extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
IFI6 Inhibits Apoptosis via Mitochondrial-Dependent Pathway in Dengue Virus 2 Infected Vascular Endothelial Cells

Dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS) is a fatal infectious disease that demands an effective treatment. Interferon (IFN)-stimulated genes (ISGs) induced by dengue virus (DENV) exert antiviral effects. Among ISGs, IFN-α inducible gene 6 (IFI6) was increased in DENV infected human umbilical vascular endothelial cells (HUVECs) by microarray analysis in our previous study. However, its function is incompletely understood. In this study, we confirmed that IFI6 was markedly induced in DENV infection of both primary HUVECs and EA.hy926 cell lines. Recombinant EA.hy926 cell lines in which IFI6 was either over-expressed (IFI6+/+) or knocked-down (IFI6-/-) were generated. The activation of caspase-3 and intrinsic apoptosis-related protein caspase-9 were down-regulated in IFI6+/+ but up-regulated in IFI6-/- cells at 24–48 hrs post-infection. After incubation with DENV for 48 hrs, the mitochondrial membrane potential (Δψ(m)) was more stable in IFI6+/+ cells but reduced in IFI6-/- cells, as assayed by fluorescence staining with JC-1. We observed that Bcl-2 expression was increased in IFI6+/+ and decreased in IFI6-/- cells. By contrast, Bax expression was decreased in IFI6+/+ and increased in IFI6-/- cells. It is presumed that the anti-apoptotic function of IFI6 is expressed by regulating the rheostatic balance between bcl-2/bax expression and inhibition of Δψ(m) depolarization during DENV infection of vascular endothelial cells(VECs). In addition, the pro-apoptotic protein X-linked Inhibitor of Apoptosis (XIAP)-Associated Factor 1(XAF1) expression had been reported to be up-regulated and led to the induction of apoptosis in DENV2-infected VECs,but the relationship between XAF1 and IFI6 dengue virus-induced apoptosis in VECs warrants further study.

Introduction
Dengue virus (DENV), which belongs to the flavivirus family, has four serotypes including DEN1 to DEN4. It is one of the most prevalent mosquito-borne viruses that cause roughly 390 million infection in humans annually [1]. Unfortunately, there is neither effective therapeutics nor approved vaccines available to prevent or treat infection [2]. Therefore, detailed understanding of the immunopathology of the interaction of DENV with the host is an urgent research need, which should contribute important knowledge in the exploration of new ways to combat this tropical communicable disease.
Clinical manifestation of DENV infection is highly variable, and ranges from unapparent infection to a mild febrile syndrome or even fatal disease with a mortality rate of 5% to 30% [3]. Although a majority of DENV infections display a self-limited course, which are resolved in several days, there remain some susceptible patients that would progress to dengue hemorrhagic fever (DHF), which is characterized by increasing vascular permeability and plasma leakage that manifests in approximately three days following infection [4]. In rare cases, a more sudden onset and extensive syndrome, might progress to shock or even death, which is referred to as dengue shock syndrome (DSS). Usually, the hinge point of the disease always appears following a temporal defervescence phase, after which patients either gradually improve or progressively decline to more severe DHF and DSS. Thus, endothelial cells are regarded as the primary cellular barrier and the major defense system of the vasculature to resist viral infection and in the determination of the prognosis of disease [5]. However, the potential mechanisms of endothelial cells during DENV infection, especially at early stages of disease, currently remain obscure.
Type I interferon (IFNα/β) is produced by most cells, and plays a crucial role in the ability of the endothelium to withstand DENV infection. Increases in gene expression and circulating levels of type I IFN were confirmed by many studies [6,7], and type I IFN is highly effective at inhibiting DENV replication during early short periods of viremia in most mild cases. However, in severe forms of DENV replication, the ability of type I IFN to inhibit viral replication is overwhelmed [8].
The canonical pathway of Type I IFN is the IFN-stimulated gene factor (ISGF)-3 pathway, which is formed by IFN-regulatory factor (IRF)-9, phosphorylation of signal transducer and activator of transcription (STAT)-1/STAT-2 and sequential activation of the IFN-stimulated response element (ISRE) that leads to transcriptional up-regulation of many IFN-stimulated genes (ISGs) [9]. IFNα inducible protein 6 (IFI6), previously known as G1P3, IFI-6-16 and IFI616, is a type I ISG that is located on chromosome 1p35 [10], and alternatively spliced transcript variants from exons two and three that encode different isoforms of IFI6 by using two downstream repeat units as splice donor sites enable transcription of three mRNA species [11]. Predominant production of IFI6 is the 14 kDa hydrophobic protein form of 130 amino acids, which may play a central role in regulating apoptosis and immunomodulation [12]. Additionally, the Janus tyrosine kinase (JAK)/ STAT signal transduction pathway regulates IFI6, which is a mitochondria-targeted protein that blocks the release of cytochrome c from mitochondria, and thus delays programmed cell death (apoptosis) that is initiated and signaled by the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/ caspase-8 pathway [13,14]. IFI6 is thus widely considered a pro-survival factor [15]. Although the functional role of IFI6 in the immune system is quite well known, its antiviral effects are poorly understood [16].
Microarray studies have assisted our understanding of the different gene expression profiles seen in DENV infected VECs [17,18]. A series of IFN-stimulated and IFN-inducible genes are  highly expressed post-infection, and include ISG15, IFI6, IFI44, IFI44L, IRF27, IFIT1, IFIT3,  and XAF1. Among these, IFI6 is not only one of the most upregulated genes [19] but is also a specific target for yellow fever virus (YFV), which is congeneric with DENV [20]. Further, IFI6 belongs to the ISG12 gene family, which is composed of four members, ISG12a, ISG12b, ISG12c and IFI6 [21]. Although they are homologous, the biological functions of the ISG12 family are quite varied, and range from preventing host susceptibility to a destructive stimulus to enhancing the sensitivity to DNA damage-induced apoptosis [16].
As a new member of the ISG12 family, most research has focused on studying the IFI6 upstream regulatory region or the function of IFI6 in cancer cells [14,16]. Moreover, its expression is extremely high in multi-drug resistant cancer cells, suggesting that a close correlation between IFI6 levels and resistance to apoptosis [16]. However, the potential role of IFI6 and its mechanism of action in infectious diseases, especially in viral infections like DENV, remain poorly defined. We hypothesized that the human ISG12 family protein member, IFI6, might be involved in regulating the intrinsic apoptotic pathway in response to DENV infection of VECs. Primary HUVECs isolated from human umbilical veins and EA.hy926 HUVEC cell line were exposed to DENV2 infection and IFI6 expression was detected on mRNA and protein levels. Then overexpression and knock-down plasmids of IFI6 were constructed and transfected into EA.hy926 cells. Annexin V-based flow cytometry assays in different groups were performed to verify the role of IFI6 in DENV-induced apoptosis and underlying mechanisms were investigated.

Cell lines
Primary HUVECs were isolated from human umbilical veins (provided by First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China) in the study and the study was approved by Medical Ethical Committee of The First Affiliated Hospital, Sun Yat-sen University. Primary HUVECs were cultured in endothelial cell growth medium (30% serum-free medium (SFM) and 60% M199, Gibco, USA) with 10% fetal calf serum (FCS), 100 U penicillin-G, 100 μg/mL streptomycin sulfate, and 30 μg/mL endothelial cell growth supplement (Millipore, USA). Samples were obtained after receiving a written informed consent document for each patient.
The EA.hy926 HUVEC cell line was obtained from the Center for Stem Cell Biology and Tissue Engineering (Sun Yat-Sen University, China). Cells were cultured in DMEM medium (Thermo Scientific, USA) with 10% FCS, 100 U penicillin-G, and 100 μg/mL streptomycin sulfate.

DENV2 preparation and infection
The DENV2 serotype originated from the New Guinea C strain was reservation in our laboratory, which was propagated and quantified as previously described [22]. All infected cells in the experiments were incubated with DENV2 at a multiplicity of infection (MOI) of 4 for 2 hrs and then the suspension medium containing DENV2 particles was removed. The culture medium was replaced with 2% FCS, followed by an additional incubation for up to 48 hrs. Vector cells were processed in parallel as controls.

Identification of EA.hy926 cells
The phenotype of EA.hy926 were confirmed by immunocytochemical staining for factor VIII and surface expression of platelet/endothelial cell adhesion molecule 1 (CD31) was detected. Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and then incubated with 3% hydrogen peroxide for 20 min at room temperature (RT) to quench endogenous peroxidase activity. Fixed cells were then blocked with 5% bovine serum albumin (BSA) for 20 min and incubated overnight at 4°C with an antibody against factor VIII-related antigen (Boster, China). Immunolabeled cells were washed thoroughly in phosphate-buffered saline (PBS), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Abcam, UK) for 1 h at RT. Immunostaining was visualized by incubation in the chromogen diaminobenzidine (DAB) for 5 min at RT. Expression of CD31 was assessed by flow cytometry. Cells were collected and stained with anti-CD31-FITC (Becton Dickinson, US) for 30 min at RT, followed by fixation for 20 min at 4°C. Samples were loaded in a FACSCalibur system (Becton-Dickinson, US) and results analyzed using FlowJo software (TreeStar, San Carlos, CA, USA).

Construction of stable EA.hy926 cell lines by over-expression or knock-down of IFI6
Retroviral-mediated stable expression of IFI6 was achieved by using EA.hy926 cells and the pBabe-Puro Retroviral Vector system (pBabe; Addgene, Cambridge, MA, USA) or the pSUPER RNAi system (pSUPER.retro.puro, pSUPERretro; Addgene) to over-express or knock-down gene expression, respectively.

MTT Assay
5000 cells/well were seeded into 96-well culture plates one night before treatment. After that, cells were infected by DENV2 with MOI 4. MTT reagent was added at 20 μL (5 mg/mL) per well at various times, and cells were then incubated for another 4 hrs at 37°C. The reaction was stopped by adding 150 μL dimethyl sulfoxide (DMSO), following which a purple formazan precipitate dissolved in the DMSO. The optical density was measured at a wavelength of 490 nm using an MRX II absorbance reader (BioRad, USA). With the background absorbance subtracted, results were described as mean values, which were measured in triplicate and repeated from three independent experiments.

TUNEL analysis and immunofluorescence
Cells were cultured on glass slides, treated as described above, and then rinsed twice in PBS. Slides were then fixed in 4% paraformaldehyde, and cells were perforated by treating them with 0.5% Triton X-100 at room temperature for 15 min. Next, cells were incubated with 0.125% pamcreatin for another 10 min, and cells were blocked with 5% BSA at room temperature. Redundant fluid was discarded 30 min later, and cells were then incubated with the primary antibody NS1 at a dilution of 1:10 (Abcam, USA) overnight at 4°C. Next, a secondary antibody anti-mouse-Cy3 at a dilution of 1:1000 (Invitrogen, USA) was added at room temperature for 30 min, followed by rinsing in PBS and staining with the Tunel reaction mixture for 60 min, following the manufacturer's guidelines. Finally, DAPI staining (37°C for 15 min) was used to visualize the nucleus, and observed by confocal microscopy.

