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J Acupunct Res > Volume 32(1); 2015 > Article
Lee and Song: Snake Venom-enhanced Cytotoxic Effect of Natural Killer Cells on A549 Human Lung Cancer Cell Growth※

Abstract

Objectives:

The purpose of this research was to investigate the cytotoxic effect of Natural Killer(NK)-92 cell and Snake Venom, and to elucidate its mechanism on human lung carcinoma cell A549.

Methods:

In order to figure out whether Snake Venom enhances the cytotoxic effect of NK-92 cell in A549 cell, Cell Viability Assay was conducted. Also, in order to observe the changes of Caspase-3 and Caspase-8, both of which are proteinases that advance apoptosis, and the changes of TNRF and DR3, which are Death Receptors of the extrinsic pathway of apoptosis, Western Blot Analysis was conducted. By conducting RT-PCR analysis, we have tried to confirm Perforin, Granzyme B, and GADPH, all of which are cytotoxic-related proteins. Lastly, in order to observe the effect of Snake Venom on NO formation within human lung carcinoma cells, NO determination was conducted.

Results:

  1. After conducting Cell Viability Assay, Snake Venom enhanced the cytotoxic effect of NK-92 cell and inhibited the growth of A549.

  2. Western Blot Analysis caused proteinases Caspase-3 and Caspase-8, which advance apoptosis, to increase in the combined treatment group, but not in treatment groups that focused only on either Snake Venom or NK-92 cell in A549 lung carcinoma cells.

  3. Western Blot Analysis caused an expression of TNFR2 and DR3, both of which are Death Receptors of the apoptosis extrinsic pathway, in the combined treatment group, but not intreatment groups that focused only on either Snake Venom or NK-92 cell in A549 human lung carcinoma cells.

  4. After conducting NO determination, NO formation within A549 cell showed no significant changes in both treatment groups that focused NK-92 cell and combined treatment group.

  5. After conducting RT-PCR, the expression of Granzyme B and Perforin, which are cytotoxic-related proteins within A549 human lung carcinoma cells, showed growth in the combined treatment group, but not the treatment group that focused only on NK-92 cell.

Conclusion:

It has been indicated that, when it comes to the A549 cell, Snake Venom enhances the increase of Death Receptor expression and continuous apoptosis reaction, leading to the enhancement of the cancer cell cytotoxic effect of the NK-92 cell. It is expected that Snake Venom can be used with the NK-92 cell for further lung cancer treatment.

Notes

This research was supported by the Gachon University Research Fund in 2015

Fig. 1.
Snake venom enhances cytotoxic effect of NK-92 cells on A549 human lung cancer cells
The A549 human lung cancer cells, and those co-cultured with NK-92 cells were seeded onto 24-well plates(5 × 104 cells/well) and subconfluent cells were subsequently treated with snake venom(4 μg/ml) for 24 hrs. The cells were then harvested by trypsinization and stained with 0.2 % Trypan blue. Relative cell survival rate was determined by counting live and dead cells. The results were expressed as a percentage of viable cells. Columns, means of three experiments, with triplicates of each experiment; bars, SD.
*: p<0.05, significantly different from co-cultured with NK-92 cells.
acupunct-32-1-79f1.gif
Fig. 2.
Snake venom enhances the expression of death receptor and its related apoptotic proteins by NK-92 cells in A549 human lung cancer cells
Expression of death receptors such as TNFR2, DR3 and apoptosis regulatory proteins such as apoptosis regulatory proteins such as bax, caspase-3, caspase-8, cleaved caspase-3 and cleaved caspase-8 was determined using western blot analysis.
A549 human lung cancer cells were co-cultured with NK-92 cells and subsequently treated with 4 μg/ml of snake venom for 24 hrs.
Equal amounts of total proteins(50 μg/lane) were subjected to 12 % or 8 % SDS- PAGE. Expression of TNFR2, DR3, Bax, caspase-3, caspase-8, cleaved caspase-3, cleaved caspase- 8 and β- actin was detected by western blotting using specific antibodies.
Each band is representative for three experiments.
acupunct-32-1-79f2.gif
Fig. 3.
Effect of co-culture of NK-92 cells on the level of NO in the A549 Human Lung Cancer Cells.
The cells were co-cultured with NK-92 cells and then treated with snake venom(4 μg/ml) for 48 hrs. Thereafter, the nitrite release in the supernatant was assessed by griess reaction as described in Materials and methods. The figures are representatives of three experiments with replicates.
Values are mean ± SD of three experiments with replicates.
acupunct-32-1-79f3.gif
Fig. 4.
Snake venom enhances the expression of NK-92 cell-related cytotoxic proteins in A549 human lung cancer cells
Expression of NK-92 cell related cytotoxic proteins such as perforin and granzyme B and GADPH was detected by RT-PCR analysis. GADPH protein was used as an internal control. Each band is representative for three experiments.
acupunct-32-1-79f4.gif
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