الفهرس 
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Abstract
Although great achievements have been made in the field of cancer therapy, chemotherapy and radiotherapy remain the mainstay cancer therapeutic modalities. Over the last decade, a growing interest in the improvement of radiation therapies has led to the development of nanomaterials as radiosensitizer.
Nanomedicine has advantages over conventional cancer therapeutics such as multi-functionality, efficient drug delivery, and controlled release of the drug cargos. It can improve therapeutic benefits by reducing systemic side effects and/or increasing drug accumulation inside tumors by using nanomaterials based on organic, inorganic, protein, lipid, glycan compounds, synthetic polymers, and viruses.
Zinc oxide nanoparticles (ZnO NPs) possess insignificant toxicity on normal healthy human cells, and demonstrating great advances in the medical field, particularly in the cancer applications. Modifications of ZnO NPs to potentiate its biological activities, were applied via conjugation with caffeic acid (CA), which is a natural phytochemical phenolic constituent in plants, which exhibits antioxidant, and scavenging power of the free radicals, due to its rich chemical structure of phenolic-hydroxyls, therefore, CA encompasses miscellaneous biological behaviors, comprising antibacterial, anti-inflammatory, immunomodulatory and anticancer activities.
The purpose of this study aimed to evaluate the ability of zinc oxide caffeic acid nanoparticles (ZnO-CA NPs) to inhibit the growth of solid Ehrlich carcinoma (EC) in female mice. The aim of the study was also extended to investigate the probable effect to augment the radiosensetivity of tumor cells to gamma (γ) radiation and to explore some of the molecular mechanisms involved in these effects.
To achieve this goal, ZnO-CA NPs were prepared by the conjugation of ZnO NPs with CA and characterized by Fourier Transform Infrared Spectra (FT-IR), X-ray Diffractometer (XRD) and Transmission Electron Microscopy (TEM). In vitro anticancer potential of ZnO-CA NPs was evaluated by assessing cell viability on human breast (MCF-7) and hepatocellular (HepG2) carcinoma cell lines. A pilot study was carried out to determine LD50. In vivo anticancer and radio-sensitizing effects of ZnO-CA NPs in solid Ehrlich carcinoma-bearing mice (EC mice) were also assessed as follows:
A total of 72 adult female Swiss albino mice were randomly divided equally into 6 groups
group 1 (Control, C): Mice left without any treatment serving as negative control group and remained for 4 consecutive weeks.
group 2 (ZnO-CA): Mice were injected intraperitoneally (ip) with 1/10 the LD50 of ZnO-CA NPs (5 mg/100 g bw) every other day for 3 consecutive weeks.
group 3 (Ehrlich, E): Mice bearing solid EC tumor that remained intact for 4 consecutive weeks.
group 4 (E+R): One week following solid EC tumor inoculation, mice were exposed to whole body γ-radiation (2 Gy/week) for 3 consecutive weeks for a total of 6 Gy.
group 5 (E+ZnO-CA): One week following solid EC tumor inoculation, mice were injected ip with ZnO-CA NPs (5 mg/100 g bw) every other day for 3 consecutive weeks.
group 6 (E+ZnO-CA+R): One week following solid EC tumor inoculation, mice were injected ip with ZnO-CA NPs (5 mg/100 g bw) every other day along with whole body irradiation (2 Gy/week) for 3 consecutive weeks.
The growth rate of solid tumors in all experimental groups was measured by Caliper at the end of the third and fourth weeks from the inoculation of Ehrlich ascites cells. At the end of the experiment, whole blood was withdrawn from animals for the analysis of biochemical parameters (AST & ALT activities, urea & creatinine concentrations, as well as complete blood picture). The solid tumors were excised and weighed. Cytochrome C, B-cell lymphoma 2 (Bcl2) and nuclear factor kappa B (NF-κB) gene expression levels were measured in solid tumors using RT-PCR. Tumor VCAM-1 level was determined by ELISA method Phosphorylated ERK1/2 protein expression was assayed in solid tumors. A part of the excised tissues (the right thigh muscle and solid EC) was kept for DNA fragmentation using agarose gel electrophoresis. In addition, cell cycle analysis was performed by flow cytometry. Finally, histological examinations were performed to confirm biochemical data.
The results obtained were statistically analyzed and can be summarized as follows:
● A gradual decrease in the viability percent of MCF7 and HepG2 cancer cells treated with different increments of ZnO NPs was observed in a dose dependent manner. A synergistic cytotoxic effect for ZnO-CA NPs against HepG2 cells was also recorded.
● A marked reduction in the tumor volume and weight of ZnO-CA NPs treated mice and a radiosensetizing effect was observed in decreasing the tumor volume and weight of γ-irradiated-EC-bearing mice coadministered with ZnO-CA NPs with respect to untreated EC-bearing mice.
