Primary parapharyngeal tumours: a review of 21 cases.

BACKGROUND: The diagnostic and therapeutic procedures performed in a series of patients with primary parapharyngeal space (PPS) tumours treated at the ENT Departments of San Giovanni Bosco Hospital, Turin, and of the Pugliese-Ciaccio Hospital, Catanzaro, Italy, in the period 2001-2010 are evaluated. MATERIALS AND METHODS: The retrospective review included 20 patients, 11 male and 9 female, average age of 41 years operated on for 21 primary PPS tumours. The most common tumours found were neurogenic neoplasms, while those of salivary origin were the next most common. RESULTS: There were 14 paragangliomas (7 originating from carotid glomus, 5 from vagal and 2 from tympanicum), 1 sympathetic chain schwannoma and 6 pleomorphic adenomas. All the tumours were benign in nature and gave rise to few signs or symptoms. Patients underwent preoperative computed tomography (CT) scan or magnetic resonance imaging (MRI) or both. Most contrast-enhanced masses were submitted to some type of angiography. Most of the surgeries were planned through imaging alone, as preoperative fine needle aspiration (FNA) biopsy was performed only in six cases. Four different approaches were adopted for tumour removal: transcervical, transcervical/transparotid, cervical-transparotid-transmandibular and infratemporal fossa approach. There was no operative mortality, though neurologic morbidity was significant. Follow-up, extended to a maximum of 11 years, did not reveal any recurrences. In conclusion, neurogenic tumours may be the most common of PPS masses. Surgery is the mainstay treatment and external approaches offer the potential for satisfactory tumour resection. Of such external approaches, transcervical and cervical/transparotid are the most often used in benign forms. CONCLUSION: The number of perioperative complications encountered in this series confirms the difficulty of performing surgery in this complex area, even in benign cases. The chances of avoiding vascular damage and saving the trunks or most of the nerve fibres involved depend not only on the skill and experience of the surgeon but also on the anatomy of the lesion, the type of connection between the tumour and the nerve from which it originates and the distribution of neural fibres in or around the tumour mass.

Antiproliferative effects of 5-fluorouracil and oxaliplatin in colon cancer cell lines: comparison of three different cytotoxicity assays.

Adjuvant therapy in colorectal cancer has evolved to become the standard of care, whereas the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy of human tumors. Therefore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells has a great importance. A number of cytotoxicity assays are currently available, each of them using a specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. The purpose of this study is to compare, under identical experimental conditions, three common cytotoxicity assays (ATP-lite, MTT and CCK-8 assays) in the assessment of the anti-proliferative effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) on three colon cancer cell lines (WiDr, SW620 and HT-29). Regarding 5-FU, the three assays were found to be significantly correlated with a moderate or high correlation coefficient, whereas in the case of OHP we found different outcomes among the assays. Our study demonstrates that the CCK-8 is the most sensitive assay for detecting changes of cell viability, suggesting that the viability measured in cells after drug exposure depends on several parameters like the drug used, the biological characteristics of the target cell and the specific approach employed by the method to detect distinct cell growth and metabolic functions.

A congenital anterior urethrocutaneous fistula in a boy whose mother was exposed to ionizing radiations: case report and literature review.

Anterior congenital urethrocutaneous fistula is a rare anomaly that may present in an isolated fashion or in association with other anomalies of the genital urinary tract or anorectal malformations. A case of congenital anterior urethrocutaneous fistula nonassociated with other congenital anomalies in a 3-year-old male whose mother has been exposed to Chernobyl's nuclear fallout is described. The patient was successfully operated with no recurrence. We report a review of the literature about etiology and surgical strategy including the role of ionizing radiations. The congenital anterior urethrocutaneous fistula represents a rare malformation. The etiopathogenesis is unknown.

Evaluation of in vitro cytotoxicity of oxaliplatin and 5-fluorouracil in human colon cancer cell lines: combination versus sequential exposure.

