I131 reinforces antitumor activity of metuximab by reversing epithelial–mesenchymal transition via VEGFR‐2 signaling in hepatocellular carcinoma

CD147 is highly expressed in hepatocellular carcinoma (HCC) and associated with the invasion and metastasis of HCC. The efficacy of I131‐metuximab (I131‐mab), a newly developed agent that targets CD147, as a radio‐immunotherapy for local HCC, has been validated in clinical practice. However, the synergistic anticancer activity and molecular mechanism of different conjugated components within I131‐mab remain unclear. In this study, the cytological experiments proved that I131‐mab inhibited the proliferation and invasion of HCC cells. Mechanically, this inhibition effect was mainly mediated by the antibody component part of I131‐mab, which could reverse the epithelial–mesenchymal transition of HCC cells partially by suppressing the phosphorylation of VEGFR‐2. The inhibitory effect of I131 on HCC cell proliferation and invasion is limited, whereas, when combined with metuximab, I131 significantly enhanced the sensitivity of HCC cells to CD147‐mab and consequently reinforced the anticancer effects of CD147‐mab, suggesting that the two components of I131‐mab exerted synergistic anti‐HCC capability. Furthermore, the experiments using SMMC‐7721 human HCC xenografts in athymic nude mice showed that I131‐mab and CD147‐mab significantly inhibited the growth of xenograft tumors and that I131‐mab was more effective than CD147‐mab. In conclusion, our results elucidated the mechanism underlying the anti‐HCC effects of I131‐mab and provided a theoretical foundation for the clinical application of I131‐mab.


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Genes to Cells WU et al. residual cancer cells are found to be more invasive and metastatic (Su, 2016;Wan et al., 2016;Yu, Park, Park, & Yoon, 2016). Thus, further studying the genetic and biological properties of HCC is important to identify specific molecular markers of HCC recurrence and metastasis, develop new anti-HCC agents and ultimately improve the quality of life of HCC patients.
Immunotherapy is an important approach of comprehensive cancer therapy. Specifically, HCC cells strongly express CD147 antigen, which is related to the invasion and metastasis of HCC . Thus, CD147, a highly glycosylated transmembrane protein and a member of the immunoglobulin superfamily, is an effective target for HCC immunotherapy. CD147 expression is up-regulated in many tumors and is especially high in HCC tissue and HCC cell lines, where it is expressed in up to 75%. Conversely, it is not expressed in normal hepatic tissue and normal hepatic cell lines (Dai et al., 2009;Gou et al., 2009). Moreover, the over-expression of CD147 can induce the expression and secretion of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), which degrade extracellular matrix (ECM) and promote tumor invasion and metastasis by interfering with mesenchymal cells (Cui et al., 2012;Wang et al., 2010). To target CD147, a highly specific monoclonal antibody, HAB18 or metuximab, was developed and conjugated to the radioisotope I 131 . The resultant I 131 -metuximab (I 131 -mab) is a radio-immunotherapy injection that was officially approved by the CFDA in 2007, being under the trade name Licartin for the treatment of local HCC (Ma & Wang, 2015;Wu, Shen, Xia, & Yang, 2016). I 131 -mab exhibits several advantages, such as its specificity, rapid response and lethality to cancer cells. The preliminary results of a phase IV clinical trial of TACE combined with I 131 -mab for the treatment of advanced HCC at eight Chinese institutions showed that I 131 -mab is a safe and effective agent for the treatment of advanced HCC (Xu et al., 2007) and can effectively delay HCC relapse after hepatic transplantation (Wu et al., 2010(Wu et al., , 2012. Moreover, the combination of TACE and I 131 -mab can significantly prolong progression-free survival and the overall survival of patients with advanced HCC according to the Barcelona clinical staging (BCLC) . Among the 167 patients with HCC in this study, the 1-year survival rate was significantly higher for patients receiving the combination treatment than that of patients only receiving the TACE treatment. The extrahepatic metastasis incidence in the combined treatment group was 2.94%, which was significantly lower than the 8.57% rate in the TACE group. This difference suggests that I 131 -mab can reduce the extrahepatic metastasis incidence in patients after TACE treatment.
The molecular mechanisms underlying the acceleration effect of CD147 on HCC invasion and metastasis and the synergistic inhibition effect of I 131 -mab on HCC progression have been reported (Bian et al., 2014;Gou et al., 2016).
Specifically, CD147 can activate the transforming growth factor-β (TGF-β) signaling pathway to promote the expression and secretion of MMPs in tumor cells and mesenchymal cells, which induces the epithelial-mesenchymal transition (EMT) in cancer cells and contributes to tumor invasion and metastasis (Ru, Wu, Chen, & Bian, 2015;Xu et al., 2007). Growing evidence shows that EMT plays an important role in the molecular mechanisms underlying HCC occurrence and development. During the process of EMT, cells of epithelial origin transform into mesenchymal cells under specific physiological and pathological conditions, and this transition is accompanied by a series of phenotypic and behavioral changes within cells (Jayachandran, Dhungel, & Steel, 2016). EMT is not only involved in embryonic development but also plays an important role in the pathology of many diseases, such as fibrosis and cancer (Nieto, Huang, Jackson, & Thiery, 2016). In cancer cells, EMT can decrease cell adhesion and increase cell motility, and these changes promote invasion, metastasis and the formation of a new tumor (Giannelli, Koudelkova, Dituri, & Mikulits, 2016;Polyak & Weinberg, 2009). I 131 -mab specifically binds to CD147, which is located on the surface of HCC cells, to inhibit TGF-β signaling and MMP secretion. This inhibition consequently interrupts EMT process and suppresses the invasion and metastasis of cancer cells.
The efficacy of TACE in combination with I 131 -mab has been confirmed, and the mechanisms underlying this effect have also been partially elucidated. However, multiple factors are involved in these mechanisms. Do the mechanisms interact? Is the effect synergistic? In addition to the I 131mab-mediated inhibition of TGF-β signaling to interrupt EMT, are other signaling pathways involved in this process? Furthermore, studies are needed to answer these questions. The work described herein shows that I 131 -mab can reverse the EMT of HCC cells, and metuximab, a component of I 131mab, plays an important role in the reversion effect. I 131 can increase sensitivity of HCC cells to metuximab to synergistically enhance the efficacy of treatment. Moreover, the mechanism by which I 131 -mab reverses the EMT of HCC cells is related to VEGFR-2 signaling. Overall, this work showed that I 131 -mab may interrupt EMT of HCC by inhibiting VEGFR-2 signaling to prevent HCC metastasis.

