Student : Rattaphong Pokkaew ( 李平 ) Student ID : 0970456 Department of Food Science

1. Physical and mechanical properties of cardboard panels made from usedbeverage carton with veneer overlay Ayrilmis, N. ; Candan,Z. ; Hiziroglu,S. 2008. 29, 1897–1903 Student : Rattaphong Pokkaew (李平) Student ID : 0970456 Department of Food Science

2. Outline 1. Introduction 2. The objective 3. Methods 4. Results 5. Conclusion

3. Introduction

5. Peanut • The peanut (Arachis hypogaeaL.) • The peanut is called as the “king” of oil seeds. • Peanuts are also known as: • Ground nuts, earthnuts, goobers, goober peas, • pindas, jack nuts, pinders, manila nuts and • monkey balls. • In the UK these are sold as monkey nuts.

6. Tocopherols in peanut • Peanuts are a good source of tocopherols, phytosterol and phospholipid • The tocopherol content of peanuts varies with variety and production location • Peanut oil mainly contains -tocopherol (50–373 ppm) and γ-tocopherols (90–390 ppm) (Firestone, 1999)

7. Tocopherols in peanut • Sturm et al. (1966) determined tocopherol content of peanut oil from 17 varieties. Runner varieties had higher levels of -, γ-and -tocopherols than the Spanish varieties • Hashim et al. (1993) reported that there were significant differences in tocopherol content among Runner- and Virginia-type peanut cultivars

8. Phospholipid in peanut • Phospholipids (PL) contribute to the smoothness, texture, and mouthfeel of foods • Phospholipidsimprove the stability of the product because of their inherent antioxidant properties

9. Breast cancer is the erratic growth of cells that originate in the breast tissue Breast cancer • A group of rapidly dividing cells may form a lump or mass of extra tissue. These masses are called tumors • The World Health Organization reported more than 1.2 millionpeople worldwide will be diagnosedwithbreast cancer this year

10. Breast cancer • The occurrence of breast cancervaries widely among womenfromdifferentcountries and cultures • Higher incidences inEuropean andNorthAmerican women • Lower in womenin lessdevelopedcountries and countries relyingmoreheavilyon vegetarian diets (American Cancer Society, 2003) • Dietary factors, specifically the proportion of animal versusplant fats, may play a role in thedevelopment of breast cancer (Messina and Barnes, 1991; Cho et al., 2003)

11. Breast cancer & Phytosterol • Plant foodstuffs contain specific phytochemicals which may offer protection from breast cancer. • An attractive hypothesis which may account for the cancerprotective action of phytosterols is that phytosterols induceapoptosis or programmed cell death in highly proliferativetumor cells. • Plant foodstuffs contain specific phytochemicals which may offer protection from breast cancer.

12. Fig 1. Structure of some representative phytosterols Source : Moreau et al., 2002; Berger et al., 2004; Kritchevsky and Chen, 2005

13. Apoptosis • Apoptosis, or programmed cell death, is associated withseveral fundamental biological processes, including celldevelopment, differentiation and response to injury. • Apoptosisis defined as a set of events that once initiated induce lethalchanges that include membrane blebbing, mitochondrial break down and DNA fragmentation • Apoptosis occurs via two main pathways • The intrinsic or mitochondrial pathway • The extrinsic or deathreceptor-mediated pathway

14. Apoptotic protease-activating factor-1 (Apaf-1) Cytochrome c Fig 2. The intrinsic or mitochondrial pathway Pro-apoptotic factors

15. Fas-associated death domain Fig 3. The extrinsic or death receptor-mediated pathway Fas ligand TNF receptorassociated death domain Fas receptor FADD

16. The objective • The objective of this study was to assess the effect of cellular supplementation with either -sitosterol or cholesterol on the extrinsic caspase-8 pathway in the two breast cancer cell lines, MCF-7 and MDA-MB-231. • Hypothesized of-sitosterol,that may potentiate Fas-death • domain signaling, leading to caspase-8 activationand ultimately • to apoptosis. • To test the hypothesis, the breast cancer cell lines were treated with -sitosterol or with cholesterol as a control and effects on cell growth, sterol incorporation in cell membranes, expression of Fas-death domain signaling proteins, and caspase-8 activity were determined.

17. Methods

18. Cell culture MCF-7 and MDA-MB-231 cells 1% antibiotic–antimycotic 2 g/l sodium bicarbonate Cultured at 37 C 5% fetal bovine serum 5%CO2/95% air as monolayers using RPMI 1640 growthmedium

19. 2-hydroxypropyl beta-cyclodextrin; CD complexes (RPMI 1640 mediasupplemented with cholesterol or b-sitosterol) Preparation of sterol supplemented media andmeasurement of cell growth Growth medium (8–16 mMsterols: 5mM CD) • Control groups • Cell were treated • with 5mM CD Studies groups

20. Cells were seeded into 6-well plates Measurement of sterol content of cell membranes by GLC harvested by scraping frozen 80 C in 350 ml 10mM Trisand 20mM mannitol buffer (pH 7.4) Samples were thawed and briefly sonicated on ice

21. Samples were then saponified at 80 C in 95% ethanolic KOH 2ml of hexane 2 ml of water Saponified samples The upper organic phase The lower phase Dried under nitrogen GLC

