Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer

Abstract

Aim: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics.

Methods: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed.

Results: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers.

Conclusion: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

Figure 1

A: A representative 2DE gel of a normal tissue. Proteins that become down-regulated in corresponding tumor (Figure 2A) are shown with arrows and capital letters. For a better visualization of spots within the box, silver stained image of another gel is shown; B: Immunodection of actin as an internal control of protein loading, the * represents the location of actin.

Figure 2

A: A representative 2DE gel of tumor tissue. Arrows and numbers indicate up-regulated proteins in comparison with their matched normal tissue (Figure 1A); B: Immunodetection of actin and the * represents the location of actin.

Figure 3

A: Verifying differential expression pattern of ß tropomyosin by RT-PCR in three separate experiments as indicated by numbers (1 to 3), normal versus tumor tissues. The amplification product (431bp) is limited to normal tissue which indicates loss or strong down regulation of this protein as observed by 2DE; B: RT-PCR amplification of ß-actin as an internal control of RT-PCR; C: Electrophoresis of the total RNA from normal and tumor tissues used for cDNA synthesis and RT-PCR. L: DNA marker; N: normal tissue; T: tumor tissue.