Flow cytometry
The frequency of DENV-induced apoptosis in both endothelial cells and recombinant cells was determined with an Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN, China). Annexin V that had bound to the externalization of phophatidylserine, which is an early apoptotic event of the cell, was analyzed by flow cytometry after incubating with DENV2 (MOI = 4) for 24 hrs, 36 hrs and 48 hrs. Approximately 5×10 5 cells were resuspended in 5 μL Annexin V binding buffer (10 mmol/L Hepes/NaOH (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl 2 ). Using double labeling for Annexin V-FITC and PI, we separated sub-populations of early apoptotic cells (Annexin V+/PI−), late apoptotic or necrotic cells (Annexin V+/PI+), and viable cells (Annexin V−/PI−) from each other.

Mitochondrial Membrane Potential (Δ ψ) analysis
Cells were seeded onto small glass slides (Orange Scientific. E.U). Culture medium was abandoned following treatment with DENV2 for 24 hrs, 36 hrs, and 48 hrs, respectively. JC-1 assay reagent (25 μmol/L) was added to the dilution buffer (500 μL/well), which was then incubated at 37°C for 20 min to stain the mitochondria. After 2 to 3 rinses with wash buffer, cells were inspected using an Axiovert 200 fluorescent inverted microscope(Zeiss, Germany). Both monomeric (excitation at 488nm, emission 500-550nm) as well as aggregation (excitation 488 nm, emission at 575-620nm) were registered using the microscope. In each slide, about 200 cells were counted to make sure the results are visualize and comparable.

Immunoblotting
Treated cells were harvested and lysed using a mammalian protein extraction reagent (Kangwei Company, China). Whole-cell lysates of a fixed quantity (30 μg/lane) were subjected to 10% SDS-PAGE and then transferred to a polyvinyldine fluoride (PVDF) membrane (Millipore, USA). We used the following specific antibodies: anti-IFI6, -caspase-3, -caspase-8, -Bax, and-Bcl-2 (all from Cell Signaling Technology, USA) and anti-XAF1 (Abcam, USA). The antibody dilutions for Western blotting primary antibodies and secondary antibodies were 1:800 and 1:2000, respectively. The vehicles for antibodies were 5% BSA. Enzyme-linked chemiluminescebetance (Kangwei Company, China) was used to detect the target band according to the manufacturer's instructions. The images were analyzed using the Image J program. Histogram of each image was first extended to saturate the gray scale. Then, the 12-bit images were converted to an 8-bit gray scale. Intensity of each band was divided by the β-actin band firstly, and then the relative value were compared.

Detection of activity of Caspase-9
3-5×10 6 cells were collected, resuspended in 50 μL of cold cell lysis buffer and incubated on ice for 10 min. Cells were centrifuged for 1 min (10, 000×g). The supernatant (cytosolic extract) was transferred to a new tube and immediately placed on ice. The BCA Protein Assay Kit was used to assay the protein concentration. Each cytosolic extract was diluted to a concentration of 50-200 μg protein per 50 μL of cell lysis buffer. Then DTT was added to the 2×Reaction Buffer immediately before use (10 mmol/L final concentration), and 50 μL of 2×Reaction Buffer (containing 10 mmol/L DTT) was added to each sample. Next, added 5 μL of 4 mmol/L LEHD-pNA substrate (Invitrogen, Shanghai, China) which is composed of the chromophore, p-nitroanilide (pNA), and asynthetic tetrapeptide, LEHD (Leu-Glu-His-Asp),. samples were then incubated in the dark at 37°C for 2 hrs. Samples were read at a wavelength of 400 nm or 405 nm in a microplate reader (BioTek, Vermont, USA).

Statistical analysis
Data are expressed as mean±s.e.m. All data groups were analyzed by Student's T test with the software program SPSS v. 16.0 (SPSS Inc., Chicago, IL, USA). An alpha value of P<0.05 was used to determine the statistical significance when interpreting the results.

IFI6 is induced in VECs in response to DENV2 infection
IFI6 up-regulated in HUVECs after infection with DENV2 for 48 hrs by GeneChip hybridization and analysis (Fig 1, panel a, top), and mRNA expression of IFI6 in primary HUVECs isolated from human umbilical veins was markedly up-regulated after 48 hrs post DENV2 infection (Fig 1, panel a, below). EA.hy926 immunostained with the antibody against factor VIII and an HRP-conjugated secondary antibody exhibited brown DAB reaction product ( Fig  1, panel b, top), while EA.hy926 cells exhibited CD31 surface staining (Fig 1, panel b, below). Immunobloting assays were employed to validate IFI6 protein levels in EA.hy926 HUVECs cells, confirming that IFI6 was increased following DENV2 infection (Fig 1c).

Generation of EA.hy926 cell lines with IFI6 over-expression or knock-down
To further understand the role of IFI6 in apoptosis that was induced by DENV2-mediated infection of VECs, we constructed two stable cell lines of EA.hy926 IFI6+/+ and IFI6-/-cells, which were transfected with IFI6 over-expression (pMSCV-neo-IFI6+) and IFI6 knock-down (pMSCV-neo-IFI6-) plasmids. Empty vectors pMSCV-neo and pSUPERretro transfected cell lines were also generated as controls, which were referred to as "vector-high" and "vector-low", respectively. Next, cells were subjected to qRT-PCR analysis of total RNA extracts and immunobloting of protein lysates in order to confirm alteration of IFI6 expression. The results were shown in Fig 2. Both mRNA and protein levels of IFI6 were significantly increased in IFI6 +/+ cells and decreased in IFI6-/-cells.

IFI6 suppresses DENV2-induced apoptosis in VECs
A number of studies had already shown that DENV2 could induce apoptosis in VECs [23][24][25]. Thus we explored the potentially important role that IFI6 might play in this pathological process of programmed cell death. With the aid of fluorescence confocal microscopy, we observed that the non-structual 1 protein (NS1) of DENV2 appeared in more than 90% of the recombinant VECs after 24 hrs post DENV2 incubation. Evidence of apoptosis was found scattered within the infected cells. However, there seemed to be almost no DNA fragments at the early stages of infection in IFI6+/+ cells, which maintained a lower level of apoptosis as compared with the vector-high cells (Fig 3a). On the contrary, DNA fragments had increased in IFI6-/-cells as compared to vector-low cells at optional time points, especially at 48 hrs after infection (Fig 3b).
Annexin V/propidium iodide (PI) staining and flow cytometry assays were also performed to distinguish the early-stages of apoptosis. The results demonstrated that after 24 to 48 hrs following DENV2 infection, Annexin V positive cells were constantly lower in IFI6+/+ cells than in the corresponding vector-high cells (left panels in Fig 3c and 3d). Concomitantly, this observation appeared much more frequent in IFI6-/-cells as compared to the vector-low cells, with statistical differences seen at 36 and 48 hrs post DENV2 infection (right panels in Fig 3c and 3d). We also enumerated cell viability by MTT assay (Fig 3e and 3f). Viability of IFI6+/+ cells was higher than vector high cells and viability of IFI6-/-cells was lower than vector-low cells. These data indicated that IFI6 may play important roles in apoptosis process induced by DENV2 infection

IFI6 inhibits DENV2-induced VECs apoptosis via caspasedependent pathway
To determine whether intrinsic or extrinsic pathways were activated during apoptosis induced by DENV2 infection, we detected protein levels of caspase-3 and 8 using immunoblotting and anzyme activity of caspase-9 using colorimetry. As shown in Fig 4a, stronger inhibition of cleaved caspase-3 occurred time-dependently in IFI6+/+ cells as compared with vector-high cells. By stark contrast, an extremely increased level of cleaved caspase-3 was observed in IFI6-/-cells as compared with vector-low cells (Fig 4b). Caspase-8 was expressed at much lower levels at the early stages of DENV2 infection in IFI6+/+ cells as compared with vectorhigh cells, but there was no marked difference at 48 hrs post-infection. Additionally, increased tendency of caspase-8 activation was also inconspicuous in vector-low cells (Fig 4a and 4b).
However, consistent lower activity of caspase-9 was observed in IFI6+/+ cells as compared with vector-high cells (Fig 4c) while much higher activity of caspase-9 was observed in IFI6-/cells as compared with vector-low cells (Fig 4d).

Mitochondria participates the apoptotic progress induced by DENV2 and IFI6 regulates mitochondrial function
It has been established that caspase-9 plays an important role in the intrinsic apoptosis pathway [26], which is mediated by activation of mitochondria. We assayed alterations in the mitochondrial membrane potential (Δψm) in situ by a cationic and voltage-sensitive vital fluorochrome, called JC-1. The lipid dye emits differential fluorescence that is depended on different polymerization forms. In healthy mitochondria with normal Δψm, JC-1 always transfers into the mitochondria and aggregates to a polymer, and emits red fluorescent signals. On damage to the mitochondria, there is depolarization of Δψm, and JC-1 is released to the cytoplasm, where the fluorescence changes to a green signal. As shown in Fig 5, the continued decline of Δψm in both vector cell lines was conspicuous from 24 hrs to 48 hrs after DENV2 infection. The infected IFI6 +/+ cells displayed a stable Δψ m as shown by a sustained aggregation of the red JC-1 fluorescence over periods of time (Fig 5a). By contrast, IFI6-/-cells appeared to be more sensitive to DENV2, which was indicated by complete vanishing of the red fluorescent signal at early-stages post-infection, and the ongoing enhanced green fluorescence of the JC-1 aggregates (Fig 5b).

IFI6 regulates apoptosis-related proteins Bcl-2/Bax and XAF1
To verify whether the change in Δψ m was associated with a balance in the expression of proapoptotic and anti-apototic proteins Bcl-2/Bax in the mitochondria or in other subcellular   structures, we performed immunoblotting assays. The infected IFI6+/+ cells provoked a much higher level of Bcl-2, but a much lower level of Bax and the nuclear protein XAF1 as compared with the vector-high cells at the outset of virus challenge (Fig 6a). Additionally, Bcl-2 expression was lower in IFI6-/-cells, while expressions of Bax and XAF1 were both higher than those of vector-low cells at all time-points measured (Fig 6b).

