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Expression of Bax protein in gastric carcinomas. A clinicopathological and immunohistochemical study
Abstract
Reduced Bax protein expression has been shown to be a negative prognostic factor in patients with breast, ovarian, colorectal, esophageal and pancreatic cancer. Our aim was to immunohistochemically study Bax protein expression in gastric carcinomas and correlate its expression with clinicopathological parameters and prognosis.
Immunohistochemistry was performed, using a monoclonal antibody against bax, in paraffin-embedded tumor specimens from 47 cases of gastric cancer.
Positive staining for the Bax protein was found in 20/47 (42.4%) adenocarcinomas examined. Negative Bax protein expression in tumour cells was correlated with lymph node metastasis (P < 0.05), and degree of differentiation (p < 0.05). Univariate analysis showed that the variables with a significant negative impact on survival were: high TNM tumour stage, depth of penetration in the gastric wall, lymph node involvement, and Bax protein expression. Multivariate analysis showed that the only variable with an impact on survival was Bax protein expression (p < 0.05, Relative Risk: 3.34). Kaplan-Meier curves showed that the 5-year survival was 36.8% in cases with positive compared with 16% in cases with negative Bax protein expression (p = 0.0427).
Negative Bax expression in gastric cancer is associated with de-differentiation, lymph node metastases, and poor clinical prognosis. Bax protein expression might play an important role in the development and phenotypic differentiation of gastric carcinomas and tumor progression.
... This finding indicated that p53 mutations usually occur before invasion during breast cancer progression, and that p53 protein expression in DCIS is an important indicator of cancer invasiveness and prognosis of DCIS. 13 In agreement with Kayaselcuk et al, 15 who found positive correlation between bcl-xl expression and grade of DCIS. This controversy reflects the complexity of bax and bcl-xl regulation and the need for more studies for these genes on both immunohistochemical and molecular levels. ...
... 37 This indicates that regulation of bax is complex, and may be explained by the presence of p53 mutations which affect its interaction with bax protein, or by intervention of other molecules which may modulate the function of either or both proteins. 36 In the current study, negative correlation between bax and bcl-2 expression was detected in DCIS (p < 0.01), which concurs with Kapucuoglu et al, (1997), 38 who found that while both proteins were expressed at the same time in normal and benign epithelium, different staining patterns were observed according to the degree of differentiation of the neoplastic epithelium. ...
... ,14 bcl-2 expression was not correlated with the grade of DCIS, which suggests that alterations in bcl-2 expression in breast carcinoma are more prominent in invasive rather than in in situ lesions.In agreement with Rehman et al, (2000),15 and Kayaselcuk et al, (2004),14 who found that bax expression did not correlate with increasing histological grades of DCIS, we found insignificant correlation between bax expression and tumor grade of DCIS. Contradictory findings were obtained byKapucuoglu et al, (1997) 16 and Anagnostopoulos et al, (2007), 17 who found that bax protein expression in DCIS was related to more aggressive neoplasms. ...
... This finding indicated that p53 mutations usually occur before invasion during breast cancer progression, and that p53 protein expression in DCIS is an important indicator of cancer invasiveness and prognosis of DCIS. 13 In agreement with Kayaselcuk et al, 15 who found positive correlation between bcl-xl expression and grade of DCIS. This controversy reflects the complexity of bax and bcl-xl regulation and the need for more studies for these genes on both immunohistochemical and molecular levels. ...
... 37 This indicates that regulation of bax is complex, and may be explained by the presence of p53 mutations which affect its interaction with bax protein, or by intervention of other molecules which may modulate the function of either or both proteins. 36 In the current study, negative correlation between bax and bcl-2 expression was detected in DCIS (p < 0.01), which concurs with Kapucuoglu et al, (1997), 38 who found that while both proteins were expressed at the same time in normal and benign epithelium, different staining patterns were observed according to the degree of differentiation of the neoplastic epithelium. ...
... ,14 bcl-2 expression was not correlated with the grade of DCIS, which suggests that alterations in bcl-2 expression in breast carcinoma are more prominent in invasive rather than in in situ lesions.In agreement with Rehman et al, (2000),15 and Kayaselcuk et al, (2004),14 who found that bax expression did not correlate with increasing histological grades of DCIS, we found insignificant correlation between bax expression and tumor grade of DCIS. Contradictory findings were obtained byKapucuoglu et al, (1997) 16 and Anagnostopoulos et al, (2007), 17 who found that bax protein expression in DCIS was related to more aggressive neoplasms. ...
... Bcl-2 was not correlated to tumor size in our study, which is consistent with the findings of Eguchi et al. (2000) and Tsutsui et al. (2006), and This finding suggests that there may be a balance between anti-apoptotic and antiproliferative activity of bcl-2 in breast carcinoma, so that the net effect of bcl-2 on tumor size is abolished. On the other hand, Anagnostopoulos et al. (2007) found positive correlations between bcl-2 expression and smaller tumor size. This difference may be explained by variable co-expression of bcl-2 with other molecules that affect tumor size e.g p53, Ki-67, or other bcl-2 family members. ...
... This controversy reflects the complexity of bax regulation and the need for more studies for this gene on both immunohistochemical and molecular levels to clarify its role (Rehman et al., 2000). Concurring with Anagnostopoulos et al. (2007), this study showed positive correlation between bax expression and tumor grade of IBC (p<0.05). ...
