Identification of 11(13)-dehydroivaxillin as a potent therapeutic agent against non-Hodgkin's lymphoma

Despite great advancements in the treatment of non-Hodgkin lymphoma (NHL), sensitivity of different subtypes to therapy varies. Targeting the aberrant activation NF-κB signaling pathways in lymphoid malignancies is a promising strategy. Here, we report that 11(13)-dehydroivaxillin (DHI), a natural compound isolated from the Carpesium genus, induces growth inhibition and apoptosis of NHL cells. Multiple signaling cascades are influenced by DHI in NHL cells. PI3K/AKT and ERK are activated or inhibited in a cell type dependent manner, whereas NF-κB signaling pathway was inhibited in all the NHL cells tested. Applying the cellular thermal shift assay, we further demonstrated that DHI directly interacts with IKKα/IKKβ in NHL cells. Interestingly, DHI treatment also reduced the IKKα/IKKβ protein level in NHL cells. Consistent with this finding, knockdown of IKKα/IKKβ inhibits cell proliferation and enhances DHI-induced proliferation inhibition. Overexpression of p65, p52 or RelB partially reverses DHI-induced cell growth inhibition. Furthermore, DHI treatment significantly inhibits the growth of NHL cell xenografts. In conclusion, we demonstrate that DHI exerts anti-NHL effect in vitro and in vivo, through a cumulative effect on NF-κB and other pathways. DHI may serve as a promising lead compound for the therapy of NHL.

an anti-pyretic, analgesic and febrifuge in traditional Chinese medicine. 20 DHI also demonstrates efficient antiplasmodial activity in vivo. 21 In this study, we report that DHI exhibits strong anti-NHL activity in vitro and in vivo. The mechanisms studies have revealed that inhibition of IKKα/β and other survival pathways such as AKT and ERK are involved in the anti-NHL effect of DHI. DHI represents a promising lead compound for the treatment of NHL.

DHI inhibits proliferation and reduces viability of human NHL cells.
To evaluate the effect of DHI (Figure 1a) on the proliferation of NHL, BL cells -Daudi and NAMALWA cellsand DLBCL cells -SU-DHL-4 (GCB-DLBCL), SU-DHL-2 (ABC-DLBCL), OCI-Ly8 (GCB-DLBCL) and U2932 (ABC-DLBCL) were treated with various concentrations of DHI (0, 5, 7, 10 μM) for 24 h. Cell proliferation was evaluated by CCK-8 assay. DHI exhibited inhibitory effect on these lymphoma cells (Figures 1b and c). Moreover, cell viability was determined by the trypan blue exclusion assay. DHI reduced cell viability and inhibited the cell proliferation in some NHL cell lines in a dose-and time-dependent manner. Among them, NAMALWA, U2932 and SU-DHL-2 cells were considered the most sensitive to DHI-induced proliferation inhibition and cell death (Figures 1d and e). However, DHI did not affect the viability of mononuclear cells isolated from umbilical cord blood cells (Figure 1f). Together, these data suggest that DHI selectively inhibits the growth of lymphoma cells.

DHI induces apoptosis in NHL cells.
