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.

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.

The effect of oral copper loading and portasystemic shunting on the distribution of copper in the liver, brain, kidney, and cornea of the rat.

In Wilson's disease, redistribution of copper from the liver to extrahepatic tissue coincides with the development of liver disease and cirrhosis. We have considered the possibility that portasystemic shunting may be a factor determining the organ distribution of copper in patients with liver disease. Thirty-two Sprague-Dawley rats were randomized into a sham-operated group, and a group subjected to partial occlusion of the portal vein (PPVO). Half the rats in each group were fed on normal diet and the remainder had copper added to the drinking water. Rats subjected to PPVO developed large portasystemic shunts. Over a 3-month period, none of the groups showed an increase in serum copper concentrations, or copper oxidase levels. Liver and kidney copper concentrations were similarly increased in copper supplemented sham-operated and PPVO rats, but brain copper content in both groups was similar to the unsupplemented controls. In neither unsupplemented nor copper supplemented rats subjected to PPVO, was the organ distribution of copper different from sham-operated controls. This study suggests that portasystemic shunting does not alter the organ distribution, or tissue concentration of copper.

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.

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.

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.

Ritodrine sulphation in the human liver and duodenal mucosa: interindividual variability.

The beta2-adrenoceptor agonist ritodrine has a bioavailability of 30% due to its presystemic metabolism and sulphation is an important metabolic route. The interindividual variability in the rate of ritodrine sulphation in 100 specimens of human liver and duodenum is reported. The final concentrations of ritodrine were 2 mM (duodenum) and 20 mM (liver). The mean estimates of ritodrine sulphation rate were 490 pmol x min(-1) x mg(-1) (duodenum) and 140 pmol x min(-1) x mg(-1) (liver). There was a 4-5-fold variation within +/- 2 SD units in the hepatic and duodenal rates of ritodrine sulphation. Statistical analysis revealed the presence of at least two subgroups of ritodrine sulphation. In the liver, 30% and 70% of the population fell into two subgroups with the mean estimates of ritodrine sulphation rate of 114 and 149 pmol x min(-1) x mg(-1), respectively (P < 0.05). In the duodenum, 25% and 75% of the population fell into two subgroups and the mean estimates of ritodrine sulphation rate were 332 and 538 pmol x min(-1) x mg(-1), respectively (P < 0.05). The rates of ritodrine and 4-nitrophenol sulphation correlated highly in the liver (r = 0.865; P < 0.001) and the rates of ritodrine and dopamine sulphation correlated highly (r = 0.914; P < 0.001) in the duodenum. In both tissues, the rates of ritodrine and (-)-salbutamol sulphation underwent a similar extent of variation and correlated highly. The intrinsic clearance of ritodrine sulphation was over one order of magnitude higher in the duodenum than in the liver suggesting that the duodenum is an important site of ritodrine sulphation.

[Roux-en-y hepatojejunostomy in benign pathology of the common duct]. / L'epaticodigiunostomia su ansa alla Roux nella patologia benigna della via biliare principale.

Personal experience is reported of 59 hepaticojejunostomies performed on an excluded Roux loop in the treatment of benign pathologies of the main bile duct. The advantages and disadvantages of this approach are discussed. Several particular cases are described as is the surgical technique adopted. Complications arising in 5 patients included 3 duodenal ulcers, one of them bleeding and 2 cases of anastomotic stenosis. In all other cases the operation was satisfactory. Follow-up involved echographic and cholescintigraphic examinations. It is concluded that Roux en Y hepaticojejunostomy is the treatment of choice in benign pathologies of the main bile duct when transduodenal papillostomy is contraindicated.

[Stenosis complications in digestive surgery: a histomorphological contribution. Comparison of mechanical and manual sutures]. / La "complicanza stenosi" in chirurgia digestiva: un contributo istomorfologico. Suture meccaniche e manuali a confronto.

BACKGROUND: Surgeons variously estimate usage of mechanical staplers in digestive surgery especially about the increase of the incidence of late complications like stricture (as some authors refer). We report a histomorphological aid to the comparison of staplers and hand-sewn sutures. Our aim was to observe at long term the healing process in the sutured tissue in the canine gastro-enteric tract. METHODS: We describe three histological pieces: one manual suture in a dog which was euthanatized and necropsied 5 months after operation and two mechanical sutures performed in two different dogs: one died because of a gastric torsion 18 months after the operation while the other was euthanatized 5 months after the operation. The histomorphological study has been carried out with a peculiar cutting (Exact) and including method which lets the staples in situ. RESULTS AND CONCLUSIONS: Compared analysis showed that cicatrization goes on better in stapled than in hand sutured tissue.

[Primary carcinoma of the gallbladder]. / Il carcinoma primitivo della colecisti.

A series of 4 patients with gallbladder cancer encountered in 1977-86 is presented. Carcinoma accounted for 2.5% of all biliary surgery with a higher incidence among female (F = 22, M = 12). Most of the tumours had reached an advanced by the time they reached the operating theatre. Given the difficulty in diagnosing tumours at an early stage, prophylactic cholecystectomy is highly recommended in patients with gallstones whether they are symptomatic or not.

[Neoplasm on cholecysto-jejunal anastomosis: an endoscopic diagnosis]. / Neoplasia su anastomosi colecisto-digiunale: una diagnosi endoscopica.

Bilio-digestive anastomoses represent a tried and tested surgical procedure in lithiasic or neoplastic type obstructions of the main biliary way. Among long-term postoperative complications the case of neoplasia in the stomy is rare. A clinical case of neoplasia on cholecystojejunal anastomosis diagnosed endoscopically is reported. The physiopathological aspects are discussed.

[Hepato-cholescintigraphy using technetium Tc 99m diethyl IDA in the follow-up of biliodigestive anastomoses]. / L'epato-colescintigrafia con 99m Tc-dietil-IDA nel follow-up delle anastomosi bilio-digestive.

The results of a study conducted on 24 patients given biliodigestive shunts are reported. Follow-up involved cholescintigraphy using 99m-IDA technetium. This examination provided valuable information about the morphodynamics of biliary flow and when the biliary peak and intestinal appearance times were lengthened, it was also able to identify significant obstructions. The technique is considered highly significant.

[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.

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.

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.