The incidence of colorectal cancer has been increasing in the last few years, while the age of diagnosis is decreasing, and today it is the third or fourth cause of death in the world. The treatment of metastatic colorectal cancer (mCRC) has changed drastically since the 1980s, when only fluorouracil (5-FU) was available for treatment and the median survival was at the most 12 months, to a time when mCRC is considered more of a chronic disease in which the median survival is now reported to be in excess of 2 years, although the 5-year survival rate is still less than 10% . The advances in the treatment of this disease include studies of single-agents vs. combination treatment with 5-FU/leucovorin, irinotecan, oxaliplatin, and capecitabine, and the role of targeted agents such as cetuximab and bevacizumab.
The platinum-based chemotherapy drugs cisplatin, carboplatin, and oxaliplatin are among the most active and widely used agents for the treatment of colorectal cancer today . Cisplatin is a third-generation platinum compound and like the rest of these agents, (oxaliplatin) kills tumor cells primarily by causing DNA damage .
Over the last few years, it has been reported that colorectal cancer is a polygenic disease in which oncogene mutation activation and tumor suppressor gene inactivation play important roles in the development of the disease and in the response to the chemotherapy.
TP73 is a gene that was described by Kaghad et. al. in 1997  and is a family member of the tumor suppressor gene TP53. TP53 and TP73 share significant structural and functional homology. Both genes contain an NH2 terminal transactivation domain, and a COOH-terminal oligomerization domain, and are capable of inducing cell cycle arrests and cell death in response to DNA damage. However, there is some evidence that shows that the roles of p53 and p73 in human tumor genesis are different.
P73 contains carboxy-terminal spliced variants known as the TA isoforms. The So-called ΔN variants also exist, which lack the transactivation domain and are transcribed from an internal promoter within exon 3 of the full-length genes . These different isoforms have been shown to have vastly different activities. The TA isoforms act similarly to p53, inducing apoptosis. In comparison, ΔN isoforms have little transactivation activity and play a role blocking target genes of p53 and their respective TAp73 isoforms . Therefore, the TA isoforms may be expected to have functions in tumor suppression while ΔN isoforms might be oncogenic.
For the first time in 2006, Dominguez et al. demonstrated an association between upregulation of ΔTAp73 isoforms and poor prognosis in colorectal cancer, specifically advanced tumor stage, suggesting that they may be of practical clinical prognostic value . Last year, some authors also demonstrated that high expression of TAp73 in colorectal cancer may be involved in the progression of colorectal cancer and may serve as a potential index to predict differentiation level and prognosis of colorectal cancer .
Although there are many reports concerning the p73 gene, some of its functions remain unclear. Little research has been reported on the relationship between p73 gene transcription and its protein expression with the response to certain drugs such as oxaliplatin and cetuximab which are drugs currently used in colorectal cancer.
Epidermal Grown Factor Receptor (EGFR) is one of the most important cell membrane receptors expressed in normal cells . The EGFR molecular structure includes an extra-cellular region, a transmembrane domain and a protein tyrosine kinase region . Epidermal Grown Factor (EGF) is a natural ligand of EGFR.
EGFR is abnormally activated in many epithelial tumors and it is frequently over expressed in colon cancer, correlating with a poor response to treatment, disease progression and poor survival .
In the early 80s the EGFR was pointed out as a potential target for cancer therapy  and two anti-EGFR strategies were adopted: monoclonal antibodies (Mabs), which bind the extracellular domain, interfering with the natural ligand, and low-molecular-weight tyrosine kinase inhibitors, which interfere with the tyrosine kinase domain . Cetuximab is a chimeric monoclonal antibody antagonist for EGFR that binds to EGFR with high affinity and prevents the ligand from adopting the conformation for dimerization and activation [14–17].
The most important mediators in EGFR signaling are K-RAS and B-RAF kinase proteins. Mutations in these effectors have been found in various cancers [18, 19].
K-Ras and B-Raf mutations are found in up to 50% and 10%, respectively of colon cancers and appear relatively early in the carcinogenesis pathway leading to constitutive activation of its proteins [20, 21]. Upon activation, RAS recruits RAF protein to the cell membrane and binds it directly, activating RAF kinase. B-RAF is considered to be the principal RAF isoform linking Ras to MEK signaling.
Several studies have indicated that the presence of mutant K-Ras in colorectal cancer correlates with a poor prognosis [21–23] and is associated with lack of response to EGFR inhibitors such as cetuximab [24, 25]. Wild type K-Ras status is currently required to administer cetuximab in monotherapy, or combined with other agents, as it has been demonstrated that this is necessary but not sufficient to confer sensitivity to Cetuximab . Some authors have recently concluded that B-Raf wild-type is also required for response to cetuximab and could be used to select patients who are eligible for the treatment . However, not all of the wild type K-Ras and B-Raf patients are responding to cetuximab.
Therefore, the identification of additional genetic determining factors of the action mechanism of EGFR-targeted therapies in colorectal cancers (CRCs) is important at least for two reasons. First, the understanding of the molecular basis of therapies could allow the rational design of alternative treatment strategies. Second, to prospectively identify patients who should not receive either treatment, this way avoiding their exposure to ineffective and expensive therapy.
As it is well known P73 cooperates with Ras in the activation of MAPK kinase signaling cascade , we investigated the relationships between TAp73 expression and K-Ras/B-Raf status as regards of the chemosensitivity. Currently there are no data published on the correlation between TAp73 and cetuximab. In an attempt to further characterize this complex pattern of expression in human colorectal cancer cell lines and to assess its role in response to chemotherapy, the purpose of this paper was to analyze TAp73 mRNA and TAp73 protein expression in colorectal cancer cell lines treated with cetuximab and oxaliplatin, using Real Time PCR and Western Blot to explore associations between p73 expression and K-Ras/B-Raf status.
For the experimental model of our study, we chose three human colon cancer cell lines: HT-29, SW-480 and Caco-2. These enterocyte cell lines were derived from human primary colon adenocarcinomas and are established cell models for the study of the biology and drug treatment of cancer. These cells lines are different in K-RAS and B-RAF pathways, as HT-29 harbors the V600E B-Raf heterozygotic mutation , SW-480 which harbors K-Ras mutation and Caco-2 is K-Ras and B-Raf wild type.
The association between the expression of TAp73 and the presence/absence of K-Ras and B-Raf mutations in response to cetuximab supports their possible apoptotic function and helps to understand the action mechanism of this drug.