One of the greatest challenges in breast cancer management is to accurately predict the outcome for each patient. To do this at present, we rely heavily on traditional pathological variables, such as tumor size, tumor grade and lymph node status. However, despite the broad applicability of clinic-pathological indices, they are still unable, to separate the 30% node-negative patients who will relapse from the 70% who will not. We aimed to: 1. Study the immunohistochemical profile of bcl-2 and bax in breast carcinoma. 2. Assess their prognostic values in relations to clinico-pathological prognostic factors. Subjects and methods: This study included 45 specimens of breast carcinoma. Patient’s age, tumor size and local aggressive changes, history of recurrence and/or presence of distant metastasis were obtained. Sections stained with H&E were evaluated for the presence of benign breast disease, tumor type and tumor grade, presence of in situ component, lymphocytic infiltration, lymphovascular invasion and axillary lymph node status. Immunostaining was done to detect the expression of bcl-2 and bax. Results: Both bcl-2 and bax were regularly expressed in areas of benign breast disease. Bcl-2 was positive in all ductal carcinoma in situ (DCIS); 100% of grade I, 89% of grade II and 87% of grade III invasive breast carcinoma (IBC). Bcl-2 was inversely correlated to tumor grade of IBC (p<0.03) and to lymphocytic infiltration (p<0.02). Bax was positive in (80%) low grade, (100%) intermediate and high grade DCIS. Bax was positive in (83%) grade I IBC and in all (100%) grade II and grade III IBC. Bax expression was positively correlated to: tumor grade (p<0.05) and lymphocytic infiltration (p<0.00). No significant difference in either bcl-2 or bax expression in lymph nodal positive or negative cases. In DCIS; negative correlation was present between bcl-2 and bax (p<0.01). In IBC; insignificant correlation was present between bcl-2 and bax. Conclusions: Bcl-2 is an indicator for good prognosis in breast cancer being negatively correlated to tumor grade and prominent lymphocytic infiltration. Bax is an indicator for poor prognosis in breast cancer being positively correlated to tumor grade and presence of prominent lymphocytic infiltration.
... 12.7–67%, 42.4–58.0%, 23.5– 72.9%, respectively [9,12,2223242526. p53 played an important role in apoptosis. ...
... This result was different from a previous research. Anagnostopoulos et al. [24] reported that negative bax expression in gastric was associated with lymph node metastasis and poor clinical prognosis. The reasons for the conflicting results were not clear. ...
The prognosis varied among the patients with the same stage, therefore there was a need for new prognostic and predictive factors. The aim of this study was to evaluate the relationship of apoptosis-related biological markers such as p53, bcl-2, bax, and c-myc, and clinicopathological features and their prognostic value.
From 1996 to 2007, 4426 patients had undergone curative D2 gastrectomy for gastric cancer at Fudan University Shanghai Cancer Center. Among 501 patients, the expression levels of p53, bcl-2, bax, and c-myc were examined by immunohistochemistry. The prognostic value of biological markers and the correlation between biological markers and other clinicopathological factors were investigated.
There were 339 males and 162 females with a mean age of 57. The percentages of positive expression of p53, bcl-2, bax, and c-myc were 65%, 22%, 43%, and 58%, respectively. There was a strong correlation between p53, bax, and c-myc expression (P=0.00). There was significant association between bcl-2, and bax expression (P<0.05). p53 expression correlated with histological grade (P=0.01); bcl-2 expression with pathological stage (P=0.00); bax expression with male (P=0.02), histological grade (P=0.01), Borrmann type (P=0.01), tumor location (P=0.00), lymph node metastasis (P=0.03), and pathological stage (P=0.03); c-myc expression with Borrmann type (P=0.00). bcl-2 expression was related with good survival in univariate analysis (P=0.01). Multivariate analysis showed that bcl-2 expression and pathological stage were defined as independent prognostic factors. There were significant differences of overall 5-year survival rates according to bcl-2 expression or not in stage IIB (P=0.03).
The expression of bcl-2 was an independent prognostic factor for patients with gastric cancer; it might be a candidate for the gastric cancer staging system.
... In the TCGA database, the expression of PCNA in gastric carcinoma tissues is higher than that in normal tissues, further confirming that downregulation of PCNA levels can inhibit gastric carcinoma cells proliferation. In addition, Anagnostopoulos GK et al. found that low expression of Bax in gastric carcinoma was associated with low 5-year survival and poor clinical prognosis [21]. ...
Bulbus Lilii, as a medicinal and edible plant, has anti-inflammatory, anti-oxidative and immunopotentiating pharmacological activities, which seems to be therapeutic on cancer prevention. The purpose of this study was to investigate the effects of total saponins from Lilium lancifolium (TSLL) on proliferation, apoptosis and migration of human gastric carcinoma cells lines SGC-7901 and HGC-27 and its underlying mechanism. The results showed that TSLL inhibited the proliferation of gastric carcinoma cells by suppressing the level of proliferating cell nuclear antigen (PCNA) and increased p21 level. TSLL induced cells apoptosis by up-regulating expression of pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2 expression. Meanwhile, TSLL remarkably inhibited cell migration and invasion, decreased matrix metalloproteinase-2 (MMP-2) expression and increased tissue inhibitor of metalloproteinases-1 (TIMP-1) expression. Notably, TSLL had stronger anti-cancer effect on undifferentiated HGC-27 cells than differentiated SGC-7901 cells. Accordingly, TSLL might be a promising candidate to prevent and suppress the growth of gastric carcinoma cells.
