Protective and therapeutic effects of sildenafil and tadalafil on aflatoxin B1-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has been classified as one of the most common forms of liver cancer occurring worldwide, and risk factors include hepatitis B & C virus, alcoholism, and dietary carcinogens like aflatoxin B1 (AFB1), which is produced by fungus Aspergillus flavus and Aspergillus parasiticus. Metabolism of AFB1 resulted into the formation of AFB1-exo-8, 9-epoxide which is largely responsible for HCC development. So far conventional cytotoxic chemotherapy has not provided much benefit in HCC, necessitating the need for newer treatment modalities. Recent reports suggest that phosphodiesterase-5 inhibitors (PDE5i) may have anticancer activity, but till date, the anticancer property of PDE5i (tadalafil & sildenafil) has not been evaluated in HCC. The present study was aimed to define the anticancer property of tadalafil and sildenafil against AFB1-induced HCC rats. Rats were randomly divided into five groups with five rats in each group. Except normal control group, rats of all other groups were fed with 5% alcohol via drinking water for 3 weeks. After 3 weeks, two successive dose of AFB1 (1 mg/kg bw, ip) was administered on subsequent days followed by the administration of PDE5i (tadalafil & sildenafil, 10 mg/kg bw) along with drinking water after 6 weeks of treatment with AFB1 for 2 weeks. An in-depth investigation into its mechanistic aspect revealed that development of HCC induced by aflatoxin B1, decreased the mRNA expression and activity of antioxidant enzyme SOD, GPx, catalase, GR and GST, and GSH content with a concomitant increase in the level of lipid peroxidation. Post-treatment with PDE5 inhibitor (tadalafil & sildenafil) restored the above parameters towards normal, and this result was more effective in case of sildenafil. Thus, results from the above studies suggest that PDE5 inhibitors may act as anticancer agents by preventing the development and progression of HCC by modulating the key parameters of antioxidant pathway.

Glutathione S-transferase pi (GST-pi) inhibition and anti-inflammation activity of the ethyl acetate extract of Streptomyces sp. strain MJM 8637.

To investigate the anti-cancer properties of soil-borne actinobacteria, MJM 8637, the glutathione S-transferase pi (GST-pi) assay, anti-tumor necrosis factor (TNF)-α assay, the level of antioxidant potential by DPPH radical scavenging activity, NO scavenging activity, and ABTS radical scavenging activity in ethyl acetate extract were determined. The 16S rDNA sequencing analysis revealed that Streptomyces sp. strain MJM 8637, which was isolated from Hambak Mountain, Korea, has 99.5% similarity to Streptomyces atratus strain NBRC 3897. The physiological and the morphological characteristics of the strain MJM 8637 were also identified. The ethyl acetate extract of MJM 8637 inhibited TNF-α production approximately 61.8% at concentration 100 µg/ml. The IC50 value of the strain MJM 8637 extract on GST-pi was identified to be 120.2 ± 1.6 µg/ml. In DPPH, NO, and ABTS radical scavenging assays, the IC50 values of the strain MJM 8637 extract were found to be 977.2 µg/ml, 1143.7 µg/ml, and 454.4 µg/ml, respectively. The ethyl acetate extract of the strain MJM 8637 showed 97.2 ± 1.3% of cell viability at 100 µg/ml in RAW 264.7 cell viability assay. The results obtained from this study suggest that the ethyl acetate extract of Streptomyces sp. strain MJM 8637 could be considered as a potential source of drug for the cancers that have multidrug resistance with its GST-pi inhibition and anti-inflammation activities, and low cytotoxicity.

Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin.

Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71% and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver-Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 µM and K i (CDNB) as 0.26 ± 0.03 µM.

The expression of MDR1, P- gp and GST- Pi biomarkers in peripheral blood from patients with refracto- ry epilepsy / 中国神经精神疾病杂志