Adjuvant therapy has evolved to become the standard care of colon cancer, but the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy. Furthermore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells could be of a great importance. As primary human colon cancer cultures from fresh tumor are technically difficult to obtain, experiments with human cancer cell lines remain essential to explore new adjuvant chemotherapy drugs, to investigate the individual responsiveness to the known agents, and particularly to clarify how these chemotherapeutic agents could be used in maximizing outcomes. In the present study we evaluate the cytotoxic effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) and of their pharmacological interaction in three human colon cancer cell lines (WiDr, HT-29 and SW620), by using an ATP luminescence assay (ATPlite; Perkin Elmer), displaying high sensitivity, linearity and reproducibility. Cell cycle, apoptosis and CD44 expression were investigated with flow cytometry. Our results show that the drug combinations inhibited the cell growth more than each drug alone in all colorectal cancer cell lines. Interestingly, the sequential exposure of OHP and 5-FU resulted in the most cytotoxic effect in all colon cancer cell lines, when compared to the simultaneous one. Our results focus on the powerful cytotoxic effect of 5-FU-OHP combination, when used in sequential exposure, suggesting interesting implications for a rational use of 5-FU, OHP combination in colon-rectal cancer therapy.

Retroperitoneoscopic lumbar sympathectomy for nonreconstructable arterial occlusive disease.

AIM: The aim of this study was to present our experience with video-assisted lumbar sympathectomy for non-reconstructive arterial occlusive disease in a series of 23 consecutive patients whose predominant symptoms were unilateral rest pain, limited skin ulcerations or gangrene of the toes. METHODS: All the procedures were performed with retroperitoneal approach, dorsal position of the patient and simple digital dissection of the retroperitoneal space. RESULTS: The operations were successfully performed in all patients except for 2, who immediately underwent open conversion. A urinoma caused by ureteral lesion was the only severe complication in this series. The mean operative time of the procedure was 55 min and the hospital stay was 2 or 3 days. No parenteral analgesics were administered postoperatively. At 1 month from operation, 20 patients out of 23 had significant relief of rest pain and improvement of ischemic lesions. After a median follow-up of 36 months, 2 patients had died, 4 underwent some type of distal amputation, 1 had recurrent rest pain and the other 16 reported persistent improvement of pain or dystrophic changes. CONCLUSIONS: Retro-peritoneoscopic technique appears the modern and less invasive version of the lumbar surgical sympathectomy.

Role of cyclooxygenases 1 and 2 in the modulation of neuromuscular functions in the distal colon of humans and mice.

BACKGROUND: Cyclooxygenase isoforms (COX-1, COX-2) may exert differential regulatory actions on enteric motor functions under normal or pathological conditions. AIMS: To examine the occurrence and functions of COX-1 and COX-2 in the neuromuscular compartment of normal distal colon using human and murine tissue. METHODS: Gene expression (human, mouse), protein expression (human), gene deletion (mouse), and the effects of dual and isoform specific COX inhibitors on in vitro motility (human, mouse) were investigated. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed mRNA expression of COX-1 and COX-2 in human and wild-type mouse colonic muscle whereas only COX-2 or COX-1 was detected in COX-1 or COX-2 knockout animals. Immunohistochemistry localised both isoforms in neurones of myenteric ganglia, COX-1 in circular layer myocytes, and COX-2 in longitudinal muscle. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor), or DFU (COX-2 inhibitor) enhanced atropine sensitive electrically induced contractions of human longitudinal muscle. The most prominent actions were recorded with indomethacin or SC-560 plus DFU. These results were confirmed under pharmacological blockade of non-cholinergic nerves. Atropine sensitive contractions evoked by carbachol in the presence of tetrodotoxin were enhanced by indomethacin or DFU but not by SC-560. In wild-type mice, contractile responses to electrical stimulation were enhanced by indomethacin, SC-560, or DFU. SC-560 potentiated electrically induced contractions in COX-2, but not COX-1, knockout mice. In contrast, DFU enhanced the contractions elicited by electrical stimuli in COX-1, but not in COX-2, knockout mice. CONCLUSIONS: These results indicate that COX-1 and COX-2 are expressed in the neuromuscular compartment of normal human colon where they modulate cholinergic excitatory control of colonic motility at prejunctional and postjunctional sites, respectively.

Curcumin is a potent inhibitor of phenol sulfotransferase (SULT1A1) in human liver and extrahepatic tissues.

