Journal of Clinical Chemistry and Laboratory Medicine
Open Access

Cytotoxicity Assessment of Sea- Star Protein SIP-Young-6His in MCF-7 Cells

Michel Leclerc^{* *}Correspondence: Michel Leclerc, Department of Immunology, Orleans University, Orleans, France, Email:

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Introduction

Cytotoxic activity of sea star axial organ, of sea star IGKappa gene have been demonstrated against Mouse tumoral cells and human ones [1,2]. In the present work, we had tried to study the cytotoxicity of sea star: SIP-Young-6His protein produced in HEK293 and provided at 0.13 mg/ml in PBS buffer [3] against MCF-7 cells when compared to doxorubicin [4].

Materials and Methods

A Cell Cytotoxicity Assay Kit from Abnova, Ref# KA4151 and lot number 187115 was used. Second, MCF-7 cell line was grown in T75 flasks with DMEM: F12 (1:1) media, with 10% Fetal Bovine Serum (FBS) and gentamicin. Detachment was performed with trypsin, after extensive washing with DPBS. Once cells were detached, trypsin was inactivated with the FBS from the cell culture media. MCF-7 is an epithelial cell line isolated from the breast tissue of a patient with metastatic adenocarcinoma. These cells were taken as targets. Doxorubicin (DOX) (Clinisciences) involved in the induction of DNA damage, inhibition of cell proliferation, impairment of mitochondria, and cell death, and was therefore used as a cell-death inducer [3] so, 2 cell-death inducers were compared: Doxorubicin and sea star SIP-Young-6His protein.

Results

Cytotoxicity assay and results

Cells from T75 flasks were seeded on 96-well plates with black walls and clear bottom in 100 uL cell culture media to reach a confluency of 70% after 48 h of culture. A first cytotoxicity test was performed to assess which is the DOX amount necessary to reach a metabolic activity decrease, or cell death, of around 50% (Tables 1-6).

Table 1: DOX was dissolved in DPBS and concentrations were adjusted so all wells were treated with 10 uL of DOX solution or DPBS (vehicle C-), in at least triplicates.

Table 2: The Cell Cytotoxicity Assay Kit is added, and the spectrophotometry reading is performed after 24 h of DOX and Cell Cytotoxicity Assay Kit incubation following manufacturer’s instructions. *The bold value is not used for the cell viability calculations since it is out of the tendency.

These results are used for the cell viability calculation, which is calculated as: Sample Mean Abs/Vehicle Mean Abs × 100.

Table 3: It is showing that higher DOX doses are related to lower cell viabilities.

The cell death effect from DOX is no longer observed for doses of 1.25 µg/well or lower, except for dose 0.31 µg/well, when lower cell viability is observed again.

Table 4: Increased DOX concentrations are tested for the final assay to reach lower cell viabilities. Also, the SIP-Young-6His protein (in PBS) is tested in different dilutions and some wells with no cells are added as background controls.

As DOX may interfere with the spectrophotometry reading, after the 24 h treatment all wells are carefully washed with DPBS twice. After that, 100 µL cell culture media and 20 µL Cell Cytotoxicity Assay Kit are added.

Table 5: The spectrophotometry reading is performed after 1, 2 and 3 h of incubation, as the Cell Cytotoxicity Assay Kit incubation time is dependent upon the cell type. No relevant differences among wells are observed after 1 and 2 hours of incubation with the reagent, so the 3 hours’ time point is used for the result analysis. *The bold values are out of the tendency for the vehicle control and are therefore dismissed.

Cell viability calculation is calculated as: (Sample Mean Abs-No cells Mean Abs)/(Vehicle Mean Abs-No cells Mean Abs) × 100.

Table 6: Cell viability of normal dose and protein dose is calculated here.

Discussion and Conclusion

Decreased cell viability is observed with higher DOXO amounts, indicating that the assay works properly. On the other hand, the treatment with sea star SIP-Young-6-His protein, after 24 h with the stated doses, does not seem to be affecting the cell viability of MCF-7 cells. We observe a weak cytotoxicity against these last cells. Further studies are necessary to complete the study of Spontaneous and Induced cytotoxicity of the invertebrate protein issued from Hek cells against cancerous vertebrate cells.

We notice that: “Spontaneously occurring cytotoxicity of sea star axial organ cells on target cells has been studied. It appears that in an allogeneic system no cytotoxic phenomenon occurs after 4 or 6 h of culture, whereas spontaneous cytotoxicity toward malignant vertebrate target cells is shown and reaches its maximum at 37 degrees C. In a certain way, the axial organ cell population includes cells, which may be compared with vertebrate natural killer cells. On the other hand, it must also be noted that axial organ cells are able to exert in a few cases, an induced cytotoxicity on MBL2 cells. This phenomenon appears at the end of the fourth day of culture”.

References

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