All chemicals were obtained from Shenyang Sinopharm Group (Shenyang, China) unless otherwise stated. ERp57 inhibitor DNTB was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Human epithelial ovarian cancer cells SKOV3 were purchased from Beijing Shijitan Hospital (Beijing, China). Cells were cultured in RPMI-1640 medium (Hyclone; GE Heathcare Life Sciences, Logan, UT, USA) supplemented with L-glutamine and 10% fetal bovine serum (TBD, Tianjin, China) in a humidified incubator (5% CO₂ at 37°C). Cell lines grew in a monolayer and were passaged when cultures were 70–80% confluent.

The paclitaxel-resistant SKOV3 (SKOV3/tax) were prepared following a standard stepwise selection procedure. SKOV3 cells were cultured in RPMI-1640 medium containing paclitaxel at a concentration of 0.1 nM for 24 h. Cells that survived were selected for the next survival selection step using a higher paclitaxel concentration. This cell culture process was repeated for several steps with an increment of 0.5 nM at each step until all cells could survive at the paclitaxel concentration of 10 nM. The survived cells were able to maintain the paclitaxel-resistance phenotype in the absence of the selection pressure and were named SKOV3/tax (13).

SKOV3 and SKOV3/tax cells were seeded on 6-well plates at the cell density of 2×10⁶ cells/ml. Following 24 h of incubation, cells were washed with PBS 3 times and fixed with methanol for 30 min at room temperature. Subsequently, cells were stained with Giemsa dye (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min at room temperature. Cells were washed with PBS once and with deionized water three times. Once they were dried, SKOV3 and SKOV3/tax cells were examined under an optic microscope (at magnifications, ×100 and ×200, respectively).

SKOV3 and SKOV3/tax cells were seeded on 96-well plates at a cell density of 5×10³ cells/well or on 6-well plates at the cell density of 1×10⁶ cells/well, respectively. Following 24 h of incubation, cell culture medium (RPMI-1640) was aspirated and replaced with fresh culture medium containing different concentrations of paclitaxel (0.01, 0.1, 1.0, 10, 100 and 1,000 nM). Following 48 or 72 h incubation, cell viability was assessed using MTT assay. Each well was aspirated and incubated with 5 mg/ml MTT reagent (in 0.01 M PBS, pH 7.4). 4 h later, extraction buffer was added to each well to resolve the MTT crystals and the optic absorbance at 570 nm was measured using an Infinite M200 PRO multiplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell viability was calculated based on the optic absorbance. Untreated cells were used as a blank control (considered as 100% viable). IC₅₀ values were estimated from concentration-viability curves.

Lentiviruses carrying ERp57 expression vectors were obtained from GeneChem (Shanghai, China). ERp57 overexpression was conducted according to the company’s instructions. Briefly, cells (0.5×10⁵ cells/well) were seeded in a 12-well plate and treated with lentiviral particles to establish ERp57 overexpression [40 µl polybrene and 2.5 µl/well containing 1×10⁸ infectious units (IFU) of ERp57 overexpression virus] and scramble control (40 µl polybrene and 2.5 µl/well containing 1×10⁸ IFU scramble virus) groups. The blank group consisted of SKOV3 cells without lentiviruses transfection. Fresh medium (RPMI-1640) without polybrene was placed on each infected well following 24 h of incubation.

ERp57-siRNA was used to target the ERp57 gene (nucleotide sequence, 5′-GGGCAAGGACUUACUUAUU-3′). For comparison, a random nucleotide sequence of 5′-UUCUCCGAACGUGUCACGU-3′ was used as a negative control (NC-siRNA). Transfection of NC-siRNA and ERp57-siRNA was carried out, respectively, in a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. The blank group was SKOV3/tax cells without siRNA transfection. After 48 h of incubation, the transfected cells were collected for cell apoptosis analysis. Total mRNAs and proteins of these cells were isolated for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively.