Discussion
IFNα/β is elicited early in DENV2 infected cells, which had been long recognized in clinical cases [27]. IFNα/β serves as an early barrier to DENV infection, and is specifically indispensable for restricting the initial replication or spreading of DENV [5]. Therefore, IFI6, which is induced by IFNα/β, may play a critical role in combating viral infection and maintaining cell survival. In our previous study, we observed enhanced IFI6 expression by differential display microarray analysis of DENV infected VECs [19]. In current study we also detected mRNA expression of IFI6 by qRT-PCR at 48 hrs post-infection in primary HUVECs, and found that IFI6 expression was dramatically increased in infected HUVECs. These observations were confirmed by immunoblotting analysis of protein levels to find that IFI6 level in infected EA.hy926 cells exceeded that of uninfected cells. For the last decade, IFI6 was generally regarded as a pro-survival gene since it is expressed at high levels in gastric cancer, colorectal cancer, breast cancer, myeloma, tongue squamous cell carcinoma, and in psoriasis [14,[28][29][30][31][32]. To know more about the role played by IFI6 in DENV infected VECs, we are committed to construct recombinant cells displaying stable overexpression or knock-down of IFI6. But HUVECs are not sensitive to the transfection,so we choose EA.hy926 cells which exhibited endothelial cell phenotype to construct the recombinant cells. We found that IFI6+/+ VECs exhibited enhanced cell viability and resistance to DENV2-induced apoptosis. On the contrary, cell death was significantly accelerated and aggravated in IFI6-/-cells. This finding is in agreement with reports of the role of IFI6 in cancer cell survival and death [14]. IFI6 also serves as a potential molecular mechanism that tumor cells might employ to resist chemotherapeutic drugs, including cycloheximide (CHX) or 5-fluorouracil (5-FU) because its expression is potently enhanced in multidrug-resistant malignant cells and attenuates apoptosis following anti-tumor therapy [10].
Many investigators have shown that DENV2 might induce apoptosis by extrinsic and intrinsic pathways [33]. Caspase-3 activation is an important event in cell death. Here, we observed impaired functional expression and activation of caspase-3 in infected IFI6+/+ cells and remarkable expression in the corresponding IFI6-/-cells. Both caspase-9 and caspase-8 are generally considered the apical caspases in the intrinsic and extrinsic pathways respectively [34]. After incubation with DENV2, both caspase-8 protein and caspase-9 activity were downregulated in IFI6+/+ cells. However, only the latter maintained the upward trend in functional activation in IFI6-/-VECs. Thus, it appears that the intrinsic-dependent pathway is more clearly targeted by expression of the IFI6 gene following DENV infection, and not necessarily the extrinsic pathway per se.
Both caspase-9, and caspase-3 activation are always engaged following altered integrity of the mitochondrial membrane potential (Δψ(m)), and both caspases are major elements of the mitochondrial pathway of apoptosis [35]. Compelling evidence obtained from the JC-1 staining assay revealed that IFI6 stabilized Δψ(m), and delayed mitochondrial dysfunction of the infected VECs. To the best of our knowledge, there is only study available from work that investigated gastric cancer cells that report the location of IFI6 in the mitochondria, and the anti-apoptotic function is expressed by inhibiting depolarization of Δψ(m) and interacting with calcium and integrin-binding protein [16]. Similarly, our study showed that the Bcl-2/Bax rheostat participated in the anti-apoptotic process of IFI6. XAF1, a well-known apoptotic protein, is induced by both TNF-α and IFN [12]. From our previous study, we found that expression of XAF1 was highly expressed in DENV2 infected VECs, with a peak occurrence at later stages (48 hrs) of viral challenge [19]. Following this enhanced expression of XAF1, as much as 28% of the cells had entered irreversible apoptosis by 72 hrs post-infection [36], at which point the balance of survival had been disrupted. In this study we found that during DENV2 infection, XAF1 was down-regulated by over-expressed IFI6. By contrast, XAF1 expression was increased in VECs in which IFI6 was knocked down.

Conclusion
Our results indicated that IFI6 might play a critical role in counteracting apoptosis during DENV infection. However, the underlying mechanisms of IFI6 function involves apoptosisrelated proteins expressed differently and their effects on caspases (Fig 7),which needed further investigation. At present we speculate that IFI6 might function as a protective gene during DENV infection in humans, it might represent a potential new therapeutic target in the setting of DHF/DSS.


text:
Cancer as a Proinflammatory Environment: Metastasis and Cachexia

The development of the syndrome of cancer cachexia and that of metastasis are related with a poor prognostic for cancer patients. They are considered multifactorial processes associated with a proinflammatory environment, to which tumour microenvironment and other tissues from the tumour bearing individuals contribute. The aim of the present review is to address the role of ghrelin, myostatin, leptin, HIF, IL-6, TNF-α, and ANGPTL-4 in the regulation of energy balance, tumour development, and tumoural cell invasion. Hypoxia induced factor plays a prominent role in tumour macro- and microenvironment, by modulating the release of proinflammatory cytokines.

Introduction
The malignancy grade of a tumour is fundamentally associated with both the development of metastatic lesions, a complex process that involves the propagation of cancer cells from the primary site to distant organs, and cancer-associated cachexia, which is dependent on factors produced by either the tumour microenvironment (tumour cells and surrounding tissues) or by the macroenvironment (comprising tumour released substances in the circulation and the secretion of proinflammatory factors by other tissues).
Cancer cachexia is an inflammatory condition, as is obesity. An excess of adipose tissue depots is associated with an increased risk of the incidence of some types of cancer and increased morbidity and mortality in cancer patients. Some factors such as insulin-like growth factors, leptin, and steroid hormones seem to connect obesity and cancer, once they contribute to the development of the chronic inflammatory state [1]. The increased estrogen synthesis associated with obesity in postmenopausal women augments the risk of development of postmenopausal breast and endometrial cancer. The constant presence of a high concentration of insulin in blood related to body fat excess and the increase of bioactive fractions of insulin, such as growth factor 1, via the downregulation of levels of insulin like growth factor-binding proteins 1 and 2, contribute to increased risk of cancer development [2,3]. Adipose stromal cells in WAT may present a role in cancer development and progression, promoting changes in the production of adipokines. Proinflammatory cytokines are involved in cancer-inducing mechanisms, while adiponectin could have anticancer effects [3,4]. The inflammatory tumour microenvironment is amplified by infiltrating macrophages, further increasing the production of cytokines, prostaglandins, angiogenic factors, and metastasis [4].
The details of the multi-step process of metastasis have been described by others [5,6]. Here, we will provide only a brief synopsis. In the primary tumour microenvironment, epithelial cancer cells are surrounded by pericytes, various extracellular matrix (ECM) proteins, and cancer-associated fibroblasts (CAF), which include fibroblasts, macrophages, endothelial cells, lymphocytes, and granulocytes. This tumour microenvironment produces various factors, such as TNF , TGF , Wnt, and HIF-1 , all of which stimulate a transient epithelial-mesenchymal transition (EMT) to promote cancer progression, invasion, and metastasis. EMT is a phenomenon wherein cells loose epithelial properties and gain mesenchymal properties, causing the loss of cell-cell contact and cell polarity. EMT increases cell migratory and invasive abilities, which are associated with the first step of cancer metastasis.
Cytokines such as TNF and interleukins, which are produced by both tumour cells and surrounding cells trigger NF-B signalling [4], consequently causing the release of TGF-, an inducer of EMT [7,8].
For the last decade, white adipose tissue has been considered an important endocrine organ producing several humoral factors including leptin, adiponectin, TNF-, IL-6, zinc-a2 glycoprotein (ZAG), IL-10, plasminogen activator inhibitor-1, and visfatin. These adipokines participate in the regulation of energy balance and a several physiological processes, including insulin sensitivity and the inflammatory response [9].
In fact, cachexia is frequently accompanied by food intake reduction and increases in proinflammatory factors such as C-reactive protein, TNF-, IL-6, and IL-1 [10].
Several reports have shown [10] that white adipose tissue (WAT) expresses and secretes proinflammatory factors in a rodent model of cancer cachexia, suggesting that these adipokines could be involved in the systemic inflammation in cancer patients.
In this review, we discuss some factors from the microand macroenvironment that could contribute to the development of metastasis and cancer cachexia.

Cancer Cachexia
Metabolic disorders that develop in anorexia and cancer cachexia are different. The weight loss in anorexia occurs mainly by the weight loss from fat, whereas cachexia involves significant weight loss from fat and muscle mass [50]. Anorectic patients respond to nutritional treatment more effectively than cachexia. In opposition, in the process of cancer cachexia, muscle mass cannot be maintained and food intake frequently cannot reverse this condition [51,52].
Cancer cachexia is a multifactorial syndrome characterized by a persistent and involuntary loss of skeletal muscle mass and fat mass, which contributes to progressive functional damage. This condition is related to a negative protein and energy balance, caused by a variable combination of abnormal metabolism and reduced food intake; conventional nutritional support is not enough to improve it. In addition, increased lipolysis and insulin resistance and reduced physical activity are present [52].
Cachexia may have a high prevalence in patients with cancer, depending on the stage of the disease and the type of tumour. The range of cachexia in patients with advanced cancer is from 60 to 80%. Pancreatic and gastric cancers are among the cancers with the highest prevalence of cachexia, approximately 85%. Over 20% of cancer patients die from cachexia [53].
The cancer in digestive tract frequently causes undernutrition related to the obstruction of alimentary tract, constipation, maldigestion, and malabsorption. However, the cachexia present in these patients was not only associated with malnutrition, since in these patients the metabolism differs from patients with anorexia, as the former present high proteolysis associated with an increase in proinflammatory protein and cytokines, aspects that will be discussed below [54].
Cancer cachexia progression comprises a variety of phases, including precachexia, cachexia, and refractory cachexia. Refractory cachexia is more difficult to reverse and is characterized by active catabolism or the presence of factors produced by the tumour and other tissues that do not allow an improvement in weight loss. This stage of cachexia is observed in patients who have a rapid progression of cancer that conventional cancer therapy cannot affect or in patients in the terminal stages of cancer, as in the cachexia consensus published in 2011 [52].
The type and stage of cancer, cancer treatment, food intake, and systemic inflammation are factors that can influence the progression from precachexia to cachexia [52,55].
Precachexia is considered the initial stage in which clinical and metabolic abnormalities, such as glucose intolerance and anorexia, already occur and precede an involuntary weight loss (≤5%). Cachexia is identified in patients with equivalent or more than 5% of body weight loss during the previous 6 months, or a BMI below 20 kg/m 2 and a continuous weight loss of more than 2%, or continuous weight loss (<2%) and sarcopenia. For many years, the majority of researches focus on muscle proteolysis in cancer cachexia. Nowadays, both muscle and adipose tissue wasting are considered important for development of cachexia in cancer patients [52].
Several factors produced during cancer development either by the micro-or the macroenviroment promote a proinflammatory condition, which are related to alterations on the regulation of energy balance. We will discuss some factors that are potentially involved in the development of cancer cachexia and metastasis.

