الفهرس 
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Abstract
Cancer is undoubtedly one of the main health concerns facing our society. It is the second leading cause of death in economically developed countries. In conventional chemotherapy treatment, the anticancer drugs get to tumor site with poor specificity and dose-limiting nephrotoxicity, hepatotoxicity. Nanotechnology is constantly being encouraged to overcome numerous limitations of conventional drug delivery systems since the later fails to efficiently distinguish between cancerous and normal cells. Nanomedicine and nano delivery systems are a relatively new but rapidly developing science where materials in the nanoscale range are employed to serve as means of diagnostic tools or to deliver chemopreventive agents to specific targeted sites in a controlled manner.
The current study aims to compare the antitumor activity of copper oxide nanoparticles, hydrazine sulphate-copper nanoparticles, nano-curcumin, curcumin capped copper nanoparticles and native curcumin with ascorbate in vivo using Ehrlich ascites carcinoma (EAC) cell line. Also, Evaluation of the cytotoxicity of different nano-complexes in vitro using breast carcinoma cell line (MCF-7).
Charaterization of different nanoparticles was performed using Transmission electron microscopy (TEM) and dynamic light scattering (DLS).
This study was divided into two sections:
The first section included evaluation of the cytotoxicity of different nano-complexes against breast carcinoma cell line (MCF-7). Cell lines were treated with the different concentrations (25, 50, 100, 200 ug/ml) of different nano-complexes for 48h. Also, apoptosis of the breast cancer cell lines (MCF-7) was screened through the alterations in nuclear morphology in response to different LD50 doses of nano-complexes treatments by using nuclear counterstain (Hoschet 33342).
The second section included the animal trial. One hundred and eighty healthy adult female Swiss albino mice weighing between 20-25g were maintained on standard commercial pellets diet and tap water ad libitum, and kept individually in stainless cages in constant conditions. CuO-NPs and HS-Cu-NPs were injected intratumorally at dose 10 mg/Kg b.wt., Cur-Cu-NPs was injected intratumorally at dose 50 mg/Kg b.wt., Nano-Cur and Native-Cur were injected intratumorally (G6, G7) or administered orally (G8, G9) at dose 50 mg/Kg b.wt. Female mice were divided into 9 groups (20 mice /group) as follow: G1: Healthy control. G2: tumor bearing mice (TBM). G3: TBM+CuO-NPs + ascorbate. G4: TBM+ HS-Cu-NPs + ascorbate. G5: TBM+Cur-Cu-NPs + ascorbate. G6, G8: TBM+Nano-Cur + ascorbate G7, G9: TBM+Native-Cur +ascorbate. The results illustrated that:
1. Concerning the evaluation of different nanocomplexes cytotoxicity, all nano complexes showed a clear cytotoxic effect on breast carcinoma cell line (MCF-7) and a clear concentration-response relationship. The calculated IC50 indicated that CuO-NCs and HS-Cu-NCs were more cytotoxic to MCF-7, then Cur-NCs followed by native-Cur-Cs. The least effect exerted by Cur-Cu-NCs. Hoschet 33342 staining indicated that different nano-complexes significantly reduced the number of colonies of breast carcinoma cell line (MCF-7) whereas, Cur-Cu-NCs was the most effective cytotoxic treatment to MCF-7 as there were less colonies and more nuclear condensation as compared to the control.
2. Regarding tumor assessment, different nanocomplexes caused a significant reduction, P<0.05 in tumor weight and tumor volume which lead to a significant inhibition of tumor growth (T/G %) in different nano-complexes treatments in G3, G4, G5, G6, G7, G8 and G9. It was 54.8%, 51.4%, 70.37%, 62.9%, 51.9%, 37.04% and 33.3% when compared to G2 in the same order. Different nanocomplexes treatments caused a remarkable prolongation of mean survival time (MST) and significant increase in life span (%ILS) as compared with G2.
3. Concerning tumor markers, different nano-complexes treatments caused a significant reduction in serum CA 15-3 levels and ALP activities. Cur-Cu- NCs treatment in G5 was the most effective treatment that caused a significant reduction in serum CA 15-3 level and ALP activity by -87.1% and -61.2%, respectively as compared to G2.
