Colon cancer is the third most common cancer among men and the second most common in women worldwide, and its treatment has always been challenging . One of the major problems in cancer treatment is the resistance of cancer cells to chemotherapy. The most important causes of cancer resistance and relapse after standard treatments has been found to be CSCs in cancerous tissues. These cells are the source of cancer and the cause of malignancy and recurrence in patients. [7–8]. This suggests that many cancer therapies, while killing the bulk of tumor cells, may ultimately fail because they do not destroy CSCs, which survive to regenerate new tumors . Therapies with CSC-specific agents could provide a strategy for successful treatment that it requires enough information about biology and molecular mechanism of these cells. Since the CSCs are a small percentage of the total cancer cells in a tumor and current cell culture techniques have some limitations for their isolation and cultivation, therefore screening of CSCs faces major challenges .
There have been different studies on CSCs drug screening  [18–20]. Many studies have been performed on the EMT process and its association with cancer  [18–20]. During EMT, cells undergo extensive changes like migration/invasion abilities and resistance to apoptosis and senescence. Also, EMT induction in normal and cancerous cells results in resistance to chemotherapeutics [8–11].
Some studies have shown EMT derived mesenchymal cells have similar attributes with CSCs and suggested that EMT induction in normal and tumor epithelial cells can result in the enrichment of CSCs [3–4] .
Mani et al. (2008) showed in human and rat breast cancer cells, CD44High / CD24Low cells that have properties of stem cells and are called breast CSCs can be increased by TGF-β‐induced EMT. Therefore, the author found a possible link between EMT and CSCs.
During EMT, markers of epithelial cells such as E-cadherin are lost and mesenchymal markers, such as Vimentin are expressed. E-cadherin is considered as the key protein of EMT, and its decreased expression leads in the collapse of cell joints and EMT induction  [13–22]. Considering the key roles of E‐cadherin in EMT and the association of EMT with CSC phenotype, Gupta et al (2009) and Onder et al (2008) in order to enrichment of CSCs used lentiviral vector carrying shRNA to knockdown of E‐cadherin in cancer cell lines to induce EMT  . Gupta (2009) used this method to develop and implement a high-throughput screening method to identify agents with specific toxicity for epithelial CSCs. The results of their screen and subsequent experiments demonstrate that it is possible to find agents with strong selective toxicity for breast CSCs .
Here, the effects of Pio and Cetuximab, were investigated on CSC enriched HT29 cell line (HT29-shE (, in which CSCs were enriched by EMT induction using lentiviral vector carrying shRNA for knockdown of E-cadherin.
Cetuximab is a monoclonal antibody that has been successful in the treatment of some cancers such as head, neck and colorectal cancer by immunotherapy. It binds to the epitopes of the human epidermal growth factor receptor (EGFR1) and prevents binding EGF to the EGFR by shutting down EGFR activity at the molecular level. Since this receptor plays a role in stimulating cell growth, proliferation and differentiation, stopping its function by Cetuximab inhibits tumor cell growth and proliferation. Studies have also shown that this drug can reduce EMT and metastasize cancerous tumors .
Pio is a thiazolidinediones family that is used clinically to treat type 2 diabetes. Recently, it has been used to treat some cancers, including colon cancer. Pio is a synthetic ligand of peroxisome proliferator-activated gamma receptor (gamma PPAR), and increases the activity of this receptor by binding to it. Activation of PPAR result in increased expression of several genes, including the gene encoding E-cadherin. Therefore, Pio can prevent cancer metastasis and invasion by increasing E-cadherin expression and effect on EMT and therefore probably on CSCs [25–27].
To investigate the effect of these drugs on CSCs after preparation and culturing of HT29 and HT29-shE, drug treatment was performed on cells.
Cells were treated with different doses of Pio (100–1000 µM) and Cetuximab (10–100 µg / ml) for 5,3 and 7 days and MTT assay was done to find the appropriate dose and duration of treatment. Finally, IC50 values for Pio and Cetuximab were obtained on day 3, 300 µM and 70 µg/ml, respectively. Then cells were treated with the drugs for 3 days based on their IC50. In the following the colon CSC markers and expression of E-cadherin and Vimentin genes were evaluated before and after drug treatment.
Prior studies showed that CD133 and CD44 are the potential markers of CSCs in HT29 colon cell line [28–29]. Therefore, we selected and analyzed these markers using flow cytometry. The expression of CD44 and CD133 markers in untreated HT29 cell line were 2.31% and 1.7%, respectively, which are nearly the same as other studies' findings [29–30]. After treatment of HT29 with Pio and Cetuximab, flow cytometry data showed decrease in expression of these markers. In untreated HT29-shE, the expression of CD44 and CD133 markers were 10.9% and 3.7% respectively and the expression of these markers decreased after cell treatment. As a result, drugs were able to reduce CSC markers. In our previous study the expression of CD44 and CD133 markers in HT29 were 1.99% and 2.07 respectively, in HT29-shE were 8.11% and 15.6%. We expected these data be similar to each other, but this may be due to different laboratory conditions and changes in cell function over time. Also rate of CSCs enrichment and rate of CSCs reduction after drug treatment are not the same with the Gupta test, this difference was probably due to differences in the type of cell lines, drugs, CD markers and other conditions that were used in the two studies. However, the expression of CSC markers in the Gupta study also decreased after the cells were treated with the Salynomysin .
To analysis the expression rate of E-cadherin (main molecular marker of epithelial cells) and Vimentin (main molecular markers of mesenchymal cells (RT-qPCR was done.
RT-qPCR results showed the increased expression of E-cadherin in addition the decreased expression of Vimentin in treated HT29-shE and HT29 cells. These results were in agreement with the other studies. Thus, the drugs were able to activate epithelial molecular markers and inhibit mesenchymal molecular markers, causing the transition from EMT to MET. [4, 6]
Since E-cadherin is involved in cellular attachments, we expected structural changes to be observed in treated HT29-shE after increasing the expression of E-cadherin. Therefore, the cells were monitored and photographed daily after treatment for 2 week using light and fluorescent microscopy. In the second week, the morphology of HT29-shE treated with Cetuximab changed from mesenchymal to epithelial. As a result, this drug was able to deformation of cells and lead to the MET process, which is the reverse of EMT. In the Gupta study, these deformations and processes also occurred. 
Finally, it might be better to study more drugs and use more techniques to confirm the results, but due to some limitations, we could not.