● Exposure of solid EC-bearing mice to γ-irradiation or treated with ZnO-CA NPs produced a marked significant upregulation in the gene expression of cytochrome C, and by contrast a marked downregulation in Bcl2 & NF-κB gene expressions and a considerable decline in VCAM level as well as a remarkable downregulation in the phosphorylated ERK1/2 protein expression in tumor tissue, compared to untreated EC-bearing mice.
● Combined treatment of ZnO-CA NPs and γ-irradiation demonstrated a synergistic potential in down-regulating tumor Bcl2 and NF-κB gene expressions, decreasing VCAM level and down-regulating phosphorylated ERK 1/2 protein expression in tumor tissues, compared to EC-bearing mice.
● Exposure of solid EC-bearing mice to γ-irradiation and/or treatment with ZnO-CA NPs induced DNA damage, as evidenced by the appearance of DNA strand breaks
● We recorded a significant increase in sub G1 population in the tumor tissue of solid EC-bearing mice treated with ZnO-CA NPs, either alone or with γ-irradiation, associated with a considerable decline in G0/G1as well as S phase and G2/M populations denoting a remarkable increase in dead cells’ population
● Furthermore, the combined therapy showed a diminution of the tumor area with increasing necrotic cancer cells in the histopathological investigations, compared to the single treatment with ZnO-CA NPs or γ-irradiated.
According to the obtained results it could be concluded that ZnO-CA NPs may be used as a potential therapeutic agent for cancer treatment and for enhancing the sensitivity of tumor cells to ionizing radiation.
Finally, our work demonstrates that ZnO-CA NPs anticancer and radio-sensitization mechanisms may consist of ROS generation, targeting of DNA damage response and repair, oxidative stress, mitochondrial dysfunction, suppression of cell cycle checkpoint machinery and promoting cell death by a variety of mechanisms such as apoptosis and necrosis, accompanied by downregulation of phosphorylated ERK 1/2 protein expression, NF-κB gene expressions and VCAM level inhibition.
In conclusion, ZnO-CA NPs is prepared in a simple method and exhibits in vitro and in vivo anticancer activity. This activity of ZnO-CA NPs is augmented upon exposure of animals to γ-irradiation. The results obtained indicate the effectiveness of ZnO-CA NPs in the treatment of EC tumor, and radiation augments this effect.
Although great achievements have been made in the field of cancer therapy, chemotherapy and radiotherapy remain the mainstay cancer therapeutic modalities. Over the last decade, a growing interest in the improvement of radiation therapies has led to the development of nanomaterials as radiosensitizer.
Nanomedicine has advantages over conventional cancer therapeutics such as multi-functionality, efficient drug delivery, and controlled release of the drug cargos. It can improve therapeutic benefits by reducing systemic side effects and/or increasing drug accumulation inside tumors by using nanomaterials based on organic, inorganic, protein, lipid, glycan compounds, synthetic polymers, and viruses.
Zinc oxide nanoparticles (ZnO NPs) possess insignificant toxicity on normal healthy human cells, and demonstrating great advances in the medical field, particularly in the cancer applications. Modifications of ZnO NPs to potentiate its biological activities, were applied via conjugation with caffeic acid (CA), which is a natural phytochemical phenolic constituent in plants, which exhibits antioxidant, and scavenging power of the free radicals, due to its rich chemical structure of phenolic-hydroxyls, therefore, CA encompasses miscellaneous biological behaviors, comprising antibacterial, anti-inflammatory, immunomodulatory and anticancer activities.
The purpose of this study aimed to evaluate the ability of zinc oxide caffeic acid nanoparticles (ZnO-CA NPs) to inhibit the growth of solid Ehrlich carcinoma (EC) in female mice. The aim of the study was also extended to investigate the probable effect to augment the radiosensetivity of tumor cells to gamma (γ) radiation and to explore some of the molecular mechanisms involved in these effects.
To achieve this goal, ZnO-CA NPs were prepared by the conjugation of ZnO NPs with CA and characterized by Fourier Transform Infrared Spectra (FT-IR), X-ray Diffractometer (XRD) and Transmission Electron Microscopy (TEM). In vitro anticancer potential of ZnO-CA NPs was evaluated by assessing cell viability on human breast (MCF-7) and hepatocellular (HepG2) carcinoma cell lines. A pilot study was carried out to determine LD50. In vivo anticancer and radio-sensitizing effects of ZnO-CA NPs in solid Ehrlich carcinoma-bearing mice (EC mice) were also assessed as follows:
A total of 72 adult female Swiss albino mice were randomly divided equally into 6 groups
group 1 (Control, C): Mice left without any treatment serving as negative control group and remained for 4 consecutive weeks.
