Induction of Apoptosis in Toxoplasma gondii Infected Hela Cells by Cisplatin and Sodium Azide and Isolation of Apoptotic Bodies and Potential Use for Vaccination against Toxoplasma gondii

Abstract

Background: Toxoplasma gondii can infect a wide range of mammalians, especially humans. It controls several intracellular signals for the inhibition of apoptosis. This study aimed to investigate the apoptogenic effect of cisplatin and sodium azide on T. gondii infected HeLa cells and isolate apoptotic bodies (blebs) as a potent stimulator of the immune system.

Methods: The cytotoxic properties of cisplatin and sodium azide (NaN3) on HeLa cells were evaluated by MTT assay. Moreover, the apoptogenic activity of cisplatin and NaN3 was studied using flow cytometry (Annexin V/PI double staining) and scanning electron microscopy (SEM). Finally, apoptotic bodies were separated by centrifugation.

Results: MTT assay data showed that the survival rate of cells treated with different concentration of NaN3 was significantly reduced, compared to negative control groups. Concerning cisplatin, only concentration of 20 μM had not a significant impact on the cell viability; however, the other concentration of cisplatin significantly reduced cell viability, compared to negative control groups. The level of early apoptosis in uninfected HeLa cells was higher compared to infected HeLa cells treated with cisplatin and NaN3. Finally, apoptotic bodies were separated from T. gondii infected HeLa cells treated with cisplatin.

Conclusion: Apoptosis was induced in both uninfected and infected HeLa cells with T. gondii and apoptotic bodies were isolated from infected cells. Therefore, further studies on apoptotic bodies are required in order to find a proper candidate for vaccine preparation against T. gondii infections.

Keywords: Apoptosis; Apoptotic blebs; Cisplatin; NaN3; Toxoplasma gondii; Toxoplasmosis.

Fig. 1:

Evaluation of cell viability using the MTT assay A) Effects of various concentrations of NaN3 on the viability of the HeLa cells after 24 h. The viability of the cells treated with various concentrations of NaN3 had significant difference compared to untreated negative control cells (^*P<0.05). Data are presented as mean±SEM of three identical repeats of each experiment B) Effects of various concentrations of cisplatin on the viability of the HeLa cells after 24 h. The viability of the cells treated with various concentrations of cisplatin had significant difference compared to untreated negative control cell except the group that treated with 20μM cisplatin (^*P<0.05).

Fig. 2:

Comparison of the early and late apoptosis in HeLa cells treated with cisplatin, Toxoplasma+Cisplatin 25μM and Cisplatin 25μM+Toxoplasma Cells stained with Annexin V represent an early stage of apoptosis. The most cells in the early apoptotic stage were seen in cisplatin-treated cells. However, cells treated with cisplatin 25μM +Toxoplasma showed the most level of the late stage of apoptosis. The early apoptosis significantly decreased in Toxoplasma +Cisplatin 25μM and Cisplatin 25μM +Toxoplasma groups vs. cisplatin group, ^**P<0.001. The late apoptosis significantly increase in Toxoplasma +Cisplatin 25μM and Cisplatin 25μM +Toxoplasma groups vs. cisplatin group, ^**P<0.001

Fig. 3:

Comparison of the early and late apoptosis in HeLa cells treated with NaN3, Toxoplasma+NaN3 1μM, and NaN3 1μM + Toxoplasma Cells stained with Annexin V represent the early stage of apoptosis. The most cells in the early stage of apoptosis were seen in NaN3 treated cells. However, cells treated with NaN3 +Toxoplasma showed the most level of the late stage of apoptosis. Early apoptosis significantly decreased in Toxoplasma+NaN3 and NaN3 +Toxoplasma groups vs. NaN3 group, ^**P<0.001. The late apoptosis significantly increase in Toxoplasma+NaN3 and NaN3 +Toxoplasma groups vs. NaN3 group, ^**P<0.001

Fig. 4:

Dot plot diagrams of Cisplatin (a) Dot plot diagrams showed in untreated the HeLa cells as control group; (b) The highest level of early apoptosis 88.9% and late apoptosis 6.6% in the HeLa cells induced with 25μM Cisplatin after 12 h; (c) Dot plot diagrams showed the level of early late apoptosis equal to 68.3% in the HeLa cells treated with the Toxoplasma + Cisplatin 25μM after 12 h; (d) The late apoptosis equal to 85.7% in the HeLa cells treated with 25 μM of Cisplatin + Toxoplasma after 12 h

Fig. 5:

SEM micrographs of surface ultra-structural characteristics of HeLa cells treated with cisplatin for 12 h (a) Control HeLa cells surface showed the restoration of a typical morphological feature of cancer cell such as numerous microvilli with several membrane connections indicated by white arrow (M). (b) Treated HeLa cells with Toxoplasma + Cisplatin (12 h, IC50: 41μgr/ml). (c and d) Treated HeLa cells with cisplatin without T. gondii showed distinct morphological changes corresponding to typical apoptosis, including cell membrane blebbing (B) and cytoplasmic extrusions (S) (b, c, d). (e) Blebs were isolated from apoptotic HeLa cells