الفهرس 
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Abstract
6.1. Summary
The aim of the current study was to compare the efficiency and cytotoxicity of zinc sulfide nanoparticles as a radio sensitizer with gold nanoparticles against breast cancer cells after irradiation with X-rays. Moreover, the mechanisms of actions for the potential cytotoxic effect for the zinc sulfide and gold nanoparticles was investigated.
To achieve our aim zinc sulfide and gold nanoparticles were prepared chemically. Then, the physical size and shape of prepared particles were determined by transmission electron microscope. The zeta potential for prepared solutions was determined by a Nano ZeTACizer particle analyzer. The optical properties of the samples were recorded using UV-Vis spectrometer with scanning range of 300-800 nm.
Prior to the evaluation of the potential radio sensitizing effect of the prepared AuNPs and ZnS Nps, the MTT cell viability assay was conducted to assess the cytotoxic effects of AuNPs, ZnS Nps and X-ray irradiation against the triple negative MDA-MB-231 and estrogen and progesterone receptor-positive MCF-7 human breast cancer cell lines and to determine the IC50 (inhibitory concentration that indicates a 50% reduction in cell proliferation) that will be used in all the subsequent analysis.
The MCF-7 and MDA-MB-231 cell viability was examined after 48 and 72 hrs of X-ray irradiation, cells were irradiated with 0, 2, 4, 6, 8, 10 Gy doses.
Similarly, both cell lines were exposed to different concentration of ZnS Nps (256- 14363 µg/ml) and AuNPs (0.5 – 250 µg/ml) and incubated for 48 hrs before MTT assay for the determination of the IC50.
The Obtained gold particles were spherical in shape with mean particle size of 12.9 nm ± 2.18 nm, having a maximum absorption at 520 nm. The zeta potential of our AuNPs was – 47 mv as the citrate gives the AuNPs a negative cap through negative carboxylic groups.
ZnS Nps were successfully prepared by the chemical precipitation method. TEM examination revealed 22.97 nm sized spherical particles with a −62.5 mV zeta potential. The spectra of ZnS nanoparticles showed a high UV absorbance at 330 nm.
The MTT assay disclosed that treating MCF-7 cells with different concentrations of AuNPs (0, 2, 4, 8, 10, 12, 14, 16, 20, 25, 30 µM) for 48 hrs resulted in gradual significant reduction in MCF-7 cell viability compared to control.
Similarly, treating MDA-MB 231 cells with different concentrations of Au Nps (0.5, 1, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125 µM) for 48 hrs resulted in significant reduction in MDA-MB 231 cell viability at concentrations higher than 0.5 µM. Collectively, the MDA-MB 231 cells was more sensitive towards AuNPs treatment since it showed a lower IC50 value (7.8±1.39) compared to MCF-7 cells in which the IC50 was 10 ±2.12 µM.
On the other hand, MCF-7 and MDA-MB-231 cells were treated with different doses of ZnS Nps (256, 513, 1026, 2052, 4104, 6155, 8207, 10259, 12311, 14363 µM) for 48hr before performing MTT assay. Treating MCF-7 cells with ZnS Nps resulted in significant reduction in cell viability at concentrations higher than 513 µM compared to control. Regression analysis of sigmoidal dose-response curves demonstrated IC50 of ZnS Nps at 14360±6.5 µM.
Treating MDA-MB 231 cells with ZnS Nps resulted in significant reduction in MDA-MB 231 cell viability at concentrations higher than 256 µM. Regression analysis of sigmoidal dose-response curves demonstrated IC50 of ZnS Nps at 12311±3.5 µM.
Therefore, collectively, the triple negative MDA-MB 231 cell line was more susceptible toward both Au Nps and ZnS Nps than the ER positive MCF-7 cells in terms of the cytotoxic dose needed to suppress the cell proliferation process by 50%. The MDA-MB-231 cells were selected to proceed in all molecular analysis.
After the determination of the IC50 values for AuNps and ZnSNps as well as X-ray irradiation in both MCF-7 and MDA-MB-231 cells, we decided to investigate the potential mechanisms leading for such cytotoxicity in MDA-MB-231 cells with and without X-ray irradiation.
Study Design:
The MDA-MB-231 cells were divided into the following groups:
group 1: Cells grown without irradiation.
group 2: Cells exposed to 10 Gy X-ray irradiation.
group 3: Cells treated with IC50 of ZnSNps (12311 µM)
group 4: Cells treated with IC50 of AuNPs (7.8 µM)
group 5: Cells treated with IC50 of ZnS Nps (12311 µM) and irradiated with 10 Gy X- ray.
group 6: Cells treated with IC50 of AuNPs (7.8 µM) and irradiated with 10 Gy X-ray
Biologically, to assess the therapeutic efficacy of the treatment protocol the following parameters were investigated
1- Evaluation of cell cycle phase distribution by Flow cytometry.
2- Evaluation of Apoptosis by Flow cytometry.
3- Evaluation of Necrosis by Flow cytometry.
4- Evaluation of DNA damage & fragmentation by gel electrophoresis.
5- Evaluation of colony formation capacity by clonogenic assay
We have investigated the effect of single and combined treatment of MDA-MB-231 cells with AuNps and ZnSNps as well as X-ray irradiation on the cell cycle status and apoptosis by flow cytometry.
Our data shows that both single ZnSNps and AuNps treatments, predominantly arrested the cell cycle at G0/G1 phase, however, ZnSNps caused a higher G0/G1 phase arrest. Interestingly, the highest G2/M phase arrest was detected in the combined treatment of MDA-MB-231 cells with ZnSNps +X-ray followed by AuNps+ X-ray
Annexin V-FITC /propidium iodide (PI) double-staining technique followed by flow cytometry analysis was used to evaluate the potential mechanism for cell death (Apoptosis Vs Necrosis) in the MDA-MB-231 cells after either single ZnSNps, Au Np and X-ray treatment or their combined treatment with X-ray irradiation.
In agreement with the cell cycle data, Au Nps combination treatment with X-ray resulted in the lowest number of viable cells with necrosis being the primary cause of cell death (up to 75%) followed by apoptosis (16%). The second most efficient treatment was ZnS Nps combination treatment with X-ray, showing up to 32% necrotic MDA-MB-231 cells and 15.5 % early+late apoptotic cells.
The presence of necrotic cells was remarkable during the X-ray treatment, implying a transition from apoptosis to necrosis in X-ray treated cells which might be considered in the treatment of apoptosis-resistant cancer cells.
Flowcytometry analysis of cell size and granularity indicated a uniformed size, shape and intracellular granularity MDA-MB-231 control cells. On the contrary, ZnS Np, combined treatments with ZnS Np + X-ray and Au Nps + X-ray have caused a significant increase in intracellular granularity and cell size suggesting a potential involvement of autophagy in addition to necrosis in their cytotoxic action.
Collectively, the cell cycle analysis and apoptosis/necrosis flowcytometry results suggest a potential radio sensitization effect for ZnSNps and AuNps against MDA-MB-231 cells.
Our DNA fragmentation assay data after various single or combined treatments of MDA-MB-231 cells did not detect any form DNA laddering which is considered as a hallmark for the apoptotic process which may be explained by the dominating necrosis-related death and potential autophagy.
Finally, we have investigated the effect of single and combined treatments on colony formation capacity for MDA-MB-231 cells. All single Au NP & ZnS NP and X-ray or their combined treatments caused a complete loss of colonies, no colonies were observed in any of the treatments compared to the control indicative of the anti-clonogenic potential for all the treatments investigated.