Ghrelin
Energy balance results from the amount of food intake and the total energy expenditure. The control of energy balance depends on a complex network system regulated by several peripheral and central signals [56]. Cancer patients with systemic inflammation show a decrease in food intake, and hypermetabolism has also been described [52].
Ghrelin is a hormone produced mainly by enteroendocrine cells and, to a lesser extent, in the colon, hypothalamus, pituitary, endocrine pancreas, placenta, lung, cardiomyocytes, ovaries, and testes. Additionally, several studies have demonstrated that ghrelin mRNA and protein are expressed in many cancer and tumour tissues (see review Chopin et al., 2012) [11]. Ghrelin was first reported as an important stimulator of hunger and an inhibitor of energy expenditure [57]. Many studies have been conducted to verify the relationship between the decreased appetite present in cancer patients and the hormones that stimulate hunger, such as ghrelin.
These studies found high levels of total ghrelin and acylated ghrelin in most cachectic cancer patients compared with cancer patients without cachexia and patients without Mediators of Inflammation 3 cancer. These results were demonstrated in patients with several types of cancer, such as breast, colon, and lung cancers [11].
However, interestingly, cancer cachexia patients did not show increased appetite, despite their increased ghrelin levels. It seems that, in these patients, ghrelin levels increased in an attempt to reverse the catabolic state, as a compensatory mechanism [11,58]. Chopin et al. (2012) [11] suggested that ghrelin resistance is similar to the GH-resistant state observed in cancer cachexia patients because GH levels are elevated, whereas IGF-I levels are not.
In contrast, Fujitsuka et al. (2011) [59] evaluated mice inoculated with AH-130 ascites hepatoma cells, which cause cachexia, and found lower plasma concentrations of ghrelin and reductions in the expression of hypothalamic Y (NPY) neuropeptide, agouti-related peptide, and proopiomelanocortin (POMC) compared with animals without cancer cachexia. It appears that the reduction in ghrelin occurs due to excessive hypothalamic interactions between serotonin (5-HT) and corticotropin-releasing factor, which are stimulated by proinflammatory cytokines. In addition, tumour-bearing rats showed attenuated responses of increasing food intake when ghrelin was administered intravenously, indicating possible ghrelin resistance. This finding could partially explain why cancer cachexia patients present hypophagia despite their low levels of leptin and high levels of ghrelin.
In addition to investigations of the effects of ghrelin on energy balance, this hormone was evaluated to determine whether it could have a role in tumour growth, because ghrelin stimulates growth hormone secretion. Accordingly, Northrup et al. (2013) [60] showed that both ghrelin and anamorelin, an active agonist of the ghrelin receptor, did not cause an increase in tumours in tumour-bearing nude mice. Despite this result, the influence of ghrelin on tumour growth remains to be elucidated. As stated by Chopin et al. (2012) [11], it is controversial whether ghrelin has tumourpromoting effects or could inhibit tumourigenesis in vivo. Further studies are needed to better understand the effects of ghrelin on cancer cachexia development.
The administration of ghrelin as a treatment for cancer cachexia patients has been evaluated [59]. In vitro evidence showed that the expression of atrogenes through PI3K -, mTORC2-, and p38-mediated pathways in myotubes and dexamethasone-induced muscle atrophy was inhibited by acylated and nonacylated ghrelin, suggesting that this peptide may be able to prevent muscular atrophy [12]. Additionally, ghrelin inhibits myostatin secretion, a negative regulator of skeletal muscle mass [61].

Myostatin
Myostatin expression is upregulated in experimental models of cancer cachexia [13,14]. In humans, blood and muscle myostatin levels are upregulated in gastric cancer patients [62]. Myostatin, also known as growth/differentiation factor-8 (GDF-8), was described in 1997 and has been shown to be a potent negative regulator of muscle growth. This protein is expressed in muscle and other tissues, such as heart, adipose tissue, and mammary gland [63].
Active myostatin binds the activin type II B receptor (ActRIIB) and, to a lesser extent, the related ActRIIA, promoting the phosphorylation and assembly with the lowaffinity type I receptor ALK-(activin receptor like-kinase-) 4 or ALK-5 [15,64].
Myostatin has been reported as a negative regulator of skeletal muscle mass: low levels of myostatin or the knockout of the myostatin gene contributes to muscle mass growth in mice [15][16][17]. Additionally, the administration of soluble ActRIIB and the overexpression of activin receptor II B (ActRIIB), a dominant-negative form of the myostatin receptor, induce muscle hypertrophy, while increased levels of myostatin lead to skeletal muscular depletion [15,17,65].
Some reports have shown that myostatin signalling is enhanced in the skeletal muscle of tumour-bearing rats and mice [13,14].
In this sense, it has been demonstrated that myostatin is abundantly secreted by C26 colon cancer cells, and it has been verified that the treatment of differentiated C2C12 myotubes with C26-conditioned medium promoted myotubular atrophy and enhanced the activity of the ubiquitin-proteasome pathway. Additionally, the addition of antagonists to myostatin prevented C26-conditioned medium-induced wasting in muscle cell cultures. The authors suggested that myostatin secretion by cachexia-inducing neoplasm would initiate the pathogenesis of cancer cachexia [66].
Likewise, myostatin inhibition by the administration of an ActRIIB/Fragment-crystallizable (ActRIIB/Fc) fusion protein or the ActRIIB soluble form by antisense oligonucleotides is able to prevent the development of muscle mass depletion in tumour-bearing mice [67,68].

Leptin
Leptin plays a central role in the control of body weight and energy homeostasis, but it is a pleiotropic cytokine with activities in many peripheral cell types. Several studies in animals and humans have demonstrated the role of leptin in the regulation of energy homeostasis, neuroendocrine function, metabolism, immune function, and bone metabolism. Despite being produced predominantly in adipose tissue, other tissues express leptin, such as placenta, ovaries, mammary epithelium, bone marrow, and lymphoid tissues [69]. Leptin binds to receptors located throughout the central nervous system and peripheral tissues, with at least six receptor isoforms identified (LepRa-f) [70].
In the cancer context, hypoxic conditions often occur in solid tumours. Cellular hypoxia induces hypoxia-induced factor-1 (HIF-1) which activates the leptin gene promoter in human adipocytes and fibroblasts [71].
There is accumulating evidence that leptin signalling might be involved in the development of several types of cancer, such as colon cancer, mammary cancer, prostate cancer, and epithelial ovarian cancer, as well as the development of several myeloid and lymphoid leukemic cell lines. In support of these data, the expression of LR was detected in all these cancer tissues and cell lines [18,72].
Indeed, leptin has been shown to regulate neoangiogenesis; enhance endothelial cell growth [19,20]; suppress apoptosis through a Bcl-2-dependent mechanism [73]; act as a mitogen, transforming factor, or migration factor for many different cell types, including smooth muscle cells [74], normal and neoplastic colon cells [75,76], and normal and malignant mammary epithelial cells [77,78]; and induce the metastasis of breast cancer, possibly in an autocrine manner [79].
In patients with cancer cachexia, the plasma levels of leptin were lower than in patients without cachexia, which could be due to reduced fat mass in these patients [80]. Because the classic effects of leptin are stimulating -MSH neurons and inhibiting NPY neurons, decreasing food intake, and increasing energy expenditure [56], a leptin-independent pathway of cancer cachexia has been proposed.
Along with low plasma leptin levels, animal models of cancer cachexia may also have an increased number of LRb receptors, which seems to be consistent with the severity of the body fat reduction present in this condition [81]. Moreover, hypoxia-induced factor 1 (HIF-1), which is elevated in cancer cachexia, induces an increase in LRb expression in tumour cells [82].
As stated by Garofalo and Surmacz (2006) [18], it is possible that the local leptin concentration and signalling could be involved in the stimulation of tumour progression. In addition, they suggested that the tumour and surrounding adipose tissue promote a leptin-rich environment, which could contribute to tumour development. Likewise, leptin could contribute to tumour metastasis.

HIF
As mentioned before, a hypoxic environment is present in different types of tumours, especially in solid tumours. To adjust to the hypoxic microenvironment, several cancer cells increase the production of hypoxia-inducible factors (HIFs). These factors are associated with increased malignancy, poor prognosis, and resistance to radiotherapy and chemotherapy [83]. However, Ranasinghe et al. (2014) [84] reported that prostate cancer cells overexpressed HIF1 even under normoxic conditions.
The literature provides studies that focus on tumour cells that alter the transcriptional profiles via hypoxia-related mechanisms to modulate glycolysis, proliferation, angiogenesis, apoptosis, and metastasis, as to persist under conditions of hypoxic stress [21]. Under hypoxia the induction of glycolysis, angiogenesis, and metastasis seems to be a tumour cell adaptation to survival, which has HIF-1 as a main regulatory factor [27].
The tumour cell in hypoxia also increases the expression of macrophage chemoattractants such as VEGF, endothelins, IL-8, and endothelial monocyte activating polypeptide II (EMAP II) which promoted an increase in monocytes infiltration and macrophages accumulation, especially in tumour avascular or perinecrotic regions [28].
In addition, HIF-1 induces myeloid-derived suppressive cells (MDSC) differentiation to tumour-associated macrophages, causes a polarization of M1/M2 type with an increase of M2 in the hypoxia tumour region, and inhibits antitumour T cells, decreasing the immune response [29] ( Figure 1).
Using animal models, Liao et al. (2007) [25] demonstrated that the depletion of HIF1-did not impair mammary tumour formation, though decreasing the tumour progression and metastasis. In spite of that, Mazumdar et al. (2010) [22], employing a KRAS-driven lung tumour model, demonstrated that HIF1 deletion presents a very small effect on tumour weight and progression, whereas the loss of HIF2 actually increased tumour growth and progression.
Studies demonstrated that HIF-1 is implicated in the regulation of several genes involved on multiple key steps of metastasis, including epithelial-mesenchymal transition (EMT), invasion, extravasation, and metastatic niche formation, mostly in solid tumours (for details see review by Liu et al. (2015)) [26].
HIF also contributes for the proinflammatory macroenviroment present in several cancer patients. It is well-known that HIF-1 increases in the adipose tissue of obese individuals inducing the expression of proinflammatory adipokines such as IL-6, leptin, TNF-alpha, and angiopoietin 4, which are involved in the promotion of cachexia and metastasis [23,24].

Cancer and Inflammation
The proinflammatory environment can increase the risk of cancer by providing bioactive molecules, including cytokines, growth factors, and chemokines that facilitate carcinogenesis programs and sustain cell proliferative rate, inhibit apoptosis, and stimulate angiogenesis, and extracellular matrixmodifying enzymes, such as metalloproteinases, which promote the epithelial-mesenchymal transition (EMT).