4. Regarding cell apoptosis, there was a highly significant increase of caspase-3 activity and the expression levels of P53 in tumor tissues of all treated groups. Cur-Cu-NCs treatment was highly apoptotic that caused a significant increase in caspase-3 activity and expression of P53 in G5 by 550% and 2686.7%, respectively as compared to TBM untreated group. Results of P53 gene expression were significantly confirmed by immunohistochemical analysis, where TBM received Cur-Cu-NCs highly expressed P53 (score 3, +++) in G5.
5. Concerning the inflammation/ immunologic markers, there was a significant increase in C-reactive protein (CRP) and interleukin-6 (IL-6) levels in untreated TBM. In contrast, Mice in G5 treated with Cur-Cu-NCs exhibited a significant decrease in CRP and IL-6 levels by -82.8% and -39.5%, respectively (P<0.05). Also, a statistically significant reduction in CRP and IL-6 levels were observed in all treated groups.
6. Regarding the hematological parameters, Markedly, Cur-Cu-NCs treatment in G5 caused a significant improvement in Hb concentration (22.4%), Hct (18.6%), RBCs count (24.4%), MCHC (3.7%) and lymphocytes % (7.8%) while there was a significant decrease in total WBCs count (-39.8%) when compared to G2. Our study revealed that intratumoral (IT) or oral treatment with Cur-NCs and native-Cur-Cs restored the Hb concentration, RBCs and WBCs counts to be near normal, Whereas, chemical treatments with CuO-NCs and HS-Cu-NCs couldn’t restore the hematological parameters to normal levels.
7. Concerning the oxidative stress markers, there was a significant increase in MDA levels in G3, G4 and G5 by 226.6%, 188.2% and 170.4%, respectively which were associated with significant decrease in GSH content and catalase activity. Hepatic MDA levels in Cur-NCs and native-Cur-Cs treatments were significantly lowered by -17.2%, -11.2%, -13.6% and -11.2% for G6, G7, G8 and G9, respectively as compared to G2. On the other side, GSH content and CAT enzyme activity were found to be significantly increased in Cur-NCs and Native Cur-Cs treated groups.
8. Regarding liver functions, Ehrlich Ascites Carcinoma (EAC) injection resulted in significant rise in the activities of liver enzymes; namely AST and ALT. CuO-NCs and HS-Cu-NCs treatments exerted hepatotoxic effect as compared with other nanocomplexes treatments. However, Cur-Cu-NCs and Cur-NCs treatments either intratumoral or oral resulted in an improved liver function in G5, G6 and G8 showing superior hepatoprotective effect when compared with chemical treatments. The results of serum ALT and AST activities in different nano-complexes treated groups indicated that Cur-Cu- NCs treatment in G5 was the most effective treatment that caused a significant reduction in serum ALT and AST activities by -63.79% and -64.56%, respectively as compared to G2 (P<0.05).
9. Concerning kidney function, CuO-NCs and HS-Cu-NCs treatments exerted a marked toxic effect on the kidney function as chemical treatments in G3 and G4 couldn’t return serum urea and creatinine levels to the normal level, whereas, there was a significant reduction in serum creatinine to the normal levels in G5 and G6 by -54.22% and -56.63%, respectively, as compared to G2. Also, there was a significant reduction in serum urea levels by -26.89%, -26.27%, -25.89% and -24.56% in G5, G6, G7 and G8, respectively as compared to G2.
10. The consequence of different nanocomplexes treatments on liver, kidney and tumor tissues was assessed by histopathological examination. Results showed that chemical treatments by CuO-NCs and HS-Cu-NCs were effective as antitumor but caused side effects on liver and kidney tissues. However, Cur-NCs and native-Cur-Cs treatments either injected or administered orally exerted no side effects on liver and kidney tissues since Cur-Cu-NCs were the most effective treatment. Microscopic examination of tumor tissues indicated that necrosis was highly observed in solid tumor treated with Cur-Cu-NCs, Cu-NCs (G6), CuO-NCs and HS-Cu-NCs while less necrosis was observed in solid tumor treated with Cur-NCs (G8) and native-Cur-Cs (G7, G9).
In general, the results of this study showed that the anticancer effect of different nanochemopreventive agents (either chemotherapy or phytochemicals) were more pronounced when nano-drug delivery system was used for treating cancer. Combination of chemopreventive approach utilizes multiple chemopreventive agents at low doses to achieve maximum chemopreventive efficacy with minimum toxicity. In conclusion, all nano-complexes treatments significantly inhibited tumor growth and reduced tumor markers but Cur-Cu nano-complex was the most effective treatment with hepatoprotective and nephroprotective effects.