Types [24,25]. Targeting STAT3 may be an effective approach to addressing tumor progression. This STAT3 effect is mediated through regulation of various STAT3 target genes, including apoptosis inhibitors and cell cycle regulators, such as Bcl-2, Mcl-1, survivin, p53, c-Myc, cyclin D1 [26?1], and vascular endothelial growth factor (VEGF) [32]. However, the role of STAT3 in AFPGC is poorly understood. Here, we examined the effect of As2O3 on proliferation and AFP and STAT3 expression in AFPGC FU97 cells. We evaluated downstream events of STAT3 signaling, Bcl-2 and Bax expression, to explore the possible mechanisms underlying this phenomenon. Furthermore, we evaluated the expression of AFP and STAT3 by immunohistochemistry in human gastric cancer samples. Down-Novel Therapy for AFP-Producing Gastric CancersTable 1. AKT inhibitor 2 chemical information primer sequences for real-time PCR.Gene GAPDH Forward Reverse AFP Forward Reverse STAT3 Forward Reverse Bcl-2 Forward Reverse BAX Forward ReversePrimer sequence GCACCGTCAAGGCTGAGAAC TGGTGGT GAAGACGCCAGT AAATGCGTTTCTCGTTGC TTTCATCCACCACCAAGC ATCACGCCTTCTACAGACTGC CATCCTGGAGATTCTCTACCACT ATGTGTGTGGAGAGCGTCAACC GAGCAGAGTCTTCAGAGACAGCC ATTGCCGCCGTGGACACAGA ATGGTGAGTGAGGCGGTGAG0.5 mL of 10 Triton X-100). Lysed cells were held at 4uC for 10 min, and supernatants were incubated with 2 mL RNase A (10 mg/mL in Tris DTA buffer) at 50uC for 30 min, then with 2 mL proteinase K (10 mg/mL in distilled water) for 45 min at 50uC. The solution was mixed with 5 M NaCl (20 mL) and 2propanol (120 mL), incubated at 20uC for 24 h, then centrifuged at 15000 rpm for 20 min. The precipitated DNA was dissolved in Tris DTA (5 mL) buffer and underwent electrophoresis with 2 agarose gel and Tris cetate DTA buffer at 50 V. The DNA fragmentation pattern was visualized with use of a UV transilluminator.Hoechst 33258 Staining Analysis of Cell ApoptosisCells grown on the glass cover-slips were fixed with 4 paraformaldehyde/PBS for 30 min, washed for 15 min in 0.1 Triton X-100/PBS and incubated in dark with Hoechst 33258 (10 mg/ml) for 15 min. After the cover-slips were washed in PBS, positive nuclei were counted. Normal nuclei and apoptotic nuclei (condensed or fragmented chromatin) were easily distinguished.doi:10.1371/journal.pone.0054774.tregulation AFP and STAT3 expression contributed to As2O3induced apoptosis and inhibition of proliferation in AFPGC.Quantitative Real-time PCRCells were cultured with As2O3 (5 mmol/L) for 72 h. Total RNA was extracted with use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by spectrophotometry. Firststrand cDNA was prepared with use of random primers following the kit instructions (Takara, Japan). Real-time quantitative PCR of AFP, STAT3 and its downstream genes involved the 7300 Realtime PCR System (ABI, USA) with Takara SYBR Premix Ex Taq reagents (Takara, Japan). Primers were designed and MedChemExpress JWH133 validated by Invitrogen. The primer information is in Table 1. PCR reactions were carried out in triplicate in a 20-mL volume for 2 min at 94uC for initial denaturing, followed by 30 cycles at 94uC for 30 s and at 60uC for 45 s. A housekeeping control gene GAPDH was used as an internal control. Each primer set was first tested to determine optimal concentrations, and products were run on a 1 agarose gel to confirm the appropriate size. Subsequently, ABI dissociation curve software was used to control for multiple species in each PCR amplification. cDNA from FU97 cells without As2O3 