Cytotoxic effects of Thai noni juice product ethanolic extracts against cholangiocarcinoma cell lines

Objective: To investigate the cytotoxic activity and molecular mechanism(s) of two Thai noni juice (TNJ) products ethanolic extracts against cholangiocarcinoma (CCA) cell lines and non-cancerous cells, and to explore phenolic acid compositions of TNJ products. Methods: Phenolic acid profiles of TNJ Chiangrai (TNJ-Cr) and TNJ Buasri (TNJ-Bs) ethanolic extracts were determined by HPLC. The cytotoxicity of TNJ ethanolic extracts on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays. Mechanism(s) underlying the anti-CCA activity of TNJ ethanolic extracts were determined by cell cycle, apoptosis, and reactive oxygen species (ROS) generation assays. The expression levels of proteins involved in apoptosis and ERK signaling were evaluated by Western blot analysis. Results: Phenolic acid profiles of both TNJ ethanolic extracts showed that the p-hydroxybenzoic, vanillic, and protocatechuic acids were the major phenolic acids in TNJ products. Cytotoxicity assays revealed that the TNJ-Cr and TNJ-Bs ethanolic extracts reduced viability of CCA cell lines through induction of apoptosis by up-regulation of p53 and Bax proapoptotic proteins. Both TNJ ethanolic extracts promoted ROS generation by activating the ERK1/2 signaling in well-differentiated CCA cells KKU-213B. Meanwhile, TNJ ethanolic extracts did not induce ROS production in poorly differentiated CCA cells KKU-100. Both TNJ ethanolic extracts showed no toxicity to human peripheral blood mononuclear cells. Conclusions: TNJ ethanolic extracts could inhibit CCA cell proliferation by inducing ROS generation and apoptosis and may be applicable for combination therapies in CCA treatment.

Cytotoxic effects of Thai noni juice product ethanolic extracts against cholangiocarcinoma cell lines

Objective: To investigate the cytotoxic activity and molecular mechanism(s) of two Thai noni juice (TNJ) products ethanolic extracts against cholangiocarcinoma (CCA) cell lines and non-cancerous cells, and to explore phenolic acid compositions of TNJ products. Methods: Phenolic acid profiles of TNJ Chiangrai (TNJ-Cr) and TNJ Buasri (TNJ-Bs) ethanolic extracts were determined by HPLC. The cytotoxicity of TNJ ethanolic extracts on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays. Mechanism(s) underlying the anti-CCA activity of TNJ ethanolic extracts were determined by cell cycle, apoptosis, and reactive oxygen species (ROS) generation assays. The expression levels of proteins involved in apoptosis and ERK signaling were evaluated by Western blot analysis. Results: Phenolic acid profiles of both TNJ ethanolic extracts showed that the p-hydroxybenzoic, vanillic, and protocatechuic acids were the major phenolic acids in TNJ products. Cytotoxicity assays revealed that the TNJ-Cr and TNJ-Bs ethanolic extracts reduced viability of CCA cell lines through induction of apoptosis by up-regulation of p53 and Bax proapoptotic proteins. Both TNJ ethanolic extracts promoted ROS generation by activating the ERK1/2 signaling in well-differentiated CCA cells KKU-213B. Meanwhile, TNJ ethanolic extracts did not induce ROS production in poorly differentiated CCA cells KKU-100. Both TNJ ethanolic extracts showed no toxicity to human peripheral blood mononuclear cells. Conclusions: TNJ ethanolic extracts could inhibit CCA cell proliferation by inducing ROS generation and apoptosis and may be applicable for combination therapies in CCA treatment.

Helicobacter pylori GroEL Seropositivity Is Associated with an Increased Risk of Opisthorchis viverrini-Associated Hepatobiliary Abnormalities and Cholangiocarcinoma

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20-3.71, P=0.008) and 2.13 (95% CI=1.21-3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.

Helicobacter pylori GroEL Seropositivity Is Associated with an Increased Risk of Opisthorchis viverrini-Associated Hepatobiliary Abnormalities and Cholangiocarcinoma

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20-3.71, P=0.008) and 2.13 (95% CI=1.21-3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.

