Marcelo Eidi Nita

5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins

Recently, apoptosis has been implicated as one of the end points of cells exposed to chemotherapeutic agents. The p53 and Bcl-2 family of proteins are involved in chemotherapy-induced apoptosis, but in a cell type-dependent manner. We sought to determine the roles played by the p53 and Bcl-2 family of proteins in 5-fluorouracil (5-FU)-induced apoptosis of human colon cancer cell lines. We first studied the p53 genetic and functional status, and then 5-FU, at inhibitory concentration of 50% (IC50) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA). Bcl-2, Bcl-X(L), Bax, Bad, Bak and p53 protein expression was analysed by Western blotting. Using five human colon cancer cell lines, we found that equitoxic (IC50) doses of 5-FU induced apoptosis in both wild-type p53 and mutant p53 cells. Analysis of the steady-state levels of Bcl-2 family proteins showed high expression of Bcl-X(L) in all of the cell lines except Colo320. Bcl-2 was expressed in two of them. Bax presented with the lowest basal expression and Bad showed homogeneous expression. On the other hand, Bak expression varied more than fivefold among these cells. In cells containing wild-type p53 (e.g. LoVo), 5-FU-induced apoptosis was accompanied by increased expression of Bax and Bak without consistent modulation of other bcl-2 family proteins. In contrast in cells containing mutant p53 (e.g. DLD1), Bak expression was remarkably increased. There was a significant correlation between chemosensitivity and Bcl-X(L) to Bax ratio, rather than Bcl-2 to Bax. In conclusion, these results suggest that some members of the Bcl-2 family of proteins, in human colon cancer cell lines, are modulated by 5-FU and that the ratio of Bcl-X(L) to Bax may be related to chemosensitivity to 5-FU.

Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522

SummaryIt is proposed that non-steroidal anti-inflammatory drugs (NSAIDs) reduce colorectal tumorigenesis by inhibition of cyclooxygenase (COX). COX is a key enzyme in the conversion of arachidonic acid to prostaglandins and two isoforms of COX have been characterized, COX-1 and COX-2. Multiple studies have shown that COX-2 is expressed at high levels in colorectal tumours and play a role in colorectal tumorigenesis. Recently it has been reported that selective inhibition of COX-2 inhibits colon cancer cell growth. In this study we investigated the effect of a selective COX-2 inhibitor (JTE-522) on haematogenous metastasis of colon cancer. For this purpose, we selected a murine colon cancer cell line, colon-26, that constitutively expresses the COX-2 protein. The subclone P expressed a high level of COX-2 and the subclone 5 expressed a low level. The colon-26 subclones were injected into the tail vein of BALB/c mice. JTE-522 was given intraperitoneally every day from the day prior to cancer cell injection, and the mice were sacrificed 16 days after cell injection. Lung metastases were compared between groups with and without JTE-522. In the mice injected with subclone P, the number of lung metastatic nodules was significantly reduced in the treated group. However, in the mice injected with subclone 5, there was little difference between the control and the treated groups. These results indicate that there may be a direct link between inhibition of haematogenous metastasis of colon cancer and selective inhibition of COX-2, and that selective COX-2 inhibitors may be a novel class of therapeutic agents not only for colorectal tumorigenesis but also for haematogenous metastasis of colon cancer.

Role of p53 and p21/WAF1 detection in patient selection for preoperative radiotherapy in rectal cancer patients

BACKGROUND: Recent studies showed that p53 and p21 may play major roles in determining tumor radiosensitivity through the apoptosis pathway. The aim of this study was to investigate the predicting value of radiosensitivity in human rectal carcinoma. METHODS: p53 and p21/WAF1 expressions in formalin fixed, paraffin-embedded, preradiation biopsy samples from 49 patients with primary rectal carcinoma were analyzed immunohistochemically. p53 and p21 expressions and their relationships with histopathologic changes after radiation and other clinical features were evaluated. RESULTS: Expressions of p53 and p21/WAF1 were 49 and 28.6 percent, respectively. In 36.7 percent of total tumors, significant histopathologic effect can be observed. There was a significant inverse expression of p53 and p21. Most of the p53(+) or p21(−) tumors were radioresistant, and the majority of p53(−) or p21(+) tumors were radiosensitive. Tumors size in the radiosensitive, p53(−), or p21(+) group decreased more significantly than in radioresistant, p53(+), or p21(−) group (P<0.01), and patients with radioresistant, p53(+), or p21(−) tumors had more local recurrence, more distant metastasis, and a shorter five-year survival rate than those with radiosensitive, p53(−), or p21(+) tumors, but without statistic significance. No statistically significant correlation can be observed between other tumor clinical features and radiosensitivity, p53, or p21 expressions. CONCLUSION: Immunohistochemistry detection of p53 and p21 expressions may be useful parameters for more radiosensitive patients selected for preoperative radiotherapy.

