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phytochemicals have been reported to activate anti-apoptotic signaling (Kelsey, Wilkins, and Linseman, 2010).
To better understand the effect of white sesame seed water-soluble fraction on neuroblast cells, we investigated its effect on CPT-induced apoptosis. To exclude the contribution of antioxidant ferulic and vanillic acids present in the water-soluble fraction, we chose to induce apoptosis via ROS-independent pathway. CPT induces apoptosis by inhibiting DNA topoisomerase I resulting in the formation of DNA double strand breaks resulting in a pro-apoptotic signaling pathway activation in different cells including neuronal cells (Morris and Geller, 1996). Pretreatment for 24 h with white sesame seed water-soluble fraction significantly reduced the percentage of apoptotic cells after treatment with different CPT concentrations (Fig. 17). Results in Fig. 17A and B showed a lower percentage of annexin-V+/PI- cells in 100 ng/mL CPT-treated cells than in 50 ng/mL CPT-treated ones.
However, results in Fig. 17C show that both CPT concentration reduced cell viability to around 70% of the control. These results might be explained by the fact that increasing CPT concentration does not increase the number of apoptotic cells, but cells treated with 100 ng/mL CPT are in a more advanced stage of apoptosis. Annexin-V is a recombinant phosphatidylserine (PS)-binding protein that interact specifically and strongly with PS residues which are translocated from inward of the cells lipid bilayer to the outer layer of the cell membrane so the apoptotic cells can be recognized by phagocytes (Elmore, 2007).
Denecker et al. (2000) reported that PS exposure to the extracellular side of cell membrane occurs in an early stage of apoptosis which precedes the release of cytochrome c from the mitochondria. On the other hand, the late apoptosis stage is characterized by cell shrinkage due to cytoplasm condensation (Elmore, 2007). This increases the probability that late-stage apoptotic cells may be discarded during cell washing and centrifugation before cell staining and flow cytometric analysis. White sesame seed water-soluble fraction also exhibited a
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concentration-dependent effect against apoptosis induced by 50 ng/mL CPT (Fig. 18). This result confirms the anti-apoptotic potential of white sesame seed water-soluble fraction.
To elucidate the anti-apoptotic mechanism of white sesame seed water-soluble fraction, we investigated its effect on Akt phosphorylation. Activation of the PI-3K/Akt kinase pathway is one of the most widely recognized anti-apoptotic signaling events and a large number of natural antioxidants have been reported to activate PI-3K/Akt pathway (Kelsey, Wilkins, and Linseman, 2010). The results showed that treatment with white sesame seed water-soluble fraction for 24 h relatively increased the expression level of both total Akt and phosphorylated Akt (pAkt) but phosphorylation ratio did not increase (Fig. 19). In addition, retinoic acid (RA, 10 μM), used as a positive control did not significantly increase Akt phosphorylation rate. Recently, Cheng et al. (2013) reported that RA protected SH-SY5Y cells from proteasome inhibition associated apoptosis mainly by inducing Akt phosphorylation. They showed that Akt phosphorylation peaked at 18 h and still elevated after 24 h treatment with 0.5 μM RA. In this paper results, total Akt level was not changed over time or sample and band intensity was relatively dense. Thus, our results might indicate that Akt was not appropriately detected by the antibody we used.
Akt phosphorylation can activate many downstream targets including Bcl-2 to inhibit apoptosis (Wu et al., 2011). Bcl-2 is the most studied and potent anti-apoptotic gene; Bcl-2 protein is ubiquitously expressed and regulates mitochondria permeability by inhibiting pro-apoptotic Bax-induce cytochrome c release (Portt et al., 2011). According to western blotting results (Fig. 20), Bcl-2 protein expression level doubled after treatment for 24 h with white sesame seed water-soluble fraction and RA; however this change was not statistically significant. In addition, Bcl-2 mRNA expression level (Fig. 21A) slightly increased after treatment with white sesame seed water-soluble fraction (2 mg/mL) and RA (10 μM) but again the increase was not significant compared to control. Taken together, Akt
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phosphorylation and Bcl-2 expression results indicate that PI-3K/Akt pathway might be involved in white sesame seed water-soluble fraction ant-apoptotic effect.
Treatment with white sesame seed water-soluble fraction and RA significantly reduced the mRNA expression level of pro-apoptotic protein Bax and the pro-apoptotic transcription regulator p53 (Fig. 21B and C). Wang et al. (2006), showed that treatment of SH-SY5Y cells with CPT increased p53 expression and Bax translocation to the mitochondrial membrane.
Thus, the down regulation of both p53 and Bax expression after treatment with white sesame seed water-soluble fraction is correlated with its protective effect from CPT-induced apoptosis (Fig. 17 and Fig. 18). Dietary antioxidants such as resveratrol protect neuronal cells from apoptosis by enhancing SIRT-1 activation (Ghosh, Liu, and Zhou, 2013). SIRT-1 pro-survival effects involve the inhibition of Bax-induced cytochrome c release from the mitochondria and the repression of pro-apoptotic transcription factors p53 and FOXO (Kelsey, Wilkins, and Linseman, 2010). Thus, white sesame seed water-soluble fraction might contain some compound that has similar effect with resveratrol. Until this step, we are unable to determine the exact mechanism of action of white sesame seed water soluble fraction since it contains several compound that might have different effects on several pathways. Therefore, the isolation and identification of the bioactive anti-apoptotic compound in white sesame seed water-soluble fraction is a crucial step to be able to elucidate the exact mechanism of action underlying the anti-apoptotic effect.