To investigate whether DHI induces apoptosis in NHL cells, Daudi, NAMALWA, SU-DHL-4 and SU-DHL-2 cells were exposed to various concentrations of DHI for 24 h. Cell population in the subG1 phase was examined by flow cytometry. In all the cell lines tested, DHI treatment induced an increase of the cell population in the subG1 phase to varying degrees (Figures 2a and b). In contrast to other cells, S phase arrest was observed in DHI-treated NAMALWA cells, which was accompanied by the reduction of cyclin A expression (Supplementary Figure S1). The apoptotic induction effect of DHI was further evaluated by Annexin V/PI staining using flow cytometry. The results demonstrated that NAMALWA and SU-DHL-2 are more sensitive than Daudi and SU-DHL-4 DHI suppresses the NF-κB transcriptional activity. Given the important role of NF-κB pathway in the survival of lymphoma cells, 11,12 we hypothesized that DHI may affect NF-κB activity. To test this hypothesis, Daudi cells stably transfected with NF-κB-dependent luciferase reporter were treated with DHI in the presence or absence of 15 ng/ml of TNFα. As shown in Figure 3a, DHI significantly inhibited TNFα-induced NF-κB activation. Consistent with this result, the immunostaining assay and nuclear and cytoplasmic extraction assay showed that DHI inhibited nuclear translocation of NF-κB p65 in HeLa cells ( Figure 3b) and SU-DHL-2 cells ( Supplementary Figures S2a and b) but did not affect the nuclear localization of p50 (Supplementary Figure S3). Moreover, TNFα-induced upregulation of NF-κB p65 target genes, 22 such as cyclin D1, Bcl-2 and IκBα, were also suppressed by DHI treatment in Daudi, NAMALWA and SU-DHL-2 cells (Figure 3c). In addition to NF-κB, we also tested other signaling pathways downstream of the B-cell receptor (BCR) that have been shown to have pivotal role in B-cell survival, such as the PI3K/AKT and ERK pathways. 23,24 In response to DHI, both activation and inhibition of AKT and ERK could be induced in different cell lines. For example, DHI inhibited the phosphorylation of AKT and ERK in Daudi, NAMALWA and U2932 cells, increased phosphorylation of AKT but decreased phosphorylation of ERK in SU-DHL-4 cells, and increased phosphorylation of AKT and ERK in SU-DHL-2 cells. DHI had no effect on the phosphorylation of AKT and ERK in OCI-Ly8 cells (Supplementary Figure S4a). Taken together, these results indicate that DHI affects PI3K/AKT and ERK pathways in a cell-type-dependent manner and the common pathway affected by DHI in NHL cells is the NF-κB signaling pathway.
DHI suppresses IKK activation. NF-κB nuclear translocation is controlled by the inhibitory IκBα proteins. Phosphorylation of IκBα by IKK leads to its proteasomal degradation, thereby allowing nuclear translocation of NF-κB. 25 Given that DHI inhibits NF-κB p65 nuclear translocation, we hypothesized that DHI may do so by inhibiting the IKK/IκBα signaling pathway. To test this hypothesis, Daudi, NAMALWA and SU-DHL-2 cells were pre-treated with various concentrations  (Figure 4c). c-Myc and cyclin D1, two NF-κB gene targets essential for lymphoma cell proliferation and survival, 26,27 were also dramatically decreased by DHI treatment (Figure 4c). Reduction of IKKα/β could be observed as early as 8 h (Figure 4d). These results indicate that DHI blocks NF-κB activation by inhibiting the IKK/IκBα signaling pathway.
DHI interacts with IKKα/β and IKKα/β knockdown enhances the effect of DHI in NHL cells. In order to investigate whether the DHI-induced inhibition of NF-κB activation was due to a direct interaction with IKKα/β, a cellular thermal shift assay (CETSA) experiment was conducted. CETSA is a newly developed method for evaluating drug-binding to target proteins in cells and tissue samples. 28,29 As shown in Figures 5a-d, compared with DMSO, addition of DHI reduced thermal stability of IKKα/β protein in Daudi cells and SU-DHL-2 cells. Moreover, increasing the concentration of DHI decreased the thermal stability of IKKα/β proteins (Figure 5e and f). As a negative control, we evaluated the thermal stability of vinculin protein in response to DHI. The thermal stability of vinculin protein was not affected by DHI in the various temperatures and concentrations tested. As a positive control, we measured the thermal stability of IKKβ in response to ainsliadimer A, a reported IKKα/β inhibitor (Supplementary Figure S6). We An equivalent number of cells were plated in 24-well plates and treated with 0, 5, 7 or 10 μM of DHI for 4 h, followed by treatment with or without TNFα (15 ng/ml) for 4 h. Luciferase activity was measured using Bright-Glo reagents (Promega). (b) HeLa cells were treated with or without the indicated concentrations of DHI for 12 h, followed by stimulation with or without TNFα (15 ng/ml) for 30 min. Immunofluorescent staining of NF-κB p65 was then performed and assayed by confocol microscopy. Green signaling represents p65 staining, whereas blue signaling indicates nuclei stained with DAPI. Scale bar, 20 μm. (c) Daudi, NAMALWA, and SU-DHL-2 cells were pre-treated with or without 5, 10 μM DHI for 6 h, followed by stimulation with TNFα (15 ng/ml) for 90 min. qRT-PCR was then used to detect the indicated mRNA. Data are representative of three or more experiments with similar results. All values represent the means ± S.D. of three independent experiments. *Po0.05; **Po0.01 versus the control observed that the thermal stability of IKKβ could be reduced in this condition. Together, these results suggest that DHI directly interacts with IKKα/β in NHL cells.