1. Curcumin has anti-carcinogen effects and is under clinical evaluation as a potential colon cancer chemopreventive agent. The first aim was to see whether curcumin inhibited phenol sulfotransferase (SULT1A1) and, if so, to study the variability of the IC(50) of curcumin for SULT1A1 in 50 human liver samples. For comparative purposes, the inhibition of catechol sulfotransferase (SULT1A3) in five human liver specimens was studied. The second aim was to measure the IC(50) of curcumin against SULT1A1 in five samples of human duodenum, colon, kidney and lung. 2. Curcumin was a potent inhibitor of SULT1A1 in human liver; the mean +/- SD and median of IC(50) were 14.1 +/- 7.3 nM and 12.8 nM, respectively. The IC(50) ranged from 6.2 to 30.6 nM between the 5th and 95th percentiles and the fold of variation was 4.9. The distribution of IC(50) was positively skewed (skewness 1.2) and deviated from normality (p = 0.0004). 3. Curcumin inhibited human SULT1A3, and the inhibition was studied in five liver specimens with an IC(50) of 4324 +/- 1026 nM. This inhibition was greater than the IC(50) of curcumin for SULT1A1 (p < 0.0001). 4. In the extrahepatic tissues, the IC(50) of curcumin for SULT1A1 was 25.9 +/- 4.8 nM (duodenum), 25.4 +/- 6.8 nM (colon), 23.4 +/- 2.2 nM (kidney) and 25.6 +/- 5.6 nM (lung). Inhibition in these tissues is greater than that of curcumin for SULT1A1 in human liver (p < 0.0001). 5. In conclusion, curcumin is a potent inhibitor of SULT1A1 in human liver, duodenum, colon, kidney and lung. The IC(50) of curcumin for SULT1A1 varied 4.9-fold in human liver. The comparison of the present data with those of the literature revealed that the IC(50) of curcumin in the liver and extrahepatic tissues is one order of magnitude lower that the peak serum concentration of curcumin after therapeutic doses of 4 g to humans.

Evaluation of telomerase in the development and progression of colon cancer.

Telomerase activity, a cardinal requirement for immortalization, is a crucial step in the development of cancer and has been studied in many kinds of malignant tumours for clinical diagnostic and/or prognostic utilities. Using a PCR-based TRAP assay, we investigated telomerase activity in 8 adenomatous polyps, 9 dysplastic polyps, and in 36 paired cancer-normal mucosa specimens, one liver and one spleen metastasis from patients resected for sporadic colorectal cancer. Telomerase was absent or very low in normal mucosa and in adenomatous polyps. Dysplastic polyps and adenocarcinoma samples showed telomerase activity, with higher levels in cancer tissues compared to dysplastic lesions. A high telomerase activity was shown to be associated with late-staged cancers and metastasis, providing arguments supporting the role of telomerase not only in the development but also in the progression of colorectal carcinoma. Moreover, telomerase evaluation may help to confirm the malignant transformation in polypoid colorectal lesions with different levels of dysplastic alterations.

7-OH-flavone is sulfated in the human liver and duodenum, whereas 5-OH-flavone and 3-OH-flavone are potent inhibitors of SULT1A1 activity and 7-OH-flavone sulfation rate.

1. The aim of this investigation was to see whether 7-OH-flavone, 5-OH-flavone and 3-OH-flavone, which are present in edible vegetables, fruit and wine, are substrates or inhibitors of human liver and duodenum sulfotransferase. 2. An assay was set up to study the sulfation of 7-OH-flavone, and using this assay, it was observed that 7-OH-flavone was sulfated and the rate of sulfation (mean +/- SD) was 324 +/- 87 pmol min(-1) mg(-1) (liver) and 584 +/- 164 pmol min(-1) mg(-1) (duodenum; p < 0.0001). 3. 7-OH-flavone sulfotransferase followed Michaelis-Menten kinetics and the K(m) (mean +/- SD) was 0.2 +/- 0.04 microM (liver) and 1.1 +/- 0.3 microM (duodenum; p = 0.008). V(max) (mean +/- SD) was 392 +/- 134 pmol min(-1) mg(-1) (liver) and 815 +/- 233 pmol min(-1) mg(-1) (duodenum; p = 0.016). 4. 5-OH-flavone and 3-OH-flavone were not sulfated and were inhibitors of human liver and duodenum SULT1A1 activity and 7-OH-flavone sulfation rate. 5. The IC50 of 5-OH-flavone for SULT1A1 was 0.3 +/- 0.06 microM (liver) and 0.3 +/- 0.1 microM (duodenum; n.s.) and those of 3-OH-flavone were 1.0 +/- 0.1 microM (liver) and 1.6 +/- 0.03 microM (duodenum; p = 0.0006). 6. There was inhibition of 7-OH-flavone sulfation rate by 5-OH-flavone and 3-OH-flavone. The IC(50) of 5-OH-flavone for the sulfation rate of 7-OH-flavone was 3.5 +/- 0.5 microM (liver) and 69 +/- 18 microM (duodenum; p < 0.0001) and for 3-OH-flavone it was 18 +/- 3.4 microM (liver) and 213 +/- 47 microM (duodenum; p < 0.0001). 7. The position of the hydroxy group confers to the molecules of OH-flavones the quality of substrate or inhibitor of sulfotransferase.

