Relationship between Helicobacter pylori virulence genes and gastroduodenal disease.

OBJECTIVE: This study aimed to identify Helicobacter pylori virulence factors and examine their associations with clinical outcomes in Thai patients. Moreover, the association between these genotypes and gastric mucosa morphological patterns was investigated. METHODS: This retrospective study enrolled patients who underwent esophagogastroduodenoscopy at Suranaree University of Technology Hospital. The presence of the cagA and vacA genes was investigated by real-time polymerase chain reaction. RESULTS: The H. pylori-specific genes ureA and 16S rRNA were detected in all 698 gastric biopsy specimens. In total, 567 (81.23%) patients with H. pylori infection were positive for the cagA gene, 443 (63.46%) were positive for the vacA gene, and 370 (53.0%) were positive for both. The cagA genotype was significantly more common in patients with chronic gastritis and peptic ulcers (78.99% and 79.41%, respectively) than the vacA gene (51.48% and 55.88%, respectively) and combined genotypes (32.34% and 47.05%, respectively). Moreover, the cagA genotype was significantly more common in patients with type 4 or 5 gastric mucosa patterns (69.49% and 76.31%, respectively). CONCLUSIONS: The cagA genotype is the main cause of serious inflammation of the gastric mucosa. The cagA gene is possibly an important factor explaining gastroduodenal disease outcomes in Thai patients with H. pylori infection.

Expression of Matrix Metalloproteinase-7 Predicts Poor Prognosis in Gastric Cancer.

To date, no potential markers have been established for predicting prognosis in gastric cancer. Matrix metalloproteinase-7 (MMP-7) has been suggested as a prognostic marker in several cancers. In this study, we aimed to determine the expression of the MMP-7 protein and its polymorphisms in gastric cancer tissues. The association between MMP-7 expression level and clinicopathological characteristics was also evaluated. MMP-7 protein expression and its polymorphisms were investigated in a total of 400 patients using immunohistochemistry and TaqMan SNP genotyping assays. The correlation of MMP-7 expression with clinicopathological characteristics, including tumor location, tumor size, histologic type, lymphatic invasion, vascular invasion, pathological T stage, pathological TNM stage, residual tumor, and CEA level, was investigated. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a multivariate Cox proportional hazards regression model. MMP-7 expression was found in 283 of 400 (70.75%) gastric cancer tissues. Expression of MMP-7 was significantly associated with poor clinicopathological characteristics, including vascular invasion (OR = 6.61, 95%CI = 4.26-9.89, p = 0.024), lymphatic invasion (OR = 8.17, 95%CI = 4.47-12.39, p = 0.017), undifferentiated histologic type (OR = 2.46; 95% CI, 1.31-4.52; p = 0.014), higher TNM stage (stage IV) (OR = 1.48, 95%CI = 1.08-3.08, p = 0.047), and high CEA level (OR = 5.96, 95%CI = 2.12-8.12, p = 0.026). We further observed a significant association of the variant genotype; gastric cancer patients carrying GG of MMP-7 (-181A/G; rs11568818) had a greater increased risk of MMP-7 expression than did wild-type (WT) carriers (AG: odds ratio (OR) = 5.67; 95%CI = 1.57-7.23; p = 0.024 and GG: OR = 8.32; 95%CI = 2.94-11.42; p = 0.016). These findings suggest that MMP-7 expression can be used to predict the prognosis of gastric cancer patients.

The mouse double minute 2 polymorphism is associated with both decreased p53 expression and poor clinicopathological outcomes of gastric cancer.

