Therapeutic Efficacy of Metronomic Doxorubicin and Low Dose of Gamma Radiation on Breast Cancer Model

Figures

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  Figure 1. The experimental design and steps of different treatments. BW, body weight; DMBA, dimethyl benz[a]anthracene; LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin.

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  Figure 2. M‐DOX and/or LDR affect related to histopathological factors. (Normal control group) viewing healthy mammary acini embedded in the surrounding adipose tissue. Positive control (tumor group) displays several empty spaces in the cribriform adenocarcinoma. The top‐left image shows adenocarcinoma; notice the several spaces within the tumor's solid masses: some are empty, and others contain pink, proteinaceous secretion. (LDR group) present cancer cell necrosis, highlighted by large eosinophilic granule areas within the tumor masses. (M‐DOX group) viewing marked cancer cells necrosis with its transformation into hyalinized eosinophilic material, as well as many pyknotic nuclei. (M‐DOX + LDR group) display necrosis and the onset of fibrous bands among the groups of cancer cells. Tumor size was determined by a standard caliper, in width and length, upon excision of all untreated and treated tumor‐bearing rats. The section's microscopic examination was carried out by the Labomed Fluorescence microscope LX400, Cat No. 9126000; US and Labomed camera software, USA. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin.

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  Figure 3. Showing the change in tumor volume (mm3) through 8 weeks in different groups. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin.

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  Figure 4. M‐DOX and/or LDR affect the histopathological features of heart tissue. The section's microscopic examination was carried out by the Labomed Fluorescence microscope LX400, Cat No. 9126000; US and Labomed camera software, USA. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin.

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  Figure 5. Effects of M‐DOX and LDR treatment on cell cycle analysis. Cell cycle histogram (A) and cell cycle chart (B) of the mammary tumor. Data are expressed as mean ± SEM, at a1 p < 0.001, a2 p < 0.01, a3 p < 0.05 versus control group; b1 p < 0.001, b2 p < 0.01, b3 p < 0.05 versus tumor group; c1 p < 0.001, c2 p < 0.01, c3 p < 0.05 versus M‐DOX group. d1 p < 0.001, d2 p < 0.01, d3 p < 0.05 versus LDR group. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin; SEM, standard error of mean.

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  Figure 6. Effects of M‐DOX and LDR treatment on cell death. Apoptotic histogram (A) and apoptotic chart (B) of Annexin‐V positive cells were evaluated in mammary tumors. Results are expressed as % of positive cells from five independent experiments. Data are expressed as mean ± SEM, at a1 p < 0.001, a2 p < 0.01, a3 p < 0.05 versus control group; b1 p < 0.001, b2 p < 0.01, b3 p < 0.05 versus tumor group; c1 p < 0.001, c2 p < 0.01, c3 p < 0.05 versus M‐DOX group. d1 p < 0.001 versus the LDR group. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin; SEM, standard error of mean.

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  Figure 7. Gating scheme for flow cytometry analysis of intracellular Ki67 expression in mammary tissues. (A) Percentage of Ki67 in different treated groups. (B) Cumulative data showing frequencies of Ki67 cells in each group. Significance was determined by one‐way analysis of variance. Data are expressed as mean ± SEM, p < 0.001 and 0.01. LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin; SEM, standard error of mean.

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  Figure 8. The change in gene expression of the regulatory P53 gene, in addition to the down‐regulation of antiapoptotic gene Bcl‐2, and the up‐regulation of apoptotic gene expression, including Bax and caspase‐3 genes. Data are expressed as relative gene expression. Bax, B‐cell lymphoma‐2‐associated X; Bcl‐2, B‐cell lymphoma‐2; LDR, low dose of radiation; M‐DOX, metronomic dose of doxorubicin.

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  Figure 9. The change in the serum biomarkers for antioxidant (GSH), oxidative stress (MDA), liver (AST and ALT) and cardiac (CK‐MB) functions. Data are expressed as mean ± SEM, at a1 p < 0.001, a2 p < 0.01, a3 p < 0.05 versus control group; b2 p < 0.01, b3 p < 0.05 versus tumor group; c2 p < 0.01, c3 p < 0.05 versus M‐DOX group; d3 p < 0.05 versus the LDR group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK‐MB, creatine kinase MB; GSH, glutathione; LDR, low dose of radiation; MDA, malondialdehyde; M‐DOX, metronomic dose of doxorubicin; SEM, standard error of mean.