| Inhibition of HCC cell proliferation by I 131 -mab
Western blotting was used to measure the expression levels of CD147 in a variety of cell lines, and CD147 was expressed in HepG2, Hep3B, SMMC-7721, MHCC97H and MHCC97L cells, with HepG2, Hep3B and MHCC97H cells expressing higher levels of CD147. MHCC97L cells expressed the lowest level of CD147, and WRL-68 cells were

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negative for the expression of CD147 (Figure 1a). Based on these results, we selected CD147-negative WRL-68 cells to investigate the cytotoxicity of I 131 -mab and CD147 antibody (CD147-mab). CD147-mab did not affect cell viability, whereas I 131 -mab exerted a dose-dependent effect on cell viability. The IC 20 and IC 50 values of I 131 -mab on WRL-68 cells were 8.09 μCi/100 μl and 12.69 μCi/100 μl, respectively ( Figure 1b). Because WRL-68 cells did not express CD147 and were not inhibited by CD147-mab, the cytotoxicity of I 131 -mab was attributed to the conjugated I 131 . Because the unit of measurement of I 131 -mab and I 131 was μCi and the unit of measurement of CD147-mab is μM, therefore, to establish the concentration equivalence relation between I 131 -mab and CD147-mab, we regarded that the concentrations of I 131 -mab corresponding to IC 20 and IC 50 could be served as the reference concentrations of I 131 and consequently calculated the molar concentrations of CD147-mab to be 5.62 μM/100 μl and 8.82 μM/100 μl, respectively.

| Inhibition of HCC cell invasion by I 131 -mab
To verify the effect of I 131 -mab on HCC cell invasion, a Transwell device was used to assess cell invasion. Both I 131 -mab and CD147-mab significantly inhibited the invasion of the HCC cell lines. Specifically, I 131 -mab inhibited HepG2, Hep3B, SMMC-7721, MHCC97H and MHCC97L cell invasion more effectively than CD147-mab, whereas the effects of CD147-mab on the invasion of Hep3B and MHCC97L did not significantly differ from the control group. These results suggested that inhibition of HCC cells by I 131 -mab or CD147-mab was closely associated with CD147 expression. I 131 alone exerted a limited effect on HCC cell invasion, and this effect only differed between the treated and control groups for MHCC97L cells (Figure 2).

