underlying mechanisms involved in the pathogenesis and the essential factors contributing metastasis might improve the therapeutic management.The signal cascade of fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) interactions can drive a variety of cellular functions, such as proliferation, migration and differentiation [3]. However, deregulated FGFR signaling may cause the pathogenesis of various malignant tumors. Especially, FGFR-1 exhibits the most confident connection than other FGFRs in cancer progression [4]. Previous statements also intensively proposed targeting the FGF-FGFR pathway might present a novel strategy for cancer therapy [4]. Interleukin-8 (IL-8), a pro-inflammatory CXC-chemokine, may elicit intracellular signaling downstream of two G-protein coupled receptors, termed CXCR1and CXCR2 [5]. Increased expression of IL-8 and/or its receptors has been characterized in tumor cells, suggesting that IL-8 is a central stimulator within the tumor microenvironment. Owing to the diversity of effectors and downstream targets, IL-8 signaling not only can initiate angiogenic responses in endothelial cells, but also enhance tumor survival. Accordingly, IL-8 signaling extremely correlates with the angiogenesis and metastasis of numerous tumors [6,7].

Integrins, a family of heterodimeric receptors, can mediate cell-cell and cell-extracellular matrix (ECM) interactions. In addition to play as a vital adhesion molecule, the integrin-ECM ligation may elicit a complex intracellular signaling and then regulate a variety of physiological responses [8,9]. The importance of integrins toward angiogenesis has been most extensively studied, such as v-integrins.
Blocking integrins v3 and v5 may typically suppress growth factor- and tumor-induced angiogenesis found in animal models [10,11]. On the other hand, antagonists of vβ3 or 5β1 can substantially decrease basic FGF (bFGF)-induced angiogenesis, implying integrins vβ3 and 5β1 may modulate a common pathway of angiogenesis [10]. Moreover, many studies also reported ligation of integrins v3 and 51 with ECMs is an important mechanism for integrins-crosstalk [10]. It is established that the cooperative integrin/growth factor receptor/cytokine receptor signaling plays a critical role in tumor growth. The receptor crosstalk-mechanisms not only may affect tumor behaviors, but also reciprocally modulate the surface expression of one another [12]. Advanced studies also proposed the mode of actions of receptor-crosstalk might have the determinate implications for tumor metastasis and the acquisition of drug resistance [9,12]. Thus, cooperation between integrin/growth factor signaling or integrin/cytokine signaling is required for tumor development. In fact, integrin ligation can extensively enhance the secretion of growth factor and/or cytokine, which thereby respectively bind to their receptors to trigger signal transduction. Furthermore, both signaling pathways of growth factor and cytokine were also proved to exert a positive regulation toward integrin functions via directly promoting integrin expression level [12].

Disintegrins, initially isolated from snake venoms, are a family of Arg-Gly-Asp (RGD)-containing, cysteine-rich polypeptides that directly interact with integrins on cell membrane. Both RGD and RGD-like sequences are the recognition motifs with the ability to influence the binding of ECMs with integrins [13,14]. Based on thenumbers of total amino-acids and disulfide-bonds, disintegrins already were grouped into six categories, including short-, medium-, long-, homodimeric-,heterodimeric-disintegrins and disintegrin-like proteins purified from the C-termini of P-III-class snake-venom metalloproteinases [15]. Native integrins acting as critical modulators can encourage proliferation, adhesion and invasion in tumor cells.Therefore, disintegrins specific targeting integrins exert the potential to disturb tumor behaviors and may be applicable to the treatment of malignant tumor. For example, acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin from Agkistrodon acutus (A. acutus) venom, has been characterized as integrins-v/-5 inhibitors of human vascular endothelial cell [16] and murine melanoma cell [17].In this work, we attempted to clarify whether acurhagin-C has the therapeutic applications for human osteosarcoma and tried to explore the signaling profiles involved in the pathogenesis of osteosacoma progression. Therefore, we hypothesized that bFGF and IL-8, two common cytokines presenting at tumor microenvironment, would promote surface interactions of membrane receptors, up-regulate gene expression of matrix metalloproteinases (MMPs) and resultantly induce malignant behaviors of MG-63 cells. Cultured human osteosarcoma MG-63 cells were thus prepared.

