Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells

Abstract

Background: Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.

Methods: OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.

Results: The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H2O2 or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H(2)O(2) or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).

Conclusion: Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.

Figure 1

Efficient knockdown of OLFM4 by RNA interference in gastric cancer SGC-7901 and MKN45 cells. A. Expression of OLFM4 protein in various gastric cancer cell lines. Expression profile of OLFM4 protein was analyzed using western blotting with β-actin as an internal control. GES-1 cells were served as normal control cells. B. OLFM4 mRNA expression in OLFM4 knockdown cells. Relative expression of OLFM4 was detected by qRT-PCR. β-actin was used as an internal control. Fold changes in OLFM4 mRNA expression were determined using the 2^-ΔΔCt method. C. OLFM4 protein expression in OLFM4 knockdown cells. OLFM4 protein expression was detected by western blotting. β-actin protein was used as an internal control. Representatives of three experiments are shown. **P < 0.01 vs. HK control cells group (n = 3).

Figure 2

knockdown of OLFM4 inhibits the growth of SGC-7901 and MKN45 cells in vitro and in vivo. A. Cell growth curve. Cell proliferation in vitro was assessed by cell growth curve, as determined by counting the cell number (WST-1 assay) in the SGC-7901 cells (left panel) and MKN45 cells (right panel). The OD value (450 nm) was counted on the indicated days and presented as the mean cell numbers (n = 3). B. Anchorage-independent growth in soft agar. Representative images of three experiments were shown (upper panel). Data represent the mean number of colonies counted at 200 × magnification for 5 random fields (lower panel). C. The mean tumor volume after subcutaneous injection of nude mice with HK control or OLFM4 knockdown cells was measured at the indicated time points. D. Representative tumors images at 35 days after subcutaneous injection of indicated cells. E. Mean tumor weight (left panel) and inhibitory rate (right panel) in tumor xenografts. Data represent the mean tumor weight of xenografts (mean ± SD, n = 10). **P < 0.01 vs. HK control group.

Figure 3

qRT-PCR and IHC detection of OLFM4 in tumor xenografts. A. qRT-PCR for OLFM4 mRNA in tumor xenografts. Fold changes in OLFM4 mRNA were determined using the 2^-ΔΔCt method. β-actin gene was used as an internal control. B. H&E staining (upper panel) and IHC for OLFM4 protein (lower panel) in tumor xenografts. Representative images (200×) are shown. N, Necrosis. C. Quantification analysis of OLFM4 expression. The average value was measured from five randomly different fields under the microscope (400×) using the Image-Pro PLUS V6.0. Data were expressed as mean ± SD. **P < 0.01 vs. HK control group.

Figure 4

Assessment of cell cycle distribution, apoptotic cells and caspase-3/9 activation in OLFM4 knock down gastric cancer cells. A. Cell cycle analysis. Changes in cell cycle distribution of SGC-7901-HK, SGC-7901-siOLFM4 and MKN45-HK, MKN45-siOLFM4 cells were analyzed by FCM. Data represent the mean percentage of cell-cycle phase distribution (n = 3). B. Cell apoptosis analysis. Apoptosis was estimated with AnnexinV-PE/7-AAD staining by FCM. Data represent the mean apoptotic cell percentage (n = 3). C. Caspase-3 and -9 activations. Caspase-3, 9 activations between indicated cells are shown. **P < 0.01 vs. HK control group.

Figure 5

Response of OLFM4 knockdown SGC-7901 and MKN45 cells to apoptosis-inducing agents H₂O₂ or TNF α. OLFM4 knockdown and HK control cells were treated with H₂O₂ (A and B) or TNF α (C and D) as indicated doses for 12 h. Cell viability was measured by WST-1 assay (A-D left panels) and expressed in the mean OD value (450 nm) (n = 3). The percentage of the apoptotic cells was determined by FCM (A-D right panels) and expressed in the mean apoptotic percentage (n = 3). *P < 0.05,**P < 0.01 vs. HK control group.

Figure 6

Involvement of caspase-3 activity in H₂O₂ or TNF α-induced apoptosis in OLFM4 knockdown SGC-7901 and MKN45 cells. (A-B) Increased caspase-3 activity in H₂O₂ or TNF α-treated OLFM4 knockdown cells. OLFM4 knockdown and HK-control cells were treated with 10 μM H₂O₂ (A) or 10 ng/ml TNF α (B) for 12 h. Caspase-3 activity was measured using colorimetric assay and expressed in fold changes. **P < 0.01 versus PBS mock group (n = 3). (C-D) Reversed cell apoptosis by caspase inhibitor in OLFM4 knockdown cells. Cells were treated with H₂O₂ or TNF α alone with indicated doses as above mentioned or pretreated with 20 μM Z-VAD-fmk 2 h before H₂O₂ or TNF α treatment. The percentage of apoptotic cells was determined by FCM. Data represent the means ± SD from three independent experiments. ** p < 0.01 vs. H₂O₂ (C) or TNF α (D)-treated OLFM4 knockdown cells group.