IL-6
There is evidence that IL-6 is implicated in promoting tumour growth metastasis and participates in the development of cancer cachexia. IL-6 is considered the prime regulator of the acute-phase response in cachectic patients.
In the 1990s, there was increasing evidence that IL-6 contributes to metastasis and that serum IL-6 levels are  [32], using in vivo and in vitro experiments, analysed the effects of IL-6 on cancer development and demonstrated that there is a positive correlation between IL-6 and human mammary tumour development and metastasis, which seems dependent on STAT-3. The authors proposed the "formation of an autocrine/paracrine IL-6/JAK/STAT3 feed-forward loop, which participates in tumour proliferation, shaping of the tumour microenvironment, and metastasis. " Recently, Guyer and Macara (2015) [92] showed that IL-6 is important for inducing STAT3 in mammary epithelial cells downstream of silencing the cell polarity protein Par3, an important regulator of mammary tissue structure, which protects the development of primary tumour growth and aggressive metastatic lesions.
IL-6 signalling involves the binding of the cytokine to the membrane-bound IL-6 receptor (mIL-6r) on target tissues, which include hepatocytes, immune cells, and skeletal muscle. The activation of mIL-6r consequently promotes the downstream activation of many signalling pathways, including JAK/STAT3 and p38. Several of these pathways have also been implicated in the regulation of muscle mass loss during cancer cachexia [93]. Batista et al. (2013) [94] evaluated the correlation between adipokine tissue expression and concentrations in cachectic and noncachectic patients with or without cancer. They found that the plasma concentration of IL-6 was 11.4 times higher in the cachectic cancer group compared with the groups without cancer and with healthy weight, in addition to demonstrating a significant correlation with the presence of cancer. The interaction between cachexia and tumours increased the amount of IL-6 in subcutaneous adipose tissue and increased the IL6/IL-10 ratio, but not in visceral adipose tissue. Thus, they suggested that subcutaneous adipose tissue is associated with changes in plasma adipokines, which can play a role as markers of cachexia. However, a more specific study of the adequacy of the IL6/IL-10 ratio in the setting of cancer cachexia is necessary. Suh et al. (2013) [95] evaluated 98 advanced cancer patients and observed that IL-6 may be a good indicator of survival time in patients with advanced cancer in later life, despite elevated IL-6 previously being considered an indicator of shorter survival in these patients. Additionally, Kim et al. (2012) [96] investigated the roles of proinflammatory cytokines in lung cancer and colorectal cancer patients with cachexia prior to treatment. They observed that patients with high levels of IL-6 showed >5% weight loss after 6 months, suggesting that IL-6 could be responsible for the induction and maintenance of cancer cachexia. Puppa et al. (2011) [97] demonstrated in tumour-bearing mice an association among increases in gut permeability, endotoxemia, and plasma IL-6 concentration with tumour growth and cachexia development.   [33] suggested that STAT3 is a primary mediator of muscle mass loss because STAT3 activation in skeletal muscle by elevated IL-6 family ligands appears to be necessary and sufficient to promote muscle mass loss, in addition to being a common characteristic observed in vivo and in vitro and for different types of cancer. However, it is not yet clear how the activation of STAT3 promotes muscle atrophy. Haddad et al. (2005) [34] showed that the infusion of IL-6 in muscle reduces the phosphorylation of S6K1, which is associated with a catabolic process. S6K1 is phosphorylated and activated by mTOR, and a reduction in the phosphorylation of S6K1 is associated with the loss of the cellular capacity to synthesize proteins. Additionally, as described above, IL-6 seems to be involved in decreasing body fat and food intake by acting on both energy expenditure and NPY release control in the hypothalamus arcuate nucleus [35].
From these reports the idea that IL-6 could be a good marker to predict the evolution of cachexia associated with cancer and could be employed in treatment strategies emerged. Moreover, it was suggested that treatments that could impair the increase in IL-6 in adipose tissue could ameliorate cachexia in cancer. More studies are essential to elucidate this issue.
In this sense, it has been demonstrated that endurance exercise ameliorates cachexia-related inflammation in a rodent model, causing a systemic effect that is also associated with adipose tissue and decreases IL-6 in mesenteric adipose tissue [98,99]. Likewise, other studies using anti-IL-6 antibodies, either in vitro or in vivo, showed improvements in cancer cachexia [100,101].
It is important to note that although IL-6 has an important effect on the development of cachexia associated with cancer, IL-6 cannot be considered the only factor contributing to the breakdown of skeletal muscle and, consequently, to the development of cachexia [38,102].
Some cancer patients present high serum levels of TNFa, IL-6, and IL-1 which correlate positively with the progression of some tumours. It has been postulated that these cytokines promote anorexia in cancer by enhancing the levels of corticotrophin-releasing hormone, a central nervous anorexigenic neurotransmitter, causing a decrease in food intake [36]. Schéle et al. (2013) [35], using IL-6 −/− and IL-1R1 −/− mice, suggested that both endogenous IL-1 and IL-6 could suppress the expression of NPY and agouti-related protein in the hypothalamic arcuate nucleus. As stated by Trayhurn and Bing (2006) [103], IL-6 is the most interesting adipokine evolved in the regulation of energy balance. IL-6 induces weight loss [104], and it is expressed with its receptor in the neurons of the hypothalamic nuclei that regulate energy homeostasis [105]. In this sense, Wallenius et al. (2002) [37] demonstrated that the chronic ICV administration of IL-6 reduces body fat through an upregulation of energy expenditure without causing an acute-phase reaction.

TNF-
Tumour necrosis factor (TNF-) is an inflammatory mediator present in the tumour microenvironment that has been implicated in carcinogenesis, especially in the early stages, including angiogenesis and invasion, versus the progression of carcinogenesis [40]. TNF-is the main inflammatory cytokine that induces a transcription factor, Snail, that is implicated in EMT induction and stabilization [106].
Furthermore, studies have proposed that systemic TNFmight also be involved in the early development of some tumours. As reported by Balkwill (2006) [107], several studies using TNF--and TNF-R1-knockout mice and a variety of cell cultures have demonstrated the role of TNF-in cancer development.
Recently, studies showed elevated TNF-plasma levels to be associated with an increased risk of colorectal adenomas and the development and progression of breast tumours [108,109].
However, the results regarding the role of TNF-in cancer are controversial; high concentrations of this cytokine induced an antitumoural response in a murine model of sarcoma [110]. In contrast, low, sustained TNF-production levels can induce a tumour phenotype [107].
Obesity is linked with chronic, subclinical inflammation characterized by elevated levels of circulating proinflammatory mediators produced by adipose tissue, such as leptin, TNF-alpha, and IL-6 [111]. This inflammatory condition could be involved in an increased risk of cancer development. In fact, several authors have reported a correlation between obesity and cancer development (see review Calle and Kaaks (2004)) [2].
Furthermore, TNF-seems to be involved in the progression of cancer cachexia. As previously noted, cachexia is related, among other factors, to decreases in fat mass and skeletal muscle associated with negative protein synthesis and positive proteolysis. Muscle atrophy is characterized by a reduced cross-sectional area of myofibers accompanied by a loss of strength and a change in the composition of muscle fibre types. During the process of cancer cachexia, rapid type II fibres are affected, contributing to a higher proportion of slow fibres compared to fast fibres. In this case, catabolic factors, such as IL-6 and TNF-, are increased, contributing to the loss of muscle mass, and it has been suggested that the tumour has a great influence on the increase in the circulation of these factors [38,39]. In fact, it has been demonstrated that TNF-acts more on type II fibres to stimulate apoptosis signalling [41].
Several factors, including TNF-and IL-6, are associated with protein degradation by ubiquitination and the proteasome pathway, the most predominant pathway among the pathways of protein degradation. TNF-is able to increase the expression of ubiquitin and to promote the accumulation of ubiquitinated proteins, contributing to the atrophy of muscle mass [112,113]. The activation of NF-kB is a major candidate as the mediator of the cellular response of TNFafter TNF-stimulates the activation and nuclear translocation of NF-kB in skeletal muscle cells, which contributes to muscle catabolism [38]. The p38/MAP kinase pathway can also be stimulated by TNF-. Both p38/MAP kinase and NF-kB upregulate the expression of genes that encode the E3 ligases MuRF1 and MAFbx, thus inhibiting protein synthesis and contributing to muscle atrophy [114].
Although the role of TNF-during the inflammatory process in cachexia induced by cancer has been extensively studied, there is no consensus about the degree of influence of this cytokine on this inflammatory process [115]. It appears that the levels of serum TNF-are related to cancer stage, which reflects the size of the tumour [116]. Corroborating the result mentioned above, Kemik et al. (2012) [117] conducted a study with the aim of evaluating acute-phase proteins, cytokines, and hormones in cachectic patients with various cancers of the gastrointestinal tract. They found higher serum concentrations of several factors, including TNFand IL-6, in patients with oesophageal, gastric, pancreatic, colon, and rectal cancers than in controls. Thus, the authors suggested an association between proinflammatory cytokines in cachectic patients with various types of gastrointestinal cancer. However, other authors have found different results in patients with other types of cancer. Gulen et al. (2012) [118] studied 63 patients with lung cancer cachexia with advanced disease with the aim of evaluating the relationship between adipokines and systemic inflammation in patients at this stage. The authors showed the absence of a relationship between adipokines or systemic inflammation and cancer cachexia in lung cancer patients: some proinflammatory factors, such as TNF-, did not differ from the control group.
Some authors have shown that TNF-correlates with body mass, but others have shown no correlation between this cytokine and anorexia in patients with advanced-stage cancer [102]. Amaral et al. (2006) [42] demonstrated in rat that the administration of TNF-in the hypothalamus promoted a decrease in 12-hour food intake by modulating the expression of neurotransmitters associated with energy balance, favoriung higher energy expenditure. Previously, Aguilera et al. (1998) [119] observed a negative correlation between NPY and TNF-in patients with nervous anorexia.
According to Arruda et al. (2010) [120], a high concentration of TNF-also acts in the expression of hypothalamusmodulating neurotransmitters and signal transduction pathways, thus increasing body temperature, oxygen consumption/carbon dioxide production, and energy expenditure and contributing to weight loss in cancer cachexia patients. Busquets et al. (1998) [121] demonstrated in rat that the administration of a single intravenous injection of TNFincreased the gene expression of uncoupling proteins 2 and 3 in skeletal muscle, suggesting this as a possible mechanism contributing to increased energy expenditure and cachexia in tumour-bearing cancers. However, weight loss in pancreatic cancer is associated with systemic inflammation and increased ubiquitin mRNA expression, but not uncoupling proteins in skeletal muscle [122].
An in vitro study showed that, in 3T3-L1 adipocytes, TNFsignificantly reduced the lipid accumulation and glucose uptake induced by adiponectin, and it increased lipolysis [123]. Previously, it was demonstrated in humans that the administration of TNF-alpha at low doses caused systemic lipolysis [124]. This finding also suggests a potential role of this cytokine in the control of adipose tissue depletion in cancer cachexia.
Another factor recently described to be involved in metabolic regulation is ANGPT4. This protein is associated with energy homoeostasis, wound repair, tumourigenesis, angiogenesis, and redox regulation [43].