... 26 Notwithstanding the variable expression of BAX in gastric carcinomas, 28 its status may be suggestive of its role being played in the development and evolution of this type of malignant tumors, since BAX-negativity has been related to shorter survival periods of patients surgically treated for their disease. 50 As regards the relatively novel member of the BCL-2 family, the BCL2L12 gene, 30 its peculiar attributes as either a pro-survival 31,32,51 or pro-apoptotic 33,34,[36][37][38]52 factor have been a pole of attraction for constant research endeavors. In fact, a series of data has demonstrated the usefulness of BCL2L12 expression in human solid tumors, including stomach apoptosis. ...
Defective apoptosis comprises the main reason for tumor aggressiveness and chemotherapy tolerance in solid neoplasias. Among the BCL-2 family members, whose mRNA or protein expression varies considerably in different human malignancies, BCL2L12 is the one for which we have recently shown its propitious prognostic value in gastric cancer. The purpose of the current work was to investigate the expression behavior of BCL2L12, BAX and BCL-2 in human stomach adenocarcinoma cells following their exposure to anti-tumor substances. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue methods assessed the impact of doxorubicin, oxaliplatin and methotrexate on AGS cells' viability and growth. Following isolation from cells, total RNA was reverse-transcribed to cDNA. Quantification of target genes' expression was performed with real-time PCR using SYBR Green detection system. The relative changes in their mRNA levels between drug-exposed and untreated cells were calculated with the comparative Ct method (2 (-ddCt) ). All three drugs, as a result of their administration to AGS cancer cells for particular time intervals, provoked substantial fluctuations in the transcriptional levels of the apoptosis-related genes studied. While BAX was principally upregulated, striking similar were the notable changes regarding BCL-2 and BCL2L12 expression in our cellular system. Our findings indicate the growth suppressive effects of doxorubicin, oxaliplatin and methotrexate treatment on stomach carcinoma cells and the implication of BCL2L12, BAX and BCL-2 expression profiles in the molecular signaling pathways triggered by chemotherapy.
... Regarding GC, the role of BAX has been studied mainly in patients undergoing curative resection, in order to show a potential correlation with pathological characteristics and risk of disease relapse and death. In small retrospective series, decreased BAX expression was strongly associated with dedifferentiation, lymph node metastases, diffuse histology and poor clinical outcome [23,24]. Recently, the largest surgical series of 501 resected GC patients failed to show any significant correlation of BAX with prognosis, while BCL2 expression was significantly and independently associated with better outcome [25]. ...
Our group and numerous others have shown in both preclinical and clinical studies that the proapoptotic mediator BAX may be deregulated through gene mutation or loss of protein expression, affecting resistance to chemotherapy and radiotherapy in several cancer types. However, BAX is also involved in cancer development and may related to prognosis, independently of treatment outcome. The clinical impact of BAX status in gastrointestinal malignancies remains controversial, although it is generally hypothesized that high expression may be a positive prognostic factor and predict increased efficacy of chemotherapy (with particular regard to platinum derivatives). The present review aims to provide updated information on BAX as potential prognostic and/or predictive biomarker in gastroesophageal and colorectal cancers, as well as in other less studied gastrointestinal malignancies.
... The prognostic role of BAX was mainly studied in series of patients with GC who underwent curative surgery. It was correlated to unfavourable pathological features such as diffuse-type, poor differentiation or lymph node metastases, leading to a poor clinical outcome in terms of disease relapse and death (58,59). At present, few data have been published about the prognostic or predictive role of BAX in advanced gastro-oesophageal cancer treated with chemotherapy. ...
Currently, therapeutic management of gastric cancer is mainly based on clinical data and histological features. Although several new treatment options have recently been introduced, inter-individual variability of response and drug resistance are still a challenge. Many promising markers have been identified to predict prognosis and likelihood of response to therapy, in order to tailor treatment regimens on the basis of patients' individual features. However, despite recent developments in gene sequencing and molecular diagnostics, many biomarkers still have a controversial role. Published data are often contradictory and at the moment, no molecular marker, other than Human epidermal growth factor receptor-2 (HER2) status for trastuzumab-based treatment, has entered the mainstream of clinical practice. The primary obstacle to the identification of reliable markers lies in technical difficulties in quantitatively assessing molecular alterations; genome-wide analyses are also often misleading due to the complexity of biological processes. Nevertheless, many biomarkers are being evaluated in clinical trials in order to identify criteria for stratifying patients and establish customized therapeutic approaches. In this review, we provide an update on promising biological prognostic and predictive markers, with a focus on growth factor signalling molecules, DNA repair systems, fluoropyrimidine metabolism and apoptotic pathways.
... In a surgical series of 57 resected gastric cancer specimens, a strong relationship between decreased Bax expression and diffuse-type gastric or poorly differentiated morphology was identified [16]. Bax-negative expression was associated with dedifferentiation, lymph node metastases, and poor clinical prognosis in a different series of 47 resected gastric cancer [17] . Moreover, the role of the low expression of Bax as a poor prognostic factor has been reported in several malignancies treated with chemotherapy or chemoradiotherapy, such as breast, ovarian, and head and neck cancer181920 . ...
Bax protein is a key mediator of apoptosis, and it might be related to chemosensitivity. The purpose of this study was to evaluate the prognostic role of Bax in patients with advanced gastric cancer treated with triplet chemotherapy COI regimen (capecitabine, oxaliplatin, and irinotecan).
Pretreatment tissue blocks were available for 23 consecutive patients, selected for good performance status (ECOG ≤ 1) and consenting for treatment with first-line COI at a single institution. Bax levels were classified as positive or negative by immunohistochemistry (bax N20; Santa Cruz Biotechnology) and related to outcome in terms of response rate, progression-free survival, and overall survival.
