Team:TecMonterrey/results.html&type=edit

Normal Cells Viability Assay

Previous studies have suggested that Apoptin and sTRAIL are toxic exclusively to cancer cells. Based on the previous statement we tested both proteins along with their fusions to the secretion peptide, Apoptin-HlyA and TRAIL-HlyA, on the non-cancerous cell line NIH-3T3.

As expected, Apoptin and TRAIL didn’t show cytotoxic activity against NIH-3T3 cells, in contrast to that shown for the cancerous cell lines. Similar results were obtained when Apoptin-HlyA and TRAIL-HlyA were tested; suggesting that the fusion does not significantly affect their selectivity against the cancer cells lines tested.

Table 1. Citotoxicity of the therapeuthic proteins NIH-3T3 cells
Protein	IC50 value
Apoptin	>6.4 µM
TRAIL	>6.4 µM
Apoptin-HlyA	>18 µM
TRAIL-HlyA	>8.9 µM

Cancerous Cell Lines Viability

Table 1. Citotoxicity of the therapeuthic proteins NIH-3T3 cells
Cell Line	Apoptin	Apoptin HlyA	sTRAIL	sTRAIL-HlyA
MCF-7	0.129 ± 0.067	0.066 ± 0.075	7.236 ± 1.770	NA
Caco-2	0.737 ± 0.127	0.077 ± 0.035	>18	NA
PC-3	1.329 ± 0.135	0.020 ± 0.005	0.013 ± 0.0088	0.044

The Table 2, summarizes the different IC50 values obtained for the four different therapeutic proteins evaluated in the cancerous cell lines. ">" denotes that the proper dose of protein to measure the IC50 value was not reached. "NA" means that the protein was not evaluated in those cell lines. The IC50 values of the different therapeutic proteins for each cell line are plotted and discussed in the following graphs.

Referring to the MCF-7 cell line, the IC50 values were much lower for Apoptin and Apoptin-HlyA, compared to those of sTRAIL. It is imperative to mention that this cancerous cell line, as well as the PC-3 and Caco-2, shows higher sensibility to the fusion Apoptin-HlyA than its counterpart Apoptin. However, in this cell line the differences of IC50 values from Apoptin and Apoptin-HlyA were not significantly different (α= 0.05).

As it was previously mentioned, Apoptin-HlyA had an unexpected increment in the cytotoxic activity on the Caco-2 cells. The decrement in the IC50 value of the Apoptin-HlyA fusion was about 10X lower than the Apoptin alone. After making an ANOVA analysis between these two variables, it was demonstrated that IC50 values are significantly different (α= 0.05). The protein sTRAIL is not shown in the Figure 2 since it didn’t reach an IC50 value with the maximum dose evaluated (18 µM).

Similarly to the results obtained with the Caco-2 cell line, the IC50 value of the Apoptin-HlyA fusion decreased dramatically in the PC-3 cell line, suggesting that the fusion enhanced the cytotoxic activity of the protein. As expected, both IC50 values are significantly different. The IC50 value of sTRAIL-HlyA is not shown in the previous graphs because it didn’t reach an IC50 value with the maximum dose evaluated on this cancerous cell line (0.044 µM). These results suggest that the fusion affected its cytotoxic activity.

Localized expression results

After 4 days in culture, the expression of GFP controlled by the pHG promoter was measured for fluorescence in a 96-well plate. Two readings were done, adjusting the amount of total protein in each well to 250ng and 500ng for each sample.

As it is shown, in the 250ng adjusted plate, the GFP induction is greater in Normoxic conditions. Also, in anoxic environment the production of GFP is low, indicating that the pHG promoter is not hypoxic specific and acts more like a constitutive.

For accuracy in the experiment a 500ng plate was measured and the results were found to be similar.

As it can be seen, the tendency in GFP expression is the same, concluding that pHG induces the production of GFP in both conditions, but in a better rate when there are higher oxygen concentrations in culture.

From this general experiment we conclude that the design of pHG was not successful because the HIP-1 promoter comes from Salmonella with the FNR native from E. coli. It is Probable that this general transcription factor was not compatible with the transcriptional machinery of E. coli and the expression of GFP was controlled by a different sequence.

pFG and pNG characterizations results.

As it is described in the methodology, to characterize the function of these two promoters a bioreactor had to be used. This device allowed us to know the concentration of oxygen at a specific time.

a) The model of bioreactor used (Sartorius Biostat ® A plus).

b) The bioreactor was opened and inoculated in the laminar flow hood. The air that entered at atmospheric conditions and could dissolve, after being closed and agitated the maximum level of oxygen was present in the liquid.

For pFG and pNG the inductions under hypoxic conditions were:

For the pNG promoter there was no induction, proving that the previous registered part is not functional BBa_K905000.

