Hesperidin induces apoptosis and triggers autophagic markers through inhibition of Aurora-A mediated phosphoinositide-3-kinase/Akt/mammalian target of rapamycin and glycogen synthase kinase-3 beta signalling cascades in experimental colon carcinogenesis  Gowrikumar Saiprasad, Palanivel Chitra, Ramar Manikandan, Ganapasam Sudhandiran  European Journal of Cancer  Volume 50, Issue 14, Pages 2489-2507 (September 2014) DOI: 10.1016/j.ejca.2014.06.013 Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 1 (A) Schematic diagram of the experimental protocol. Mice received AOM once a week for three consecutive weeks for inducing colon cancer. Hesperidin was given orally starting a week before AOM injection and continued till the last AOM injection (initiation) or were administered hesperidin one week after the end of the last AOM injection and continued till the end of experiment (post-initiation). (B) The levels of CEA (carcino embroyonic antigen) were estimated in serum of control and experimental groups of animals. Each value is expressed as mean±S.D. of three separate experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 2 (A) Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts for Aurora-A in the colonic tissues of control and experimental groups of animal. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as internal control. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B) The quantitative data represent the band intensity levels assessed using densitometry. (C) Immunoblots of Aurora-A in the colonic tissues of control and experimental groups of animal. β-Actin served as internal control. (D) The quantitative data represent the corresponding protein levels assessed using densitometry. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significant at P<0.05. Values sharing the following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 3 Representative photographs for the immunohistochemical staining of Aurora-A, 20× (Scale bar – 50μm). (A) Colon tissue sections of control animals (control); the square with enlarged image highlights scarce expression of Aurora-A. (B) Colon tissue sections of AOM induced animals (AOM); the square with enlarged image highlights the significantly increased expression of Aurora-A. Colonic tissue sections of hesperidin treated animals at both initiation (HES+AOM) (C) as well as post-initiation (AOM+HES) phase (D); the square with enlarged image highlights the markedly decreased immunoreactivity of Aurora-A. (E) Colon sections of hesperidin alone administered animals (HES) showing scarce expression of Aurora-A similar to control. (F) The Aurora-A expression was quantified by averaging positive cells across 20 randomly selected fields. Each value is expressed as mean±S.D. of three separate experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 4 (A) Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts for Bcl-2 and Bax in the colonic tissues of control and experimental groups of animals. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as internal control. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B and C) The quantitative data represent the corresponding levels of mRNA transcripts assessed using densitometry. (D) Immunoblotting data of mitochondrial expressions of Bcl-2, Bax, cytochrome c in the colonic tissues of control and experimental groups of animals. (E) Immunoblotting data of cytoplasmic expressions of Bax, cytochrome c and caspase-3 and 9 in the colonic tissues of control and experimental groups of animals. VDAC1 served as internal control for mitochondrial protein analysis and β-Actin served as internal control for the cytoplasmic protein analysis. (F and G) The quantitative data represent the corresponding protein levels assessed using densitometry. (H) The graphical data represent the ratio between Bax/Bcl-2 (protein expression) assessed using densitometry. (I) DNA fragmentation analysis of the corresponding genomic DNA of control and other experimental group of animals. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significant at P<0.05. Values sharing the following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 5 (A) Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts for p53 and p21 in the colonic tissues of control and experimental groups of animals. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as internal control. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B) The quantitative data represent the corresponding levels of mRNA transcripts assessed using densitometry. (C) Immunoblotting data of p53 and p21 in the colonic tissues of control and experimental groups of animals. β-Actin served as internal control. (D) The quantitative data represent the corresponding protein levels assessed using densitometry. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 6 (A) The immunoblotting data of p-PI3K, p-AktSer-473, p-Akt Thr-308, total Akt and phosphatase and tensin homologue (PTEN) from the colon tissues of control and experimental groups of animals. β-Actin served as internal control. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B) The quantitative data represent the corresponding protein levels assessed using densitometry. (C) The graphical data represent the ratio between p-Akt/total Akt assessed using densitometry. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 7 Confocal microscopic analysis of p-PI3K expression in the colonic sections of control and experimental groups of animals. Images shown are representative of three separate experiments (n=3). Tissue sections were immunostained with anti-p-PI3K antibody and FITC conjugated secondary antibody (green). Tissue sections were counter stained using PI (red) to stain nucleus (Scale bar – 50μm). Slides were visualised under the confocal microscope (Leica TCS-SP2 XL) using excitation/emission wavelength for PI – 529nm/620nm and FITC – 494nm/525nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 8 (A) Representative immunoblotting data for p-mTOR, Beclin-1 and LC3-II from the colonic tissues of control and experimental groups of animals. β-Actin served as internal control. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B) The quantitative data represent the corresponding protein levels assessed using densitometry. (C) Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts for Beclin-1 and LC3 in the colonic tissues of control and experimental groups of animals. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as internal control. (D) The quantitative data represent the corresponding levels of mRNA transcripts assessed using densitometry. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and hesperidin alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 9 (A) The immunoblotting data of p-GSK-3βSer-9, Cyclin-D1 and β-catenin from the colon tissues of control and experimental groups of animals. β-Actin served as internal control for the cytoplasmic protein and Histone H2B served as internal control for the nuclear protein analysis. Lane 1 – Control, Lane 2 – AOM, Lane 3 – HES+AOM (initiation), Lane 4 – AOM+HES (post-initiation), Lane 5 – HES. (B) The quantitative data represent the corresponding protein levels assessed using densitometry. (C) Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts for c-myc and c-jun in the colonic tissues of control and experimental groups of animals. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as internal control. (D) The quantitative data represent the corresponding levels of mRNA transcripts assessed using densitometry. Y-axis represents relative intensity (arbitrary units)/gene expression (fold change). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included a one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Results are statistically significance at P<0.05. Values sharing following symbols differ significantly, ∗ – versus control; # – versus AOM; † – versus AOM+HES (post-initiation). No significant difference was observed between control and HES alone administered group of animals (AOM – Azoxymethane, HES – Hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions

Fig. 10 Representative scanning electron microscopic images of the colonic tissues of control and experimental groups of animals. (A, H) Control, H - enlarged image of A, (B–D) AOM induced animals, (E, G) AOM induced animals treated with hesperidin at initiation phase (HES+AOM), G - enlarged image of (E, F) AOM induced animals treated with hesperidin at post-initiation phase (AOM+HES) (AOM – Azoxymethane, HES – hesperidin). European Journal of Cancer 2014 50, 2489-2507DOI: (10.1016/j.ejca.2014.06.013) Copyright © 2014 Elsevier Ltd Terms and Conditions