Bax-negative and -positive samples were 26% and 74%, respectively. Bax expression was associated with significantly higher response rate (87% vs 33%), progression-free survival (8.7 vs 4.9 months, P = .016), and overall survival (23.8 vs 12.7 months, P = .025). In multivariate analysis including Bax and performance status, low Bax independently predicted worse outcome, along with suboptimal performance status.
In advanced gastric cancer, Bax expression was related to clinical benefit with COI regimen. Whether Bax is a prognostic or mixed prognostic/predictive factor warrants prospective confirmation. It is to be defined if Bax predicts sensitivity to platinum analogs or to whatever chemotherapy regimen.
... BAX gene encodes a protein belonging to the BCL family members. Negative BAX protein expression has been associated with de-differentiation, lymph node metastasis and shorter survival, suggesting that BAX status may play a role in the development and differentiation of gastric cancer and tumor progression [99] . ...
Gastric cancer is one of the leading causes of cancer-related deaths worldwide, although the incidence has gradually decreased in many Western countries. Two main gastric cancer histotypes, intestinal and diffuse, are recognised. Although most of the described genetic alterations have been observed in both types, different genetic pathways have been hypothesized. Genetic and epigenetic events, including 1q loss of heterozygosity (LOH), microsatellite instability and hypermethylation, have mostly been reported in intestinal-type gastric carcinoma and its precursor lesions, whereas 17p LOH, mutation or loss of E-cadherin are more often implicated in the development of diffuse-type gastric cancer. In this review, we summarize the sometimes contradictory findings regarding those markers which influence the progression of gastric adenocarcinoma.
Gastric cancer (GC) is the fifth most common cancer in the world and the third most frequent cause of neoplasm-related deaths. Despite the gradually decreasing incidence of GC, its diagnosis is still associated with one of the highest mortality rates worldwide. The GC symptoms are nonspecific, and this makes it difficult to establish an early diagnosis. Patients with locally advanced or metastatic GC who fail to have a radical gastrectomy performed represent a significant group of patients. Metastatic cancer diagnosis is immensely important since it helps to avoid aggressive and extensive surgery, as well as it allows systemic therapy to be employed. This chapter will concentrate on the clinical aspects of metastatic GC. It will also look at trends in research with regard to the above issues, paying special attention to the role of positron emission tomography/computed tomography. Aspects with regard to prognosis, predictive factors, and treatment of metastatic GC will also be discussed.
Objective: To investigate the effect of BAX on the apoptosis of human laryngeal squamous cell carcinoma Hep2 cells and their sensitivity to 5-aza-2′-deoxycitydine (5-Aza-CdR). Methods: The proliferation of Hep2 cells was detected by methyl thiazolyl tetrazolium (MTT) colorimetry after treatment with 5-Aza-CdR in vitro. The plasmid containing BAX cDNA was transfected into Hep2 cells. The mRNA level of BAX gene was detected by RT-PCR. Flow cytometry was used to analyze the apoptosis and cell cycle of Hep2 cells. Results: After treatment with different concentrations of 5-Aza-CdR, the proliferation of Hep2 cells was inhibited by 5-Aza-CdR in vitro in a dose- and time-dependent manner, and the IC50 was 4μmol/L. The results of flow cytometry detection showed that both BAX and 5-Aza-CdR could block Hep2 cells in G1/S phase and promote apoptosis. The percentage of cells in G 0/G1 and the apoptotic rate of Hep2 cells treated with 5-Aza-CdR were increased from 51.57% to 71.17% and from 1.67% to 13.96%, respectively. The RT-PCR results showed that transfection of BAX significantly increased the expression of BAX gene level in Hep2 cells (P<0.05). After transfection, the percentage of Hep2 cells in S phase were decreased from 33.29% and 32.42% to 16.07% and 13.58% in blank control group and empty vector transfected group, respectively. The apoptotic rate of Hep2 cells transfected with plasmid containing BAX cDNA was increased from 7.13% to 23.74%, with a significant difference from that of the blank control group and empty vector transfected group (P<0.05). Conclusion: Transfection of BAX can induce apoptosis of Hep2 cells and increase their sensitivity to the killing effect of 5-Aza-CdR.
Cancers of the esophagus, stomach and colon contribute to a major health burden worldwide and over 20% of all cancer deaths. Biomarkers that should indicate pathogenic process and are measureable in an objective manner for these tumors are rare and not established in the clinical setting. In general biomarkers can be very useful for cancer management as they can improve clinical decision-making regarding diagnosis, surveillance, and therapy. Biomarkers can be different types of molecular entities (such as DNA, RNA or proteins), which can be detected, in different tissues or body fluids. However, more important is the type of biomarker itself, which allows diagnostic, prognostic or predictive analyses for different clinical problems. This review aims to systematically summarize the recent findings of genetic and epigenetic markers for gastrointestinal tumors within the last decade. While many biomarkers seem to be very promising, especially if used as panels, further development is urgently needed to address practical considerations of biomarkers in cancer treatment.
Although the incidence and mortality of Gastric Cancer (GC) have fallen over the past 70 years, GC continues to be the second
leading cause of cancer death and the fourth most common malignant neoplasia across the world. GC still represents a major
clinical challenge because it has a poor prognosis and limited treatment options due to its relative resistance to radiotherapy
and chemotherapy. At present, tumour stage provides the major prognostic variables used in clinical management of GC patients.
However, GC with similar morphology may display different biological aggressiveness, prognosis and response to therapy.