VEGFR-2 signaling
To elucidate the mechanism by which I 131 -mab inhibits HCC cells, we compared some markers related to EMT in HCC cells treated with I 131 -mab, I 131 and CD147-mab. Ecadherin expression was significantly increased, whereas F I G U R E 2 Inhibition of HCC invasion by I 131 -mab. The upper Transwell chambers were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum, and the Transwell chambers were placed in a well of a 24-well plate. HCC cells were seeded into the upper chamber of the Transwell (1 × 10 5 cells/well) and cultured for 24 hr; the cells were treated with I 131 -mab, I 131 (8.09 μCi/100 μl) and CD147-mab (5.62 μM/100 μl) were used to treat cells for 48 hr. The Transwell chambers were collected, and the cells were stained with 0.1% crystal violet for 20 min. Positive cells were counted. Compared with the control group: *p < .05, **p < .01 and ***p < .001

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Genes to Cells WU et al. the expression levels of N-cadherin and vimentin were decreased in all HCC cells treated with I 131 -mab and CD147mab. Conversely, I 131 did not affect the expression levels of these proteins (Figure 3a). These results suggested that I 131mab reversed HCC cell EMT via the CD147 antibody in the conjugated molecules but not via I 131 .
VEGFR expression was further studied in HCC cells treated with I 131 -mab, CD147-mab and I 131 . The results showed that VEGFR-2 phosphorylation (p-VEGFR-2) significantly decreased in HCC cells treated with I 131 -mab and CD147-mab, whereas the levels of VEGFR-1, p-VEGFR-1 and VEGFR-2 did not change. I 131 alone did not affect the expression levels of these proteins in HCC cells (Figure 3b). These results suggest that I 131 -mab reversed EMT by inhibiting the phosphorylation of VEGFR-2 in HCC cells.

| I 131 increases the sensitivity of HCC cells to the cytotoxicity of CD147-mab
By MTT assay, we found that I 131 did not inhibit the proliferation of all HCC cells at doses <8.09 μCi/100 μl, whereas the cytotoxicity of 5.62 μM/100 μl CD147-mab (concentration corresponding to the I 131 dose) varied among HCC cell lines. We cotreated cells with I 131 (8.09 μCi/100 μl) and CD147-mab (5.62 μM/100 μl) to compared the effect of this treatment to that of CD147-mab alone. We found that the viability of all HCC cell lines significantly decreased in response to the combined treatment (Figure 4a). The results of cell invasion capability were coincident with the MTT assay (Figure 4b,c). The results suggested that I 131 increases the sensitivity of HCC cells to CD147-mab and consequently enhances the cytotoxicity of this agent.

