Thology. Clinical and histopathological data, including sex, histology, tumor site, stage, histological grade and lymph node metastasis were all collected. Clinical characteristics of the purchase NHS-Biotin patients were summarized in Table 1.Patient SamplesAll specimens and relevant clinical data were obtained from the department of oncology and general surgery, Drum Tower Hospital Affiliated to Medical School of Nanjing University during the period from February 2012 to November 2012. TheHDRAHDRA procedures were performed as described previously [15]. The procedure of histoculture drug response assay was shown in Figure 3. Briefly, the fresh tumor tissues were washed and minced into small pieces to approximately 10 mg, which wereFigure 2. Flow chart showing patient disposition and experiments performed. doi:10.1371/journal.pone.0065164.gSynergistic Anticancer Effects of PPI and EVOTable 1. Patient characteristics.CharacteristicPatients N = 40, N( )Inhibition rate of PPI Mean ?StdInhibition rate of EVO Mean ?StdAge .61 #61 Sex Male Female Histology Adenocarcinoma Mucinous Signet ring cell Tumor Site Distal MedChemExpress Bexagliflozin stomach Proximal stomach Whole stomach Stage I,II III, IV Histological grade 2 3 Mixed 2? Lymph node metastasis No Yes doi:10.1371/journal.pone.0065164.t001 3 (7.5 ) 37 (92.5 ) 27.11 617.37 20.12 614.12 22.97 624.06 20.99 612.76 16985061 8 (20.0 ) 27 (67.5 ) 5 (12.5 ) 18.31 617.22 18.48 69.47 21.71 615.20 18.44 614.23 22.23 69.90 21.31 624.54 11 (27.5 ) 29 (72.5 ) 21.27 613.51 20.40 614.75 21.28 613.81 21.08 613.53 11 (27.5 ) 14 (35 ) 15 (37.5 ) 17.42 67.68 16.13 616.45 27.21 614.05 16.78 611.06 20.26 614.17 25.15 613.99 28 (70 ) 6 (15 ) 6 (15 ) 22.01 615.65 18.28 610.87 16.62 610.31 21.38 613.52 23.29 614.75 17.83 613.56 32 (80 ) 8 (20 ) 21.91 615.27 15.56 67.84 21.99 613.64 17.70 612.83 20 (50 ) 20 (50 ) 22.80 616.35 18.25 611.46 23.29 613.90 18.75 612.84then placed on prepared collagen surfaces in 24-well microplates. There were 8 parallel culture wells for each drug sensitivity testing and 8 parallel culture wells for control. After incubation for 7 days at 37uC (in a humidified atmosphere containing 95 air 25 CO2) in the presence of drugs dissolved with RPMI 1640 medium containing 20 fetal calf serum, 100 ml type I collagenase (0.1 mg/ml, Sigma) and MTT (5 mg/ml, Sigma) were added to each culture well and incubated for another 16 hours. Concentration of Pt, 5-FU, CPT-11, PPI and EVO was 20 mg/ml, 300 mg/ml, 20 mg/ml, 200 mg/ml and 200 mg/ml respectively, according to its peak plasma concentration (ppc) in patients [16]. Pt, 5-FU and CPT-11 were obtained from Jiangsu Hengrui Medicine Company (Jiangsu, China). PPI and EVO were supplied as powder with purity of .98 by the School of Pharmacy in China Pharmaceutical University. After extraction with dimethyl sulfoxide (DMSO, Sigma), absorbance of the solution in each well was read at 540 nm. Absorbance per gram of cultured tumor tissue was calculated from the mean absorbance of tissue from 8 parallel culture wells, and the tumor-tissue weight was determined before culture. The inhibition rate was calculated by using the following formula:T is the mean absorbance of treated tumor /Weight. C is the mean absorbance of control tumor / Weight.Synergy AnalysisMedian effect analysis using the combination index (CI) method of Chou and Talalay [17] was employed to determine the nature of the interaction observed between PPI or