Peanut testa extracts enhance anticancer effect of cisplatin against human cholangiocarcinoma cells via modulation of histone deacetylase inhibitory activity

Objective: To investigate the effect of combination treatments of cisplatin and KK4 and ICG15042 peanut testa extracts against cholangiocarcinoma cells in vitro. Methods: The growth inhibition, cell cycle arrest and apoptosis of cholangiocarcinoma cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry analysis, respectively. The levels of proteins involved in apoptosis were assessed using Western blotting assays. The caspase activity was assessed using a colorimetric caspase activity assay. Results: Cisplatin and peanut (KK4 and ICG15042) testa extracts inhibited the growth of cholangiocarcinoma cell lines (KKU-M214 and KKU-100 cells) in a dose- A nd time-dependent manner. The combination treatments reduced cell viability and induced apoptosis of cholangiocarcinoma cells more efficiently than singledrug treatments. Cancer cell death synergistically mediated by cisplatin and peanut testa extracts was observed in KKU-M214 cells (combination index 1.0). The combination treatments also increased the sub-G1 population and caused KKU-M214 cell cycle arrest at S and G2/M phases, which were the combined effects of cisplatin (S phase arrest) and peanut testa extracts (G2/M phase arrest). In addition, pERK1/2, Ac-H3, Bcl-2 and proteins related to apoptosis, including Bax and caspases 3, 8, 9, exhibited enhanced expression in KKU-M214 cells. The combination treatments caused down-regulation of p53, whereas the expression of p21 was fairly constant when compared with cisplatin single drug treatment. Conclusions: Peanut testa extracts in combination with cisplatin synergistically reduce cell viability and induce apoptosis through stimulation of caspases 3, 8 and 9 in KKU-M214 cells.

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF−, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1β, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF− and APF+) cases. Polymorphisms in genes encoding IL-1β-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α−308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α−308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 −174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Chronic Opisthorchis viverrini Infection and Associated Hepatobiliary Disease Is Associated with Iron Loaded M2-like Macrophages

Chronic Opisthorchis viverrini-induced hepatobiliary disease is associated with significant leukocyte infiltration, including activated macrophages; however, the polarization of infiltrating macrophages remains to be fully characterized. In this study, we characterized macrophage polarization and phenotype in chronic O. viverrini-induced hepatobiliary disease in humans and hamsters using gene expression and histochemical analysis. Chronic O. viverrini infection and associated hepatobiliary diseases were associated with iron loaded M2-like macrophages in both humans and hamsters. This study provides suggestive evidence that iron loaded M2-like macrophages promote hepatobiliary disease in chronic O. viverrini infection.

Effect of Mallotus repandus onVascular Endothelial and Cholangiocarcinoma Cells Migration

Background and objectives: Mallotus repandus (Euphorbiaceae), a widely distributed plant in South-East Asia, used as medicinal herb in many countries, including Thailand which is commonly used in the treatment of muscle and joint pain. Many active ingredients were found in Mallotus repandus, especially triterpenoids previously reported in various pharmacologic effects including anti-inflammatory and anticancer activities. The objective of this study was to study the potential of the methanol extract of the stem bark of M. repandus on the migration of vascular endothelial cells and cholangiocarcinoma (CCA) cells in comparing with to anti-mitotic drug, paclitaxel (Taxol)Methods: Non-cytotoxic concentrations of M. repandus extract, paclitaxel and vehicle were determined by MTT assay. Co-culture technique was performed to test for anti-migration. The cells were pre-treated with the non-cytotoxic concentrations of paclitaxel, extracts, or vehicle for 30 min before being added to insert (upper chamber), then further incubated for 18 h at 37oC in 5% CO2 incubator. The number of cells migrated to well (lower chamber) were counted under a microscope and the percentage of inhibition was calculated.Results: The findings revealed that the non-cytotoxic dose of the M. repandus extract could inhibit the migration of both vascular endothelial and CCA cells in dose-dependent manners.Conclusions: Our results suggested that the methanol extract of the M. repandus stem bark of had anti-tumor metastatic activity.Key words: Mallotus repandus, Cholangiocarcinoma, Anti-migration

Comparison of Italic cytoxicity of generic paclitaxel and irinotecan formulations with their reference formulations on seven human intrahepatic cholangiocarcinoma cell lines

