Decreased fragile histidine triad expression in colorectal cancer and its association with apoptosis inhibition

Abstract

Aim: To detect the expression of fragile histidine triad (FHIT) in normal colorectal tissue, colorectal adenoma and colorectal cancer (CRC) tissue, and to analyze its relationship with the clinicopathological features of CRC, and apoptosis-associated proteins (Bcl-2, Bax, survivin) and apoptosis in colorectal cancer.

Methods: FHIT mRNA analysis was performed by nested reverse transcription-polymerase chain reaction (RT-PCR) assay. Tissue microarray (TMA) was established to detect the expression of FHIT, Bcl-2, Bax and survivin genes in 80 CRC tissue specimens, 16 colorectal adenoma tissue specimens and 16 hemorrhoid (PPH) tissue specimens during the same period of time as the control. Citrate-microwave-SP was used as immunohistochemical method. The relationship between clinicopathological factors, such as differentiation grades and 5-year survival rate was observed. TUNEL assay was used to detect the apoptosis index in 80 CRC tissue specimens.

Results: Ten out of 26 (38.5%) CRC tissue specimens expressed aberrant FHIT transcripts, none of the aberrant FHIT transcripts was observed in the matched normal tissue and colorectal adenoma tissue by nested RT-PCR assay. The positive rate of FHIT gene expression in normal colorectal tissue, colorectal adenoma and carcinoma tissue was 93.75%, 68.75% and 46.25%, respectively. Clinicopathological analysis of patients showed that the decreased FHIT gene expression was not associated with age, sex, serum CEA levels, tumor site and size, histological classification. However, the expression of FHIT was correlated with differentiation grades, pathological stages, lymph node metastases and 5-year survival rate after operation. The positive rate of apoptosis-associated proteins (Bax, Bcl-2 and survivin) in CRC tissue was 72.50%, 51.25% and 77.50%, respectively. The expression of these apoptosis-associated proteins in CRC tissue was correlated with the expression of FHIT. The mean apoptosis index in FHIT negative tumors was significantly lower than that in FHIT positive tumors (5.41 +/- 0.23 vs 0.56 +/- 0.10, P < 0.01).

Conclusion: The FHIT gene plays an important role in the regulation of apoptosis and decreased FHIT expression plays a key role in the initiation and progression of colorectal carcinoma.

Figure 1

Frequency of intragenic deletions in FHIT transcripts in human CRC. Gel photo of FHIT RT-PCR products showing that the full length FHIT was the predominant transcript in samples 1-3, 5, 7 and 9. Both full length FHIT and shorter fragments representing transcripts containing deletions in FHIT could be seen in samples 4, 6 and 7. The arrow shows the gel position of splice variants.

Figure 2

Sequencing analysis of aberrant FHIT transcripts. A: Deletion of exons 4-6 in the FHIT gene; B: Deletion of exons 4-8 in the FHIT gene.

Figure 3

TMA-IHC assay showing significantly decreased FHIT expression in normal colonic mucosal samples (A), CRC tissue samples (B), and magnified views of the respective samples A and B (C and D).

Figure 4

Relationships between FHIT expression and CRC differentiation grade (A) and stage (B), and survival rate (C) CRC patients.

Figure 5

TUNEL assay showing significantly decreased apoptosis in FHIT-negative CRC cells (A), FHIT-positive CRC cells (C), and magnified views of the respective samples A and C (B and D).

Figure 6

Decreased FHIT expression and apoptosis inhibition in human CRC. Reduced apoptosis index was detected in the same number of cells per field in CRC with FHIT-negative expression.

Figure 7

TMA-IHC showing significantly decreased Bax expression in FHIT positive CRC tissue samples (A), FHIT-negative CRC tissue samples (C), and magnified views of the respective samples A and C (B and D).

Figure 8

TMA-IHC showing significantly increased Bcl-2 expression FHIT positive CRC tissue samples (A), FHIT-negative CRC tissue samples (C), and magnified views of the respective samples A and C (B and D).

Figure 9

TMA-IHC showing significantly increased survivin expression in FHIT- positive CRC tissue samples (A), FHIT-negative CRC tissue samples (C), and magnified views of the respective samples A and C (B and D).

Figure 10

Tree-view of differential protein expression data from 80 CRC tissue samples. Tree-view demonstrated the FHIT protein expression related to Bax, Bcl-2 and survivin protein expression. Red indicates up-regulation, green indicates down-regulation, and yellow indicates no significant change.