The effects of acurhagin-C tested alone and in combination with two antagonists against FGFR and CXCR in bFGF/IL-8-activated MG-63 cells were evaluated by flow cytometric and real-time polymerase chain reaction (RT-PCR) analyses. Additionally, functional studies were also executed.MG-63 osteosarcoma cells were purchased from American Type Culture Collection (Manassas, VA, USA). Eagle’s minimum essential medium (EMEM), bovine serum albumin (BSA), a CXCR1/2 inhibitor reparixin [18] and recombinant human IL-8 were purchased from Sigma Chemical (St Louis, MO, USA). Human bFGF was purchased from ProSpec (East Brunswick, NJ, USA). Matrigel-precoated chambers were purchased from BD Biosciences (Bedford, MA, USA). The kit for the determination of viable cell number was purchased from Promega (Madison, USA). Anti-human IgG fluorescein isothiocyanate (FITC)-HP6017, anti-human IgG phycoerythrin (PE)-HP6017, anti-integrin 2 monoclonal antibody (mAb) FITC-AK7 [19], anti-integrin 5 mAb FITC-NKI-SAM-1 [20] and anti-integrin 1 mAbPE-TS2/16 [19] were purchased from BioLegend (San Diego, CA, USA), FGFR-1inhibitor AZD4547 [21], anti-integrin v mAb PE-P2W7 [19], anti-integrin 3 mAb FITC-SAP [19], anti-human CXCR1 mAb FITC-B1 [22] and anti-human CXCR2 mAb PE-E2 [23] were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC-FGFR-1 mAb M19B2 [24] was purchased from Novus Biologicals (Littleton, CO, USA). All other chemicals were of analytical grade.Human osteoblastic MG-63 cells were thawed and grown to 80-90% confluence in EMEM containing 10% fetal bovine serum (FBS). Cells were cultured at 37°C in the presence 5% CO2 and serially passed in cell culture flasks.

Confluent cultures were washed with phosphate-buffered saline (PBS), harvested with 0.025% trypsin and 0.01% EDTA, and counted with a haemocytometer before functional assays.As described by our previous method [19]. Briefly, MG-63 cells pretreated with different reagents were fixed for 30 min and then washed with PBS. Subsequently, cells were incubated with fluorescence-conjugated antibody for 30 min on ice. Following the pre-treatments of various inhibitors with MG-63 cells in an aliquot of 2% FBS-containing medium for 15 min, PBS, or activators (bFGF/50 ng/ml +IL-8/50 ng/ml), was respectively added as indicated and co-incubated for additional 24 h to evaluate their effect on cell proliferation. The number of living cells was measured by the method described previously [19].RT-PCR was performed on RT-PCR detection system CFX96 (BioRad) using the LabStar Sybr green PCR master mix in a 25 l-volume containing 30 ng cDNA, 12.5l of 2X PCR master mix and 1 l of primer mix (10 M forward primer, 10 Mreverse primer). The reactions were undertaken using the following programs: 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C [25]. The primers used in this study are listed below: MMP-2: forwardprimer-5′-GGGGCCTCTCCTGACATT-3’/reverse primer-5′- TCAC AGTCCGCCAAATGAA -3′; β-actin: forward primer-5′-GGAGCAATGATCTTGAT CTTCATTG-3’/reverse primer-5′-AGATCATGTTTGAGACCTTCAACAC-3′.As described by our previous method [19]. Briefly, cultured MG-63 cells were incubated in the absence and presence of bFGF/IL-8 for 15 min. After 4 h-incubation in the plates, unattached cells were gently washed away. Thereafter, the attached cells were fixed and stained. The stained adherent cells were examined by microscope and photographed before lysis for ELISA.The invasion assay was initiated by inoculating the upper chamber with MG-63 cells that were treated with PBS, or variable inhibitors, and the lower chamber was filled with PBS, or bFGF/IL-8. Functional inhibitors tested were respectivelypre-incubated with MG-63 cells for 15 min. Following 18 h-incubation, those cells migrated onto the lower membrane were measured by our previous method [19].Data were expressed as mean ± standard error mean (SEM). Student’s t-tests were used to assess the significance of differences between means.

Results and discussion
The crude venom of A. acutus was separated into five fractions by gel-filtration chromatography on a BioSep-SEC-s2000 column. The second fraction exhibiting caseinolytic activity was collected for further purification. The desalted and concentrated fraction was further re-fractionated by anion-exchange chromatography on a BioSep-DEAE-PEI column. The active fractions were then collected, filtered and concentrated for functional analyses. In previous investigations, acurhagin-C, at 0.2M without inducing human endothelial cell death [16], can typically promote murinemelanoma cell apoptosis [17]. We therefore applied this concentration to characterize the effects of acurhagin-C on MG-63 cell functions. In addition, our preliminary experiments also demonstrated the concentration used at 50 ng/ml for bFGF and IL-8 as the optimal setting with more efficient activity to elicit MG-63 cells proliferation. Surface exposure of integrin, fibroblast growth factor receptor and CXC-chemokine receptor on MG-63 cell membrane by flow cytometric analysisTo investigate the presence, or absence, of native receptors on resting (PBS-added) and bFGF/IL-8-activated MG-63 cell membrane, formalin-fixed cell suspension and the fluorescence-labeled antibodies as probes were subsequently prepared and utilized to detect the surface expression of essential receptors presented on MG-63cell-membrane by flow cytometry.