To investigate whether targeting IKKα/β contributes to DHI's ability to inhibit cell proliferation, IKKα/β was knocked down in lymphoma cells. As shown in Figures 6a, d and e, knockdown of IKKα/β in Daudi and SU-DHL-2 cells specifically reduced the protein level of IKKα/β. Interestingly, silencing only IKKα or IKKβ increased the protein level of IκBα and decreased p-IκBα, whereas silencing both of them decreased IκBα (Supplementary Figures S7a and b). Though the underlying mechanism of decreased IκBα in both IKKα/IKKβ silenced cells is currently not known, one possibility is that by reducing the expression of both IKKα and IKKβ, the protein level of IκBα is regulated in an IKKα/β kinase-independent manner. Compared with the vectortransfected cells, knockdown of either IKKα or IKKβ alone significantly inhibited cell growth (Figures 6b and f.) and enhanced DHI-induced inhibition of cell proliferation (Figures 6c and g). This effect could be further enhanced by knockdown of both IKKα and IKKβ (Figures 6f and g).
Overexpression of p65, p52 or RelB partially abrogates the anti-proliferation effect of DHI on NHL cells. Both the classical and alternative NFκB pathway could be regulated by IKKα/β. P65, p50, RelB are the important downstream substrates of IKKα/β. Intriguingly, knockdown of IKKβ reduced the protein level of phosphorylated p65 in cytoplasm but did not alter the amount of nuclear p-p65 protein in NHL cells DHI inhibits lymphoma tumor growth in xenograft mice models.. To further explore the effect of DHI in vivo, xenograft mice models were established by the subcutaneous injection of Daudi and SU-DHL-2 cells in B-NSG mice which are NOD-SCID IL-2 receptor gamma null mice. When tumors grew to 100 mm 3 , mice were injected intraperitoneally with DHI (50 mg/kg) or vehicle daily for 10 days. Both tumor volume and mouse body weight were measured every other day. DHI significantly inhibited tumor growth (Figure 8b). At the end of treatment, tumor weight of the DHI-treated group was significantly lower than that of the vehicle-treated group (Figure 8c) and just a slight body weight loss was observed in DHI-treated mice (Figure 8a). IHC analysis showed that protein levels of IKKα/β and the proliferating nuclear cell antigen (PCNA), a marker of proliferation, were significantly decreased in the DHI-treated tumor (Figure 8d). We also observed an increase of TUNEL-positive cells in DHI-treated tumors compared to those from the vehicle condition, which indicates the induction of apoptosis (Figure 8d). These results suggest that DHI has anti-tumor activity in NHL xenograft models.

Discussion
Aberrant NF-κB activation underlies the development of many cancers 30 and has been associated with tumor cell survival, proliferation, invasion and angiogenesis, 31 especially in lymphoid malignancies. Targeting NF-κB signaling pathways represents a promising strategy in the treatment of NHL. 14 In the present study, we demonstrated that DHI, a small natural compound, induces proliferation inhibition and apoptosis of NHL cells in vitro and in vivo. Moreover, we provide evidence that the anti-NHL effect of DHI is associated with inhibition of IKKα/β and other survival pathways.