Background: Several chemotherapies have now been introduced for the treatment of cholangiocarcinoma (CCA).  Although it has previously been reported that two new chemotherapeutic agents, paclitaxel and irinotecan, showed strong cytotoxic effect to human CCA cell lines the treatment cost currently using reference formulations of these two drugs are very expensive.  To date, the generic drugs for both paclitaxel and irinotecan are now commercially available in Thailand with relatively low price compared to reference formulations.  However, the study demonstrating the efficacy of the innovator and generic formulation of these two agents has never been reported.Objective: To determine and compare the cytotoxic activity of generic paclitaxel and irinotecan formulations with the reference formulations on seven human intrahepatic CCA cell lines.Design: In vitro studySetting: Faculty of Medicine, Khon Kaen UniversityMaterial and Method: Cytotoxic activitiy of chemotherapeutic agent on CCA cellines was determined by sulforhodamine B (SRB) assay.  The IC50 value expressed as the concentration of drug that caused a 50% growth inhibition comparing with no drug treated control.  The cytotoxic activity of the generic formulation was compared to the reference formulation using Student’s unpaired t-test.Results: Paclitaxel exhibited strong potency towards most CCA cell lines with IC50 values ranging from 0.001-1.40 mM whereas irinotecan showed IC50 values ranging varied from 0.02 to 69 mM. KKU-M055 was the most sensitive cell line to paclitaxel and KKU-OCA17 showed the highest sensitivity to irinotecan whereas KKU-M156 was the least sensitive cell line to both drugs. IC50 values of the generic product (IntaxelÒ, Dabur, India) and reference product (TaxolÒ, Bristol-Myers Squibb, USA) formulations were not significantly different (P \> 0.05).  Similary, the cytotoxicity of the generic formulation of irinotecan (IrinotelÒ, Dabur, India) was not statistically significant different from the reference formulation (CamptoÒ, Aventis Pharma, UK).Conclusion: The cytotoxic activity of paclitaxel towards six CCA cell lines was more potent than irinotecan except for KKU-OCA17.  No different in the IC50 values of the generic and reference formulations of paclitaxel and irinotecan against seven CCA lines suggest that the in vitro efficacy of generic and reference formulations of these two drugs are very similar.  

Biliary Cytology of Patients with Cholangiocarcinoma in Northeast Thailand

Background: Cholangiocarcinoma, the cancer of biliary epithelium, is highly prevalent in Northeast Thailand and is associated with the liver fluke, Opisthorchis viverrini, infection and the consumption of carcinogen contaminated daily food. Diagnosis of this cancer is practically based on clinical and utrasonography. However, several markers can be used in conjunction with the standard procedure to increase diagnosis accuracy including biliary cytology as described in carcinoma of other pancreatobiliary diseases. On the basis of cancer cells can be exfoliated into the bile and they can be detected by routine Papanicolaou staining. In endemic areas of cholangiocarcinoma and opisthorchiasis, there is no report about biliary cytology and percentage of positive malignant cells presented in the bile, here we examined biliary cytology of the gallbladder and/or hepatic bile to clarify its usefulness potential for using in diagnosis.Objective: To study biliary cytology and examine Opisthorchis-ova in the gallbladder/hepatic bile in patients with cholangiocarcinoma in Northeast Thailand.Patients and Methods: Aspirated gallbladder and/or hepatic bile from 100 histological proven cholangiocarcinoma cases who admitted to Srinagarind Hospital during December 1996 to July 1998 were studied. Four Papanicolaou\’s stained smears per bile sample per case were examined for the presence of malignant or atypical cells and O. viverrini ova. Positive specimens for malignancy contained singly, numerous malignant cells and/or in three dimensional clusters. Liver fluke positive cases contained ova with distinct bilaminar walls and prominent shoulder.Results: Overall, the positivity for malignant cells and O. viverrini ova was 51 % (51/100) and 27 % (27/100), respectively. Five cases were suspicious for malignancy. Paired gallbladder and hepatic bile specimens were obtained in 32 cases. Of these, the positivity for cancer cells was significantly higher in the hepatic bile than in the gallbladder bile (2-test, p\<.01). Five cases with gallstone were negative for malignant cells.Conclusion: The results indicate that cancer cells are frequently detected in the bile, particularly in hepatic bile from patients with cholangiocarcinoma. This study implicates the potential application of biliary cytology in diagnosis of this cancer, i.e. in the bile from ERCP or duodenal drainage which is less invasive, in conjunction with other investigations in endemic areas.

