Porntip Pinlaor

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.

iNOS-dependent DNA damage via NF-κB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation‐mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535–42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV‐infected hamsters. We also examined the expression of nuclear factor‐κB (NF‐κB), which functions as a tumor promoter in inflammation‐associated cancer. Our results showed that although 1‐week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV‐induced 8‐nitroguanine and 8‐oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8‐oxodG level in the liver of OV‐infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF‐κB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV‐induced cholangiocarcinoma by inhibiting iNOS‐dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.

Curcumin decreases cholangiocarcinogenesis in hamsters by suppressing inflammation-mediated molecular events related to multistep carcinogenesis

Cholangiocarcinoma (CCA) is a highly metastatic tumor linked to liver fluke infection and consumption of nitrosamine‐contaminated foods and is a major health problem especially in South‐Eastern Asia. In search for a suitable chemopreventive agents, we investigated the effect of curcumin, a traditional anti‐inflammatory agent derived from turmeric (Curcuma longa), on CCA development in an animal model by infection with the liver fluke Opisthorchis viverrini and administration of N‐nitrosodimethylamine and fed with curcumin‐supplemented diet. The effect of curcumin‐supplemented diet on histopathological changes and survival were assessed in relation to NF‐κB activation, and the expression of NF‐κB‐related gene products involved in inflammation, DNA damage, apoptosis, cell proliferation, angiogenesis and metastasis. Our results showed that dietary administration of this nutraceutical significantly reduced the incidence of CCA and increased the survival of animals. This correlated with the suppression of the activation of transcription factors including NF‐κB, AP‐1 and STAT‐3, and reduction in the expression of proinflammatory proteins such as COX‐2 and iNOS. The formation of iNOS‐dependent DNA lesions (8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine) was inhibited. Curcumin suppressed the expression of proteins related to cell survival (bcl‐2 and bcl‐xL), proliferation (cyclin D1 and c‐myc), tumor invasion (MMP‐9 and ICAM‐1) and angiogenesis (VEGF), and microvessel density. Induction of apoptotic events as indicated by caspase activation and PARP cleavage was also noted. Our results suggest that curcumin exhibits an anticarcinogenic potential via suppression of various events involved in multiple steps of carcinogenesis, which is accounted for by its ability to suppress proinflammatory pathways.

Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model

Peribiliary fibrosis caused by chronic infection with Opisthorchis viverrini (OV) is a risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Matrix metalloproteinases (MMPs) are enzymes capable of degrading and remodeling the extracellular matrix in the process of fibrosis and carcinogenesis. We examined MMPs expression and their role in fibrogenesis and cholangiocarcinogenesis in hamsters treated with OV and N‐nitrosodimethylamine (NDMA). We assessed the time profiles of MMPs, inducible nitric oxide synthase (iNOS), Rac1, α‐smooth muscle actin (α‐SMA) and DNA lesions (8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine, 8‐oxodG) in relation to fibrosis and CCA development. Histopathology revealed OV and NDMA synergistically induced peribiliary fibrosis time‐dependently, and CCA occurred at 3 months, whereas OV or NDMA alone induced less fibrosis. Hydroxyproline levels in the liver and plasma were positively associated with the expression of collagen I and α‐SMA. MMP‐9 expression was significantly increased and correlated with the accumulation of myofibroblast, fibrosis levels and cholangiocarcinogenesis. MMP‐9 activity was correlated with iNOS, and immunocolocalization was observed in inflammed tissues, early and invasive CCA. OV and NDMA synergistically induced MMP‐9 expression in association to Rac1. In addition, Rac1 was colocalized with iNOS, and 8‐nitroguanine, in inflammed tissues and CCA. Formation of 8‐nitroguanine and 8‐oxodG increased with tumor progression. The results suggest that MMP‐9 expression is associated with the accumulation of peribiliary fibrosis in conjunction to the induction of iNOS and Rac1 that may potentiate DNA damage and cholangiocarcinogenesis.

Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

Background The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Methodology/Principal Findings Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of ∼3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size. Conclusions/Significance A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Protective effect of melatonin against Opisthorchis viverrini-induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract:  The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.