This study aimed to determine the mouse double minute 2 (MDM2) SNP309 polymorphism and to evaluate MDM2 and p53 expression and the association of MDM2 positivity in gastric cancer and clinicopathological outcomes. A total of 400 patients with chronic gastritis, precancerous lesions, and gastric cancer were used to identify the MDM2 SNP309 polymorphism by using the Taq Man SNP Genotyping assay. Immunohistochemistry was performed to evaluate MDM2 and p53 expression. The associations of polymorphisms, protein expression, clinicopathological outcomes, and gastric cancer risk were calculated by multivariate Cox proportional hazards regression model analysis and expressed by odds ratios (ORs) and 95% confidence intervals (CIs). The MDM2 SNP309 G/G homozygous polymorphism was significantly associated with expressed MDM2 in gastric cancer (OR = 1.57, 95% CI = 1.39-2.03, P = 0.039). Moreover, in gastric cancer, p53 was significantly decreased compared to MDM2 (P = 0.007). However, MDM2 and p53 expression were not significantly different among genotypes, and the G/G genotype can result in the altered protein expression of p53 in gastric cancer. Clinicopathological outcome was significantly associated with MDM2 expression, including tumor location in the upper gastric region (OR = 1.48, 95% CI = 1.25-3.54, P = 0.037), undifferentiated type (OR = 2.47, 95% CI = 1.38-4.14, P = 0.016), presence of lymphatic invasion (OR = 1.96, 95% CI = 1.22-3.19, P = 0.014), and unresectable tumor (OR = 3.39, 95% CI = 1.61-4.94, P = 0.017). Our study indicated associations of the MDM2 SNP309 G/G homozygous polymorphism, MDM2 and p53 expression. Therefore, G/G-associated MDM2 revealed that P53 expression was decreased in gastric cancer and poor clinicopathological outcomes. Understanding the genetic polymorphisms and expression of MDM2 may help explain gastric cancer risk.

Co-Expression of LGR5 and CD133 Cancer Stem Cell Predicts a Poor Prognosis in Patients With Gastric Cancer.

BACKGROUND: The LGR5 and CD133 have been identified as cancer stem cells (CSCs) marker and prognostic marker in several cancers including gastric cancer. The purpose of the present study was to determine the association between co-expression of CSCs marker LGR5 and CD133 in patients with gastric cancer and their clinicopathological outcomes; to analyze the efficacy of co-expression of both markers in evaluating the prognosis of gastric cancer. METHODS: LGR5 and CD133 expression were investigated in a total of 400 patients by using immunohistochemistry. Results were analyzed in association with patient characteristics outcomes. Overall survival was performed using Kaplan-Meier Curve analysis. RESULTS: LGR5 and CD133 were found positive in 219/400 (54.75%) and 251/400 (62.75%) respectively in gastric cancer tissues. Co-expression of LGR5 and CD 133 were significantly associated with poor clinicopathological outcomes, including lymphatic invasion, vascular invasion, higher pathological T stage, and higher TNM staging (stage IV) (P < .05). The overall survival of patients who were positive for LGR5 and CD133 had shorter than that of LGR5 and CD133-negative gastric cancer, especially in patients who were positive for both markers. CONCLUSION: Our finding indicates that co-expression of LGR5 and CD133 could be used as a marker indicating poor prognosis, which can provide information for selected effective treatment and carried out of intensive follow-up in gastric cancer patients.

Precancerous Gastric Lesions with Helicobacter pylori vacA +/babA2+/oipA + Genotype Increase the Risk of Gastric Cancer.

OBJECTIVE: The clinical outcomes of gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer have been attributed to the interplay of virulence factors of Helicobacter pylori (H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of cagA, vacA, iceA2, babA2, and oipA genes and their association with clinical outcomes. METHODS: Chronic gastritis, atrophic gastritis, and intestinal metaplasia specimens were obtained from patients who underwent endoscopy and surgical resection between January 2017 and December 2018; specimens from gastric cancer patients treated between January 2014 and December 2018 were also added. H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of cagA, vacA, iceA2, babA2, and oipA genes and their association with clinical outcomes. H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of. RESULTS: H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of vacA, babA2, and oipA genes and their association with clinical outcomes. vacA, babA2, and oipA genes and their association with clinical outcomes. P=0.033, OR = 2.64; 95% CI = 1.44-4.82, P=0.033, OR = 2.64; 95% CI = 1.44-4.82, P=0.033, OR = 2.64; 95% CI = 1.44-4.82, H. pylori vacA +/babA2, and oipA genes and their association with clinical outcomes. P=0.033, OR = 2.64; 95% CI = 1.44-4.82. CONCLUSION: In this present study, we reported on the virulence genes of H. pylori infection to reveal their association with increased risk of chronic gastritis, precancerous gastric lesions, and gastric cancer. Precancerous gastric lesions with H. pylori vacA+/babA2+/oipA+ genotype increased the risk of gastric cancer.H. pylori), host genetic susceptibility, and host immune responses. This study investigated the presence of H. pylori vacA +/babA2, and oipA genes and their association with clinical outcomes.