Objective The pathogenesis of intractable epilepsy was explored by examining the expression of the P-gp , GST-Pi as well as MDR1 in peripheral blood of the patients with intractable epilepsy. The potential of the above mentioned three genes as the biomarkers for treatment of intractable epilepsy was investigated. Methods Thirty-one sub?jects with refractory epilepsy, 33 subjects under good circumstances by antiepileptic drugs, and 37 healthy subjects were included in the present study. fluorescence quantitative polymerase chain reaction and flow cytometry were used to detect mRNA levels of MDR1 and GST-Pi and P-gp of MDR1 in the peripheral blood of the patients, respectively. Results The expression levels of MDR1 and GST-Pi were significantly higher in the AEDs intractable group(1.36±0.14,0.585±0.257) than in the treatment group(0.82±0.15,0.309±0.217, P<0.05)The expression levels of MDR1 and GST-Pi were signifi?cantly higher in the AEDs treatment group than in the normal group(0.27±0.07,0.134±0.223,P<0.05). The expression levels of P-gp were significantly higher in the AEDs of the intractable group(0.104±0.084)than in the treatment group (0.063 ± 0.030, P<0.05). The GST-Pi gene expression levels were significantly higher in three(0.535 ± 0.256)or two (0.425±0.254)kinds of antiepileptic drugs combination therapy than in single drug treatment(0.267±0.265, P<0.05). Leucocyte P-gp levels were significantly higher in combination therapy of three kinds of antiepileptic drugs(0.141 ± 0.096)than in combination therapy of two kinds of antiepileptic drugs(0.071±0.020)or in monotherapy(0.050±0.020, P<0.05). Conclusion MDR1 and GST-Pi gene expression levels of peripheral blood can be used as the reference in?dex for treatment of intractable epilepsy and the resistant index of combination treatment for intractable epilepsy.

Effect of Ginkgo biloba extract on the expressions of Cox-2 and GST-Pi in rats with hepatocellular carcinoma risk.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers worldwide, and the pathogenesis is complicated at present. There iare few effective therapeutic measures, and novel therapeutic strategies are urgently required to improve clinical outcome. Ginkgo biloba extract (EGb) is reported to have an anti-cancer activity. OBJECTIVES: To explore the effect of EGb on expressions of cyclooxygenase-2 (Cox-2) and glutathione S-transferase Pi (GST-Pi) in the pathogenesis of HCC. METHODS: 120 Wistar rats were divided into three groups at random: normal control group (control group), HCC risk group without treatment (HCC risk group), HCC risk group treated with EGb (EGb group); n=40, respectively. The HCC risk in rat was induced by aflatoxin B1 injection. At the end of 13-week, 33-week, 53-week and 73-week, 10 rats in each group were killed and the relevant samples were collected. RESULTS: The mRNA and protein expressions of Cox-2 and GST-Pi were measured by real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and western-blot. When compared with those in the control group in 73-week, the mRNA and protein expressions of GST-Pi in EGb group were weaker than those in HCC risk group in 73-week. However, the mRNA and protein expressions of Cox-2 in HCC risk group were increased than that of control group, and there was no statistical difference for mRNA and protein expressions of Cox-2 between HCC risk group and EGb group. CONCLUSION: EGb can regulate the expression of GST-Pi, but it does not seem to have an effect on Cox-2 expression in the liver of HCC risk rats.

Morphoproteomic confirmation of an activated nuclear factor-кBp65 pathway in follicular thyroid carcinoma.

The role of nuclear factor (NF)-кBp65 pathway in the pathogenesis of follicular thyroid carcinoma (FTC) has not been fully investigated. We retrieved 10 cases of FTC from our file. Tissue microarrays (TMAs) were constructed using 2.0 mm cores from formalin-fixed, paraffin-embedded tissue blocks. TMA sections were immunohistochemically stained for phosphorylated (p)-NF-кBp65 (Ser 536), cyclooxygenase-2 (COX-2), IL-8, and glutathione S-transferase (GST)-pi. Staining intensity (0-3+), extensiveness (0-100%) and subcellular compartmentalization were evaluated. Both nuclear and cytoplasmic immunoreactivities with p-NF-кBp65 (Ser 536) antibodies were observed in all 10 cases, including moderate to strong nuclear staining intensity with a range of extensiveness in 20% - 100% of tumor cells. Moderate (2+) or strong (3+) cytoplasmic expressions of COX-2 and IL-8 were present in 60-100% and 50- 100% of tumor cells, respectively, in all cases. GST-pi was diffusely (70-100%) and moderately or strongly staining the tumor cytoplasm in all cases (except one case with insufficient tissue) with three of them demonstrating nuclear positivity as well. Morphoproteomic analysis reveals the constitutive activation of the NF-кBp65 pathway in follicular thyroid carcinomas as evidenced by phosphorylation at Ser 536 with nuclear translocation and with correlative expression of transcriptionally activated gene products (COX-2, IL-8, and GST-pi). This observation may provide a molecular basis for the tumor biology and targeted therapies for follicular thyroid carcinoma.