22. The media were replaced with sterol-supplemented or control media Cells were seeded in T-75 flasks; 24 h (10,000 cells/cm2) Determination of caspase-8 activity 3 day treatment Cells were scraped Washed in PBS (pH 7.4)

23. Lysed by suspension on iceof 107 cells/200 mlin buffer After 30 min The lysates were aspirated Centrifuged at 10,000g ; 4 C ; 30 min

24. The supernatants (cell lysates) were analyzed by the Bio-Rad DC protein assay Cell lysates (100 mg protein) were assayed caspase-8 activity

25. Results

26. Results Fig. 4. Effect of sterol supplementation on growth of MCF-7 cells. Cells were grown in RPMI-1640 growth medium supplemented with different concentrations of sterols

27. Results Fig. 5. Effect of sterol supplementation on growth of MDAMB-231 cells. Cells were grown in RPMI-1640 growth mediumsupplemented with different concentrations of sterols

28. Supplementation Cholesterol -Sitosterol Total sterol -Sitosterol (mg/mg protein) (mg/mgprotein) (mg/mg protein) (% totalsterol) CD vehicle 432a0.0 0a43 2a0a Cholesterol 49 2b0.0 0a49 2a0a -Sitosterol 40 1a50 5b90 5b56b Table 1. Effect of 2 d-sterol supplementation on sterol content of MCF-7 cell membranes* *MCF-7 cells were treated for 2 d with 16 mM sterol or vehicle and sterol contents of membranes determined by gas–liquid chromatography as described. Data are means SEM (n = 3) and letters (a, b) of values in each column are significantly different (p<0.05).

29. Supplementation Cholesterol -Sitosterol Total sterol -Sitosterol (mg/mg protein) (mg/mgprotein) (mg/mg protein) (% totalsterol) CD vehicle 497a0.0 0a497a0a Cholesterol 73 4b0.0 0a734b0a -Sitosterol 332a516b848b61b Table 2. Effect of 2 d-sterol supplementation on sterol content of MDA-MB-231 cell membranes* *MDA-MB-213 cells were treated for 2 d with 16 mMsterol or vehicle and sterol contents of membranes determined by gas–liquid chromatographyas described. Data are means SEM (n = 3) and letters (a, b) of values in each column are significantly different (p<0.05)

30. Supplementation Caspase-8 activity of Caspase-8 activity of MCF-7 lysates MDA-MB-231 lysates CD vehicle 6000 800a4600 200a Cholesterol5000 200a4600 200a -Sitosterol9800 400b8100 800b Table 3. Effect of 3 d-sterol supplementation on caspase-8activity of MCF-7 and MDA-MB-231 cell lysates* *Cells were treated for 3 d with 16 mM sterol or vehicle and caspase-8 activities of cell lysates determined. Data are RFU/100 mg lysate protein and represent means  SEM (n = 3). Letters (a, b) of values in each column are significantly different (p<0.05).

31. SIT CONT CHOL Fas FasL FADD p-FADD Caspase-8 Actin Fig. 6. Effect of sterol supplementation on expression levels of Fas-related signaling proteins in MCF-7 cells. MCF-7 cells were treated with sterols for 24 h as described. Expression of Fas-related signaling pathway proteins was quantified by immunoblot.

32. SIT CONT CHOL Fas FasL FADD p-FADD Caspase-8 Actin Fig. 7. Effect of sterol supplementation on expression levels of Fas-related signaling proteins in MDA-MB-231 cells. MDA-MB-231cells were treated withsterols for 24 h as described. Expression of Fas-related signaling proteins wasquantified by immunoblot.

33. Conclusion

34. Conclusion • -sitosterolcan be affect the amounts and activity of components ofthe extrinsic apoptotic pathway in human breast adenocarcinoma cells • -sitosterol induces a reductionin membrane sphingomyelin and an increase theceramide levels in some tumor cells • The effect of -sitosteroltreatment to increasecaspase-8 activity andapoptosis in these cells may be mediated, at least in part,by changes in membrane sterol content and effects onthe Fas apoptotic pathway

35. THANK YOU FOR YOUR ATTENTION !

37. Arachis hypogaeaL Source : http://upload.wikimedia.org/wikipedia/commons/1/1f/Koeh-163.jpg http://en.wikipedia.org/wiki/Image:Peanuts.jpg

38. Preparation of sterol supplemented media andmeasurement of cell growth Studies groups incubated for 24 h cells were seeded at 5000 cells/cm2 into 24-well plates media were replaced with that containing -sitosterol or cholesterol in graded concentrations or CD vehicle

39. Preparation of sterol supplemented media andmeasurement of cell growth Media were similarly changed on days 3 and 5 Cells were trypsinized and counted on days 2, 4 and 6 by Coulter Counter using the electrical sensing zone method Growth curves were generated from the Coulter Counter data

40. buffer containing • 10mM HEPES,pH 7.4 • 2mM EDTA • 0.15 CHAPS • 0.1% Triton X-100 • 5mM DTT

41. Fig 1. Structure of some representative phytosterols Source : Moreau et al., 2002; Berger et al., 2004; Kritchevsky and Chen, 2005