To improve the clinical outcome of patients with gastric cancer, intensified combination strategies are currently in clinical development, including combinations of more extensive surgery, (neo)adjuvant chemotherapy, and radiotherapy. The present study used DNA copy number profiling to identify subgroups of patients with different clinical outcomes. We hypothesize that, by identification of subgroups, individual treatment strategies can be selected to improve clinical outcome and to reduce unnecessary treatment toxicity for patients with gastric cancer.
DNA from 206 gastric cancer patients was isolated and analyzed by genomewide array comparative genomic hybridization. DNA copy number profiles were correlated with lymph node status and patient survival. In addition, heat shock protein 90 (HSP90) expression was analyzed and correlated with survival in 230 gastric cancer patients.
Frequent (>20%) DNA copy number gains and losses were observed on several chromosomal regions. Losses on 5q11.2-q31.3 and 14q32.11-q32.33 (14% of patients) were correlated with good clinical outcome in univariate and multivariate analyses, with a median disease-free survival interval of 9.2 years. In addition, loss of expression of HSP90, located on chromosome 14q32.2, was correlated with better patient survival.
Genomewide DNA copy number profiling allowed the identification of a subgroup of gastric cancer patients, marked by losses on chromosomes 5q11.2-q31.3 and 14q32.11-q32.33 or low HSP90 protein expression, with an excellent clinical outcome after surgery alone. We hypothesize that this subgroup of patients most likely will not benefit from (neo)adjuvant systemic treatment and/or radiotherapy, whereas anti-HSP90 therapy may have clinical potential in patients with HSP90-expressing gastric cancer, pending validation in an independent dataset.
Understanding of the biology and clinical behavior of ductal carcinoma in situ (DCIS) is currently inadequate. The aim of this comprehensive review was to identify important molecular biological markers associated with DCIS and candidate markers associated with increased risk of ipsilateral recurrence after diagnosis of DCIS. A comprehensive systematic review was performed to identify studies published in the past 10 years that investigated biological markers in DCIS. To be included in this review, studies that investigated the rate of biological expression of markers had to report on at least 30 patients; studies that analyzed the recurrence risk associated with biomarker expression had to report on at least 50 patients. There were 6,252 patients altogether in our review. Biological markers evaluated included steroid receptors, proliferation markers, cell cycle regulation and apoptotic markers, angiogenesis-related proteins, epidermal growth factor receptor family receptors, extracellular matrix-related proteins, and COX-2. Although the studies in this review provide valuable preliminary information regarding the expression and prognostic significance of biomarkers in DCIS, common limitations of published studies (case-series, cohort, and case-control studies) were that they were limited to small patient cohorts in which the extent of surgery and use of radiotherapy or endocrine therapy varied from patient to patient, and variable methods of determining biomarker expression. These constraints made it difficult to interpret the absolute effect of expression of various biomarkers on risk of local recurrence. No prospective validation studies were identified. As the study of biomarkers are in their relative infancy in DCIS compared with invasive breast cancer, key significant prognostic and predictive markers associated with invasive breast cancer have not been adequately studied in DCIS. There is a critical need for prospective analyses of novel and other known breast cancer molecular markers in large cohorts of patient with DCIS to differentiate indolent from aggressive DCIS and better tailor the need and extent of current therapies.
Gastric epithelial turnover is a dynamic process. It is characterized by continuous cell proliferation, which is counterbalanced by cell loss. The biological principle that mediates the homeostasis of epithelium is programmed cell death, or apoptosis. Currently, several subtypes of apoptosis are distinguished, which are mediated by different mechanisms. Various subtypes of apoptosis also occur in the gastric epithelium under various conditions. In the normal stomach, apoptosis due to cell isolation (anoikis) mediates the physiological epithelial turnover. Albeit rarely seen in routine histology, approximately 2% of epithelial cells in the normal stomach are apoptotic. In Helicobacter pylori-induced gastritis, apoptosis and epithelial proliferation are moderately increased, with approximately 8% apoptotic epithelial cells. In gastritis, factors such as CD95 ligand or tumor necrosis factor (TNF) alpha act as death factors. They bind to specific receptors, CD95 and TNF-R, which are induced either by other cytokines, such as interferon gamma, or by Helicobacter pylori itself. In addition to CD95, H.pylorican also induce upregulation of CD95 ligand expression. Taken together, the upregulated expression of CD95, and the presence of CD95L in the close proximity to apoptotic gastric epithelial cells suggest a functional role of the CD95-CD95L system in the induction of apoptosis in H.pylori-gastritis. The role of other pathways to apoptosis is currently under study. Apart from being a biological phenomenon, apoptosis in the stomach may also have direct clinical consequences. An extreme example is given in gastric graft-vs.-host disease when epithelial denudement occurs.
Dysregulation of apoptosis may be closely related to the development of cancer and its chemoresistance. Overexpression of Bax, an inducer of apoptosis, has led to increased cell death in a variety of cancer cell lines. In this study, we investigated the effect of Bax overexpression in two gastric cancer cell lines, MKN-28 and MKN-45, using a Cre-loxP-mediated inducible expression system. After induction of bax, both cell lines showed decreased proliferation, partially due to increased cell death. Furthermore, Bax-expressing MKN-28 cells were more sensitive to cisplatin. These results indicate that up-regulation of the bax gene may provide a novel strategy for the treatment of gastric cancer.
Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.