Sulfation of R(-)-apomorphine in the human liver and duodenum, and its inhibition by mefenamic acid, salicylic acid and quercetin.

1. The aims were to study the sulfation of R-(-)-apomorphine (hereafter apomorphine) in the human liver and duodenum, and to study the rate of inhibition of apomorphine sulphation by mefenamic acid, salicylic acid and quercetin also in the human liver and duodenum. 2. A rapid and sensitive method was developed to measure the sulfation rate of apomorphine in the human liver and duodenum. The method was based on the use of 0.4 micro M 3'-phosphoadenosine-5'-phosphosulfate-[(35)S] (PAPS) and 50 micro M apomorphine. The unreacted PAPS was precipitated with barium hydroxide, barium acetate and zinc sulfate. 3. The rate of apomorphine sulfation (mean +/- SD and median) was 261 +/- 82 and 242 pmol min(-1) mg(-1), respectively (liver), and 433 +/- 157 and 443 pmol min(-1) mg(-1), respectively (duodenum). The apomorphine sulfation rate was higher in the duodenum than in the liver (p = 0.0005). 4. Apomorphine sulfation was correlated with SULT1A1 activity in the liver (r(2) = 0.363, p = 0.005) and duodenum (r(2) = 0.494, p = 0.0005), but it did not correlate with SULT1A3 activity both in the liver and duodenum. 5. The K(m) estimate of apomorphine sulfation rate was 20 +/- 3.6 (liver) and 6.5 +/- 0.2 microM (duodenum, p = 0.024), and the V(max) estimate was 248 +/- 99 (liver) and 636 +/- 104 pmol min(-1) mg(-1) (duodenum, p = 0.018). 6. Mefenamic acid, salicylic acid and quercetin were potent inhibitors of apomorphine sulfation rate in the liver, and the IC(50) estimates were 16 +/- 0.2 nM, 54 +/- 8.6 microM and 18 +/- 2.8 nM, respectively. These compounds were poor inhibitors of apomorphine sulfation in the duodenum. 7. Apomorphine is sulfated by the human liver and duodenum, the highest activity being associated with the duodenum. The K(m) of apomorphine sulfotransferase is in the order of micro M both in the liver and duodenum. The non-steroidal anti-inflammatory drug mefenamic acid and the natural flavonoid quercetin inhibit the hepatic sulfation of apomorphine with an IC(50) in the order of nM.

Evaluation of telomerase mRNA (hTERT) in colon cancer.

Telomerase activation, a cardinal requirement for immortalization, is a crucial step in the development of malignancy and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. By reverse transcription-PCR, using primers within the reverse transcriptase domain of hTERT, we investigated telomerase messenger in 8 adenomatous and 9 dysplastic polyps, and in 32 paired cancer-normal mucosa specimens, one liver and one spleen metastasis from patients resected for sporadic colorectal cancer. Telomerase messenger was absent or very low in normal mucosa and in adenomatous polyps. Dysplastic polyps and adenocarcinoma samples showed hTERT mRNA, with higher levels in cancer tissues compared to dysplastic lesions. A high telomerase messenger level was shown to be associated with late-staged cancers and with metastasis; thus, detection of telomerase messenger may be useful in the early diagnosis of colon cancer, and telomerase may be a new target for therapeutic intervention.

Neoangiogenesis in colon cancer: correlation between vascular density, vascular endothelial growth factor (VEGF) and p53 protein expression.

Angiogenesis is an essential requirement for the development, progression and metastasis of malignant tumors. Vascular endothelial growth factor (VEGF) plays an essential role in the development of angiogenesis of numerous solid malignancies, including colon cancer. The tumor suppressor gene p53 is a potent transcriptional regulator of genes which are involved in many cellular activities, including cell-cycle arrest, apoptosis and angiogenesis. In order to better understand the relation among p53 status, VEGF expression and microvessels count (MVC) in colon cancer, we evaluated immunoreactivity for CD34 endothelium-associated antigen, VEGF and p53 proteins in 43 cases of colon adenocarcinoma. Our results demonstrated an association between VEGF expression, p53 status and angiogenesis, suggesting that mutant p53 plays a central role in promoting angiogenesis in colon cancer progression.