EVO and chemotherapeuti.Thology. Clinical and histopathological data, including sex, histology, tumor site, stage, histological grade and lymph node metastasis were all collected. Clinical characteristics of the patients were summarized in Table 1.Patient SamplesAll specimens and relevant clinical data were obtained from the department of oncology and general surgery, Drum Tower Hospital Affiliated to Medical School of Nanjing University during the period from February 2012 to November 2012. TheHDRAHDRA procedures were performed as described previously [15]. The procedure of histoculture drug response assay was shown in Figure 3. Briefly, the fresh tumor tissues were washed and minced into small pieces to approximately 10 mg, which wereFigure 2. Flow chart showing patient disposition and experiments performed. doi:10.1371/journal.pone.0065164.gSynergistic Anticancer Effects of PPI and EVOTable 1. Patient characteristics.CharacteristicPatients N = 40, N( )Inhibition rate of PPI Mean ?StdInhibition rate of EVO Mean ?StdAge .61 #61 Sex Male Female Histology Adenocarcinoma Mucinous Signet ring cell Tumor Site Distal stomach Proximal stomach Whole stomach Stage I,II III, IV Histological grade 2 3 Mixed 2? Lymph node metastasis No Yes doi:10.1371/journal.pone.0065164.t001 3 (7.5 ) 37 (92.5 ) 27.11 617.37 20.12 614.12 22.97 624.06 20.99 612.76 16985061 8 (20.0 ) 27 (67.5 ) 5 (12.5 ) 18.31 617.22 18.48 69.47 21.71 615.20 18.44 614.23 22.23 69.90 21.31 624.54 11 (27.5 ) 29 (72.5 ) 21.27 613.51 20.40 614.75 21.28 613.81 21.08 613.53 11 (27.5 ) 14 (35 ) 15 (37.5 ) 17.42 67.68 16.13 616.45 27.21 614.05 16.78 611.06 20.26 614.17 25.15 613.99 28 (70 ) 6 (15 ) 6 (15 ) 22.01 615.65 18.28 610.87 16.62 610.31 21.38 613.52 23.29 614.75 17.83 613.56 32 (80 ) 8 (20 ) 21.91 615.27 15.56 67.84 21.99 613.64 17.70 612.83 20 (50 ) 20 (50 ) 22.80 616.35 18.25 611.46 23.29 613.90 18.75 612.84then placed on prepared collagen surfaces in 24-well microplates. There were 8 parallel culture wells for each drug sensitivity testing and 8 parallel culture wells for control. After incubation for 7 days at 37uC (in a humidified atmosphere containing 95 air 25 CO2) in the presence of drugs dissolved with RPMI 1640 medium containing 20 fetal calf serum, 100 ml type I collagenase (0.1 mg/ml, Sigma) and MTT (5 mg/ml, Sigma) were added to each culture well and incubated for another 16 hours. Concentration of Pt, 5-FU, CPT-11, PPI and EVO was 20 mg/ml, 300 mg/ml, 20 mg/ml, 200 mg/ml and 200 mg/ml respectively, according to its peak plasma concentration (ppc) in patients [16]. Pt, 5-FU and CPT-11 were obtained from Jiangsu Hengrui Medicine Company (Jiangsu, China). PPI and EVO were supplied as powder with purity of .98 by the School of Pharmacy in China Pharmaceutical University. After extraction with dimethyl sulfoxide (DMSO, Sigma), absorbance of the solution in each well was read at 540 nm. Absorbance per gram of cultured tumor tissue was calculated from the mean absorbance of tissue from 8 parallel culture wells, and the tumor-tissue weight was determined before culture. The inhibition rate was calculated by using the following formula:T is the mean absorbance of treated tumor /Weight. C is the mean absorbance of control tumor / Weight.Synergy AnalysisMedian effect analysis using the combination index (CI) method of Chou and Talalay [17] was employed to determine the nature of the interaction observed between PPI or EVO and chemotherapeuti.