For the pFG promoter it can be observed that there are two induction points, one between 8.5 and 8.0% of oxygen and the other at 5.0%, proving that this part is totally functional and specific for the desirable conditions. The genetic part designed was appropriate and now it is established that the induction oxygen concentration of the pFG promoter is between 6.9 and 7.2% of the total amount of oxygen (from the air that entered when it was inoculated) dissolved in the liquid at a constant agitating speed of 250rpm. This is an improvement to the Nirb promoter (BBa_K905000).

Secretion Results: Apoptin, Apoptin+HlyA

Considering the lack of results for the secretion of Apoptin-HlyA (AH), and the abnormal secretion of Apoptin(A), we decided to repeat the secretion protocol for both of them. The double transformants A+Secretion system (HlyB and HlyD) and AH+Secretion system, were induced following the same conditions from past experiments. Soluble fraction from cell lysates were purified as previously mentioned and the medium was concentrated by ultrafiltration, both samples were treated and evaluated by Western blot analysis.

Figure X: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from the growth media and from the lysates of E.coli BL21 (DE3) co-transformed with the secretion complex, and purified using HisPur Ni-NTA Purification Kit (Thermo Scientific). Lane1: : Purified HIS-TAT-APOPTIN (from soluble fraction); Lane2: : no treatment; Lane3: : HIS-TAT-APOPTIN (from medium); Lane4: : No treatment; Lane5: Purified HIS-TAT-APOPTIN-HlyA (from soluble fraction); Lane6: : no treatment; Lane7: : HIS-TAT-APOPTIN-HlyA (from medium) ; Lane8: : Positive control (previously confirmed HIS-GFP); Lane9: Molecular Weight Marker; Lane 10: : No treatment.

For this experiment we had some trouble during the electro-blotting procedure, but managed to conclude some results from the obtained data. As image (X) shows, A is being produced internally, and apparently, some of the protein is present in the medium. On the other hand, there is less amount of AH being produced intracellularly with a small portion being detected in the medium.

From the previous experiments, evidence suggests A has the ability to be secreted without the secretion peptide (HlyA), as long as HlyB and HlyD are also expressed. On the other hand, AH presented less overall production than A, but secretion was also evident when HlyB and HlyD were co-induced.

Internalization in vivo assay

A second assay was performed in a different cell line, this time using CHO-S cells growth in suspenssion to prove the functionality of TAT as a cell penetrating peptide. As in the past experiment, protein samples previously confirmed by Western blot analysis were sterilized by filtration; the Internalization Assay protocol Version II was followed as indicated in the Internalization methods. Finally, CHO-S cell lysates and the supernatants were analyzed by Western blot using an anti-HIS6x antibody HRP conjugated.

Figure 4 shows the Western blot from CHO-S cell lysates and supernatants that were incubated with 1.3 µg/mL of purified HIS-GFP and HIS-TAT-GFP. Protein bands can be appreciated in both the lysates and the supernatants at the expected molecular weight. However, the negative control sample (no treatment with heterologous proteins) also presents a band at the same molecular weight.

Figure 4: Western Blot, probed with anti-His6x antibody HRP conjugated. CHO-S cell lysates and supernatants treated with 1.3 µg/mL of purified HIS-GFP and HIS-TAT-GFP expressed in E.coli BL21. Lane1: No treatment; Lane2: Positive control (previously confirmed HIS-GFP); Lane3: Molecular Weight Marker; Lane4: No treatment; Lane5: cell lysates with 1.3µg/mL HIS-TAT-GFP treatment; Lane6: supernatants with 1.3µg/mL HIS-TAT-GFP treatment; Lane7: No treatment; Lane8: cell lysates with 1.3µg/mL HIS-GFP treatment; Lane8: supernatants with 1.3µg/mL HIS-GFP treatment; Lane9: negative control, cell lysates without protein treatment. Lane 10: No treatment.

From the given results in our previus Western blot, we can conclude that native proteins in CHO-S cells of similar molecular weight to TAT-GFP and HIS-TAT-GFP are capable of binding to the anti-His6x antibody HRP conjugated. These conclusions are corroborated by Natalie Bertz (2010), who showed that endogenous Ni-binding proteins in CHO-S cell lines bind to some anti-His primary antibodies. Therefore, further assays have to be performed with different cell lines in order to prove the efficiency of the internalization of proteins fused with the TAT peptide. Cell and protein concentrations can also be optimized, so that the background signal from unwashed heterologous proteins is reduced as much as possible.

Reference:

Betz, N. (2005). Purification of Recombinant Polyhistidine-Tagged Proteins From Mammalian Cells Using the MagneHis Protein Purification System . Promega, 1, 1.