| Inhibition of SMMC-7721 human hepatoma xenografts by I 131 -mab
The inhibition of HCC by I 131 -mab and the molecular mechanisms underlying this inhibition were verified in nude mice harboring SMMC-7721 human hepatoma xenografts. Treating these xenografts with I 131 -mab injection markedly inhibited tumor growth speed; specifically, the tumor volume was significantly smaller in the I 131 -mab treatment group than that in the control group 7 days after the first injection, and this difference increased over time. The efficacy of CD147-mab group was evident 14 days after the start of treatment, whereas no noticeable effects were observed in the I 131 treatment group, even at the end of the observation period (Figure 5a). After 21 days of treatment, the tumor volume in the control group exceeded the standard limit, and the observation was ended. The tumors were dissected and weighed, and the tumor weight was lowest in the I 131 -mab group, followed by the CD147-mab group. There is no difference in tumor weight between the I 131 group and the control group (Figure 5b). Tissue sections were obtained from the hepatoma xenografts, and immunohistochemistry staining was used to measure E-cadherin and p-VEGFR-2 expression. The results showed that E-cadherin expression was up-regulated, whereas p-VEGFR-2 expression was down-regulated in the I 131 -mab and CD147-mab groups (Figure 5c).
TACE is an important treatment for HCC but has been clinically associated with increases in distant metastasis due to residual cancer cells in many patients with HCC (Xue et al., 2016). This phenomenon has greatly compromised the long-term efficacy of TACE. Moreover, TACE can induce tissue hypoxia, which down-regulates E-cadherin and up-regulates of N-cadherin in residual cancer cells. Thus, hypoxia may mediate the negative effects of TACE by inducing EMT in cancer cells (Fransvea, Angelotti, Antonaci, & Giannelli, 2008;Xue et al., 2015). EMT enhances the invasion and metastasis of cancer cells, reduces the sensitivity or increases the resistance of HCC to chemotherapeutic agents (Bae et al., 2014;Nishida, Kitano, Sakurai, & Kudo, 2015) and contributes to the immune escape of HCC cells (Chockley & Keshamouni, 2016;Ye et al., 2016).
The studies of the relationship between EMT and the metastasis of HCC have provided an effective target for HCC treatment. LY2109761, a TGF-β 1 receptor kinase inhibitor, inhibits EMT to reduce vascular invasion and metastasis in 40 | Genes to Cells WU et al.

F I G U R E 3
Impact of I 131 -mab on EMT and VEGFR-2 expression in HCC cells. (a) HCC cells were seeded in 24-well plates (1 × 10 5 cells/100 μl/well) and cultured for 24 hr. I 131 -mab and CD147-mab were diluted in serum-free medium. The cells were then treated with I 131mab, I 131 (8.09 μCi/10 0 μl) or CD147-mab (5.62 μM/100 μl) for 48 hr. The cells were harvested, total protein was extracted, and the protein concentration was measured. Western blotting was used to measure the expression of E-cadherin, N-cadherin and vimentin. GAPDH was used as a loading control. Compared with the parental cell group: *p < .05, **p < .01 and ***p < .001. (b) Cells were cultured and treated as described above. Western blotting was used to measure the expression of VEGFR-1, p-VEGFR-1, VEGFR-2 and p-VEGFR-2. GAPDH was used as a loading control. Compared with the parental cell group: **p < .01 and ***p < .001

