Expression and Underlying Roles of IGFBP-3 in Paclitaxel-Treated Gastric Cancer Sgc-7901 Cells

Surgery remains the first option for the treatment of gastric cancer. However, chemotherapy is nevertheless indispensable, especially for advanced gastric cancer. Paclitaxel, which arrests tumor cells in G2/M phase and induces apoptosis, is one of the most effective drugs for malignant tumor (Vallon et al., 2012; Hossein G et al., 2013). Several signaling pathways were involved in the anti-tumor effect of paclitaxel, including cyclins and cyclin-dependent kinases (CDKs). Although as first line chemotherapeutic agent for gastric cancer, paclitaxel reamains restricted for its drug resistance and side effects during clinical application. A previous study showed that RhoA signaling pathway was associated with paclitaxel resistance by regulating Cdk2 and PCNA expression (Kang et al., 2005). Furthermore, the tumor cell sensitivity had an inverse relationship with Ki-67 expression (Wang et al., 2014). Therefore, it is warranted to further investigate the underlying mechanisms of paclitaxel resistance in gastric cancer cells. Recent studies showed that insulin-like growth factor binding proteins (IGFBPs), which specifically bind to insulin-like growth factor (IGF), were closely related to cell cycle and apoptosis. Among the six members of IGFBPs, IGFBP-2, -3 and -5 stood at most studied. In both breast cancer and colon cancer, the expression of IGFBP-2 was elevated while the expression of IGFBP-3 was down-regulated (Soubry et al., 2012; Sunderic et al., 2012; Foulstone et al., 2013; Duggan et al., 2013;


Introduction
Surgery remains the first option for the treatment of gastric cancer.However, chemotherapy is nevertheless indispensable, especially for advanced gastric cancer.Paclitaxel, which arrests tumor cells in G2/M phase and induces apoptosis, is one of the most effective drugs for malignant tumor (Vallon et al., 2012;Hossein G et al., 2013).Several signaling pathways were involved in the anti-tumor effect of paclitaxel, including cyclins and cyclin-dependent kinases (CDKs).Although as first line chemotherapeutic agent for gastric cancer, paclitaxel reamains restricted for its drug resistance and side effects during clinical application.A previous study showed that RhoA signaling pathway was associated with paclitaxel resistance by regulating Cdk2 and PCNA expression (Kang et al., 2005).Furthermore, the tumor cell sensitivity had an inverse relationship with Ki-67 expression (Wang et al., 2014).Therefore, it is warranted to further investigate the underlying mechanisms of paclitaxel resistance in gastric cancer cells.
Recent studies showed that insulin-like growth factor binding proteins (IGFBPs), which specifically bind to insulin-like growth factor (IGF), were closely related to cell cycle and apoptosis.Among the six members of IGFBPs, IGFBP-2, -3 and -5 stood at most studied.In both breast cancer and colon cancer, the expression of IGFBP-2 was elevated while the expression of IGFBP-3 was down-regulated (Soubry et al., 2012;Sunderic et al., 2012;Foulstone et al., 2013;Duggan et al., 2013; RESEARCH ARTICLE

Expression and Underlying Roles of IGFBP-3 in Paclitaxel-Treated Gastric Cancer Sgc-7901 Cells
Gang Huang 1,2 , Zhong-Feng Dang 3 , Ya-Mei Dang 1 , Wei Cai 1 , Yuan Li 1 , Yi-Rong Chen 1,4 , Xiao-Dong Xie 2 * Dang et al., 2014) Moreover, the overexpression of IGFBP-5 indicated a poor prognosis for bladder cancer patients (Liang et al., 2013).Since IGFBPs were closely related to cell proliferation and apoptosis, their roles on chemoresistance have also been investigated.In prostate cancer cells, IGFBP2 played a pivotal role in the hyperglycemia-induced chemoresistance (Biernacka et al., 2013).While in non-small cell lung cancer, the deficiency of IGFBP-3 mediated the chemoresistance to cisplatin (Cortes-Sempere et al., 2013).Bcl-2, one of important anti-apoptotic factors, overexpressed in various tumor cells was also considered to be possibly related to chemoresistance (Eichhorn et al., 2014).Suppression of Bcl-2 expression by HDAC inhibitor could attenuate the chemoresistance to concanamycin A1 in human oral squamous cells (Kiyoshima et al., 2013).Herein, we aimed to investigate whether IGFBPs were involved in the chemoresistance of paclitaxel in SGC-7901 cells, and its underlying mechanisms

Gene-silencing by small interfering RNA (siRNA)
Anti-sense siRNA against human IGFBP-3 was designed and synthesized by GenePharma Co., Ltd.(Shanghai, China).The transfection of siRNA was performed using EntransterTM-R (Engreen Biosystem, Beijing, China) at a final concentration of 100 nM according to the manufacturer's instruction.Forty-eight hours after transfection, the gene-silencing effect was determined in both mRNA and protein level.

Cell viability assay
The viability of SGC-7901 cells was detected by MTT assay.Normal and IGFBP-3-silenced SGC-7901 cells seeded in 96-well plates were incubated with paclitaxel (10 nM) for 48 h.Four hours before the end of the incubation, aliquots (15 μL) of MTT were added into each well.The medium were aspirated and 100 μL DMSO were added to dissolve the formazan crystals.The absorbance at 490 nm presents cell viability.

Cell cycle analysis
After indicated treatment for 48 h, cells were harvested and washed by ice cold PBS.The cells were further fixed in pre-cold 70% ethanol for 12 h.After that, 500 μL propidium iodide (PI, 50 μg/mL) was added and incubated away from light for 30 min.The cell cycle was determined by flow cytometry immediately.