Relation between thymidylate synthase expression and survival in colon carcinoma, and determination of appropriate application of 5-fluorouracil by immunohistochemical method

BackgroundThymidylate synthase (TS) is regarded as a parameter of 5-fluorouracil (5-FU) chemosensitivity for colorectal carcinoma. Recent researchers indicate that the chemosensitivity of 5-FU for colorectal carcinoma with low expression of TS is better than tumors with high expression of TS. But the relation between TS expression and overall survival of curatively resected colorectal cancer patients has been less studied.MethodsSpecimens of curatively resected colon carcinoma from 148 patients were included in this study. TS expression in the tumor was assessed by immunohistochemical staining technique, and the patients were categorized into TS-(+) and TS-(−) groups. First, the relation between TS expression and survival of patients was examined. Next, for each group, we compared survival between the chemotherapy-(+) and the chemotherapy-(−) subgroup.ResultsOverall survival was significantly better in the TS-(−) group (n=107) than in the TS-(+) group (n=41) (P=.0003). In the TS-(−) group, there was little difference between the chemotherapy-(+) and the chemotherapy-(−) subgroup. In the TS-(+) group, the survival of the chemotherapy-(+) subgroup was significantly better than the chemotherapy-(−) subgroup (P=.0439).ConclusionsTS, itself, may be a prognostic factor for colon carcinoma; and 5-FU adjuvant chemotherapy may be appropriate for colon carcinoma with high expression of TS.

p21Waf1/Cip1 Expression Is a Prognostic Marker in Curatively Resected Esophageal Squamous Cell Carcinoma, but Not p27Kip1, p53, or Rb

Background: p21Waf1/Cip1 (p21), p27Kip1 (p27), p53, and Rb play critical roles in cell cycle regulation and may influence the clinical behavior of tumors. We examined whether their expression is useful to predict survival of patients with esophageal squamous cell carcinoma (ESC).Methods: Expression of p21, p27, p53, and Rb was studied by the immunohistochemical method in specimens from 62 patients with curatively resected ESC tumors and scored by a computerized image analysis system.Results: The median expression scores of p21, p27, p53, and Rb (14, 12, 27, and 50, respectively) were used as cut-off points to define low and high expression groups for each protein. The 5-year survival rate for the high p21 expression group was 68%; that for the low expression group was 31% (P = .0062). p27, p53, and Rb were not correlated with overall survival. When patients were categorized into four groups based on p21 expression level and lymph node involvement (pN), the survival curves were significantly different (P = .0017). Thus, patients without lymph node involvement but with low p21 expression had survival similar to that of patients with lymph node involvement and high p21 expression. Multivariate analysis showed that age (P = .0102), lymph node involvement (P = .0076), and p21 (P = .0276) were independent prognostic factors.Conclusions: Expression of p21 is an independent prognostic factor in curatively resected ESC. Definition of new subgroups of patients based on p21 expression may help to enhance the stratification of stage.