SKOV3/tax cells were treated with ERp57-siRNA for 48 h, followed by 10 nM paclitaxel (the dosage commonly used for treatment of SKOV3 cells) for 48 h and 100 nM paclitaxel (the concentration near the IC₅₀ value of SKOV3/tax cells) for 48 h, respectively. Subsequently, cells were pelleted by centrifugation (500 x g for 5 min at 4°C) and washed with cold PBS. Cell pellets were resuspended in the radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing protease inhibitor (protease inhibitor cocktail; Roche Diagnostics, Basel, Switzerland), and lysed by bath sonication (4 times for 30 sec on/off). Lysates were centrifuged (15,000 x g for 30 min at 4°C) and the concentration of proteins were diluted to 3 µg/µl with 5X sample loading buffer (as determined by BCA assay). Samples were boiled at 100°C for 5 min. Following this, the extracted proteins (30 µg per lane) were separated by SDS-PAGE on Bis-Tris 4-12% gradient gels, transferred to polyvinylidene difluoride membranes and detected using specific antibodies. The membranes were blocked using 5% skimmed milk at room temperature for 2 h and incubated with appropriate primary antibodies at 4°C overnight. The following antibodies were used: Monoclonal anti-ERp57 (cat. no. sc80648), anti-TUBB3 (cat. no. sc-69966), anti-STAT3 (cat. no. sc8019) and anti-phospho(p)-STAT3 (Tyr705) antibodies (cat. no. sc81523) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); monoclonal anti-phospho-glycoprotein (P-gp) antibodies (cat. no. ab170904) from Abcam (Cambridge, UK); polyclonal anti-p53 (cat. no. 10442-1-AP), anti-nucleolin (cat. no. 10556-1-AP) and monoclonal anti-GAPDH antibodies (cat. no.60004-1-lg) from ProteinTech Group Inc. (Wuhan, China); polyclonal anti-proliferating cell nuclear antigen (PCNA; cat. no. WL01804), anti-B-cell lymphoma B-cell lymphoma-extra large (Bcl-xl; cat. no. WL01558), anti-matrix metalloproteinase (MMP)1 (cat. no. WL01201), anti-MMP2 (cat. no. WL01579a), anti-MMP9 (cat. no. WL01580) and anti-vimentin antibodies (cat. no. WL00742) from Wanlei Biotechnology Inc. (Shenyang, China); and anti-B-cell lymphoma-2 (Bcl-2; cat. no. D260117) and anti-Bcl-2-associated X protein (Bax; cat. no. D120073) from Sangon Biotechnology Inc. (Shanghai, China). The primary antibodies were diluted to 1:800. Thereafter, the membranes were incubated with the secondary antibodies (anti-rabbit cat. no. ZB-2301 or anti-mouse cat. no. ZB-2305; ZSGB-Bio, Beijing, China) for 1 h at room temperature. The secondary antibodies were diluted to 1:10,000. Finally, the immune reactive proteins were detected using an enhanced chemiluminescence kit (cat. no. WLA003a; Wanlei Life Science, Shenyang, China) and the enhanced chemiluminescence detection system (Tanon-5200; Tanon Science and Technology Co., Ltd., Shanghai, China).

SKOV3 and SKOV3/tax cells were incubated at the density of 2×10⁶ cells/well in 4 ml of RPMI-1640 supplemented with 10% fetal bovine serum for 48 h. Once cells were collected and washed with PBS, total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was obtained with RT using a PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed using SYBR Premix Ex Taq II (Takara Bio, Inc.) according to the manufacturer’s protocols. The following primers were used in the present study: ERp57, forward 5′-GAGCAATGATGGGCC TGTGA-3′ and reverse 5′-TGACGATATTTGGGTCTTTGC TGA-3′; and GAPDH, forward 5′-TGCACCACCAACTGCTT AGC-3′ and reverse 5′-GGCATGGACTGTGGTCATGAG-3′. The PCR process was performed using an ABI PRISM 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was performed as follows: 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec; and 1 cycle of 95°C for 15 sec, 60°C for 1 h and 95°C for 15 sec. RT-qPCR data were normalized using GADPH as an internal standard and analyzed using the 2^−ΔΔCq method (14).