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HCC cells (Baldassarre et al., 2012). Blocking the effects of upstream loop regulatory factors of EMT, such as inhibition of NF-κB/miR-448, can improve the response of cancer cells to chemotherapeutic agents . CD147, under the regulation of slug at transcriptional level, can promote EMT process in HCC via TGF-β signaling (Wu et al., 2011). Furthermore, hypoxia caused by TACE can up-regulate CD147 expression in HCC cells (Gou et al., 2016), and the over-expression of CD147 enhances EMT in cancer cells by activating TGF-β signaling. Thus, I 131 -mab, which targets CD147, may specifically inhibit EMT to attenuate tumor development and metastasis. In nude mouse HCC models, I 131mab effectively inhibited the growth and metastasis of HCC and inhibited MMPs and VEGF expression in the para-tumor microenvironment (Wu et al., 2011). We conducted a prospective controlled clinical study in which the combination of TACE and I 131 -mab was used to treat intermediate-stage HCC (BCLC staging). Specifically, 68 patients with intermediatestage HCC were included in the combined treatment group, and 70 patients were included in the TACE alone group. The median survival time was 26.7 months in the combined treatment group but only 20.6 months in the control group, and the overall survival in the combined treatment group was significantly better than that in the control group (p = .038). The median time to progression was 18.6 months in the combined treatment group and superior to only 12.5 months in the control group (p = .046). In particular, the extrahepatic metastasis rate was 2.94% in the combined treatment group and 8.57% in the TACE group. These results suggested that I 131mab reduces the risk of extrahepatic metastasis after TACE treatment (Wu et al., 2012).
In this work, the cytological experiments proved that I 131 -mab inhibits the proliferation and invasion of HCC cells, and this inhibition is closely related to the level of CD147 expression. HCC cells expressing high levels of CD147 are more sensitive to I 131 -mab. The inhibition effect of I 131 -mab on HCC cells was mostly attributed to the CD147 antibody within the conjugated molecule. Although the inhibition of HCC cell proliferation and invasion by I 131 alone is limited, when combined with CD147-mab, I 131 significantly enhanced the sensitivity of cancer cells to CD147-mab and consequently enhanced the cytotoxicity of anticancer antibodies. This finding suggested that the two components of I 131 -mab synergistically inhibited HCC. Both I 131 -mab and CD147-mab can reverse the EMT of HCC cells partially by inhibiting the phosphorylation of VEGFR-2 and then reduce the capabilities of proliferation and metastasis of HCC cells. The experiments using SMMC-7721 human hepatoma xenografts in athymic nude mice also proved that I 131 -mab and CD147-mab F I G U R E 4 I 131 enhanced the inhibition of CD147-mab on HCC cells. (a) HCC cells were seeded in 96-well plates (1 × 10 4 cells/100 μl/well) and cultured for 24 hr. I 131 (8.09 μCi/100 μl) and CD147-mab (5.62 μM/100 μl) were combined to treat cells. The control group was treated with the same dose of I 131 alone or CD147-mab alone. The cell viabilities were carried out as described in Figure 1b. Compared with the parental cell group: # p < .05 and ## p < .01; compared with the parental cell group, the I 131 group or the CD147-mab group: ***p < .001. (b) The transwell assay was carried out as described in Figure 2 to compare the capability of HCC cell invasion after treated with I 131 , CD147-mab or combination of them. (c) The invasive cells were counted within five high-magnification fields by three persons. Compared with the parental cell group: **p < .01 and ***p < .001 WU et al.
significantly inhibited xenograft tumors, and I 131 -mab was more effective than CD147-mab in this inhibition. The synergistic effect of conjugated I 131 -mab was attributed to I 131 because I 131 alone did not significantly inhibit the proliferation and invasion of HCC. Our results elucidated the mechanism underlying the antiproliferative and antimetastatic effects of I 131 -mab and provided a theoretical foundation for the clinical application of I 131 -mab.

| Cells and reagents
Human hepatoma cell lines (HepG2, Hep3B, SMMC-7721, MHCC97H and MHCC97L) and normal hepatic cells (WRL-68) were provided by Cell Bank, Institute of Cell Biochemistry, Chinese Academy of Sciences Shanghai F I G U R E 5 Inhibition of SMMC-7721 human hepatoma xenografts by I 131 -mab. (a) SMMC-7721 cell suspension (5 × 10 5 cells/100 μl per mouse) was injected subcutaneously into the right flanks of the nude mice. Tumors formed 12 days after inoculation. The mice were then randomly divided into four groups of five mice each (the I 131 -mab group, CD147-mab group, I 131 group and control group). The injections were carried out every other day for 5 times. Each mouse in the I 131 and I 131 -mab groups received 12.69 μCi/100 μl I 131 or I 131 -mab, respectively, whereas each mouse in the CD147-mab group received 8.82 μM/100 μl CD147-mab. The control group mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the tumor volume was calculated with the following formula: "maximum diameter × minimum diameter 2 × 0.5." Compared with the control group: *p < .05, **p < .01 and ***p < .001. (b) At the end of observation, the mice were killed, and the tumor specimens were harvested and weighed. Compared with the control group: **p < .01 and ***p < .001; (c) sections of hepatoma xenografts were obtained. The expression levels of E-cadherin and p-VEGFR-2 were measured by immunohistochemical staining. Stained cells were counted in five high-magnification fields, and the percentages of positive cells were determined. Compared with the control group: *p < .05 and ***p < .001 Institutes for Biological Sciences (Shanghai, China

| Cell proliferation assay
A tetrazolium colorimetric assay (MTT assay) was used to assess the effects of I 131 -mab, CD147-mab and I 131 on the proliferation of HCC and normal cells. Briefly, cells were harvested during the logarithmic growth phase and seeded in 96-well plates at 1 × 10 4 cells/100 μl/well, and then cultured for 24 hr; serum-free culture medium was used to dilute I 131 -mab, and cells were treated with various concentrations of this agent, with eight duplicates per concentration. After incubated for 2 hr, the medium was then replaced with medium containing 10% serum (100 μl/well). After 48 hr of culture, the medium was replaced with 0.1 M PBS solution (100 μl/well), and MTT labeling reagent (10 μl/well for a final concentration of 0.5 mg/ml; Roche Diagnostics GmbH, Shanghai, China) was then added. After 4 hr of incubation, the solubilization solution (10% SDS in 0.01 mol/L HCl, 100 μl/well) was added for overnight, the absorbance was measured at 490 nm, and the values were plotted to assess cell viability and calculate the IC 20 and IC 50 of each concentration of I 131 -mab and its controls.