treatment was used to construct a.Types [24,25]. Targeting STAT3 may be an effective approach to addressing tumor progression. This STAT3 effect is mediated through regulation of various STAT3 target genes, including apoptosis inhibitors and cell cycle regulators, such as Bcl-2, Mcl-1, survivin, p53, c-Myc, cyclin D1 [26?1], and vascular endothelial growth factor (VEGF) [32]. However, the role of STAT3 in AFPGC is poorly understood. Here, we examined the effect of As2O3 on proliferation and AFP and STAT3 expression in AFPGC FU97 cells. We evaluated downstream events of STAT3 signaling, Bcl-2 and Bax expression, to explore the possible mechanisms underlying this phenomenon. Furthermore, we evaluated the expression of AFP and STAT3 by immunohistochemistry in human gastric cancer samples. Down-Novel Therapy for AFP-Producing Gastric CancersTable 1. Primer sequences for real-time PCR.Gene GAPDH Forward Reverse AFP Forward Reverse STAT3 Forward Reverse Bcl-2 Forward Reverse BAX Forward ReversePrimer sequence GCACCGTCAAGGCTGAGAAC TGGTGGT GAAGACGCCAGT AAATGCGTTTCTCGTTGC TTTCATCCACCACCAAGC ATCACGCCTTCTACAGACTGC CATCCTGGAGATTCTCTACCACT ATGTGTGTGGAGAGCGTCAACC GAGCAGAGTCTTCAGAGACAGCC ATTGCCGCCGTGGACACAGA ATGGTGAGTGAGGCGGTGAG0.5 mL of 10 Triton X-100). Lysed cells were held at 4uC for 10 min, and supernatants were incubated with 2 mL RNase A (10 mg/mL in Tris DTA buffer) at 50uC for 30 min, then with 2 mL proteinase K (10 mg/mL in distilled water) for 45 min at 50uC. The solution was mixed with 5 M NaCl (20 mL) and 2propanol (120 mL), incubated at 20uC for 24 h, then centrifuged at 15000 rpm for 20 min. The precipitated DNA was dissolved in Tris DTA (5 mL) buffer and underwent electrophoresis with 2 agarose gel and Tris cetate DTA buffer at 50 V. The DNA fragmentation pattern was visualized with use of a UV transilluminator.Hoechst 33258 Staining Analysis of Cell ApoptosisCells grown on the glass cover-slips were fixed with 4 paraformaldehyde/PBS for 30 min, washed for 15 min in 0.1 Triton X-100/PBS and incubated in dark with Hoechst 33258 (10 mg/ml) for 15 min. After the cover-slips were washed in PBS, positive nuclei were counted. Normal nuclei and apoptotic nuclei (condensed or fragmented chromatin) were easily distinguished.doi:10.1371/journal.pone.0054774.tregulation AFP and STAT3 expression contributed to As2O3induced apoptosis and inhibition of proliferation in AFPGC.Quantitative Real-time PCRCells were cultured with As2O3 (5 mmol/L) for 72 h. Total RNA was extracted with use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by spectrophotometry. Firststrand cDNA was prepared with use of random primers following the kit instructions (Takara, Japan). Real-time quantitative PCR of AFP, STAT3 and its downstream genes involved the 7300 Realtime PCR System (ABI, USA) with Takara SYBR Premix Ex Taq reagents (Takara, Japan). Primers were designed and validated by Invitrogen. The primer information is in Table 1. PCR reactions were carried out in triplicate in a 20-mL volume for 2 min at 94uC for initial denaturing, followed by 30 cycles at 94uC for 30 s and at 60uC for 45 s. A housekeeping control gene GAPDH was used as an internal control. Each primer set was first tested to determine optimal concentrations, and products were run on a 1 agarose gel to confirm the appropriate size. Subsequently, ABI dissociation curve software was used to control for multiple species in each PCR amplification. cDNA from FU97 cells without As2O3 treatment was used to construct a.