SKOV3 and SKOV3/tax cells were transfected with ERp57-siRNA, respectively, for 48 h, then harvested and washed with PBS. Similarly, SKOV3 and SKOV3/tax cells were also transfected with NC-siRNA for comparison. A total of 500 cells/well were plated for 1-2 weeks at 37°C. Cells were fixed with 10% paraformaldehyde at room temperature for 30 min. Colonies were stained with 0.25% of crystal violet at room temperature for 30 min and counted using ImageJ (version 1.46r) software (National Institutes of Health, Bethesda, MD, USA).

The cell migration capability was examined using the Transwell assay (24-well insert; pore size, 8 µm; Corning Inc., NY, USA). Cells transfected with ERp57-siRNA or NC-siRNA for 48 h were harvested, suspended (5×10⁴ cells/well) in 100 µl serum-free RPMI-1640 medium, and loaded on the upper chamber. A total of 500 µl complete RPMI-1640 medium (containing 10% fetal serum albumin) was added in the lower chamber. Following 24 h of incubation, cells were fixed with 10% paraformaldehyde at room temperature for 30 min and free cells were removed carefully from the upper surface of the filter with a cotton swab. Migrated cells on the lower side of the filter were stained with 0.5% crystal violet for 1 h at room temperature and counted from five random fields under a optic microscope (magnification, ×200) using ImageJ (version 1.46r) software.

Cells were subjected to paclitaxel treatment and compared with those without this treatment. In the non-treatment group, SKOV3/tax cells were transfected with NC-siRNA, ERp57-siRNA and blank for 48 h, respectively, and then harvested directly without paclitaxel treatment. In the paclitaxel-treated group, SKOV3/tax cells were treated with NC-siRNA, ERp57-siRNA and blank for 48 h, respectively, followed by treatment with 10 nM paclitaxel for 24 h, and then harvested. For comparison, one extra sample was prepared: SKOV3/tax cells were treated with 1 mM DNTB for 48 h, followed by 10 nM paclitaxel treatment for 24 h, and then harvested. After the cells were harvested, all the samples were resuspended with 100 µl binding buffer (140 mmol/l NaCl, 5 mmol/l CaCl₂ and 10 mmol/l HEPES buffer) and washed three times with PBS (pH 7.4). A total of 5 µl Annexin V-fluorescein isothiocyanate and 10 µl propidium iodide (Beijing Biosea Biotechnology, Co., Ltd., Beijing, China) were added and the cell suspension was incubated at room temperature in dark for 10 min. Following centrifugation, the cell pellet was resus-pended in 200 µl binding buffer and analyzed using a FACSort flow cytometer (BD Biosciences, San Jose, CA, USA). The percentage of apoptotic and necrotic cells was determined using FCS express software (version 3.0; DeNovo Software, Los Angeles, CA, USA).

The protein-protein interaction (PPI) network was established using the online tool STRING (string-db.org) (15). The gene/protein-gene/protein interaction network was generated with GeneMANIA (genemania.org) (16). Biological process and gene co-occurrence analysis was performed using COREMINE (coremine/medical) (17). Pathway enrichment analysis was performed using DAVID (david.abcc.ncifcrf.gov) (18).

Data analysis was performed using SPSS software 17.0 (SPSS, Inc., Chicago, IL, USA). All experimental data were expressed as the mean ± standard deviation and statistically analyzed. The statistical significance of the results was assessed using one-way analysis of variance followed by Tukey’s post hoc multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference. All the measurements were repeated at least three times.

SKOV3/tax cells were characterized. The difference in cell morphology between SKOV3 and SKOV3/tax cells was clarified under an optic microscope and with Giemsa staining (Fig. 1A and B). Notably, more vesicles and vacuoles were observed in SKOV3/tax cells compared with SKOV3 cells.

Cell proliferation was examined using the clonogenic assay. Equal numbers of SKOV3 and SKOV3/tax cells were cultured for 2 weeks and colony numbers were subsequently counted. SKOV3/tax cells grew significantly slower compared with SKOV3 cells (P<0.05; Fig. 1C). Cell population doubling times were estimated to be ~22 h for SKOV3 cells and 36 h for SKOV3/tax cells, respectively (by a factor of 1.6; Fig. 1D). The IC₅₀ values of SKOV3 and SKOV3/tax cells to paclitaxel were determined based on the growth curves in Fig. 1E to be 3.24±0.03 and 101.06±0.99 nM, respectively. The drug-resistance index of SKOV3/tax to SKOV3 was calculated to be >30-fold. These data confirmed the paclitaxel-resistant characteristics of SKOV3/tax cells.