Ranitidine bismuth citrate-based triple therapy for seven days, with or without further anti-secretory therapy, is highly effective in patients with duodenal ulcer and Helicobacter pylori infection.

OBJECTIVE: To compare the efficacy of two protocols for the eradication of Helicobacter pylori infection and the healing of active duodenal ulcer: (i) ranitidine bismuth citrate (RBC) plus two antibiotics for 7 days, and (ii) the same triple therapy followed by 3 weeks of anti-secretory drug treatment. METHODS: The study comprised 102 patients with active duodenal ulcer and H. pylori infection; the patients were randomized to open treatment with either RBC 400 mg b.d. plus amoxycillin 1 g b.d. and clarithromycin 500 mg b.d. for 7 days, or the same treatment followed by 3 weeks of RBC 400 mg b.d. alone. Ulcer healing was confirmed by endoscopy. H. pylori eradication was assessed by endoscopy, rapid urease test and histology. RESULTS: The ulcer healed in 48/50 patients on RBC-based triple therapy alone (96.0%) and in 51/52 patients on triple therapy plus further anti-secretory treatment (98.1%). On an intention-to-treat basis, H. pylori had been successfully eradicated in 42/50 patients on triple therapy (84.0%) and in 44/52 patients on triple therapy plus anti-secretory treatment (84.6%), while by per protocol analysis the H. pylori eradication rates were 91.3% (42/46) and 89.8% (44/49), respectively. CONCLUSIONS: One-week triple therapy with RBC, amoxycillin and clarithromycin is highly effective in eradicating H. pylori and healing duodenal ulcers, even if not followed by anti-secretory drug treatment.

[Clinical significance of a carcinogenesis model of colorectal carcinoma]. / Importanza clinica di un modello di carcinogenesi nel carcinoma del colon-retto.

OBJECTIVE: Carcinoma of the rectal colon begins as a small neoplastic polyp which gradually increases in size and, after passing through various degrees of dysplasia, develops into an overtly malignant carcinoma. Clinical experience suggests that patients may be divided into subgroups based on the aggressivity of the tumour. The genetic mutations associated with colorectal cancer have been studied and it is known that the genes primarily responsible for biological changes in the tumour cell, in the early stages, are APC, hMSH2, k-ras2 and, in particular, p53. Indeed, the mutation at the level of gene p53 has been recognized as the most common mutation in tumour cells. The aim of this study was investigate the role of p53 and CD34 in colorectal cancer. METHODS: We studied p53 positivity using immunohistological methods and compared our results with the site, stage (using the TNM system) and histological grade of the tumour. We evaluated CD34 positivity using the same methods in order to detect and quantity the presence of angiogenesis in colorectal cancer. RESULT: P53 was found to be markedly raised in the T3 stage of colorectal cancer, while its expression was decreased in stage T2 and stage T1 carcinomas and it was not detectable in adenomas. These results suggest a close correlation between the tumour stage and the expression of p53. An analogous correlation was found between CD34 expression and angiogenesis. CONCLUSION: The overexpression of p53 in epithelial cells and raised angiogenesis (as reflected in CD34 levels) in stromal cells could represent useful prognostic factors in the management of colorectal cancer.

Differential inhibition of human liver and duodenum sulphotransferase activities by quercetin, a flavonoid present in vegetables, fruit and wine.