Angiopoietin-Like 4 (ANGPTL4)
A new protein with similarity to members of the ANG (angiopoietin) family was identified by three independent research groups in 2000 [125,126]. ANGPTL-4 was also named fasting-induced adipose factor (FIAF) [127].
The protein ANGPTL4 was classified as an adipokine because it was expressed predominantly in adipose tissues and liver; thus, it was believed to be involved in lipid metabolism [43,128]. ANGPTL4 primarily showed an inhibitory effect on LPL activity [125,129].
An analysis of recent studies makes it clear that the specific form of ANGPTL4 associated with the microenvironment tumour influences the clinical impact of this protein, along with other factors [44,130,133].
Over the past decade, there has been increasing recognition that this protein participates in numerous physiological and pathological processes [130]. ANGPTL-4 has been implicated as an important factor involved in energy homoeostasis, redox regulation, angiogenesis, the development of cancer and cachexia, and the development of metastasis and inflammation; however, the studies are still incomplete and often contradictory [43]. Galaup et al. (2006) [134] found in an experimental model that Angptl4 prevents tumour metastasis through the inhibition of vascular permeability, tumour cell motility, and invasiveness. Similarly,   [131] demonstrated that cANGPTL4 inhibits angiogenesis. Because angiogenesis plays an important role in the progression of cancer, the authors suggested that cANGPTL4 has an antitumoural effect, thus decreasing tumour growth and metastasis.
In contrast, hypoxia stimulates the expression of ANGPTL4 in several tumour types [43,135]. ANGPTL4 mRNA is increased in the perinecrotic areas of many human tumours [135,136]. Moreover, elevated ANGPTL4 expression increased as tumours progressed from benign to metastatic states, thus implying a role of ANGPTL4 in tumour growth [45,46].
The Angptl4 protein is more often expressed in colorectal cancer (CRC) tissues than that in normal tissues, and it is related to cell migration through the cytoskeletal signalling pathway [44]. The overexpression of Angptl4 promotes colon cancer cell migration through the cytoskeletal signalling pathway, while the downregulation of ANGPTL4 impairs tumour growth and metastasis [44,46,47].
Inflammatory factors, such as IL-1b and IL-6 or hypoxia alone, increased ANGPTL4 protein levels, but the role of hypoxia was more significant. It was also observed that increasing tumour size and increasing degree of hypoxia in the tumour mass promoted the upregulation of ANGPTL4, especially in the hypoxic areas surrounding the necrotic area [138]. Studies have shown that ANGPTL4 is a target gene of PPARs, which are involved in the development of tumours [139,140]. However, the role of PPARs in the regulation of ANGPTL-4 in tumour cells is controversial. Zhu et al. [46] (2011) did not detect a correlation between PPAR , , and and the expression of ANGPTL-4. In contrast, Girroir et al. (2008) [137] demonstrated that the addition of PPAR and to tumour cell culture (MCF7 and UACC903) reduced cell proliferation and increased ANGPTL-4 gene expression.
The metabolic effects of ANGPTL-4 support the idea that this protein contributes to cancer cachexia development: fat-specific Angptl4 overexpression caused a 50% reduction in adipose tissue weight, partly by enhancing fatty acid oxidation and lipolysis [48]; the central administration of ANGPTL4 lowered food intake and body weight gain and enhanced energy expenditure [49].

Conclusion
The grade of malignancy of tumour is primordially associated with both the development of metastatic lesions and cancer-associated cachexia, which are multifactorial conditions depending on factors present in the micro-and macroenvironments in the tumour-bearing patient.
In this review, we point out some factors that are released and/or act in the tumour and other tissues related to the development of metastasis and cancer cachexia. Hypoxiainduced factor plays a prominent role in inducing tumour macro-and microenvironment release of proinflammatory cytokines. The cross talk among factors released by the tumour, adipose tissue, muscle, and other tissues seems to contribute to the difficulty of treating the cancer patient, once a multifactorial alteration occurs in the micro-and macroenvironment of the individual (Figure 2) (Table 1). Furthermore, there is still some controversy in the literature, related to the role of ghrelin, ANGPT4, HIF-1, and TNF-in cancer cachexia and metastasis development, indicating the necessity of more studies. ANGPTL-4 Contribute to angiogenesis [43] Contribute to metastasis [43][44][45][46][47] ↑ Lipolysis and fatty acid oxidation [48] ↓ Food intake and body weight gain [49]


text:
Quantitative evaluation of cell death response in vitro and in vivo using conventional-frequency ultrasound.

Previous studies using high-frequency ultrasound have suggested that radiofrequency (RF) spectral analysis can be used to quantify changes in cell morphology to detect cell death response to therapy non-invasively. The study here investigated this at conventional-frequencies, frequently used in clinical settings. Spectral analysis was performed using ultrasound RF data collected with a clinical ultrasound platform. Acute myeloid leukemia (AML-5) cells were exposed to cisplatinum for 0–72 hours in vitro and prepared for ultrasound data collection. Preclinical in vivo experiments were also performed on AML-5 tumour-bearing mice receiving chemotherapy. The mid-band fit (MBF) spectral parameter demonstrated an increase of 4.4 ± 1.5 dBr for in vitro samples assessed 48 hours after treatment, a statistically significant change (p < 0.05) compared to control. Further, in vitro concentration-based analysis of a mixture of apoptotic and untreated cells indicated a mean change of 10.9 ± 2.4 dBr in MBF between 0% and 40% apoptotic cell mixtures. Similar effects were reproduced in vivo with an increase of 4.6 ± 0.3 dBr in MBF compared to control, for tumours with considerable apoptotic areas within histological samples. The alterations in the size of cells and nuclei corresponded well with changes measured in the quantitative ultrasound (QUS) parameters.

INTRODUCTION
Assessing the efficacy of cancer treatments on an individual patient basis is presently limited from a clinical perspective. This is mainly because of the fact that currently even imaging-based monitoring of cancer therapy effects is frequently performed at macroscopic levels, relying on the standard clinical practice to measure the size of tumour [1]. Changes in tumour size, however, can take several weeks to months to become apparent, and do not always occur even when the treatment is effective [2,3]. In order to overcome this problem, cancer therapy response monitoring at microscopic physiological levels has gained much attention in cancer research and imaging [3][4][5]. Functional imaging techniques are thus commonly proposed based on the observations that development of response to cancer therapy introduces micro-structural, morphological, and functional alterations within tumour, which can be detected non-invasively early on after the start of treatment using different imaging modalities [2].
Different imaging modalities including positron emission tomography (PET) [6], single photon emission www.impactjournals.com/oncoscience computed tomography (SPECT) [7], and magnetic resonance imaging (MRI) [8] have been investigated for the evaluation of responses to cancer treatment, based on changes in cell metabolism and blood perfusion [5], or the development of apoptotic cell death within tumours [9]. However, these methods have two drawbacks, i.e., being presently expensive and requiring exogenous contrast agents frequently. The agents' costs, radiation dose involved, and the potential for side effects including allergic reactions limits their applicability as a standard method to be used routinely in clinic. Amongst other imaging techniques, quantitative ultrasound (QUS) techniques coupled with high-frequency high-resolution ultrasound have shown promise in detecting development of cell death in response to cancer therapies [3,4,10,11]. Such techniques, if translated into conventional clinicallyrelevant frequency ranges, could provide a cost-effective and rapid framework to assess cancer therapy effects. These methods also alleviate the requirement for injecting external contrast agents since in these methods, the alterations in bio-acoustic properties of dying cancerous cells are the main source of changes in imaging contrast [10,12].
In this context, early investigations combining well controlled biological experimentation and high-frequency QUS techniques have provided valuable information to detect and potentially quantify cell death [10,11]. Ultrasound has been used successfully as a clinical diagnostic tool for many years because it is safe, realtime, non-invasive, and relatively inexpensive. Ultrasonic scattering in biological tissues is a complex process. It is primarily affected by the acoustic impedance mismatches and the size and density of scattering structures within tissue, in relation to the ultrasound wavelength. The application of ultrasound in cancer imaging is based on the foundation that pathological or therapeutic processes alter physical characteristics of tissue such as compressibility, density, and geometry of potential scatterers, and these alterations cause observable changes in acoustic scattering properties. Such physical characteristics of ultrasound make it a very attractive tool to monitor changes in tumours in response to treatment. Based on these principles, high-frequency ultrasound has been used to monitor structural changes at the cellular level in vitro and in vivo (described further below).
High-frequency ultrasound has been used to detect apoptosis, necrosis and other forms of cell death in vitro, in situ, and in vivo [10,11,[13][14][15]. It was demonstrated that cell death could be detected in vivo using high-frequency QUS spectral analysis techniques in preclinical animal models, where xenograft tumours were treated using a number of different modalities. The first preclinical use of high-frequency QUS spectral analysis for monitoring cancer therapy effects was to evaluate responses of xenografted melanoma tumours to photodynamic therapy (PDT) in vivo [15]. Banihashemi et al. observed a time-dependent increase in QUS spectral parameters, namely mid-band fit (MBF) and spectral slope, after treatment. The MBF is a measure of ultrasound backscatter power, and the spectral slope parameter has been demonstrated to be influenced by the size of effective acoustic scatterers [16,17]. One other parameter, the 0-MHz intercept can be related mathematically to the concentration of acoustic scatterers. The observed increases in these spectral parameters correlated with morphologic findings, indicating increases in apoptotic cell death after PDT. Analyses of changes in spectral slope strongly correlated with changes in mean nuclear size over time, associated with apoptosis induced by the therapy. In another study, Vlad et al. used high-frequency ultrasound in a similar manner to track the responses of xenograft tumours in vivo to radiotherapy [14]. Three mouse models grafted with head and neck carcinomas (FaDu, C666-1, and Hep-2) exhibited large hyperechoic regions after radiotherapy. The ultrasound integrated backscatter increased by 6.5 to 8.2 dBr (p < 0.001) and spectral slopes increased from 0.77 to 0.90 dBr/MHz for C666-1 tumours and from 0.54 to 0.78 dBr/MHz for FaDu tumours (p < 0.05), compared with pre-irradiated tumours. The hyperechoic regions in the ultrasound images corresponded in histology to areas of cell death.
Whereas high-frequency ultrasound (20-50 MHz) has the main advantage of providing higher resolution compared with conventional-frequency ultrasound (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), it is limited by a low penetration depth, which makes it unsuitable for deeper structures such as those in the abdomen and in breast imaging. This limits the application of high-frequency ultrasound to research and very superficial tissues such as the eye [18]. Conventional-frequency ultrasound penetrates deeper into body structures and hence is used with most clinical ultrasound equipment. Therefore, for a broad adoption of QUS methods in clinical applications for cancer response monitoring, the extension of these methods to conventional-frequency range is highly desired.
In this study, conventional-frequency (~7 MHz) QUS spectral analysis techniques were investigated for the assessment of cancer therapy effects in vitro, and in vivo, in order to demonstrate an efficacy for the noninvasive detection of cell death. Ultrasound data was collected at conventional frequencies in vitro from acute myeloid leukemia (AML-5) cell samples before and at different times after cisplatinum chemotherapy exposure. Experiments in vivo were conducted on AML-5 tumourbearing mice undergoing chemotherapy and tumours were imaged before and after treatment. Ultrasound data were analyzed using spectral analysis techniques with normalized power spectra derived from ultrasound RF data. Time-based in vitro experiments indicated statistically significant changes in spectral parameters 48 hours after the administration of chemotherapy (p < 0.05). Moreover, concentration-based experiments here demonstrated an average increase of 10.9 ± 2.4 dBr in MBF between 0% and 40% mixtures of apoptotic and viable cells. Similar confirmatory results were obtained in in vivo experiments. Obtained results demonstrated that QUS techniques at conventional-frequencies can be applied to detect cell death in response to anti-cancer therapies. This work here therefore suggests good promise for the use of QUS techniques in a clinical setting for the monitoring treatment response in cancer patients [2].