Cell apoptosis analysis
Cell apoptosis was analyzed by using Annexin V-FITC/PI method.Cells were treated as indicated for 48 h and were further stained with Annexin V-FITC and PI according to the manufacturer's protocol (Sigma, St. Louis, MO).

Statistical analysis
All data were expressed as mean±standard deviation (SD).Differences between control and experimental groups were analyzed by one-way analysis of variance with Dunnett's test post hoc, and p<0.05 was considered as statistically significant.All calculations were performed using SPSS 18.0 statistical software (SPSS, Chicago, IL, USA).

IGFBPs expression after paclitaxel treatment
After paclitaxel (10 nM) treatment, the expression of IGFBP-2 and -5 in mRNA level were similar to control group (0.94±0.11 and 1.08±0.15fold, respectively, both p>0.05).However, the mRNA level of IGFBP-3 was significantly elevated (1.82±0.10fold compared to control group, p<0.05).Results from Western blot analysis were consistent with real time PCR assay (Figure 1).

Effect of IGFBP-3 silencing on cell cycle arrest of paclitaxel-treated SGC-7901 cells
As shown in (Figure 3) and (Table 1), paclitaxel (10 nM) treatment could arrest SGC-7901 cells to G2/M phase (from 7.6±2.2% to 19.2±4.2%before and after paclitaxel incubation).Single silencing of IGFBP-3 did not affect cell cycle without paclitaxel treatment.However, there was a decline of cells in G2/M phase after IGFBP-3 silencing when cells are treated with paclitaxel (12.5±3.6%,p<0.05).

Effect of IGFBP-3 silencing on apoptosis of paclitaxeltreated SGC-7901 cells
As shown in (Figure 4) and (Table 1), paclitaxel incubation could significantly induce apoptosis in SGC-

Bcl-2 expression after paclitaxel and/or IGFBP-3 silencing
Compared to control group, the mRNA level of Bcl-2 decreased to 0.65±0.06fold after paclitaxel (10 nM) incubation.While the expression of Bcl-2 increased to a level of 0.86±0.07fold in IGFBP-3 knockdown SGC-7901 cells after paclitaxel treatment.Results from Western blot analysis of Bcl-2 expression in protein level exhibited a similar trend (Figure 5).

Discussion
Most of the currently available chemotherapeutic agents exhibit their anti-tumor effect by inducing cell cycle arrest and apoptosis.IGFBPs, closely related to cell cycle regulation and apoptosis, have been reported to modulate cancer cell chemoresistance to various anti-tumor agents.Biernacka et al demonstrated that the overexpression of IGFBP-2 could attenuate docetaxel-induced prostate cancer cell apoptosis (Biernacka et al., 2013).Further, IGFBP-3 expression could enhance the anti-tumorigenic effect of irinotecan possibly via inhibiting of Akt phosphorylation (Alami et al., 2008).Moreover, a study from Zeng and colleagues showed elevated IGFBP-3 was found in 5-Aza-2'-deoxycytidine (AZA) treated in breast cancer cells.Inhibition of IGFBP-3 could palliate the anticancer effect of AZA (Zeng et al., 2013).
In our current study, paclitaxel (10nM) treatment could significantly up-regulate IGFBP-3 expression in SGC-7901 gastric cancer cells, while having no effect on either IGFBP-2 or IGFBP-5 expression.Similar to a previous study (Zeng et al., 2013), silencing of IGFBP-3 by RNA interference could attenuate the effect of paclitaxel on cell viability, cell cycle arrest and apoptosis.Our result indicated that up-regulation of IGFBP-3 might enhance gastric cancer cell sensitivity to paclitaxel.More investigations on various chemotherapeutic agents and their relationship to IGFBP-3 in different tumor cell lines are warranted.
However, there are nevertheless some disputes concerning IGFBP-3 expression and chemoresistance.A study from Holdaway's group suggested overexpression of IGFBP-3 would lead to chemoresistance in breast cancer cells (Holdaway et al., 2003).While in our study, we tended to interpret IGFBP-3 overexpression as an adjuvant to paclitaxel in inducing cell apoptosis.The apoptotic effect of IGFBP-3 has been extensively studied, which could be categories into IGF-dependent and IGF-independent.For the former one, IGFBP-3 could specifically bind to IGF-1, resulting reduced free IGF-1 level, and thus induce cell apoptosis (Kalluri et al., 2011).On the other hand, IGFBP-3 itself could directly or indirectly induce apoptosis via NF-κB, p38 MAPK and Akt signaling pathway (Peters et al., 2006;Williams et al., 2007;Koyama et al., 2010).Further, Bcl-2 is a classical anti-apoptotic factor, which exerts its anti-apoptotic effect via NF-κB signaling pathways (Adams, 1998;Zhang et al., 2013).Our observation of Bcl-2 level also indicated IGFBP-3 could induce cell apoptosis via down-regulating Bcl-2 expression.The underlying mechanisms should be elucidated in future studies.
In conclusion, IGFBP-3 expression is closely related to chemoresistance of paclitaxel in SGC-7901gastric cancer cells.Clinical monitoring of IGFBP-3 level in patient might be beneficial when applying paclitaxel.

Figure 1 .
Figure 1.IGFBPs mRNA Expression After Paclitaxel Treatment.Relative fold change was determined by 2 -ΔΔCt method with GAPDH as reference gene.*p<0.05,compared with control group

Table 1 . Values of Cell Cycle Analysis and ApoptoticProportion by Flow Cytometry
of G1 phase (%) of G2/M phase (%) proportion (%) *compared with Control p<0.05.# compared with the group only treated with Paclitaxel p<0.05.