Effects of periodontal therapy on eradication and recurrence of Helicobacter pylori infection after successful treatment.

OBJECTIVES: This study aimed to evaluate the effects of periodontal therapy on the efficacy of Helicobacter pylori eradication and on the recurrence of infection after eradication. METHODS: We conducted a prospective randomized trial on 698 gastric H. pylori-infected patients, of whom 347 received gastric H. pylori treatment alone and 342 received gastric H. pylori treatment plus periodontal therapy. The presence of H. pylori and associated virulence genes were detected by real-time polymerase chain reaction. RESULTS: After eradication of gastric H. pylori infection, the recurrence of gastric H. pylori was significantly lower in the gastric H. pylori treatment plus periodontal therapy group than in the group receiving gastric H. pylori treatment alone (OR 0.67; 95% CI 0.45 to 0.99), whereas the eradication rate was not significantly different (OR 0.87; 95% CI 0.68 to 0.98). There was a close relationship between the presence of H. pylori in saliva and its presence in the stomach. CONCLUSIONS: The oral cavity is an important reservoir for gastric H. pylori infection. Adjunctive periodontal therapy could enhance the efficiency of H. pylori treatment and reduce the recurrence of gastric H. pylori infection.

Toll-like receptor 2 and 4 polymorphisms associated with Helicobacter pylori susceptibility and gastric cancer.

BACKGROUND/AIMS: Genetic polymorphisms in Toll-like receptors (TLRs) are important influence on gastric lesion development and Helicobacter pylori susceptibility. MATERIALS AND METHODS: TLR2 rs3804099 and rs3804100 and TLR4 rs10759932 were determined in a total of 400 patients. The association among genotypes and the risk of gastric lesion development and H. pylori susceptibility were evaluated by the odds ratios (ORs) and 95% confidence intervals (95% CIs) from logistic regression analyses. RESULTS: TLR4 rs10759932, C/C homozygous genotype was associated with an increased risk of premalignant/malignant (OR=2.48, 95% CI=1.96-4.62, p=0.015). The recessive model of TLR4 rs10759932 showed a decreased risk of H. pylori susceptibility (adjusted OR=0.52, 95% CI=0.38-0.82, p=0.046). Meanwhile, the recessive model was associated with an increased risk of non-malignant (OR=3.46, 95% CI=2.25-5.67, p=0.001). In subjects with H. pylori infection, the recessive model was associated with an increased risk of non-malignant (OR=2.28, 95% CI=1.24-3.57, p=0.001) and premalignant/malignant (OR=1.83, 95% CI=1.16-2.84, p=0.027). CONCLUSION: TLR4 rs10759932, but not TLR2 rs3804099 and rs3804100, was associated with risk of premalignant and/or malignant and H. pylori susceptibility. H. pylori infection seems to contribute to chronic gastritis, and premalignant/malignant supported the development of the premalignant/malignant lesions involved in H. pylori infection that is critical to gastric cancer in Thai patients.

Expression of CD44 and MDM2 in cholangiocarcinoma is correlated with poor clinicopathologic characteristics.

The aim of the study was to investigate the expression of cluster of differentiation 44 (CD44) and mouse double minute 2 (MDM2) in cholangiocarcinoma, in addition to evaluating their association with clinicopathologic characteristics and overall survival time. Paraffin-embedded tumor tissues from 128 patients from 3 study centers in Thailand were evaluated using immunohistochemistry. The results demonstrated that positive expression of CD44 was associated with high histologic grade (P=0.013), large tumor size (P=0.027), lymph node metastasis (P=0.037), and distant metastasis (P=0.031). MDM2 expression was related to high histologic grade (P=0.013), lymph node metastasis (P=0.025), and distant metastasis (P=0.016). Furthermore, multivariate analyses revealed that combined expression of CD44 and MDM2 was significantly associated with worse overall survival time (OR=1.52; 95% CI=1.04-2.26; P=0.041) in patients with cholangiocarcinoma. CD44 and MDM2 significantly indicate poor clinicopathologic outcomes in patients with cholangiocarcinoma.

Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.

Role of toll-like receptor 10 gene polymorphism and gastric mucosal pattern in patients with chronic gastritis.