In vitro experiments
Exposure to the chemotherapeutic agent, cisplatinum, produced constant and reproducible changes in ultrasound backscatter and the derived spectral parameters as a function of time after exposure (described further below). To better illustrate these effects, Figure 1 shows representative ultrasound B-mode images and normalized power spectra from a control untreated and a treated cell sample, demonstrating an increase in echogenicity within cell samples 24 hours after treatment. This also affected the corresponding normalized power spectra where an increase in backscatter power was apparent across the entire bandwidth of the transducer. These changes in ultrasound backscatter and the spectral parameters during treatment are known to be linked to structural and morphological changes that occur throughout apoptosis in AML-5 cells [15]. Figure 2 demonstrates light microscopy images of H&E and TUNEL stained slides at high magnification, obtained from representative control and treated cell samples. Considerable changes were observed within the H&E slides in terms of cellular and nuclear structure and morphology after chemotherapy. A noteworthy point is the large number of cells without any nuclei or nuclear fragments at 72 hours after treatment, indicative of latestage apoptosis. The TUNEL stained slides also confirmed that a considerable number of cells within the samples underwent apoptosis in response to chemotherapy. Figure 3 demonstrates results of time-dependent histological analysis on AML-5 cell samples. Figure 3A illustrates light microscopy images of the representative H&E stained slides of AML-5 cell samples for each specific experimental time after treatment. Structural and morphological alterations were evident in cells and nuclei throughout the apoptosis process in response to treatment. The number of cells with degraded nuclear material accounted for 46% of the total number of cells imaged at 72 hours after chemotherapy exposure. Figures 3B and 3C demonstrate the changes in the average size of the cells and nuclei, and the average number of nuclear fragments, respectively, as functions of time (0, 6, 12, 24, 48, and 72 hours) after exposure to cisplatinum. Initial increases were observed in the average size of cells and nuclei, before steady and more homogeneous size decreases after 24 hours of treatment. This implies substantial morphological changes in cells and nuclei such as cellular blebbing and nuclear coalescence at early stages after   the MBF parameter (a measure of ultrasound backscatter power) up to 48 hours after treatment, very similar to the trend observed previously with high-frequency ultrasound [11]. Specifically, the average values obtained for MBF at 0, 24, 48, and 72 hours after treatment were −46.5 ± 1.4 dBr, −44.4 ± 1.3 dBr, −42.1 ± 0.6 dBr, and −48.5 ± 1.3 dBr, respectively. In particular, the MBF increased by 4.4 ± 1.5 dBr at 48 hours and decreased substantially at 72 hours after treatment. Statistical tests of significance (t-test, unpaired, two-sided) demonstrated the measured change at 48 hours to be significant (p < 0.05), compared to pre-treatment. Figure 4B presents the corresponding spectral slope parameter (influenced by effective scatterer size) which exhibited a similar trend. The spectral slope increased from 0.7 ± 0.5 dBr/MHz prior to treatment to 2.0 ± 0.2 dBr/MHz at 48 hours, and then decreased to 0.8 ± 0.1 dBr/MHz at 72 hours after treatment. Similarly, Figure 4C demonstrates mean increases in the 0-MHz intercept parameter (influenced by acoustic concentration) up to 48 hours after chemotherapy and subsequently a decrease at 72 hours. The changes observed in the QUS spectral parameters corresponded well with the trend of changes seen in the nuclear and cellular sizes and ratio, and number of nuclear fragments in the cell samples after treatment.
To measure the sensitivity of QUS parameters to detect apoptosis at this frequency, mixtures of apoptotic and untreated cells were made and imaged. Figures 4D-F demonstrate mean values obtained for the MBF, spectral slope and the 0-MHz intercept parameters acquired for mixtures of apoptotic and viable AML-5 cells with proportions ranging from 0 to 100% apoptotic cells. The maximum ultrasound backscatter power as represented by the MBF parameter occurs when 40-60% of the AML-5 cells were apoptotic. The MBF parameter increased by 10.9 ± 2.4 dBr on average between 0% and 40% mixtures of apoptotic cells. A similar pattern was observed with the spectral slope and 0-MHz intercept parameters. The spectral slope parameter increased from the baseline of 2.4 ± 0.3 dBr/MHz (0% mixture) to 3.7 ± 0.4 dBr/MHz at 40% apoptotic cell mixture. Similarly, the 0-MHz intercept demonstrated an average increase of 7.0 ± 1.9 dBr from 0% to 40% mixtures.

In vivo experiments
Chemotherapy produced appreciable and consistent changes in ultrasound backscatter and the spectral parameters in vivo. Increased backscatter power was observed in tumour xenografts 24 hours after chemotherapy exposure using low-frequency ultrasound. Results obtained via the in vivo study on AML-5 tumour xenografts are presented in Figure 5. Figure   demonstrates representative ultrasound B-mode images with ROI parametric overlays of the MBF parameter obtained before and after the treatment. The MBF parametric images demonstrate a considerable increase in ultrasound backscatter power within the tumour region in response to treatment. Figures 5B-5E present light microscopy images of H&E and TUNEL stained slides at low and high magnifications, respectively. Similar morphological and structural changes in cells and their nuclei as observed in vitro were also detected in the histology slides of the tumour xenografts. Specifically, nuclear condensation and fragmentation was readily detectable in the H&E stained slides after treatment. At higher magnification, extensive changes in morphology were observable in the treated area, including blebbing in the cellular membrane, nuclear coalescence and fragmentation, and structural disorganization. These alterations were a strong indication of dominant apoptotic cell activity that occurred in response to treatment, as also confirmed by the TUNEL staining. The TUNEL stained slides demonstrated areas of apoptotic cell death in treated tumours. This was clearly visualized in low and high magnification microscopic analysis. Average values measured for the size of the cells and their nuclei from the histology slides before and after the treatment have been presented in Figure 5F. Remarkable decreases are observed in size of cells and nuclei after the treatment, consistent with the observations in vitro. Figure 5G depicts mean values of the MBF parameter obtained before and after the treatment. The MBF parameter increased from −9.6 ± 0.2 dBr prior to treatment to −5.0 ± 0.2 dBr after treatment, a change that was found to be statistically significant (t-test, unpaired, two-sided, p < 0.05), compared to pre-treatment. Similarly, the 0-MHz intercept parameter demonstrated a statistically significant increase of 9.0 ± 1.5 dBr, on average, after treatment ( Figure 5H). However, the spectral slope parameter did not show any statistically significant increases after treatment. The alterations in the size of cells and nuclei observed through the histological analysis corresponded well with the changes measured in the QUS parameters, specifically the MBF and the 0-MHz intercept.