As excluding integrins-2/3 and CXCR1 based upon their low MFI-value (Fig.1a,e,g), those receptors of integrins-5/-v/-1, FGFR-1 and CXCR2 are highly expressed on the membrane of resting MG-63 cells (Fig. 1b-d,f,h). By contrast, only integrins-5/-1, FGFR-1 and CXCR2 are significantly exposed on the membrane of bFGF/IL-8-activated MG-63 cells. It is implied those activated receptors may possibly participate in the complicated interactions of MG-63 cells with exogenous effectors.The inhibitory effect toward surface exposure of integrin-5, FGFR-1 and CXCR2 on bFGF/IL-8-activated MG-63 cell by distinct drug-pretreatmentsTo further evaluate whether combined use of three antagonists had the synergistic effect to decrease the surface exposure of targeted receptors on activated MG-63 cells, three specific antagonists were thus administrated by separation and combination modes, as well as respectively pre-incubated with MG-63 cell prior to administration of bFGF and IL-8 for 15 min-incubation. The surface exposure of integrins-5/-1, FGFR-1 and CXCR2 on bFGF/IL-8-activated MG-63 cells were then detected by flow cytometry. As shown in Fig. 2a,c,d, the combined mode of three antagonists exhibited the strongest suppression on the surface exposure of integrin-5 (31%inhibition, P  0.01), FGFR-1 (42% inhibition, P 0.01) and CXCR2 (20% inhibition, P  0.05), but no influence that of integrin-1. However, the FGFR-1 antagonist AZD4547 used alone at 1 M as referred by AstraZeneca [21] had a significant suppression toward the expression of integrin-5 (25% inhibition, P 0.05) and FGFR-1 (34% inhibition, P 0.01).

Additionally, the CXCR1/2 antagonist reparixin, at 50 nM as suggested by Cayman Chemical [18], also could disturb the expression of CXCR2 (17% inhibition, P 0.05) on activated cell membrane. Interestingly, the ECD-disintegrin acurhagin-C [17] at 0.2 M was able to respectively suppress thesurface expression of integrin-5 (13% inhibition, P 0.05), FGFR-1 (17% inhibition, P 0.05) and CXCR2 (14% inhibition, P 0.05). These findings implied the signaling cascade of receptors cross-talk should occur in the complex networks among the interactions of integrin-5, FGFR-1 and CXCR2.The anti-proliferative effect of various drug-combinations on bFGF/IL-8-activated MG-63 cell by ELISAPrevious study reported that integrin crosstalk with FGFR-1 and/or CXCR1/2 may reciprocally cooperate to up-regulate tumor development [11]. We therefore evaluated whether the co-administration of three inhibitors exerted better inhibition onactivated-MG-63 cells proliferation. As shown in Fig. 3A, the combined use of AZD4547, reparixin and acurhagin-C, indeed, had stronger suppression (73% inhibition, P 0.01) than the single-administration of AZD4547 (37% inhibition, P 0.05) and acurhagin-C (54% inhibition, P 0.05) on diminishing cells proliferation.It is well-known that integrins may evoke the activation of specific MMPs.Particularly, MMP-2 and MMP-9 have the strongest cleavage activity to digest collagen-IV [26].