Immunohistochemical Expression of Placental Form of Glutathione S-transferase (GST-pi) in Fetal Skin / 대한피부과학회지

BACKGROUND: Glutathione S-transferases(GST) are a family of multi-functional enzymes involved in cellular detoxification and excretion of a variety of exogenous and endogenous toxic or carcinogenic compounds. The GST family has been divided into three classes, alpha, mu, and pi, based on substrate specificity and sequence homology. GST-pi is an acidic type and predominant in skin, small intestine, breast, lung and prostate. The overexpression of GST-pi associated with skin tumor and tumor-like lesion suggests that GST-pi is a major detoxifying enzyme in skin tumors. OBJECTIVE: The purpose of this study was to observe the expression and the distribution pattern of GST-pi in the human fetal skin. METHODS: Skin was obtained from the scalp, chest, and sole of 49 human fetuses, ranging from 8th to 40th weeks of gestational age. Immunohistochemical staining was performed using avidin biotin peroxidase complex method on paraffin embedded tissue using antirabbit polyclonal antibody against the human GST-pi. RESULTS: GST-pi was expressed in intermediated layer of epidermis at 8th week, and gradually increased in strength of expression stronger in suprabasal layer. In hair unit, GST-pi was expressed in sebaceous gland, bulge, hair matrix cell and outer root sheath cell from 15th week. In eccrine gland, also GST-pi was expressed in central differentiated cells of intradermal eccrine duct from 18th week, and in terminal duct and acini from 26th week of fetal age. CONCLUSION: GST-pi was expressed from the 8th week of gestation suggesting that GST-pi plays an important role in detoxification for the protection of the skin in fetal stage from the various toxic agent.

Multidrug Resistance-Related Gene Expressions in Germ Cell Tumors in Testis / 대한비뇨기과학회지

The development of drug resistance is a major obstacle in effective cancer chemotherapy. Multidrug resistance(MDR) is a widely studied phenomenon of interest to both clinicians and research workers because many different cancer chemotherapeutic agents are involved and the genetic basis of MDR is understood to a large extent. Several studies show that the P-glycoprotein (P-gp), multidrug resistance-associated protein(MRP), glutathione-s-transferase-pi(GST-pi), and DNA topoisomerase II(topo II) have a complex role for the malignant phenotypes and MDR. Clearly, there is a need to investigate links between the diverse characteristics of tumors and the emergence of drug resistance. We have therefore used reverse transcription-polymerase chain reaction(RT-PCR) assay to analyze expressions of MDR-related genes including the mdr1, MRP, topo II and GST-t gene in normal testis and testis tumors. The results are as follows: 1. The expression levels of topo II and GST-n genes in testis tumors, especially in the nonseminomatous germ cell tumor(NSGCT), were significantly higher than in normal testis(p=0.015 and 0.025, respectively). 2. The MDR-related gene expressions in testis tumors did not appear to be correlated with stage(p>0.05 in each case) and chemotherapy status(p>0.05 in each case). 3. MRP expression levels in primary tumors were much higher than in metastatic tumors. 4. In NSGCT, the coexpressions of the topo II and GST-r or MRP genes were significantly correlated but, seminoma showed no correlation between MDR-related genes in the same sample. Although the mechanism of these connection are not known, the results suggest that these expression patterns and higher GST-rexpression in NSGCF compared to seminoma confer diverse characteristics including difference in the presentation of tumor markers and the responsiveness to chemotherapy on NSGCF and seminoma.

Immunohistochemical Observation of Placental Form of Glutathione S-Transferase in Squamous Cell Carcinoma

Glutathione S-Transferase (GST) is a conjugation enzyme in the metabolism of exogenous and endogenous lipophilic compounds for their excretion and detoxification. Acidic isozyme of GST, GST-Pi, has been recognized as a preneoplastic marker in the experimental hyperplastic nodules of liver in rats, and GST-Pi is abundant in the squamous cells of the skin, also. This histochemical study was carried out to evaluate the distribution and the relationship between the differentiation status of squamous cells in dysplastic or neoplastic epithelium in various organs. The human placental form of glutathione S-transferase (GST-Pi) were stained immunohistochemically with specific anti GST-Pi rabbit antibody in 23 cases of human squamous cell carcinomas. The patients consisted of 14 cases from the uterine cervix, 3 cases from the esopahgus, 3 cases from the lung and 3 cases from the larynx. The results obtained were as follows; 1. Basal cells in normal mucosa were stained negative for GST-Pi while superficial keratinocytes were stained moderately positive. Basal dysplastic cells were stained negatively or weakly positive. Carcinoma cells especially large cells either keratinizing or nonkeratinizing were stained moderately to strongly. Carcinoma cells surrounding keratin pearl were strongly reacted with GST-Pi than other carcinoma cells. 2. Differentiated cells of squamous cell carcinoma showed moderate to strong positive reaction to GST-Pi staining irrespective of its site of origin. 3. Therefore, Immunohistochemical staining pattern of GST-Pi in various squamous carcinoma cells showed similar immunohistochemical reaction to the GST-pi, which is closely correlated to the degree of differentiation, keratinigation and also suggested that squamous carcinoma cells had abundant GST-Pi related detoxifying system.