Bcl‐XL Antisense Sensitizes Human Colon Cancer Cell Line to 5‐Fluorouracil

Resistance to 5‐fluorouracil (5‐FU) has been frequently found in the treatment of digestive tract cancer patients. Our previous study suggested that high expression of endogenous Bcl‐XL, might be associated with resistance to 5‐FU in colorectal cancer. The aim of this study is to analyze the role of Bcl‐XL in 5‐FU resistance and to explore a new therapeutic strategy using Bcl‐XL antisense. First, western blot analysis shows that Bcl‐XL rather than Bcl‐2 is overexpressed in primary adenocarcinoma of colon. Second, when Colo320 cells, with undetectable endogenous Bcl‐XL expression, were transfected with Bcl‐XL gene, they acquired high resistance to 5‐FU. Finally, antisense oligodeoxynucleotides (ODNs) that targeted the start codon of Bcl‐XL mRNA (ASI) prove to be the most effective in DLD1 cells with high endogenous Bcl‐XL expression. Bcl‐XL protein expression was decreased in a dose‐dependent manner when the cells were treated with AS1 ODNs, while non‐sense and sense controls and 5‐FU had no effect on Bcl‐XL protein. 5‐FU treatment induced a level of apoptosis 10‐fold higher in DLD1 cells than in untreated control cells, while the same dose of 5‐FU induced a 55‐fold higher level of apoptosis in DLD1 cells treated with Bcl‐XL antisense oligodeoxynucleotides (P=0.0003). Moreover, AS1 ODNs coupled with 5‐FU decreased viable colon cancer cells 40% more than did 5‐FU alone (P < 0.05). These results suggest that Bcl‐XL is an important factor for 5‐FU resistance and the suppression of Bcl‐XL expression by the specific antisense ODNs can increase the sensitivity of colon cancer cells to 5‐FU.

Expressions of Cell Cycle Regulators in Human Colorectal Cancer Cell Lines

To study the altered mechanisms of cell cycle regulation in colorectal cancer, the expressions of cyclins, cyclin‐dependent kinases (CDKs), CDK inhibitors, p53 and retinoblastoma (Rb) protein were analyzed by western blotting in a series of human colorectal cancer cell lines. The colorectal cancer cell lines exhibited various expression patterns of cell cycle regulators, which may reflect differences in the biological characteristics of cancer cells and in the genetic backgrounds of carcinogenesis. A correlation was found between p53 gene alteration and p21 expression, suggesting that p53 gene mutation usually suppresses p21 expression, though p21 expression could be induced via both ap53‐dependent and a p53 ‐independent pathway in colorectal cancer. None of the cell lines studied expressed p16 protein, suggesting that inactivation of p16 may be a common alteration in colorectal cancer. Moreover, all the D‐type cyclins, especially D2 and D3, were expressed at a high level in most of the cell lines. Loss of p16 expression and increased expression of D‐type cyclins promote CDK‐mediated Rb phosphorylation. All of the colorectal cancer cell lines studied herein expressed Rb protein, but the growth‐suppressive properties of Rb may be inactivated by the loss of p16 expression and increased expressions of D‐type cyclins. In view of the pivotal role of Rb in cell cycle regulation, loss of p16 expression and overexpression of D‐type cyclins may be critical alterations in colorectal cancer.

Screening for APC Mutations Based on Detection of Truncated APC Proteins

Mutation of the adenomatous polyposis coli (APC)gene is frequently found in colorectal tumors from bothfamilial adenomatous polyposis (FAP) and non-FAPpatients. Analysis of APC mutation is time-consuming and costly due to the large size of the APCgene. As the majority of APC mutations result in thetruncation of gene products, the detection of truncatedAPC proteins may be used as a screening method for APC mutations. The aim of this study is toestablish a practical method of detecting truncated APCproteins for the screening of APC mutations. APCproteins in human colorectal cancer cell lines wereanalyzed by western blotting. Truncated APC proteinswere expressed in all of the colorectal cancer celllines studied. Two species of truncated APC proteinswere expressed in two cell lines. Western blotting is a rapid, reliable screening method for APCmutations and provides information on bothalleles.

