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Student : Rattaphong Pokkaew ( 李平 ) Student ID : 0970456 Department of Food Science Ayrilmis, N. ; Candan, Z. ; Hiziroglu, S. 2008. 29, 1897–1903 Physical.

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Presentation on theme: "Student : Rattaphong Pokkaew ( 李平 ) Student ID : 0970456 Department of Food Science Ayrilmis, N. ; Candan, Z. ; Hiziroglu, S. 2008. 29, 1897–1903 Physical."— Presentation transcript:

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

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

3 Introduction

4

5  The peanut (Arachis hypogaea L.)  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. Peanut

6  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) Tocopherols in peanut (Firestone, 1999)

7  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 Tocopherols in peanut

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

9 Breast cancer  Breast cancer is the erratic growth of cells that originate in the breast tissue  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 million people worldwide will be diagnosed with breast cancer this year

10  The occurrence of breast cancer varies widely among women from different countries and cultures  Higher incidences in European and North American women  Lower in women in less developed countries and countries relying more heavily on vegetarian diets (American Cancer Society, 2003) Breast cancer  Dietary factors, specifically the proportion of animal versus plant fats, may play a role in the development of breast cancer (Messina and Barnes, 1991; Cho et al., 2003)

11  Plant foodstuffs contain specific phytochemicals which may offer protection from breast cancer.  An attractive hypothesis which may account for the cancer protective action of phytosterols is that phytosterols induce apoptosis or programmed cell death in highly proliferative tumor cells. Breast cancer & Phytosterol  Plant foodstuffs contain specific phytochemicals which may offer protection from breast cancer.

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

13  Apoptosis, or programmed cell death, is associated with several fundamental biological processes, including cell development, differentiation and response to injury. Apoptosis  Apoptosis is defined as a set of events that once initiated induce lethal changes that include membrane blebbing, mitochondrial break down and DNA fragmentation  Apoptosis occurs via two main pathways The intrinsic or mitochondrial pathway The extrinsic or death receptor-mediated pathway

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

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

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 activation and 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 5% CO 2 / 95% air as monolayers using RPMI 1640 growth medium 2 g/l sodium bicarbonate 5% fetal bovine serum 1% antibiotic–antimycotic Cultured at 37  C

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

20 Measurement of sterol content of cell membranes by GLC Cells were seeded into 6-well plates harvested by scraping frozen 80  C in 350 ml 10mM Tris and 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 Saponified samples The upper organic phase Dried under nitrogen 2 ml of water 2ml of hexane The lower phase GLC

22 Determination of caspase-8 activity Cells were seeded in T-75 flasks; 24 h (10,000 cells/cm 2 ) Cells were scraped Washed in PBS (pH 7.4) The media were replaced with sterol- supplemented or control media 3 day treatment

23 Lysed by suspension on ice of 10 7 cells/200 ml in buffer The lysates were aspirated Centrifuged at 10,000g ; 4  C ; 30 min After 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 medium supplemented with different concentrations of sterols

28 Supplementation Cholesterol  -Sitosterol Total sterol  -Sitosterol (mg/mg protein) (mg/mg protein) (mg/mg protein) (% total sterol) CD vehicle 43  2 a 0.0  0 a 43  2 a 0 a Cholesterol 49  2 b 0.0  0 a 49  2 a 0 a  -Sitosterol 40  1 a 50  5 b 90  5 b 56 b 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/mg protein) (mg/mg protein) (% total sterol) CD vehicle 49  7 a 0.0  0 a 49  7 a 0 a Cholesterol 73  4 b 0.0  0 a 73  4 b 0 a  -Sitosterol 33  2 a 51  6 b 84  8 b 61 b 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 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)

30 Supplementation Caspase-8 activity of Caspase-8 activity of MCF-7 lysates MDA-MB-231 lysates CD vehicle 6000  800 a 4600  200 a Cholesterol 5000  200 a 4600  200 a  -Sitosterol 9800  400 b 8100  800 b Table 3. Effect of 3 d-sterol supplementation on caspase-8 activity 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 Fas FasL FADD p-FADD Caspase-8 Actin SIT CHOLCONT 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 Fas FasL FADD p-FADD Caspase-8 Actin SIT CHOLCONT Fig. 7. Effect of sterol supplementation on expression levels of Fas-related signaling proteins in MDA-MB-231 cells. MDA-MB-231 cells were treated with sterols for 24 h as described. Expression of Fas-related signaling proteins was quantified by immunoblot.

33 Conclusion

34 Conclusion   -sitosterol can be affect the amounts and activity of components of the extrinsic apoptotic pathway in human breast adenocarcinoma cells   -sitosterol induces a reduction in membrane sphingomyelin and an increase the ceramide levels in some tumor cells  The effect of  -sitosterol treatment to increase caspase-8 activity and apoptosis in these cells may be mediated, at least in part, by changes in membrane sterol content and effects on the Fas apoptotic pathway

35 THANK YOU FOR YOUR ATTENTION !

36

37 Arachis hypogaea L 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 and measurement of cell growth Studies groups cells were seeded at 5000 cells/cm 2 into 24-well plates incubated for 24 h media were replaced with that containing  -sitosterol or cholesterol in graded concentrations or CD vehicle

39 Preparation of sterol supplemented media and measurement of cell growth 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 Media were similarly changed on days 3 and 5

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

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


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