Aspirin-induced apoptosis is one of the important mechanisms for its antitumour effect against gastric cancer. We aimed at investigating the involvement of bcl-2 family members in the apoptotic pathway in gastric cancer. Gastric cancer cell line AGS and MKN-45 were observed as to cell growth inhibition and induction of apoptosis in response to treatment with aspirin. Cell proliferation was measured by MTT assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole staining. Protein expression was determined by western blotting. We showed that aspirin activated caspase-8, caspase-9 and capase-3, cleaved and translocated Bid, induced a conformational change in and translocation of Bax and cytochrome c release. In addition, suppression of caspase-8 with the specific inhibitor z-IETD-fmk, as well as the pan-caspase inhibitor z-VAD-fmk, prevented Bid cleavage and subsequent apoptosis. The caspase inhibitors failed to abolish the effects on Bax activation. In conclusion, our results identify a role of caspase-8/Bid and activation of Bax as a novel mechanism for aspirin-induced apoptosis in gastric cancer.
Apoptosis and the genes regulating this process have recently become a focus of interest in the study of cancer development and progression. Both Bcl-2 and Bax are transcriptional targets for the tumor supressor protein, p53, which induces cell cycle arrest or apoptosis in response to DNA damage. The coordinate performance of these molecules is crucial for controlling life or death of a cell. Correlations between apoptosis and protein expression of genes controlling this process including Bcl-2, Bax and p53 in gastric cancer were here investigated with gastric tumor samples of forty patients . DNA ploidy pattern was analyzed using flow cytometry and Bcl-2, Bax, and p53 were immunohistochemically localized using specific monoclonal antibodies. In addition, serum Bcl-2 protein was estimated by enzyme linked immunosorbant assay (ELISA). The obtained data showed that the mean serum Bcl-2 protein concentration demonstrated a significant increase (P<0.0001) in positive cases (61.5+/-11.0 unit/ml) compared to the negative ones (47.5+/-3.5 unit/ml). Serum Bcl-2 protein positivity was detected in 13/40 of gastric cancer patients. Immunohistochemical positivity for Bcl-2, Bax, and p53 was shown in 45%, 68%, and 63% of samples, respectively. Positive Bcl-2 and p53 immunostaining was significantly linked with the histological grade (P<0.02 and P<0.009 respectively) and lymph duct invasion (P<0.02 and P<0.001 ). On the other hand, Bax was significantly differed with lymph duct invasion and the ploidy pattern (P<0.03 and P<0.002). In conclusion, the apoptosis-related genes p53, Bcl-2, and Bax are all linked to the occurrence of gastric cancer. Therefore, analysis of their expressions may add useful information concerning tumor behavior.
Aspirin- and non-steroidal anti-inflammatory drug (NSAID)-induced apoptosis is one of the important mechanisms for their anti-tumour effect in gastric cancer. We aimed at determining the role of bcl-2 family proteins and caspases in the apoptotic process. Gastric cancer cell lines AGS (wild-type p53) and MKN-28 (mutant p53) were used. Cell proliferation was measured by MTT assay. Apoptosis was determined by acridine orange staining. Protein expressions were determined by western blotting. Aspirin and indomethacin inhibited cell proliferation and induced apoptosis in both cells. AGS cells were more sensitive compared with MKN-28 cells. The pro-apoptotic proteins bax and bak were overexpressed after treatment, while the protein level of bcl-2 remained unchanged. Apoptosis was accompanied by an increase in caspase-3 activity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. Inhibition of caspase-3 rescued aspirin-induced apoptosis. Our results suggest that one of the major pathways which mediates the anti-tumour response of aspirin and indomethacin in gastric cancer cells is through up-regulation of bax and bak and activation of caspase-3. Bax and bak are important in the chemoprevention of gastric cancer.
Background/Aims:Helicobacter pylori induces the apoptosis of gastric epithelial cells in vivo and in vitro. However, the molecular mechanism has not been clarified. The aim of this study was to investigate the effect of H pylori on the apoptosis of gastric epithelial cells and the expression of apoptosis related genes in vitro. Methods: Human gastric adenocarcinoma SGC-7901 cells were co-cultured with a cytotoxic H pylori strain, NCTC 11637, at various densities ranging from 3.2 × 104 to 1.0 × 108 colony forming units (CFU)/ml for 48 hours. Apoptosis in gastric cells was determined by transmission electron microscopy, Hoechst 33258 fluorochrome staining, and flow cytometry. The expression of apoptosis related proteins, Bcl-2, Bax, and c-Myc, was measured by an immunohistochemical method, and c-Myc mRNA expression was determined by the reverse transcription-polymerase chain reaction. Results:Helicobacter pylori induces morphological changes typical of apoptosis. Both fluorochrome staining and flow cytometry showed that the apoptotic index began to increase when H pylori were at a density of > 1.6 × 104 CFU/ml, and in a density dependent manner (p < 0.01; one way ANOVA). The expression of the Bax and c-Myc proteins and of c-Myc mRNA was increased, whereas Bcl-2 expression was decreased after co-culture for 48 hours. Conclusions:Helicobacter pylori induced apoptosis in gastric epithelial cells is mediated by altered expression of the products of the Bcl-2, Bax, and c-Myc genes.
ApoptosisThree separate research groups report in this issue of Science ([pages. 782][1],[784][2], andp. [787][3]) that a cellular protein called nuclear factor kappa B (NF-κ B)blocks the programmed form of cell suicide known as apoptosis in several cell types. This finding has led to the suggestion that blocking NF-κ B in tumor cells might make them more vulnerable to drugs or radiation.