Furthermore, several proteins associated with apoptosis (Bcl-2, Bax, Bcl-xl and p53), migration (MMP1, MMP2, MMP9 and vimentin), cell proliferation (PCNA and nucleolin) and drug-resistance (P-gp and TUBB3) were compared in SKOV3 and SKOV3/tax cells using western blot analysis to assess paclitaxel-resistant behavior (Fig. 1F). The expression levels of MDR phenotype biomarkers P-gp and TUBB3 were increased in SKOV3/tax cells compared with SKOV3 cells. Protein PCNA and nucleolin expression levels were considered biomarkers for cell proliferation. Notably, PCNA expression levels were reduced in SKOV3/tax cells compared with SKOV3 cells. By contrast, nucleolin expression levels were similar in SKOV3/tax and SKOV3 cells. Weak p-STAT3 expression was indicated in SKOV3 cells; however, p-STAT3 protein expression levels were increased in SKOV3/tax cells. In SKOV3/tax cells, apoptosis-inhibiting proteins Bcl-2 and Bcl-xl were highly expressed, whereas the apoptosis promoting protein Bax was expressed in lower levels. The SKOV3 cell is a p53-mutant cell line that does not express p53 (19); however, p53 was highly expressed in SKOV3/tax cells. MMP1, MMP2 and MMP9 proteins, which are associated with metastasis (20), were expressed in lower levels in SKOV3/tax cells compared with SKOV3 cells, suggesting a lower invading ability of SKOV3/tax cells. Furthermore, an epithelial-mesenchymal transition (EMT) protein marker, vimentin, was expressed to almost at the same level in SKOV3 and SKOV3/tax cells.

ERp57 mRNA and protein expression levels were compared in SKOV3 and SKOV3/tax cells using RT-qPCR and western blot analysis. As indicated in Fig. 2A, mRNA expression levels of ERp57 in SKOV3/tax cells were >2-fold higher than that of SKOV3 cells (P<0.05). In addition, western blot analysis results also revealed that the expression level of ERp57 protein in SKOV3/tax cells was upregulated (Fig. 2B). These data were consistent with a previous report (21), which indicated that ERp57 is strongly associated with the paclitaxel-resistant ovarian cancer cells SKOV3/tax.

To examine the effect of ERp57 overexpression on the paclitaxel sensitivity of SKOV3 cells, ERp57 was overexpressed in SKOV3 cells (Fig. 2C and D). As indicated in Fig. 2E, the IC₅₀ values of untransfected SKOV3 cells and scramble control were 3.33±1.18 and 3.897±1.39 nM. However, the IC₅₀ value of ERp57-overexpressed SKOV3 cells was increased to 90.59±1.13 nM. These data indicated that ERp57 overexpression could increase drug resistance of SKOV3 cells.

The effect of ERp57-siRNA silencing on the sensitivity of SKOV3/tax cells to paclitaxel was examined. As indicated in Fig. 3A, ERp57-siRNA silencing significantly inhibited the expression levels of ERp57 mRNA (P<0.05). Similarly, western blot analysis results demonstrated that ERp57 protein expression levels were also inhibited (Fig. 3B). As indicated in Fig. 3C, ERp57-siRNA silencing significantly downregulated the viability of SKOV3/tax cells at 24, 48 and 72 h (P<0.05), and the cell viability percentages were determined to be 87, 76 and 71% at the respective time-points. By contrast, NC-siRNA did not significantly alter the cell viability of SKOV3/tax cells.