1. Quercetin is a natural flavonoid present in vegetables, fruit and wine, and is known to inhibit sulphotransferase. Drugs are often taken orally and the intestinal mucosa is an early site of drug metabolism. The aims of this investigation were to study the inhibition of dopamine, (-)-salbutamol, minoxidil and paracetamol sulphation by quercetin in the duodenal mucosa and liver and to compare the IC50 in these tissues. 2. The rates (pmol min(-1) mg(-1)) of sulphation of 4-nitrophenol were 343+/-92 (liver) and 164+/-22 (duodenum; p = 0.031), of dopamine were 15+/-11 (liver) and 656+/-516 (duodenum; p = 0.049), of (-)-salbutamol 153+/-31 (liver) and 654+/-277 (duodenum; p = 0.018), of minoxidil were 156+/-47 (liver) and 105+/-7 (duodenum; n.s.), and of paracetamol were 229+/-86 (liver) and 328+/-187 (duodenum; n.s.). 3. The IC50 of quercetin for 4-nitrophenol was 48+/-11 nM (liver) and 56+/-1 nM (duodenum, n.s.), for dopamine was 5.7+/-0.7 microM (liver) and 170+/-12 microM (duodenum, p < 0.0001), for (-)-salbutamol was 54+/-4 nM (liver) and 16+/-8 microM (duodenum; p = 0.025), for minoxidil was 134+/-22 nM (liver) and 3+/-0.3 microM (duodenum, p = 0.013), and for paracetamol was 57+/-7 nM (liver) and 35+/-1 microM (duodenum; p = 0.0002). 4. Quercetin inhibited the sulphation of 4-nitrophenol, dopamine, (-)-salbutamol, minoxidil and paracetamol both in liver and duodenum. With dopamine, (-)-salbutamol, minoxidil and paracetamol as substrates, quercetin was a more potent inhibitor in the liver than the duodenum. Such a difference may reflect the different composition of sulphotransferase forms in the liver and duodenum.

Differential inhibition of hepatic and duodenal sulfation of (-)-salbutamol and minoxidil by mefenamic acid.

OBJECTIVE: The aim of this investigation was to determine whether mefenamic acid and salicylic acid inhibit the sulfation of (-)-salbutamol and minoxidil in the human liver and duodenum, and if so, to ascertain whether the 50% inhibitory concentration (IC50) estimates are different in the two tissues. METHODS: Sulfotransferase activities were measured for 10 mM (-)-salbutamol and 5 mM minoxidil, and the concentration of 3'-phosphoadenosine-5'-phosphosulphate-[35S] was 0.4 microM. RESULTS: The IC50 estimates for (-)-salbutamol and minoxidil sulfation of mefenamic acid were 72 +/- 5.4 nM and 1.5 +/- 0.6 microM (liver), respectively, and 161 + 23 microM and 420 +/- 18 microM (duodenum), respectively. The figures for the liver were significantly lower (P < 0.0001) than those for the duodenum. The IC50 estimates for (-)-salbutamol sulfation of salicylic acid were 93 +/- 11 microM (liver) and 705 +/- 19 microM (duodenum, P < 0.0001). Salicylic acid was a poor inhibitor of minoxidil sulfation. CONCLUSION: The IC50 estimates for (-)-salbutamol sulfation of mefenamic acid and salicylic acid are lower than their unbound plasma concentrations after standard dosing, suggesting that mefenamic acid and salicylic acid should inhibit the hepatic sulfation of (-)-salbutamol in vivo.

Sulphation of resveratrol, a natural compound present in wine, and its inhibition by natural flavonoids.

1. Resveratrol, a polyphenolic compound present in grape and wine, has beneficial effects against cancer and protective effects on the cardiovascular system. Resveratrol is sulphated, and the hepatic and duodenal sulphation might limit the bioavailability of this compound. The aim of this study was to see whether natural flavonoids present in wine, fruits and vegetables inhibit the sulphation of resveratrol in the human liver and duodenum. 2. In the liver, IC50 for the inhibition of resveratrol sulphation was 12+/-2 pM (quercetin), 1.0+/-0.04 microM (fisetin), 1.4+/-0.1 microM (myricetin), 2.2+/-0.1 microM (kaempferol) and 2.8+/-0.2 microM (apigenin). Similarly, in the duodenum, IC50 was 15+/-2 pM (quercetin), 1.3+/-0.1 microM (apigenin), 1.3+/-0.5 microM (fisetin), 2.3+/-0.1 microM (kaempferol) and 2.5+/-0.3 microM (myricetin). 3. The type of inhibition of quercetin on resveratrol sulphation was studied in three liver samples and was determined to be non-competitive and mixed in nature. Km (mean+/-SD; microM) was 0.23+/-0.07 (control), 0.40+/-0.08 (5 pM quercetin) and 0.56+/-0.09 (10 pM quercetin). Vmax (mean+/-SD; pmol min(-1) x mg(-1)) was 99+/-11 (control), 73+/-15 (5 pM quercetin) and 57 +/- 10 (10 pM quercetin). Kj and Kies estimates (mean+/-SD) were 3.7+/-1.8 pM and 12.1+/-1.7 pM respectively (p = 0.010). 4. Chrysin was a substrate for the sulphotransferase(s) and an assay was developed for measuring the chrysin sulphation rate in human liver. The enzyme followed Michaelis-Menten kinetics and Km and Vmax (mean+/-SD) measured in four livers were 0.29+/-0.07 microM and 43.1+/-1.9 pmol x min(-1) x mg(-1) respectively. 5. Catechin was neither an inhibitor of resveratrol sulphation nor a substrate of sulphotransferase. 6. These results are consistent with the view that many, but not all, flavonoids inhibit the hepatic and duodenal sulphation of resveratrol, and such inhibition might improve the bioavailability of this compound.