Hepatitis C: facts in numbers

Despite the technical development to detect hepatitis C virus (HCV) in the serum had decreased substantially the risk of acquiring the virus through blood transfusion, at least 3 to 4 million are infected every year and it is estimated that 3% of the population is infected worldwide. The anti-HCV prevalence in blood donors in Latin America varies from 0.2%-0.5% in Chile to 1.7%–3.4% in northeast of Brazil. Indeed, anti-HCV prevalence in blood donors varies from 0.8%–2.8% in southeast to 1.7%-3.4% in northeast of Brazil. It is estimated that the frequency to be of 1.23%. Most patients with hepatitis C are asymptomatic, having discovered the virus during blood donation, routine check-ups or during diagnostics exams for other reasons. The infection becomes chronic in approximately 80% of the cases. HCV seems to develop mechanisms through its proteins to survive in host cells. Despite the studies to determine the natural history of hepatitis C be controversial, it is estimated that at least 20% of patients chronically infected develop cirrhosis within 20 years. Thus, chronic hepatitis C can be considered worldwide as “the silent epidemic”. The HCV infection is also an important risk factor for hepatocellular carcinoma (HCC) development. HCV proteins have been correlated with hepatic carcinogenesis. Usually, HCC develops after two or more decades of hepatitis C infection and the risk is increased with advanced fi brosis. It is a frequent complication in the eastern countries, but it is already observed an increasing trend in its frequency in the western countries. Risk factors for HCV infection are blood transfusion before 1992, intravenous drug use, intra-nasal cocaine use, surgery, sexual promiscuity, tattoo, blood exposure and hemodialysis. However, in some cases, no risk factor can be identifi ed. In this number of the Archives of Gastroenterology, three active research groups in Brazil bring to us important contributions emphasizing the hepatitis C high prevalence in specifi c population groups in Brazil. In the fi rst, TOVO et al., describe the frequency of HBV-HIV and HCV-HIV co-infections, being important the association of HCV. Among those who tested for anti-HCV, 126/330 (38.2%) were positive, being the use of intravenous drug abuse (75.3%) the main risk factor in this group. In the second, GALPERIM et al. report the hepatitis C virus prevalence in alcoholic addicted patients. Among 114 alcohol addicted patients, 17 (15%) were positive for the anti-HCV. And again, the use of intravenous drugs abuse (13/17) was the main risk factor in this group. And in the third, PAROLIN et al. report the HCV prevalence in type 2 diabetes mellitus (DM) patients. In comparison to the virus prevalence in the healthy adult local population, defi ned by blood donors (0.65%), type 2 DM patients presented more anti-HCV positive tests (2%, P = 0.04). In chronic hepatitis C, progression of fi brosis is what determines prognosis and thus the need for treatment and its urgency. The fi brogenesis is a dynamic process, where it is mediated by necroinfl ammatory activity and stellar cells activation. The rate of progression of fi brosis seems to vary among patients and the main factors associated with this progression seem to be the acquired infection age, masculine sex and excessive alcohol consumption. Immunosuppression (HIV infection), hepatic steatosis, obesity and diabetes seem also to contribute for progression of fi brosis. Interferon alpha has been being the base of all the regimens for effective hepatitis C treatment. During the last decade, researchers have been focusing their attentions in the attempt to improve the doses and time of treatment, testing different interferons alpha and other drugs. Ribavirine, that has little effect on HCV when used in monotherapy, has been doubling the treatment effectiveness when combined with interferon. Therefore, currently the combination of interferon alpha or peguilatedinterferon alpha and ribavirine have been the treatment choice for the hepatitis C 7, 11, 12, . However, these treatments present limited effectiveness, because many of the patients who receive this treatment will persist with the virus after stopping the treatment. In general, patients with hepatic steatosis due to the obesity, alcohol consumption or DM respond to treatment less frequently than patients without steatosis and in HIV co-infected patients the virologic response frequency is lower than the patients without co-infection. The epidemiological results presented by TOVO et al., GALPERIM et al. and PAROLIN et al. are important for public health decision making, because they refl ect the reality found in daily clinical practice. In Brazil, where most high cost medical treatment has been defrayed by the government it is important to perform local pharmacoeconomical studies. In this setting, the development of the National Network for Clinical Trials is a positive contribution, because it will be able to identify forms of allocating resources more cost-effi ciently,

Avaliação de tecnologia em saúde: II. A análise de custo-efetividade

New health technologies have made an impact in clinical and economic outcomes. Therefore, research methodologies that allow to evaluate the efficiency of these new technologies such as cost-effectiveness analysis are necessary. Cost-effectiveness analysis assess the value of health care interventions or drugs, the technology. Cost-effectiveness analysis is also deemed a determinant of modern health care practice, because the therapeutic options available at public (SUS) or private health care system must go through a formal health technology assessment in Brazil; thus, both the health care system and the health care professionals have to reevaluate the clinical consequences and costs of their actions to assure that the most efficient technologies are the one used in the practice. In this second article about health technology assessment we review the concepts of cost-effectiveness analysis, the steps involved in performing such analysis, and the criteria most frequently used to critically review the results.