group 2 (ZnO-CA): Mice were injected intraperitoneally (ip) with 1/10 the LD50 of ZnO-CA NPs (5 mg/100 g bw) every other day for 3 consecutive weeks.
group 3 (Ehrlich, E): Mice bearing solid EC tumor that remained intact for 4 consecutive weeks.
group 4 (E+R): One week following solid EC tumor inoculation, mice were exposed to whole body γ-radiation (2 Gy/week) for 3 consecutive weeks for a total of 6 Gy.
group 5 (E+ZnO-CA): One week following solid EC tumor inoculation, mice were injected ip with ZnO-CA NPs (5 mg/100 g bw) every other day for 3 consecutive weeks.
group 6 (E+ZnO-CA+R): One week following solid EC tumor inoculation, mice were injected ip with ZnO-CA NPs (5 mg/100 g bw) every other day along with whole body irradiation (2 Gy/week) for 3 consecutive weeks.
The growth rate of solid tumors in all experimental groups was measured by Caliper at the end of the third and fourth weeks from the inoculation of Ehrlich ascites cells. At the end of the experiment, whole blood was withdrawn from animals for the analysis of biochemical parameters (AST & ALT activities, urea & creatinine concentrations, as well as complete blood picture). The solid tumors were excised and weighed. Cytochrome C, B-cell lymphoma 2 (Bcl2) and nuclear factor kappa B (NF-κB) gene expression levels were measured in solid tumors using RT-PCR. Tumor VCAM-1 level was determined by ELISA method Phosphorylated ERK1/2 protein expression was assayed in solid tumors. A part of the excised tissues (the right thigh muscle and solid EC) was kept for DNA fragmentation using agarose gel electrophoresis. In addition, cell cycle analysis was performed by flow cytometry. Finally, histological examinations were performed to confirm biochemical data.
The results obtained were statistically analyzed and can be summarized as follows:
● A gradual decrease in the viability percent of MCF7 and HepG2 cancer cells treated with different increments of ZnO NPs was observed in a dose dependent manner. A synergistic cytotoxic effect for ZnO-CA NPs against HepG2 cells was also recorded.
● A marked reduction in the tumor volume and weight of ZnO-CA NPs treated mice and a radiosensetizing effect was observed in decreasing the tumor volume and weight of γ-irradiated-EC-bearing mice coadministered with ZnO-CA NPs with respect to untreated EC-bearing mice.
● Exposure of solid EC-bearing mice to γ-irradiation or treated with ZnO-CA NPs produced a marked significant upregulation in the gene expression of cytochrome C, and by contrast a marked downregulation in Bcl2 & NF-κB gene expressions and a considerable decline in VCAM level as well as a remarkable downregulation in the phosphorylated ERK1/2 protein expression in tumor tissue, compared to untreated EC-bearing mice.
● Combined treatment of ZnO-CA NPs and γ-irradiation demonstrated a synergistic potential in down-regulating tumor Bcl2 and NF-κB gene expressions, decreasing VCAM level and down-regulating phosphorylated ERK 1/2 protein expression in tumor tissues, compared to EC-bearing mice.
● Exposure of solid EC-bearing mice to γ-irradiation and/or treatment with ZnO-CA NPs induced DNA damage, as evidenced by the appearance of DNA strand breaks
● We recorded a significant increase in sub G1 population in the tumor tissue of solid EC-bearing mice treated with ZnO-CA NPs, either alone or with γ-irradiation, associated with a considerable decline in G0/G1as well as S phase and G2/M populations denoting a remarkable increase in dead cells’ population
● Furthermore, the combined therapy showed a diminution of the tumor area with increasing necrotic cancer cells in the histopathological investigations, compared to the single treatment with ZnO-CA NPs or γ-irradiated.
According to the obtained results it could be concluded that ZnO-CA NPs may be used as a potential therapeutic agent for cancer treatment and for enhancing the sensitivity of tumor cells to ionizing radiation.
Finally, our work demonstrates that ZnO-CA NPs anticancer and radio-sensitization mechanisms may consist of ROS generation, targeting of DNA damage response and repair, oxidative stress, mitochondrial dysfunction, suppression of cell cycle checkpoint machinery and promoting cell death by a variety of mechanisms such as apoptosis and necrosis, accompanied by downregulation of phosphorylated ERK 1/2 protein expression, NF-κB gene expressions and VCAM level inhibition.
In conclusion, ZnO-CA NPs is prepared in a simple method and exhibits in vitro and in vivo anticancer activity. This activity of ZnO-CA NPs is augmented upon exposure of animals to γ-irradiation. The results obtained indicate the effectiveness of ZnO-CA NPs in the treatment of EC tumor, and radiation augments this effect.