## 3 Results
### Effect of He-Ne Laser Pre-Sowing Seed Treatment on the Bioactivities of S. marianum Fruit Extracts
#### 3.5.2 Effect of _S. marianum_ Fruit Extracts on _Dictyostelium discoideum_ Development Life Cycle  
_Dictyostelium_ cells are unicellular, feeding on bacteria and dividing by mitosis. When the food source is removed, cells start a development phase that begins with cAMP secretion and GPCR activation that links to heterotrimeric G proteins in order to initiate cell chemotaxis within the first 6 h. The _D. discoideum_ development cycle completes with fruiting body formation after 24 h [18]. Chemotaxis is a very dynamic mechanism that is crucial for cancer metastasis. To determine the role of 6-min laser-treated _S. marianum_, the multicellular development cycle of _D. discoideum_ was tested. The AX2 cell line was used for this study. The cells were washed with DB buffer and starved onto a DB agar plate along with 600 \(\upmu\)g of either 6-min laser-treated _S. marianum_ extract or laser-untreated _S. marianum_ extract. The results showed that treatment with 600 \(\upmu\)g of laser-treated _S. marianum_ extract caused a noticeable delay in the development cycle and fruiting body formation  
Figure 2: Cell growth of _Dictyostelium discoideum_. Cells were grown in a 24-well plate, as described in Section 2.6.2. The AX2 cells (WT) were treated with a fruit extract of either 6-min laser-treated _S. marianum_ (LSM) or untreated _S. marianum_ (USM). Treatment with the solvent (DMSO) at a compatible percentage was used as a control. (**A**) AX2 cells treated with the extracts at the lowest concentration (100 \(\upmu\)g). (**B**) AX2 cells treated with extracts at a concentration of 300 \(\upmu\)g. (**C**) AX2 cells treated with the highest concentration (600 \(\upmu\)g) of the extracts. (**D**) wild-type AX2 cell densities over three days compared to the DMSO-treated samples for the three concentrations used. The values represent three independent experiments +SD (*_p_ < 0.05, **_p_ < 0.01).  
(Figure 3G-I) compared to the laser-untreated _S. marianum_ extract sample (Figure 3D-F) and the control sample (1.5% agar + 0.6% DMSO) (Figure 3B). To emphasize the effect of laser-treated _S. marianum_ extract on the development cycle of _D. discoideum_, we doubled the concentrations of the treatments. Using 1200 ug of the extracts confirmed the conclusion reached. Adding 1200 ug of laser-treated _S. marianum_ extract to the agar plate caused severe developmental delays (Figure 3M-O) compared to the sample treated with 1200 ug laser-untreated _S. marianum_ extract (Figure 3J-L) and the control sample (1.5% agar + 1.2% DMSO) (Figure 3C). This effect was not due to the solvent (DMSO) concentration, as the results showed no differences between the development cycle of the AX2 cells on DB containing 1.5% agar only (Figure 3A) and the control samples (1.5% agar + 0.6% DMSO, Figure 3B, or 1.5% agar + 1.2% DMSO, Figure 3B). We also observed that treatment with 1200 ug of 6-min laser-treated _S. marianum_ extract did not inhibit cells streaming; however, it caused a delay in this stage, as some of the population began to aggregate after 24 h of starvation (Figure 3M).  
Figure 3: Development phenotypes of _Dictyostelium discoideum_ after 24 h of starvation. (**A**) Ax2 cells were washed with DB buffer and plated onto a DB (1.5% agar) plate at a density of 2 \(\times\) 10\({}^{7}\) cells per plate (90 mm dia.). Ax2 cells were starved onto a DB agar plate (1.5%) containing 0.6% DMSO (**B**) or 1.2% DMSO (**C**) as controls. (**D–F**), Ax2 cells were starved onto a DB agar plate (1.5%) that contained 600 ug of laser-untreated _S. marianum_ extract. (**G–I**), Ax2 cells were starved onto a DB agar plate (1.5%) containing 600 ug of laser-treated _S. marianum_ extract. Images show the development phenotype of AX2 cells starved onto a DB agar plate (1.5%) containing 1200 ug of laser-untreated _S. marianum_ extract (**J–L**) or 1200 ug of laser-treated _S. marianum_ extract (**M–O**).