Sulphation of resveratrol, a natural product present in grapes and wine, in the human liver and duodenum.

1. Resveratrol, a polyphenolic compound present in grapes and wine, has beneficial effects against cancer and protective effects on the cardiovascular system. It is present in the diet, and the hepatic and duodenal sulphation might limit the bioavailability of this compound. The aim was to study the sulphation of resveratrol in the human liver and duodenum. 2. A simple and reproducible radiometric assay for resveratrol sulphation was developed. It employed 3'-phosphoadenosine-5'-phosphosulphate-[35S] as the sulphate donor and the rates of resveratrol sulphation (mean +/- SD, pmol/min/mg cytosolic protein) were 90 +/- 21 (liver, n = 10) and 74 +/- 60 (duodenum, n = 10, p = 0.082). 3. Resveratrol sulphotransferase followed Michaelis-Menten kinetics and Km (mean +/- SD; microM) was 0.63 +/- 0.03 (liver, n = 5) and 0.50 +/- 0.26 (duodenum, n = 5, p = 0.39) and Vmax (mean +/- SD, pmol/min/mg cytosolic protein) were 125 +/- 31 (liver, n = 5) and 129 +/- 85 (duodenum, n = 5, p = 0.62). 4. Resveratrol sulphation was inhibited by the flavonoid quercetin, by mefenamic acid and salicylic acid, two commonly used non-steroidal anti-inflammatory drugs. IC50 of resveratrol sulphation for quercetin was 12 +/- 2 pM (liver) and 15 +/- 2 pM (duodenum), those for mefenamic acid were 24 +/- 3 nM (liver) and 11 +/- 0.6 nM (duodenum), and those for salicylic acid were 53 +/- 9 microM (liver) and 66 +/- 4 microM (duodenum). 5. The potent inhibition of resveratrol sulphation by quercetin, a flavonoid present in wine, fruits and vegetables, suggests that compounds present in the diet may inhibit the sulphation of resveratrol, thus improving its bioavailability.

Effect of IL-6 overexpression on the metastatic potential of rat hepatocellular carcinoma cells.

BACKGROUND: Previous studies demonstrated that excess IL-6 production correlated with the metastatic potential of rat hepatocellular carcinoma cells. In the work reported here a retroviral construct containing the gene for murine IL-6 was introduced into otherwise nonmetastatic tumor cells to directly determine the effect of IL-6 overexpression on tumor metastatic potential. METHODS: The clonal cell lines 1682.C.2.9.L0 (L0, poorly metastatic) and 1682.C.2.9.L10 (L10, highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet. Viral supernatant was used to infect the PA317 amphotropic cell line, and retrovirus produced from these cells infected the poorly metastatic L0 hepatocellular carcinoma cell line. Neomycin-resistant cells were selected in G418 and designated L0-IL-6. RESULTS: As determined by bioassay, L0 cells produce 10 +/- 1.2 U/mL IL-6 in culture, whereas L10 cells release 95 +/- 11 U/mL (P < 0.01, Student's t-test). Retroviral-mediated IL-6 gene transfer resulted in the production of 1266 +/- 48 U/mL IL-6 by L0-IL-6 cells under identical culture conditions. When an inoculum of 5 x 10(6) cells is injected subcutaneously, both L0 and L10 cell lines result in primary tumors with equivalent rates of growth; only L10 cells metastasize to the lung, however. A similar inoculation of L0-IL-6 cells produced local tumors in all 24 animals tested. Interestingly, 15 of 24 (62%) animals presented with metastatic nodules in the abdominal cavity, whereas no such tumors were found in animals receiving L10 cells. CONCLUSION: Overexpression of IL-6 increases metastatic potential of tumor cells, with preferential metastases to the abdominal cavity when compared with tumor cells elaborating endogenous IL-6.