The IKK complex is composed of IKKα and IKKβ catalytic subunits and a regulatory subunit, IKKγ/NEMO. Its key function is to phosphorylate IκBs and the NF-κB precursors, p105 and p100, which then leads to the activation of NF-κB. 25 In recent years, an IKK kinase-independent function was also found. 32,33 As this complex has a crucial role in cancer, as well as inflammatory and autoimmune disease, significant efforts have been made to identify IKK inhibitors. 25,34 Many compounds have now been introduced as IKK inhibitors, such as celastrol, PS1145, BMS345541 and others. [35][36][37][38][39][40] Inhibition of IKK can inhibit the proliferation of cancers or sensitize cancer cells to chemotherapeutic drugs. 41,42 In this study, we demonstrated that IKKα and IKKβ are direct targets of DHI, which was supported with the following evidence: CETSA results unambiguously showed that DHI interacts with IKKα and IKKβ. CETSA, a recently developed method to assess drug engagement in complex environments, 28,29 builds on the discovery that ligand induced protein thermal shift can also be measured in the context of cell lysates. [43][44][45] Compared with in vitro assays using recombinant proteins, CETSA keeps target proteins in their native, local environments, maintaining their original post-translational modifications and expression level. Using this method, we found that DHI reduces the thermal stability of IKKα/β, indicating that DHI can interact with IKKα/β in cells. Consistent with this result, DHI treatment inhibited the phosphorylation and degradation of IκBα and also inhibited the phosphorylation of NF-κB, which in turn inhibited the activation of NF-κB and thus the related, downstream In addition to the classical pathway, IKKα plays an important role in regulating the activation of the alternative NF-κB pathway, it is possible that DHI can also inhibit the alternative pathway. In support of this opinion, we found that both total and nuclear p52, the critical component of alternative pathway, were significantly downregulated by the treatment of DHI ( Supplementary Figures S10a and b). In addition, similar to the inhibition induced by DHI, IKKα/β knockdown suppress NHL cells proliferation. Unexpectedly, IKKβ knockdown decreases p65 in nucleus, but did not change the nuclear p-p65 and overexpression of p65 partially abrogates the proliferation inhibitory effect of DHI on NHL cell lines, these data suggest that the inhibitory effect of knockdown of IKKα/β might be p65 phosphorylation independent.
Similar observations were reported that the phosphorylation of serine 536 was not required for p65-mediated protection from TNFα cytotoxicity and Traf1 induction in fibroblasts. 46 One interesting observation is that DHI treatment decreased the protein level of IKKα/β, which could not be reversed by proteasome inhibitor MG132 or chloroquine (Supplementary Figure 11), indicating that other ways beyond proteasome system or lysosomal system participates in this process. The underlying mechanism need to be further explored. Compared with the other inhibitors that only impede kinase activity, DHI may not only block IKK kinase activity but also abrogate the kinase-independent activity of IKK. 32,33 Moreover, it has been shown that mutation of IKK can confer cancer cell resistance to an IKK inhibitor. 47 Therefore, DHI may provide an alternative approach to overcome this drug resistance. Most importantly, we demonstrate that DHI exhibits a significant anti-tumor effect in vivo with only mild liver toxicity (Supplementary Figure S9). Thus, targeting IKKα/β and thereby inhibiting the activation of NF-κB may contribute to the tumoricidal properties of DHI. DHI has not been previously reported to have any anti-tumor effect, our results indicate that DHI is a novel potent compound in the treatment of NHL. Another interesting finding in this work is that NAMALWA and SU-DHL-2 cells are more sensitive to DHI treatment than Daudi and SU-DHL-4 cells. We assumed that the degree of sensitivity might be associated with differing NF-κB signaling components in each cell. Notably, the expression pattern of NF-κB signaling components is similar in NAMALWA and SU-DHL-2 cells (Supplementary Figure S12). In the BL cell lines, IKKα/β, p65 and p50 were more highly expressed in NAMALWA cells than in Daudi cells, whereas IκBα exhibited lower expression in NAMALWA cells (Supplementary Figure S12a), which suggests that the level of NF-κB activation was higher in NAMALWA cells. In the DLBCL cell lines, the expression of NF-κB components (IKKα, IKKβ, p65, p-p65, p50 and p-IκBα) was more highly upregulated in SU-DHL-2 cells than in SU-DHL-4 cells (Supplementary Figure S12b), indicating that NF-κB was constitutively activated in SU-DHL-2  24 Overall, these results support the idea that the different sensitivity of NHL cells to DHI treatment depends on their respective NF-κB activity. It has been previously shown that SU-DHL-2 (ABC-DLBCL) cells are more resistant to R-CHOP treatment than are SU-DHL-4 (GCB-DLBCL) cells. 48 Our results suggest that DHI might be used to overcome the R-CHOP resistance that arises in ABC-DLBCL cells. Besides the NF-κB, BCR signaling pathways also have an important role in the survival of lymphoma cells. As overexpression of components of NF-κB only partially abrogate the effect of DHI, we have also tested whether the BCR signaling pathway are also involved. It was found that DHI could evoke or repress the activity of AKT or ERK in a cell type dependent manner, meanwhile, the CETSA indicates that DHI does not directly interact with AKT and ERK protein (Supplementary Figure S4b). Further, we found that combined treatment with either AKT inhibitor MK2206 or ERK inhibitor U0126 enhance the inhibitory effect of DHI on SU-DHL-2 and Daudi cells ( Supplementary Figures S4c and d). Taken together, we propose that the anti-NHL effect of DHI may be a combined effect from its influence on the NF-κB signaling pathway, PI3K/ AKTand ERK pathways or other unknown signaling pathways.