| Cell invasion assay
The upper Transwell chambers (8 μm pore size, Corning, Tewksbury, USA) were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum. The Transwell chambers were placed in a well of a 24-well plate. Harvested cells were seeded into the upper Transwell chamber at 1 × 10 5 cells/well and cultured for 24 hr; I 131mab was diluted in serum-free culture medium and added to the wells for a variety of final concentrations. The cells were incubated for 2 hr, and the medium was replaced with medium containing 10% serum (100 μl/well) after 48 hr of incubation. The Transwell inserts were removed, and the cells were stained with 0.1% crystal violet for 20 min before being counted under a microscope; three random fields were photographed (200× magnification).

| Immunoblotting
The above-studied cells were seeded in a 24-well plate at 1 × 10 5 cells/well and cultured for 24 hr. I 131 -mab was diluted in serum-free culture medium and added to the culture wells at various concentrations (based on the protocol). These cells were cultured for 2 hr. Medium containing 10% serum (100 μl/well) was used to replace the previous medium. After 48 hr of incubation, the cells were harvested. Western blotting was used to measure the expression of the target proteins. The antibodies used in the experiments are listed below: rabbit anti-CD147 (Abcam Trading Company Ltd., Shanghai, China); mouse anti-E-cadherin, mouse anti-N-cadherin, mouse antivimentin, rabbit anti-VEGF-C antibodies (Cell Signaling Technology, Danvers, MA, USA); and mouse anti-VEGFR-1, mouse anti-VEGFR-2, rabbit anti-phospho-VEGFR-1 and rabbit anti-p-VEGFR-2 (Cell Applications Inc., CA, USA).

| Human hepatoma xenografts in nude mice
A total of 25 healthy, purebred BALB/C male mice aged 4 weeks were purchased from the SLAC Experimental Animal Centre of the Chinese Academy of Sciences (Shanghai, China). SMMC-7721 cells were harvested during the logarithmic growth phase, and cell suspension was prepared. The cells (5 × 10 5 cells in 100 μl per mouse) were subcutaneously injected into the right flanks of nude mice, and tumors formed 12 days after inoculation. The three mice with the largest tumors and the two mice with the smallest tumors were excluded. The remaining 20 mice were randomly divided into four groups (the I 131 -mab group, the CD147-mab group, the I 131 group and the control group). Animals in the I 131 and I 131 -mab groups received injections of I 131 or I 131mab at multiple sites (dose: 12.69 μCi/100 μl per mouse); animals in the CD147-mab group received CD147-mab at a dose of 8.82 μM/100 μl per mouse. The animals were injected every other day for a total of five injections. The control mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the following formula was used to calculate tumor volume: "maximum diameter × minimum diameter 2 × 0.5." The experiment was terminated immediately when the mean tumor volume exceed 2,000 mm 3 in any group, as defined by the Animal Ethics Committee of the Second Military Medical University. At the end of observation, the mice were killed with over-doses anesthesia. The tumor specimens were harvested and weighed, and the tumor tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and immunohistochemically stained.

| Immunohistochemistry
The sections of paraffin-embedded SMMC-7721 hepatoma xenografts were subjected to streptavidin-peroxidase immunohistochemistry to detect the expression of E-cadherin and p-VEGFR-2. The antibodies used for staining were the WU et al.
same as those used for Western blotting. The percentages of positive cells in all sections were determined by counting the cells in five high-magnification fields.

| Statistical analysis
The cytological experiments were repeated three times independently, and a total of five animals were included in each group. The data are expressed as the mean ± SD. An analysis of variance (ANOVA) and a t test were used for the statistical analysis with the PASW Statistics 18 software. p value less than .05 was considered significant.