Determination of Growth Inhibitory Effect of Gemcitabine on Human Intrahepatic Cholangiocarcinoma Cell lines and Comparison of its Inhibition Between the Generic and Reference Formulation

Background and Objective: Gemcitabine is one of the most popular drug-of-choices that is currently used for the treatment of cholangiocarcinoma (CCA).\  However, the study revealing the inhibitory effect of this agent in the series of CCA cell lines established from Thai patients has not been reported. We aim to determine and compare the growth inhibitory effect of generic gemcitabine formulation with the reference formulation on CCA cell lines.Methods: Seven CCA cell lines established in Srinagarind Hospital, Khon Kaen University were used. A cell growth inhibition by gemcitabine was determined by sulforhodamine B.\  The IC50 value was expressed as the concentration of drug that caused a 50% growth inhibition comparing with untreated control.\  The IC50 values of those two formulations were compared using independent t-test.Results: Growths of KKU-M055, KKU-OCA17 and KKU-M139 CCA cell lines were highly inhibited by gemcitabine (IC50 = 13.35-16.0 M) whereas KKU-M214 was moderately inhibited by this drug (IC50 = 36.7 M). These least inhibited growths were found on KKU-100, KKU-M156 and KKU-M213 (IC50 = 406-4629 M).\  The generic (Gramagen) and the reference product (Gemza) formulations were not significantly different in their inhibitory effects on the all seven CCA cell lines.\ Conclusions: Although the inhibitory effect of gemcitabine was varied towards seven CCA cell lines, there was no difference in the IC50 values of the generic and reference formulations. Our findings indicate that the in vitro efficacy of these two formulations is similar.\ Keywords: growth inhibitory effect, gemcitabine, cholangiocarcinoma, generic formulation

Antimigration of Cancer Cells by Derris scandens on Cholangiocarcinoma Cells

Background: Derris scandens (Leguminosae) is used as traditional remedies in Thailand for arthritis. Phytochemicals obtained from the stems are mainly isoflavones such as genistein and their derivatives. Many anticancer activities of isoflavone have been reported, including antimigration effect.Objectives: To assess antimigration activity of D. scandens extract on cholangiocarcinoma (CCA) cell lines compared to other human cancer cell lines and standard antimitotic drug (e.g., paclitaxel).Methods: Non-cytotoxic concentrations of D. scandens ethanol extract, paclitaxel and vehicle were determined by MTT assay. For antimigration assay, co-cultured technique using CCA cell lines (KKU-100, KKU-M139 and KKU-M213), hepatoma cell line (HepG2) and breast cancer cell line (MCF-7) was employed. The cells (2.5x104 cells) were pre-treated with non-cytotoxic concentrations of tested drug or herbal extract for 30 min before adding to insert (upper chamber), then further incubated for 18 h at 37oC in 5% CO2 incubator. Non-migrating cells were removed using cotton swab, the number of cells migrated to well (lower chamber) were counted under a microscope and percent inhibition was calculated.Results: From MTT assay, in comparison to vehicle (0.25-1% DMSO), the non-cytotoxic concentrations were up to 800 g/ml 0.5% DMSO and 10-9 M for D. scandens and paclitaxel, repectively. Antimigration activity of D. scandens was clearly demonstrated with nearly all of the human cancer cell lines, except KKU-100 which is derived from the poorly differentiated adenocarcinoma tissue. The migratory inhibition effect of paclitaxel was observed in all cell lines.Conclusions: The ethanol extract of D. scandens shows antimigration in most many cancer cell lines. For CCA cell lines, the extract showed potent inhibitory effect especially with squamous cell carcinoma (KKU-M139) and adenosquamous carcinoma (KKU-M213). Therefore, it is interesting that the extract may have a potential as antimetastasis on CCA cells, at least in part, mediated through antimigration activity.Key words: Derris scandens, Cholangiocarcinoma, Antimigration, Cytotoxicity\ 

Novel Genes of Opisthorchis viverrini Identified by Immunoscreening with Cholangiocarcinoma Associated Opisthorchiasis Serum

The liver fluke, Opisthorchis viverrini (OV), is an important human pathogen distributed in northeast Thailand. The linkage between the liver fluke and CCA is strongly supported by previous literature. The molecular mechanisms by which OV induces CCA in humans are unknown. Opisthorchis antigens are found in the bile ducts (in regions where parasites reside as well as those in smaller ducts where they do not) as well as damaged liver cells and various inflammatory cell infiltrates, further incriminating ES antigens in cholangiocarcinogenesis. Characterization of OV molecules associated with OV infection and cholangiocarcinoma using the immunoscreening method was performed. The adult cDNA library screening was done with cholangiocarcinoma associated opisthorchiasis serum. The results show that 6 protein encoding genes including asparaginyl endopeptidase, egg protein, actin, testis enhanced gene transcript and 2 unknown function genes were positive with strong reaction to human serum. Further investigation on OV asparaginyl endopeptidase show that the full length of the OV asparaginyl endopeptidase gene was 1,302 bp, encoded 408 amino acid residues predicted molecular weight at 47 kDa and predicted cleavage site between amino acid residure 17 and 18.