In summary, our findings highlight the potential clinical benefit of DHI as a chemotherapeutic agent against NHL, particularly for those patients with IKK-related aberrant NF-κB activation.
Cell viability assay. The effect of DHI on cell viability was determined by trypan blue exclusion assay. Trypan blue exclusion assay measuring cell viability is based on the principle that live cells with intact membranes are able to exclude the dye, whereas dead cells without an intact membrane accept the dye. NHL cells (5 × 10 5 /ml per well) were seeded in 6-well plates and culture medium containing vehicle or DHI was added to each well. Cells were incubated at 37°C. At the indicated time points (0, 24, 48 h), cells were suspended with 0.05 % trypsin-EDTA and stained with trypan blue dye (0.4%). The unstained (viable) and stained (dead) cells were counted in the Neubauer hemocytometer. The cell viability (%) was calculated as a ratio, the number of live cells relative to the total number of cells. Experiments were repeated three independent times, and the data were presented as bar diagram with standard deviation.
Cell proliferation assay. Cell proliferation activity was determined by CCK-8 assays, according to the manufacturer's instructions. NHL cells were plated at a certain density in 96-well plates with either the proper vehicle control or with different concentrations of DHI for 24 h. Each condition was run in three duplications. Next, 10 μl of CCK-8 solution (CK04, Dojindo Molecular Technologies, Kumamoto, Japan) were added to the wells, and the plates were incubated for 4- Cell apoptosis assay. The apoptosis rate of NHL cells was determined using Annexin V-FITC and PI (BD Pharmingen, San Jose, CA, USA) staining. NHL cells (5 × 10 5 /ml) in 6-well plates were treated with different concentrations of DHI for 24 h. Cells were collected, washed with PBS three times, and resuspended in 400 μl of 1 × binding buffer. Next, 5 μl of Annexin V-FITC and 5 μl of PI were added to each sample, and the samples were incubated in the dark for 15 min. Cells were analyzed by fluorescence-activated cell sorting using a BD LSRFortessa cell analyser (BD Biosciences). Cells that stained positive for early apoptosis (Annexin V-FITC-stained only) and for late apoptosis (Annexin V-FITC-and PI-stained) were combined for analysis, and the results were analyzed using FlowJo 7.0 software (Tree Star, Ashland, OR, USA).
ShRNA-mediated IKKα/ β knockdown. The shRNA or NC plasmids were cotransfected with the lentiviral packaging vectors PSPAX2 and pMD2G and introduced into HEK293T cells to produce lentivirus. After 48 h, the viral supernatant was collected and added to lymphoma cells in six-well plates with medium containing 8 μg/ml polybrene (Sigma). The infected cells were spun at 1000 × g for 100 min, and fresh media was added. After 2 days' infection, stably transfected cells were selected with puromycin. The targeting sequences of shIKKα/β were shown to be the following: shIKKα#1, 5′-AAGCAGAAGATTATTGATCTA-3′; shIKKα#2, 5′-CAGGAGAAGTTCGGTTTAGTA-3′; shIKKβ#1, 5′-CGCTTAGATACCTTCATGAAA-3′; shIKKβ#2, 5′-CTGGATGA CATCTTAAACTTA -3′ Plasmid p65, p52, RelB and overexpression. P65 gene was amplified from HEK293T cells by reverse transcription PCR and the PCR product was cloned into PMSCV vector to create p65 plasmid. P52 gene was amplified from the p100 plasmid (kindly gift from Dr. Xiaodong Yang, Shanghai Jiaotong University School of Medicine) and then cloned into Plvx-IRES-ZsGreen1 vector to form p52 plasmid. RelB were purchased from the DNA library of Shanghai Jiao Tong University School of Medicine. The p65 or vector plasmids were cotransfected with retrovirus packaging vectors Gag-Pol and VSVG and introduced into HEK293T cells to produce retrovirus. P52, RelB and its corresponding vector were cotransfected with lentiviral packaging vectors PSPAX2 and pMD2G and introduced into HEK293T cells to produce lentivirus. The infection of NHL cells was performed as described above. Luciferase assay. Daudi cells were stably infected with NF-κB Luciferase reporter plasmid (Promega, Madison, WI, USA). Equal numbers of cells were plated in 24-well plates, then treated with various concentrations of DHI for 4 h, followed by a 4-h stimulation with or without TNFα (15 ng/ml). Cells were lysed in passive lysis buffer (Promega) for 15 min. The transcriptional activity was determined by measuring the activity of firefly luciferase in a multiwell plate luminometer (Tecan, Durham, NC, USA) using a luciferase reporter assay system (Promega) according to the manufacturers' instructions.