Following ERp57-siRNA silencing, the numbers and the size of the colonies of SKOV3/tax cells were significantly reduced compared with the control (P<0.05; Fig. 3D, top left vs. lower left). Furthermore, after ERp57-siRNA silencing, a total of 10 nM paclitaxel decreased the colony formation and number of SKOV3/tax colonies by ~60% when compared with the control with no paclitaxel treatment (P<0.05; Fig. 3D, top left vs. lower right). The Transwell assay was used to examine the migration ability of SKOV3 and SKOV3/tax cells. As indicated in Fig. 3E, the migration ability of SKOV3 cells was significantly increased compared with SKOV3/tax cells (P<0.05). ERp57-siRNA silencing could significantly reduce the cell migration of SKOV3 and SKOV3/tax cells (P<0.05).

The effects of ERp57-siRNA silencing on SKOV3/tax cell apoptosis were examined using Annexin V and PI double staining. The apoptosis rate of the ERp57-silenced cells was 17.16%, which was significantly higher than the apoptosis rate in the control (3.61%) and NC-siRNA cells (5.95%; P<0.05; Fig. 3F). Furthermore, in SKOV3/tax cells treated with paclitaxel, the apoptosis rates of the control and NC-siRNA cells were 5.67 and 7.63%, whereas the apoptosis rate of ERp57-siRNA cells was significantly increased to ~38% (mean of the three measurements; P<0.05). For comparison, the effect of the ERp57 inhibitor DNTB was examined, and the results indicated that the apoptosis rate of SKOV3/tax cells was 29.7%.

Cell viability of SKOV3/tax cells under different conditions was assessed using the MTT assay. Fig. 3G and H revealed the growth curves and the calculated IC₅₀ values of the different samples. The IC₅₀ values of SKOV3 and SKOV3/tax cells were ~3.33±1.18 and 102.8±1.17 nM, respectively. The IC₅₀ values of SKOV3/tax cells were reduced to 3.5±1.15 and 0.44±1.3 nM following treatment with ERp57-siRNA or DTNB, respectively (Fig. 3H). These data indicated that ERp57-siRNA silencing could restore the sensitivity of SKOV3/tax cells to paclitaxel.

The expression levels of the selected protein biomarkers were assessed in cells treated with ERp57-siRNA and paclitaxel. As indicated in Fig. 4, when SKOV3/tax cells were treated with ERp57-siRNA alone, protein P-gp expression was not significantly impacted (columns 1 and 2). However, once paclitaxel was applied, P-gp expression levels were decreased in a concentration-dependent manner (columns 4 and 6), which suggested that pre-treatment of ERp57-siRNA effectively inhibited P-gp protein expression and increased paclitaxel efficacy. The MDR biomarker TUBB3 also exhibited a similar trend. The expression level of PCNA was reduced and that of nucleolin was marginally altered in response to paclitaxel (columns 1, 3 and 5). When ERp57-siRNA was applied, PCNA and nucleolin expression levels were further reduced in the presence of paclitaxel in a concentration dependent-manner (columns 2, 4 and 6). Furthermore, western blot analysis revealed that co-treatment of ERp57-siRNA and paclitaxel reduced the p-STAT3 expression levels in a dose-dependent manner (columns 2, 4 and 6). Additionally, Bcl-2 and Bcl-xl expression levels were downregulated when ERp57-siRNA and 100 nM paclitaxel were used (columns 4 and 6), whereas the apoptosis promoting protein Bax was highly expressed.

Paclitaxel treatment alone did not impact p53 expression, even at 100 nM, due to the drug-resistant phenotype of SKOV3/tax cells (columns 1, 3 and 5). In addition, p53 expression was not affected by ERp57-siRNA alone (column 2). However, when ERp57-siRNA was combined with 10 nM paclitaxel, the p53 band was markedly reduced, and its isoform p53/p47 became merged (column 4). When ERp57-siRNA was combined with 100 nM paclitaxel, the p53 band was further reduced and the p53/p47 band was highly expressed (column 6). Notably, MMP1 and MMP2 protein expression levels were reduced when paclitaxel and/or ERp57-siRNA were applied. However, MMP9 protein expression was unchanged with ERp57-siRNA treatment alone (columns 1 and 2) and a minor reduction in expression was observed with treatment of paclitaxel alone (10 or 100 nM) (columns 1, 3 and 5). When ERp57-siRNA was combined with paclitaxel, MMP9 expression was markedly reduced with 10 nM paclitaxel and completely eliminated with 100 nM paclitaxel (columns 4 and 6). No notable changes were indicated with regards to the expression of the EMT marker vimentin with paclitaxel and/or ERp57-siRNA treatment; however, a reduction was observed with 100 nM paclitaxel and ERp57-siRNA treatment (column 6).