Furthermore, previous study indicated IL-8 has the activity to enhance cell migration and invasion by increasing the levels of MMPs and integrins in trophoblast cell, which also expresses two receptors of CXCR1 and CXCR2 [27]. We thereafter performed the antibody-based array to investigate the gene expression profile in bFGF/IL-8-activated MG-63 cell. Notably, only the expression level of MMP-2, but not that of MMP-9 and integrins-5/-1, had significantly amplified (P 0.01, as compared with control value) after the pre-treatment of cells with bFGF and IL-8 (data not shown). To confirm the gene expression profile of MMP-2 in MG-63 cells, RT-PCR analysis was conducted. However, exogenous bFGF and IL-8 had the constitutive activity to potently induce the gene expression of MMP-2 (83% increment, P  0.001) in activated-MG-63 cells (as shown in Fig. 3B). As a FGFR-1 inhibitor, AZD4547 used alone could inhibit the MMP-2-gene expression (36% inhibition, P  0.05). As compared with single-administration (24% inhibition, P 0.05), acurhagin-C in combination with AZD4547 and reparixin remained to exhibit the synergistic inhibition (52% inhibition, P 0.01). This result clear indicated the signaling events in activated-MG-63 cells may involve the cross-talk betweenFGFR-1 and integrin-5. Hence, snake venom disintegrin acting as a therapeutic adjuvant in cancer therapy might become an effective and a promising approach available for the eradication of osteosarcoma.Functional assessment for various drug-combinations toward activated-MG-63 cells adhesion and invasion by ELISA and microscopic examinationTo determine whether various administration modes might substantially affect MG-63 cell behaviors, two assays of cell adhesion and invasion were thus executed. As compared with BSA by ELISA, those bFGF/IL-8-activated MG-63 cells adheringonto collagen-IV and fibronectin, respectively, showed to have a statistical increment of 31% (P 0.01) and 63% (P 0.001) (Fig. 4A).

In general, various pre-treatments of inhibitors as indicated had statistically inhibition toward cells adhesion ontocollagen-IV and fibronectin. It is established that improper integrin/ECM interactions may typically induce cell apoptosis. For example, the integins 51- andv3-expressing cells would undergo programmed cell death at the absence ofpreferential binding of ECMs with suitable integrins [28]. As shown in Fig. 4B, the attachment of MG-63 cells onto BSA, as a negative-control matrix, showed to spontaneously elicit a morphological change with round appearance (shown as indicated). It represented MG-63 cells without extensive spreading might already initiate apoptotic pathway through caspase activation, leading to most of the apoptotic cells had been washed away after washing process. Interestingly, such a response also could be additionally enhanced under the pretreatment of AZD4547 alone. However, in other cases of single pretreatment of cells using reparixin and acurhagin-C, MG-63 cells specially elicited to form large multi-cellular aggregates (as indicated). This finding might be possibly due to undergo apoptosis of MG-63 cells via a sequential blockade of CXCR2 and integrin-5. That may strengthen aggregate formation by increasing physical stretch from neighboring cells.Also, our morphological analysis by microscopy found MG-63 cells adhering onto collagen-IV and fibronectin with extensive spreading that is different from those cells on BSA (Fig. 4B).

Parallel with our previous result found in cell proliferation assay, the combined use of three antagonists also had a maximal synergism, causing almost completely suppress MG-63 cells adhering onto ECMs. For further evaluating theanti-invasive effect of acurhagin-C used alone or in combination on MG-63 cells, the three-dimensional barrier Matrigel-coated membrane in screening chamber was used. As shown in Fig. 4Ca,b, bFGF and IL-8 in 2% FBS-containing EMEM stronglypromoted MG-63 cell invasion to the lower surface of filter membrane. As expected, the respective use of three antagonists under the same concentration within proliferation assay remained effectively to suppress bFGF/IL-8-induced MG-63 cell invasion (Fig. 4Cc-e). However, the mode of combination use also could maximize the synergistic suppression on MG-63 cell invasion via Matrigel-barrier, leading to a little cells migrating into the lower surface of the filter membrane (as shown in Fig. 4Cf). Indeed, this result is consistent with our previous finding in the assay of cell proliferation. Since acurhagin-C, acting as both the inhibitors of integrins 5 and v [17], showed to have potent inhibition to interrupt cell invasion, the anti-invasive function of acurhagin-C may be attributable to its integrin-blocking activity [17] (Fig. 4Ce,f).

In conclusion, therapeutic interventions against integrin/FGFR/CXCR-mediated tumor development became a novel, promising strategy to terminate tumor growth. Acurhagin-C, an ECD-disintegrin, is a potential candidate for its multiple applications by affecting tumor behaviors. In this study, acurhagin-C targeting integrin-5 was proved to be an effective adjuvant upon combined used with a FGFR-1 blocker and a CXCR2 antagonist. The strategy of three-in-one therapy was found to exert a significant attenuation toward human MG-63 cell functions, such as proliferation, adhesion and invasion, as well as the MMP-2-gene expression. Imaginably, the downstream of cross-talk mechanism following the complicated interactions of integrin-5/FGFR-1/CXCR2 signaling may involve the specific amplification of MMP-2 gene and might thereby encourage the invasive action of MG-63 AZD4547 cells.