Real time quantitative RT-PCR. Total RNA was isolated using a TRIzol kit (Invitrogen, Carlsbad, CA, USA), and RNA was treated with DNase (Promega). cDNA was reverse transcribed using MMLV reverse transcriptase (Promega). Real time quantitative PCRs were performed with SYBR Green PCR Master Mixture reagents (Roche, Basel, Switzerland). The primer sequences are shown in Supplementary Table S1.

CETSA.
Daudi cells or SU-DHL-2 cells were harvested and diluted in PBS supplemented with protease inhibitor cocktail (Calbiochem, Billerica, MA, USA). The cell suspensions were freeze-thawed three times in liquid nitrogen. The soluble fraction (lysates) was separated from the cell debris by centrifugation at 20 000 × g for 20 min at 4°C. The cell lysates were divided into two aliquots. One aliquot was treated with DMSO and the other aliquot with DHI (100 μM) or ainsliadimer A (10 μM). After a 30 min incubation at room temperature, the respective lysates were divided into separate aliquots (30 μl) and heated individually at different temperatures for 3 min (Veriti thermal cycler, Applied Biosystems/Life Technologies, Foster City, CA, USA). This was followed by cooling for 3 min at room temperature. The appropriate temperatures were determined in preliminary CETSA experiments (data not shown). The heated lysates were centrifuged at 20 000 × g for 30 min at 4°C in order to separate the soluble fractions from the precipitates. The supernatants were transferred to fresh microtubes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting analysis. Dose effect of DHI on the thermal stability of IKKα/β was evaluated as follows. After the cell lysates solution was obtained via the above method, we divided the lysates solution into six uniform parts and then added equal volumes of the corresponding concentration of DHI, followed by 30 min incubation. Subsequently, we heated the lysates solution incubated with various concentrations of DHI at 54°C for 3 min (Veriti thermal cycler, Applied Biosystems/Life Technologies), followed by cooling for 3 min at room temperature. The heated lysates were centrifuged at 20 000 × g for 30 min at 4°C to separate the soluble fractions from the precipitates. The supernatants were transferred to new microtubes and analyzed by SDS-PAGE followed by western blotting analysis.
Animal experiments. B-NSG mice, NOD-SCID IL-2 receptor gamma null mice, 6-8 weeks old were obtained from Jiangsu Biocytogen Co., Ltd (Nantong, China). Daudi (1 × 10 7 ) cells and SU-DHL-2 (2 × 10 7 ) cells were injected into the right flank of the B-NSG mice, respectively. After 5-10 days of inoculation, mice were randomly divided into two groups. One group was dosed intraperitoneally with DHI (50 mg/kg) and the other was injected with vehicle (administrated daily). The treatment period lasted for 10 days. Tumor volumes and mice body weights were monitored every two days. At the end of DHI treatment, mice were killed, and tumors were excised and weighed. Tumor volume was calculated by using the formula mm 3 = 1/2 a × b 2 , where a is the length and b is the width. All animals were handled according to the protocols approved by the Committee for the Humane Treatment of Animals at Shanghai Jiao Tong University School of Medicine.
Immunohistochemical analysis. The tumors of the mice treated with vehicle or DHI were formalin fixed, paraffin embedded and sectioned into slices. Tissue sections were stained with hematoxylin and eosin, and then the immunohistochemistry for IKKα/β, PCNA and TUNEL were performed on the sections.
Statistical analysis. All graph preparations and statistical calculations were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Student's t-test was used to evaluate differences between two groups. P-valueo0.05 was regarded as statistically significant.