[1]: /lookup/doi/10.1126/science.274.5288.782
[2]: /lookup/doi/10.1126/science.274.5288.784
[3]: /lookup/doi/10.1126/science.274.5288.787
REVIEW
A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome
c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and
participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger
or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.
The execution phase of apoptosis is comprised of those processes that commit cells to apoptotic death. Many independent studies have implicated mitochondria as playing a critical role in apoptotic execution. The activation of caspase-3 and subsequent late stage degradative events are probably triggered by the release of proteins (such as cytochrome c) from the intermembrane space of mitochondria. The mechanisms responsible for this release are controversial but may include mitochondrial permeability transition and bcl-2-regulated swelling of the mitochondrial matrix. Two theoretical models of execution are discussed. It is important to note that some critical features of these models are largely based on data acquired from cell-free studies. Further studies with intact cells are urgently needed to test the physiological validity of these models.
Proapototic gene is an important target to enhance the chemotherapeutic effect in cancer cells. Based on in vitro study showing that the introduction of bax gene enhanced the sensitivity to anticancer drugs, we examined whether the intratumoral administration of bax gene could enhance the anti-tumor effect in combination with anticancer drugs in gastric cancer. The human gastric cancer cell line, MKN45 was transplanted into nude mice, and the intratumoral administration of bax gene was performed using the bax cDNA plasmid complexed with a cationic lipopolyamine. The enhancement of antitumor effect was examined in the combination with 5-fluorouracil (5-FU) and cisplatin (CDDP). The anticancer drugs were administered intraperitoneally with one third of LD50 four times at 4-day intervals, and the antitumor effect was assessed by the NCI protocol. The expression of bax gene was analyzed by RT-PCR and the apoptotic cell death was assessed by TUNEL method. The intratumoral administration of bax gene alone showed slight anti-tumor effect as compared to that of control tumor injected with vector alone. The antitumor effect of 5-FU and CDDP was significantly enhanced in the combination with intra-tumoral administration of bax gene as compared to that of CDDP and 5-FU alone (p<0.05, Student's t-test). The enhancement of antitumor effect was associated with the constitutive overexpression of bax gene and with the induction of apoptosis in the tumor treated with anticancer drug and bax gene. These results indicate that the combination therapy of intratumoral administration of bax gene complexed with a cationic lipopolyamine and anticancer drugs may provide us a new strategy for cancer chemotherapy to enhance its therapeutic efficacy in gastric cancer as termed with gene-chemotherapy.
Bcl-2 family proteins are key regulators of programmed cell death or apoptosis that is implicated in many human diseases, particularly cancer. In recent years, they have attracted intensive interest in both basic research to understand the fundamental principles of cell survival and cell death and drug discovery to develop a new class of anticancer agents. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions in either inhibiting or promoting cell death. High expression of anti-apoptotic members such as Bcl-2 and Bcl-XL commonly found in human cancers contributes to neoplastic cell expansion and interferes with the therapeutic action of many chemotherapeutic drugs. The functional blockade of Bcl-2 or Bcl-XL could either restore the apoptotic process in tumor cells or sensitize these tumors for chemo- and radiotherapies. This article reviews the recent progress in the design and discovery of small molecules that block the anti-apoptotic function of Bcl-2 or Bcl-XL. These chemical inhibitors are effective modulators of apoptosis and promising leads for the further development of new anticancer agents.
Two major intracellular apoptosis signaling cascades have been characterized, the mitochondrial pathway and the death receptor pathway. The mitochondrial pathway is regulated by members of the Bcl-2 protein family. The members of this family can be subdivided into anti- and pro-apoptotic proteins. The pro-apoptotic members are further divided into two groups, the multidomain and the 'BH3 domain only' proteins. When cells are exposed to apoptotic stimulation, pro-apoptotic proteins are activated through post-translational modifications or changes in their conformation. The main site of action of the multidomain proteins are the mitochondria, where these proteins induce permeabilization of the outer membrane resulting in the release of proteins, including cytochrome c, from the intermembrane space. In the cytosol cytochrome c activates caspase cascades ultimately leading to cell death. Mounting evidence indicates that apoptosis is involved in a wide range of pathological conditions. Recent studies suggest that the mitochondrial signaling pathway is involved in several diseases. Although, so far, with the exception of C. elegans, most studies on apoptosis have been performed in mammalian systems, recently homologues to the Bcl-2 family members, including pro-apoptotic members, have been identified in Drosophila and zebrafish. Here the structure and function of the various pro-apoptotic Bcl-2 family members, their effects on mitochondria, and their involvement in diseases are discussed.
AIM:To study the expression of proapoptotic gene Bax in human gastric carcinoma and its significance.METHODS:Using immunohistochemistry methods, the Bax protein expression in 57 specimens of gastric carcinoma and its relationship with clinical status and pathomorphological parameters were observed.RESULTS:Thirty- three (57.9%) cases were positive for Bax protein staining which was mainly located in the cytoplasm of tumor cells. The rate of Bax protein expression was not correlated with the tumor size, lymph node metastasis, depth of invasion, clinical stages of tumors and age and sex of patients (P >0.05), but strongly associated with the morphological type and differentiation degree of tumors. It was significantly higher in intestinal type and well or moderately differentiated gastric carcinoma than in diffuse type and poorly differentiated gastric carcinoma (P < 0.05 and P <0.01).CONCLUSION:The proapoptotic gene Bax is differently expressed in most of gastric carcinoma and may take part in the modulation of apoptosis in gastric carcinoma. The expression of Bax might be associated with the occurrence of intestinal type gastric carcinoma and the differentiation of gastric carcinoma.
