C agents. The CI is determined by the following equation:CI

C agents. The CI is determined by the following equation:CI ? = x? z ? = x? za ? ? = x? x? , in which (Dx)1 and (Dx)2 are the concentrations for D1 (PPI or EVO) and D2 (chemotherapeutic agent) alone that gives x inhibition rate, whereas (D)1 and (D)2 in the numerators are the concentrations of PPI or EVO and another drug that produce the identical level of effect in combination. a = 0 when the drugs are mutually exclusive, while a = 1 of they are mutually non-exclusive. CI .1 indicate antagonism, CI ,1 indicate synergy, and CI = 1 indicate additivity.Total RNA Extraction from FFPE TissueSix 7-mm sections were prepared from FFPE tumor blocks that contained at least 80 tumor cells. After hematoxylin-eosin staining, the cancerous parts were microdissected and transferred into a microcentrifuge tube. RNA was isolated in accordance with a proprietary procedure (European patent number EP1945764B1) [18]. Briefly, paraffin was removed by xylene, and microdissected cancerous parts were lysed in a proteinase K-containingInhibitionrate( ) (1 ?T=C)|100 .Synergistic Anticancer Effects of PPI and EVOFigure 3. The procedure of histoculture drug response assay (HDRA). doi:10.1371/journal.pone.0065164.gbuffer at 60uC for 16 h. RNA was purified by phenol and chloroform extractions followed by precipitation with isopropanol in the presence of sodium acetate at 220uC. The RNA pellet was washed in 70 ethanol and resuspended in 53 ml of RNase-free water followed by treatment with DNase I (Life Technologies).Results Patient Characteristics and Sensitivity of Freshly-removed Tumor Tissues to Different DrugsThe method for sensitivity test of freshly-removed tumor tissues was successfully established and carried out (Figure 3). We first examined the cytotoxicity of each drug on freshly-removed tumor tissues of 40 testing samples, including PPI (200 mg/ml), EVO (200 mg/ml), Pt (20 mg/ml), 5-FU (300 mg/ml), CPT-11 (20 mg/ ml).Characteristics of all patients are shown in Table 1. The mean age of those 40 patients was 61 (range: 34?1). The majority of patients were males (80 ), and the majority histology of every sample was adenocarcinoma (70 ). In 11 (27.5 ) patients, the tumor was located in the distal stomach, in 14 (35 ) in the Essed between all patients (groups HAT-1 and HAT-2) and the control proximal stomach, and in 15 (37.5 ) in the whole stomach. Twenty-nine (72.5 ) patients had stage III and IV disease. Lymph node metastasis was S the disease progresses it transitions into being hormone independent and present in 37 (92.5 ) patients. The Figure 4 shows the tumor inhibition rates for these tissues following exposure to the five agents. The average inhibition rate of the five drugs were 20.64 614.25 for PPI, 21.14 613.43 for EVO, 50.57 622.37 for Pt, 53.54 622.03 for 5-FU, and 39.33 624.79 for CPT-11, respectively. It seems that although PPI and EVO exhibited certain anticancer effect, their efficiency were lower than chemotherapeutic agents (PPI vs. Pt:P,0.001;PPI vs. 5-FU:P,0.001;PPI vs. CPT-11: P = 0.013; EVO vs. Pt:P,0.001;EVO vs. 5-FU:P,0.001;EVO vs. CPT-11: P = 0.037). There were no statistically significant differences in inhibition rates between PPI and EVO (P.0.05), and between different therapeutic agents (P.0.05). As shown in Table 1, there were neither no statistically significant differences in inhibitionQPCR Assessment of Gene ExpressionM-MLV Reverse Transcriptase Kit (Invitrogen) was applied to generate cDNA for Quantitative polymerase chain reaction (qPCR) to detect the b-actin (ACTB), aprataxin (APTX), excision repair cross-complementing 1 (ERCC1), thymidylate synthase.C agents. The CI is determined by the following equation:CI ? = x? z ? = x? za ? ? = x? x? , in which (Dx)1 and (Dx)2 are the concentrations for D1 (PPI or EVO) and D2 (chemotherapeutic agent) alone that gives x inhibition rate, whereas (D)1 and (D)2 in the numerators are the concentrations of PPI or EVO and another drug that produce the identical level of effect in combination. a = 0 when the drugs are mutually exclusive, while a = 1 of they are mutually non-exclusive. CI .1 indicate antagonism, CI ,1 indicate synergy, and CI = 1 indicate additivity.Total RNA Extraction from FFPE TissueSix 7-mm sections were prepared from FFPE tumor blocks that contained at least 80 tumor cells. After hematoxylin-eosin staining, the cancerous parts were microdissected and transferred into a microcentrifuge tube. RNA was isolated in accordance with a proprietary procedure (European patent number EP1945764B1) [18]. Briefly, paraffin was removed by xylene, and microdissected cancerous parts were lysed in a proteinase K-containingInhibitionrate( ) (1 ?T=C)|100 .Synergistic Anticancer Effects of PPI and EVOFigure 3. The procedure of histoculture drug response assay (HDRA). doi:10.1371/journal.pone.0065164.gbuffer at 60uC for 16 h. RNA was purified by phenol and chloroform extractions followed by precipitation with isopropanol in the presence of sodium acetate at 220uC. The RNA pellet was washed in 70 ethanol and resuspended in 53 ml of RNase-free water followed by treatment with DNase I (Life Technologies).Results Patient Characteristics and Sensitivity of Freshly-removed Tumor Tissues to Different DrugsThe method for sensitivity test of freshly-removed tumor tissues was successfully established and carried out (Figure 3). We first examined the cytotoxicity of each drug on freshly-removed tumor tissues of 40 testing samples, including PPI (200 mg/ml), EVO (200 mg/ml), Pt (20 mg/ml), 5-FU (300 mg/ml), CPT-11 (20 mg/ ml).Characteristics of all patients are shown in Table 1. The mean age of those 40 patients was 61 (range: 34?1). The majority of patients were males (80 ), and the majority histology of every sample was adenocarcinoma (70 ). In 11 (27.5 ) patients, the tumor was located in the distal stomach, in 14 (35 ) in the proximal stomach, and in 15 (37.5 ) in the whole stomach. Twenty-nine (72.5 ) patients had stage III and IV disease. Lymph node metastasis was present in 37 (92.5 ) patients. The Figure 4 shows the tumor inhibition rates for these tissues following exposure to the five agents. The average inhibition rate of the five drugs were 20.64 614.25 for PPI, 21.14 613.43 for EVO, 50.57 622.37 for Pt, 53.54 622.03 for 5-FU, and 39.33 624.79 for CPT-11, respectively. It seems that although PPI and EVO exhibited certain anticancer effect, their efficiency were lower than chemotherapeutic agents (PPI vs. Pt:P,0.001;PPI vs. 5-FU:P,0.001;PPI vs. CPT-11: P = 0.013; EVO vs. Pt:P,0.001;EVO vs. 5-FU:P,0.001;EVO vs. CPT-11: P = 0.037). There were no statistically significant differences in inhibition rates between PPI and EVO (P.0.05), and between different therapeutic agents (P.0.05). As shown in Table 1, there were neither no statistically significant differences in inhibitionQPCR Assessment of Gene ExpressionM-MLV Reverse Transcriptase Kit (Invitrogen) was applied to generate cDNA for Quantitative polymerase chain reaction (qPCR) to detect the b-actin (ACTB), aprataxin (APTX), excision repair cross-complementing 1 (ERCC1), thymidylate synthase.