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Chapter V
Conclusion
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Sesame (Sesamum indicum L.) is an important oil seed crop with around 70% of the annual world’s production used for the extraction of oil. Defatted sesame seed flour is recovered as a byproduct after oil extraction; it is usually considered as a waste and not appreciated for its potential benefits for health. Previously, we showed that water-soluble fractions, purified from defatted white and gold sesame seed flour, exhibited a good antioxidant activity in vitro as measured by DPPH free radical scavenging and ORAC. In the present study, we focused on the potential physiological functions of these water-soluble fractions.
In the first part of this study, we demonstrated that white and gold sesame seed water-soluble fractions protect cells from both extracellular and intracellular oxidative stress. Co-treatment with water-soluble fractions and AAPH protected B lymphocyte Raji cell and neuroblast SH-SY5Y cell from peroxyl radicals-induced oxidative damage. The ineffectiveness of pretreatment with water-soluble fractions against AAPH-induce oxidative damage suggests that water-soluble fractions protect cell by directly scavenging generated free radicals in the extracellular medium and therefore inhibits free radical-induced cell membrane damage.
On the other hand, pretreatment with white and gold sesame seed water-soluble fractions protected SH-SY5Y cells from H2O2-induce oxidative damage. This suggests that sesame seed water-soluble fractions can also protect cells from hydroxyl radicals-induced intracellular oxidative stress by enhancing cell resistance to oxidative stress. In fact, treatment with white and gold sesame seed water-soluble fractions increased HO-1 mRNA expression level as well as Nrf-2 protein level in the nucleus.
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Results also showed that ferulic and vanillic acids, previously identified as bioactive antioxidants in sesame seed water-soluble fractions, contribute to the protective effect of these fractions against both peroxyl and hydroxyl radicals-induced oxidative stress.
In the second part of this study, we focused on the neuroprotective effect of white sesame seed soluble fraction. Results showed that treatment white sesame seed water-soluble fraction significantly increased neuroblast SH-SY5Y cell viability in a dose-dependent manner; while gold sesame seed water-soluble fraction as well as ferulic and vanillic acids did not significantly affect SH-SY5Y cell viability. Furthermore, white sesame seed water-soluble fraction effect on cell viability is specific to neuroblast cells, since treatment with white sesame seed water-soluble fraction did not significantly affect the viability of Raji cells or intestinal Caco-2 cells.
Pretreatment with white sesame seed water-soluble fraction protected SH-SY5Y cells from apoptosis when cells were treated with different CPT concentrations. White sesame seed water-soluble fraction also exhibited a dose-dependent effect against CPT-induced apoptosis.
These results suggest that white sesame seed water-soluble fraction may enhance SH-SY5Y cell viability by inhibiting apoptotic cell death.
To better understand the anti-apoptotic mechanism induced by white sesame seed water-soluble fraction, we investigated its effect on anti-apoptotic signaling. Treatment with white sesame seed water-soluble fraction relatively increased phosphorylated Akt level and Bcl-2 protein and mRNA expression level compared to control. Thus, it is still unclear whether white sesame seed water-soluble fraction induces anti-apoptotic PI3K/Akt pathway or not. In addition, treatment with white sesame seed water-soluble fraction significantly decreased pro-apoptotic Bax and p53 mRNA expression levels. These results indicate that other anti-apoptotic pathway may be involved in white sesame seed water-soluble fraction
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anti-apoptotic mechanism. Therefore, considering the complex composition of sesame seed water-soluble fraction, it is important to isolate and identify the potential anti-apoptotic bioactive compound in this fraction in order to be able to understand its anti-apoptotic mechanism.
In conclusion, our results show that sesame seed water-soluble fractions have a good potential to protect cells from oxidative stress. In particular, thanks to its good antioxidant potential in addition to its anti-apoptotic effect in neuroblast cells, white sesame seed water-soluble fraction might have a significant effect on preventing neurodegeneration. Studies on the exact mechanism of action of this fraction as well as its effect on neurodegenerative diseases in both in vitro and in vivo models should be performed to determine the potential use of defatted sesame seed water-soluble fraction as a functional food ingredient.
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Acknowledgements
First, I would like to acknowledge the contribution of Shinsei Co. Ltd. in this study by providing sesame seed flour raw material, funds for the research, and valuable discussion.
I want to express my sincere gratitude to:
Professor Tomio YABE, my main supervisor, for guiding me through my research, his kind help and support.
Professor Kohji KITAGUCHI, for his valuable discussion and useful critiques.
Professor Shingo KAWAI, for his interest and constructive discussions during mid-term presentations.
Professor Tohru MITSUNAGA, for accepting to review my PhD dissertation as well as his support during the PhD program.
Professor Sachi SRI KANTHA, for his valuable lectures and tips concerning scientific English writing and for the enjoyable and inspiring discussions we had during our meetings.
I also wish to extend my thanks to all previous and actual members of Laboratory of Functional Food Biochemistry for their help during experiments, valuable discussion during meetings and always maintaining such a friendly and happy working environment.
Finally I would like to dedicate this work to my family. To my husband and best friend, Riadh Aloulou, without whom I would never have been able to achieve this work. To my parents, brother, and sister, for always continuing to support me despite the distance and time difference between us.
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