Seminal studies on the proto-oncogene bcl-2 have first demonstrated that mutations that inhibit programmed cell death (apoptosis) can promote lymphomagenesis and influence the sensitivity of tumour cells to chemotherapy or radiotherapy. It is now widely believed that neoplastic transformation of many, perhaps even all, cell types requires mutational changes that interfere with the cell death programme. In this review, we describe current knowledge of the molecular control of cell death and discuss the role of pro- and anti-apoptotic members of the Bcl-2 protein family in tumourigenesis and anti-cancer therapy.
Products of the arachidonic acid metabolizing enzyme, 5-lipoxygenase (5-LOX), stimulate the growth of several cancer types. Inhibitors of 5-LOX and 5-LOX-activating protein (FLAP) induce apoptosis in some cancer cells. Here, the authors investigated the effect of a FLAP inhibitor, MK-886, on the inhibition of proliferation and induction of apoptosis in gastric cancer.
Cell proliferation in gastric cancer cells was measured using an 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was measured using acridine orange staining and flow cytometry. Protein expression of apoptosis-related genes p53, p21waf1, p27kip1, bcl-2 families, cytochrome c, and the caspases were examined using Western blotting. Caspase-3 activity was measured using colorimetric assay of substrate cleavage.
MK-886 inhibited cell growth in a dose- and time-dependent manner. Apoptosis was induced in gastric cancer cells and was characterized by upregulation of p27kip1 and bax, with release of cytochrome c from mitochondria into cytosol, which initiated caspase-3 activation. Specific caspase-3 inhibitors partially blocked MK-886-induced apoptosis.
The present results suggest that MK-886 induces apoptosis in gastric cancer cells through upregulation of p27kip1 and bax, and that MK-886 is a potentially useful drug in gastric cancer prevention and therapy.
Bcl-2 protein prolongs cell survival in the face of classical apoptotic stimuli, and is considered to be a suppressor of apoptosis. Bax plays a key role in apoptosis by accelerating cell death after an apoptotic stimulus. The aim of our study was to determine the roles of the Bax proapoptotic gene and the Bcl-2 antiapoptotic gene in the carcinogenesis of gastric cancer.
One hundred and forty-five gastric biopsy specimens of chronic gastritis, atrophic gastritis, intestinal metaplasia and gastric dysplasia were studied. Using immunohistochemical methods, Bax and Bcl-2 protein expression was observed.
Bax was expressed in epithelial cells in all cases of chronic gastritis. Bax was not detected in 26% of specimens of atrophic gastritis. As intestinal metaplasia develops, Bax is further suppressed. In biopsy samples with dysplasia, Bax expression was demonstrated only in 12% of biopsy samples. Although Bcl-2 protein was not detected in chronic gastritis, aberrant expression was found in gastric epithelial intestinal metaplasia and dysplasia.
The suppression of Bax and overexpression of Bcl-2 protein is an early event in gastric tumorigenesis, before gastric dysplastic changes occur.
Apoptosis proteins may play a role in prognosis and therapy response; however, they have not been fully investigated in gastric cancer. We aimed to assess the expression of proteins in the Bcl-2 family. Immunohistochemistry was employed to examine the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic proteins Bad, Bak, Bax, Bid, Bim, and p53 in 21 cases of gastric cancer. Immunopositivity was observed in 12/21 (57%) cases for p53, 16/21 (76%) cases for Bcl-XL, and 5/21 (23%) cases for Bcl-2. For the proapoptotic members of the Bcl family, loss of protein expression was observed: Bid (14/21 cases; 66%), Bad (13/21 cases; 61%), Bax (12/21 cases; 57%), Bak (9/21 cases; 42%), and Bim (4/21 cases; 19%). This study identified apoptosis proteins that exhibit heterogeneous expression between primary gastric carcinomas.
Evasion of apoptosis is one of the hallmarks of cancer. Bax-interacting factor-1 (Bif-1) interacts with both Bax and Bak that are essential for the intrinsic pathway of apoptosis, and enhances apoptosis. The aim of this study was to explore whether alteration of Bif-1 expression might be a characteristic of gastric cancer.
We analysed the expression of Bif-1 protein in 60 gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach.
In normal gastric mucosal cells, surface and glandular epithelial cells strongly expressed Bif-1. While Bif-1 expression was detected in 24 cases (40%) of the gastric carcinomas, there was no Bif-1 immunostaining in the remaining 36 cancers. Even in the 24 cases with positive Bif-1 immunostainings, 10 cancers showed decreased intensity of immunostaining compared with the normal mucosal epithelial cells.
The decreased expression of Bif-1 in malignant gastric epithelial cells compared with the normal mucosal cells suggests that loss of Bif-1 expression may play a role in gastric tumorigenesis, possibly by inhibiting the apoptosis mediated by Bif-1.
Matrine, one of the main active components from the dry roots of Sophora flavescence, was known to induce apoptosis in a variety of tumor cells in vitro. However, the molecular mechanism of cell apoptosis induced by Matrine remains elusive. Here, we investigated the apoptosis in Matrine-treated human gastric cancer MKN45 cells. The results showed that Matrine could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Further immunoblots revealed that in Matrine-treated cells, caspase-3, -7 were activated and the pro-apoptotic molecules Bok, Bak, Bax, Puma, and Bim were also up-regulated. Our results suggested that Matrine induced gastric cancer MKN45 cells apoptosis via increasing pro-apoptotic molecules of Bcl-2 family.
Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax).
To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53.
The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment.
Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment.
Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.