DISCUSSION
This study demonstrated the applicability of QUS spectral analysis techniques at conventional low frequencies to detect cell death in vitro and in vivo within hours after the start of treatment. Quantitative ultrasound spectral parameters derived from ultrasound RF data were demonstrated to be sensitive to changes in tissue microstructure that are associated with cell death. The results were confirmatory across in vitro and in vivo studies where AML-5 cell samples and tumour xenografts underwent chemotherapy. As such, this study can be considered as an important extension to previous works that suggested a very good potential of QUS spectral analysis techniques at high-frequency ranges for evaluating cell death [11,13,14].
Particularly, time-and concentration-dependent in vitro experiments revealed changes in spectral parameters at conventional frequencies that were linked to cell death as indicated in standard histological analysis. Most chemotherapeutic agents such as cisplatinum mainly induce apoptotic cell death in tumour cells [28]. Apoptosis causes significant structural and morphological changes in cells and nuclei, which begins with nuclear coalescence, condensation, and fragmentation and eventually leads to nucleus degradation and loss [29,30]. In our experiments, these changes were apparent in histological images of in vitro cell samples at different times after treatment. Nuclear condensation and fragmentation were observed up to 48 hours after treatment, whereas at 72 hours after treatment about half of the cells lost their nuclear structure. Also, average nucleus:cell size ratio decreased from 0.7 prior to treatment to 0.4 at 72 hours after exposure to cisplatinum. According to the results obtained, the MBF, spectral slope and the 0-MHz intercept parameters demonstrated changes that were consistent with these structural and morphological alterations in cells and their nuclei. In particular, these parameters demonstrated increases up to 48 hours after treatment and consequently start to decrease. Previous studies that applied QUS techniques at high-frequencies to detect cell death support the observations of this study with similar trends being identified albeit in more limited experiments [10,11,15]. Specifically, those studies demonstrated that early stages of apoptosis generally lead to large increases in ultrasound backscattered signal intensity acquired from in vitro cell aggregates [10,13,31] and in vivo tumour xenografts [14,15]. However, it has been shown that the ultrasound backscattered signal intensity decreases in advanced stages of apoptosis [11,32]. In particular, the early studies using high-frequency ultrasound showed that alterations in nucleus is the major cause for changes in ultrasound backscatter [11]. Early-stage cell-death (nucleus condensation and fragmentation) results in increases in ultrasound backscattered signal intensity. On the other hand, late-stage cell-death (chromatin dissolution/nucleus degeneration) has two contradictory effects on ultrasound backscatter. It initially increases the randomness in fairly regular distributions of scatterers, resulting in a larger backscattered signal. However, when a large fraction of the nuclei scatterers become disintegrated due to DNA cleavage (advanced necrosis), the amplitude of backscattered signal is reduced [11,32,33]. The results obtained from both time and concentration-based in vitro experiments in this study confirmed that a consistent trend is observed using QUS spectral parameters at conventional frequencies. The experiments conducted by mixing the two types of cells suggest an increase in backscatter from two effects. For 100% apoptotic cells an increase occurred purely as a result of the change in the nature of scatterers. For mixtures of viable and these same apoptotic cells where the backscatter was higher than that for the 100% sample it is possible that mixed scatterer types and their randomization contributes to the further increased backscatter detected. The effects of cell packing have been dealt elsewhere [10,11] and a discussion on the nature of cellular causes of backscatter associated with cell death can be found in previous studies [15].
The changes observed in QUS spectral parameters with cell death progress in vitro were also similar to those obtained in the in vivo experiments. Particularly, the MBF and 0-MHz intercept parameters demonstrated a significant increase after chemotherapy in AML-5 tumour xenografts. Similar results were previously reported using high-frequency ultrasound in vivo [11,[13][14][15]. The spectral slope parameter did not show any statistically significant increases in AML-5 tumours in vivo after the treatment. The spectral slope parameter has been demonstrated to be linked to the size of acoustic scatterers in tissue micro-structures [16,34]. Previous studies on cancer therapy response monitoring using high-frequency QUS techniques have shown that a time-dependent increase in spectral slope after treatment is correlated with changes in average nuclear size as results of apoptotic cell death [15]. In another study, however, Vlad et al. reported no considerable changes in the spectral slope parameter at high frequencies after radiotherapy, that induced both apoptotic cell death and mitotic arrest within AML-5 cells [13]. They explained this observation based on two opposite changes observed in size of potential acoustic scatterers due to nuclear condensation and fragmentations in apoptosis, and cell enlargement in mitotic arrest. In the context of conventional-frequency ultrasound applied in this study, bulk changes in tissue are believed to be related to ensembles of cells and nuclei smaller than the wavelength of the ultrasound being used. These ensembles influence acoustic properties and thus ultrasound backscatter characteristics. As such effects of different alterations in potential acoustic scatterers (cellular and nuclear condensation and fragmentation versus cell aggregation and development of patches of response) on the spectral slope parameter can be more likely superimposed, resulting in non-significant overall changes. www.impactjournals.com/oncoscience The results in this study demonstrated that development of cell death in response to anti-cancer therapies (in particular apoptosis in response to chemotherapy) could be detected by QUS techniques at conventional frequencies, as they result in detectable alterations in ultrasound backscatter power at these frequencies. These results are in correspondence with previous investigations performed in vitro and in vivo using high-frequency ultrasound in order to evaluate tumour response to various types of cancer-targeting treatments [11,[13][14][15]. Although both conventional and high-frequency ultrasound demonstrate similar trends of change in the spectral parameters in response to treatment, the predominant type of scattering expected at these two frequencies is potentially different. At the conventional frequencies, Rayleigh scattering (backscatter intensity proportional to f 4 , where f is frequency) [35] is expected to predominate, since the cellular components are considerably smaller than the ultrasound wavelengths. In contrast, with high-frequency ultrasound, the cellular components are approximately proportional to the ultrasound wavelength, with a more complex relationship between backscatter intensity and frequency. As stated before, the ultrasound backscatter at conventional-frequencies is mainly impacted by bulk changes in ensemble of cells and nuclei, forming a speckle pattern of change due to alterations in bioacoustic properties of cancerous cells after treatment. The interactions among the scatterers in cell ensembles have been shown previously to have a great impact on ultrasound backscatter properties at high frequency [32]. This can be an important factor at conventional frequencies as well.
The present study complements other imagingbased approach such as those based on magnetic resonance imaging or positron emission tomography being investigated for cancer therapy response monitoring [2,5,8]. Unlike these methods, the QUS techniques applied in this study rely on intrinsic contrast alterations arising from changes in bio-acoustic characteristics as cancer cells respond to treatment, hence do not require any injection of exogenous contrast agents. Ultrasound imaging has also the advantage of portability, low cost, rapid imaging speed, and high spatial resolution.
This study provides promise that QUS spectral analysis techniques can advance the monitoring of cancer treatment in the near future. QUS techniques at conventional frequencies applied here in vitro and in vivo can potentially be adopted for clinical use [36], permitting an early assessment of treatment response to cancer-targeting therapies on an individual patient basis. The research findings here form a basis for such applications of QUS spectral methods for therapy response monitoring.

In-vitro experiments
Cell preparation and treatment AML-5 cells (Ontario Cancer Institute, Toronto, ON, Canada) were cultured at a density of 3 × 10 5 cells/ml in in α-minimum essential medium (GIBCO 11900, Rockville, MD, USA) supplemented with 5% fetal bovine serum (Cansera International, Etobicoke, ON, Canada) and 1% penicillin/streptomycin (Invitrogen Canada Inc., Burlington, ON, Canada). The cells were maintained in 150 ml suspensions in tissue culture flasks (Sarstedt, Nümbrecht, Germany) in a 37°C thermo-incubator maintained at 5% CO 2 . The cells were treated with 1.5 ml of cisplatinum, which is a chemotherapeutic agent that forms platinum-DNA adducts [19]. This disrupts DNA replication and transcription thus leading to apoptosis. The flasks were treated in sets of five, with a parallel set of untreated flasks (control), forming six sets of 0, 6, 12, 24, 48, and 72-hour time points. Treated AML-5 cells from 48-hour time-point set (100% apoptotic in this line) were mixed with untreated AML-5 cells to obtain proportions of apoptotic to healthy cells at 0%, 10%, 20%, 40%, 60%, and 100%.
At each time after treatment, two sets of six (treated and untreated) flasks were processed to form centrifuged samples for ultrasound imaging. Each flask was divided into 500 ml centrifuge containers and spun at 500 g in a fixed angle centrifuge at 4°C for 10 minutes. The condensed pellet of cells was re-suspended with phosphate buffer saline (PBS) and the concentrated suspensions were spun again in 500 ml conical tubes with a swinging bucket centrifuge at 1000 g. After a second re-suspension, the cells were spun once more at 2000 g in a three well (8 mm diameter) stainless steel container. The AML-5 samples mimic the close packing of cells as seen in solid malignancies. All samples (n = 5 per group) were approximately the same size with a height of 5 mm each. Consistency of packing between the viable and apoptotic cells has been demonstrated previously [10].

Ultrasound data acquisition and analysis
Conventional-frequency ultrasound data were acquired with a Sonix RP (Ultrasonix, Vancouver, BC, Canada) system utilizing an L14-5/38 linear transducer with a center frequency of ~7 MHz, focused at 1 cm depth, and data sampled at 40 MHz. The AML-5 centrifuged samples were scanned at room temperature in stainless steel wells, at 6, 12, 24, 48, and 72 hours after exposure to cisplatinum. Ultrasound data were analyzed across multiple scan planes for each sample within a standardized region of interest (ROI), using an in-house software in MATLAB (MathWorks, Natick, MA, USA) and the results were averaged subsequently. The ROIs were determined manually on B-mode ultrasound images of the AML-5 sample in the well. The power spectrum was calculated using a Fourier transform of the raw RF data for each scan line through the ROI and subsequently averaged. The averaged power spectrum from each ROI was normalized to that of a reference signal from the base of a water-filled steel well, scanned with the same setting used for the cell sample scans, in order to remove system and transducer transfer functions. Linear regression analysis was performed on the normalized power spectrum data within a -6 dB window from the transducer center frequency, in order to provide a best-fit line. The MBF, 0-MHz intercept and spectral slope parameters were then calculated [13][14][15][16][17][20][21][22][23] and reported for all samples.

Histological analysis
To investigate the morphology of cells, AML-5 cell samples were fixed with formalin, embedded in paraffin and stained with haematoxylin & eosin (H&E). Slides of AML-5 cells were prepared for the 0, 6, 12, 24, 48, and 72-hour time-point sets. Eighteen images from each slide were obtained at both 20× and 40× objective magnifications using a Leica DC100 microscope and a Leica DC100 camera connected to a 2 GHz PC equipped with Leica IM1000 software (Leica GmbH, Wetzlar, Germany). The sample on the slide was divided into 3 bands, i.e., top, middle, and bottom and 6 pictures of the AML-5 cells were taken from each band. The images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA), where a few thousand cells and nuclei were measured from untreated and treated slides. From the outset, 30 cells were measured from each image. Cell and nucleus size and the number of nuclear fragments (apoptotic cells) within each selected cell were measured. Cell and nucleus size was considered as the largest diameter of a cell or nucleus, respectively. Cells were selected if they had a nucleus and part or whole of the cell membrane was distinct from surrounding cells. Nuclei or nucleus fragments were measured (and counted) only if they were within a cell membrane. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was also performed to confirm that the treated AML-5 cells were undergoing apoptosis.

Ethics statement
This study has been conducted in accordance with the ethical standards, according to the Declaration of Helsinki and national and international guidelines, and has been approved by the authors' institutional review board.

Animal model and treatment
In vivo experiments were conducted on ten severe combined immunodeficiency disease (SCID) mice. One hind leg of each animal was subcutaneously injected with 10 6 in 50 μl of AML-5 cells. Tumours reached a size of 7-10 mm at 4-5 weeks after cell injection. The mice were then divided into two groups of untreated control and treated (n = 5 per group). The treatment was administered using dexamethasone (0.246 mg/kg) [24] and cisplatinum (75 mg/m 2 ) via intravenous tail vein injection.

Ultrasound data acquisition and analysis
All animals were imaged 24 hours after treatment, and subsequently sacrificed. Ultrasound data collection consisted of acquiring B-mode images in addition to raw RF data for quantitative spectral analysis of backscattered signal. After depilatory hair removal, animals were anaesthetized (Ketamine 100 mg/kg, Xylazine 5 mg/ kg, Acepromazine 1 mg/kg; CDMV, St Hyacinthe, QC, Canada) and imaged using high viscosity ultrasound gel (ATL Inc., Reedsville, PA, USA) as coupling agent over the skin in tumour-bearing areas. The same ultrasound system and transducer was used as applied in the in vitro experiments. The ROIs (approximate size of 5 × 5 mm) were located at the tumour center which was consistently positioned at the transducer focal depth (1.5 cm). The same spectral analysis method was used as described in the in vitro experiments with an glass-bead-embedded agar-gel phantom model [25,26] used in place of stainless steel as a reference, and scanned with the same setting as used for the tumour scans. Ten to fifteen ROIs were analyzed for each tumour sample and the results were averaged subsequently. In addition to the averaged QUS parameters, parametric images of these ultrasound-based biomarkers were also generated for a visual representation of the RF data analysis. Parametric images were generated utilizing a sliding window analysis within the ROI using a Hamming function. The size of the sliding window was selected to cover approximately 10 wavelengths in order to obtain robust parametric maps independent of the window length [27].