Reduction of periductal fibrosis in liver fluke-infected hamsters after long-term curcumin treatment

Chronic infection with the liver fluke, Opisthorchis viverrini, induces advanced periductal fibrosis and is a relative risk factor for cholangiocarcinoma in Southeastern Asia. We examined the reducing effect of curcumin on hepatobiliary fibrosis using O. viverrini-infected hamsters supplemented with dietary 1% curcumin (w/w) as an animal model. The expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), cytokines, and collagens was assessed in relation to liver fibrosis. Histopathological studies revealed that curcumin had no effect on fibrosis at the short-term infection (21 days and 1 month); however, peribiliary fibrosis was significantly reduced after the long-term curcumin treatment for 3 months, compared to the untreated group. Expression of alpha-smooth muscle actin was associated with the reduction of liver fibrosis. A decrease in hepatic hydroxyproline level and mRNA expression of collagen I and III supported the reduction of fibrosis. The expression of TIMP-1, TIMP-2, and tumor necrosis factor-alpha genes was also decreased after curcumin treatment. In contrast, curcumin increased mRNA expression of MMP-13, MMP-7 (at 6 months), interleukin-1 beta, and transforming growth factor beta, implying that increased MMPs activity contributes to extracellular matrix degradation. These results suggest that curcumin reduces periductal fibrosis after long-term treatment by tissue resorption via inhibition of TIMPs expression and enhancement of MMPs expression mediated by cytokines. In conclusion, curcumin may serve as a promising nutraceutical agent exerting antifibrotic effect in O. viverrini-infected patients and contribute to cholangiocarcinoma prevention.

PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients

The polymerase chain reaction (PCR) technique was compared with Wright-Giemsa (WG), Gomori methenamine silver (GMS) stains and an immunofluorescence assay (IFA) for detection of Pneumocystis carinii in immuno-compromised patients. Specimens of 21 bronchoalveolar lavages (BAL) and 139 sputum samples, were obtained from 157 patients (38 with AIDS and 119 with HIV) from four hospitals in Khon Kaen, Thailand. A true positive required at least two positives by techniques considered gold standard tests. Eleven (52.38%) BAL and 13 (9.35%) sputum specimens were positive. PCR produced the highest sensitivity and negative predictive values for the BAL (100% for each) vs. sputum samples at 84.62 and 98.41 percent, respectively. The specificity of PCR was 90% and 98.41% for BAL and sputum samples, respectively. We suggest PCR is an important tool for the epidemiological study of P. carinii in high-risk individuals.

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.

Effect of praziquantel treatment on the expression of matrix metalloproteinases in relation to tissue resorption during fibrosis in hamsters with acute and chronic Opisthorchis viverrini infection.

Praziquantel has been used for the treatment of diseases caused by infection with various parasites. Praziquantel treatment in Opisthorchis viverrini-infected patients reduces the hepatobiliary fibrosis due to tissue resorption, however, some fibroses are irreversible. To clarify the effect of praziquantel treatment on hepatobiliary fibrosis, we examined the expression of matrix metalloproteinases (MMPs) in relation to fibrolysis on praziquantel-treated hamsters after O. viverrini acute infection (AI) for 21 days and chronic infection (CI) for 4 months. The reduction rate of hydroxyproline content in the livers of the AI group with 6-month praziquantel treatment (71%) was higher than that of corresponding praziquantel-treated CI group (13%), similar to the decrease in the thickness of peribiliary fibrosis. Hepatic mRNA levels of collagen I significantly decreased compared with untreated control after praziquantel treatment both in AI and CI groups. Expression of collagen III significantly decreased in the AI group but unchanged in the CI group. MMP-9 and MMP-13 (except 3 months in CI group) levels significantly decreased in both groups. Notably, expression of MMP-7 level increased at 1 and 6 months in both AI and CI groups, respectively. MMP-2 level did not change in both groups. Gelatinase activity of MMPs-2 and -9 was associated with their transcriptional levels. Tissue inhibitors of MMP (TIMP)-1 and TIMP-2 significantly decreased in the AI group, whereas TIMP-1 levels did not change and TIMP-2 level was significantly increased in CI group. Praziquantel treatment increased the expression of TNF-alpha in both groups, suggesting an association with MMP-7 expression. TGF-beta expression was significantly increased only in the CI group indicating its may involve in TIMPs expression. These results suggest that in animals with chronic O. viverrini infection, praziquantel treatment induces the expression of TGF-beta, and TIMPs and resultant inhibition of MMP activity, leading to slow resorption of hepatic fibrosis.