In order to understand the underlying mechanisms of ERp57 in drug-resistant ovarian cancer, STRING was used to construct a PPI network between ERp57 (PDIA3) and STAT3, P-gp (ABCB1), TUBB3, p53 (TP53), Bcl-2, Bax, Bcl-xl (BCL2L1), vimentin, PCNA, nucleolin, MMP1, MMP2 and MMP9. As indicated in Fig. 5A, ERp57 (referred as PDIA3 in STRING) has direct interactions with STAT3 and vimentin. ERp57 can have the indirect interactions with p53, Bcl-2, Bax, Bcl-xl, PCNA, MMP1, MMP2 and MMP9 proteins via STAT3, and indirect interactions with p53, Bcl-2, PCNA, MMP1, MMP2 and MMP9 via vimentin. Furthermore, ABCB1 can be regulated indirectly by ERp57 via p53 and Bax. GeneMANIA was used to construct a gene/protein-gene/protein interaction network between ERp57 (PDIA3) and various other components, including calnexin (CANX), calreticulin (CALR), transporter 1 (TAP1), transporter 2 (TAP2), TAP binding protein (TAPBP), protein disulfide isomerase family A member 4 (PDIA4), protein disulfide isomerase family A member 6 (PDIA6) and heat shock protein 90 kDa beta family member 1 (HSP90B1). As indicated in Fig. 5B, ERp57 had strong interactions with 20 proteins/genes. Co-expressed ERp57 had strong physical interactions and shared a pathway with CANX and CALR. Furthermore, CANX was associated with ATP transporter-associated proteins/genes (ABCB1, ABCC1 and ABCC3) and apoptosis-associated proteins/genes [Bcl-2, poly (ADP-ribose) polymerase 1 (PARP1), caspase-3 (CASP3), p53]. ABCB1, ABCC1, ABCC3, Bcl-2, PARP1 and CASP3 expression changes have been associated with drug resistance (22-26). The findings suggested that inference of ERp57 must be associated with drug resistance in ovarian cancer.

The associated biological processes of ERp57 with ovarian cancer and drug resistance were assessed using COREMINE. The results indicated that a total of 25 biological processes were associated with ERp57, ovarian cancer and drug resistance (P<0.01), including cell death (autophagy, cell killing, cell death and the apoptotic process), cell growth (cell proliferation, cell growth, growth, cell division and angiogenesis), the cell cycle (cell cycle, cell cycle arrest and S phase), gene expression regulation (RNA interference, gene silencing, gene expression, DNA methylation, methylation, phosphorylation, mismatch repair, DNA repair and signal transduction), platelets (platelet activation and platelet aggregation), cell migration, metabolism and protein folding-associated processes (Fig. 5C). These results suggested that either ERp57 may be a regulator of these processes or these processes contribute the development of drug resistance phenotype of ovarian cancer.

Fig. 5D indicated that a total of 11 molecular functions, including folic acid binding, protein binding, catalytic activity, motor activity, protein kinase C activity, urokinase plasminogen activator receptor activity, phospholipase C activity, DNA binding, epidermal growth factor binding, ATPase activity and active transmembrane transporter activity were predicted to be associated with ERp57, ovarian cancer and drug resistance (P<0.05). Furthermore, pathway enrichment analysis was performed using DAVID, which revealed a total of 31 genes (finding by Coremine Medical) co-occurred with ERp57 and drug resistance in ovarian cancer (Table I). Many familiar genes were indicated, including BCL2, PARP1, STAT3, CASP3, CASP9, vimentin, phosphatase and tensin homolog, Erb-B2 receptor tyrosine kinase 2, heat shock protein 90 β1 and epidermal growth factor receptor. As indicated in Table I, ERp57 may be associated with drug resistance in ovarian cancer through its regulation on the four pathways: The cell death-associated pathways (apoptosis and p53 signaling pathway), the focal adhesion signaling pathway and the cancer related pathway.