BACKGROUND/AIMS: Helicobacter pylori stimulates the host's toll-like receptors (TLRs). Single-nucleotide polymorphism (SNP) of TLRs is related to the manipulation of regulatory cytokines and also implicated in the varied outcomes of the inflammatory response, including the development of precancerous lesions of gastric mucosa and disease progression. We hypothesized that TLR10 rs10004195 polymorphism is associated with gastric mucosal patterns. MATERIALS AND METHODS: TLR10 rs10004195 polymorphisms were identified in a total of 400 gastritis patients using the TagMan SNP genotyping assay. Gastric mucosal patterns were classified by Conventional Narrow Band Imaging gastroscopy (C-NBI gastroscopy). Logistic regression was used to analyze the association. RESULTS: The gastritis patients was Type 1, 37.5% of Thai patients. The T/T homozygous genotype was exhibited by the highest percentage (46.5%) of patients, and the A/A homozygous and A/T heterozygous genotypes were exhibited by 20.25% and 33.25%, respectively, of patients. TLR10 rs10004195 was significantly associated with gastric mucosal patterns. After adjusting for confounding factors, patients with the A/A homozygous genotype showed a significantly increased risk of severe inflammation (OR=1.35, 95% CI=0.97-2.13, p=0.028). Patients with the A/T heterozygous and T/T homozygous genotypes showed a significantly increased risk of mild inflammation (OR=1.24, 95% CI=0.78-2.07, p=0.042 and OR=1.78, 95% CI=0.51-3.35, p=0.001, respectively). CONCLUSION: Our results indicate that the presence of TLR10 rs10004195, A/T heterozygous, and T/T homozygous genotypes is associated with type 1, 2, and 3 whereas that of the A/A homozygous genotype is associated with type 4 and 5 of gastric mucosal patterns. This suggests that the A/A homozygous genotype contributes to severe inflammation in H. pylori-associated gastritis in Thai patients.

Expression of Cancer Stem Cell Marker CD44 and Its Polymorphisms in Patients with Chronic Gastritis, Precancerous Gastric Lesion, and Gastric Cancer: A Cross-Sectional Multicenter Study in Thailand.

Here we investigated CD44 protein expression and its polymorphisms in patients with chronic gastritis, precancerous gastric lesions, and gastric cancer; and we evaluated our result with the risk of CD44 protein expression and clinicopathological characteristics. Our results obtained by analyzing 162 gastric cancer patients, 125 chronic gastritis, and 165 precancerous gastric lesions from three study centers in Thailand showed that CD44 expression was significantly higher in patients with precancerous gastric lesions and gastric cancer while patients with chronic gastritis were negative for CD44 staining (p = 0.036). We further observed the significant association of variant genotype; gastric cancer patients carrying AG or GG of CD44 rs187116 had more increased risk of CD44 expression than wild-type (WT) carriers (AG: odds ratio (OR) = 5.67; 95% CI = 1.57-7.23; p = 0.024 and GG: OR = 8.32; 95% CI = 2.94-11.42; p = 0.016), but no significant difference in the risk of CD44 expression due to polymorphism in patients with precancerous gastric lesions. Our results suggested that CD44 expression could be used as a marker for the prediction of gastric cancer development, particularly in patients with precancerous gastric lesions carrying AG or GG, who were selected to surveillance follow-up for gastric cancer prevention.

Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.

To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU­M139 and KKU­M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU­M139/GEM and KKU­M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU­M139/GEM and KKU­M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU­M214/GEM supporting the view of its higher GEM resistant characteristics. These findings indicate that long-term exposure of CCA cell lines to gemcitabine induce not only multidrug resistance but also enhance their invasiveness.

Terpenoids from the root bark of Pterolobium macropterum.

Five new compounds, including pteroloterins A-C (1, 3, and 4), 1ß-acetoxytaepeenin C (2), and 8aα-hydroxycadinenal (5), and 11 known compounds were isolated from the root bark of Pterolobium macropterum. All compounds were evaluated for cytotoxicity against the cholangiocarcinoma cell lines. Compound 9 showed weak cytotoxicity against the KKU-M139 cell line with an IC50 value of 23.24 ± 0.18 µM and showed no activity against normal cells.

Synergistic effects of isomorellin and forbesione with doxorubicin on apoptosis induction in human cholangiocarcinoma cell lines.

BACKGROUND: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines. METHODS: KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis. RESULTS: Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups. CONCLUSION: The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.