Apoptotic pathways in human breast cancer cell models (MCF-7 and MDA-MB-231) induced by rice bran derived pentapeptide Ruiqi Li1, Navam Hettiarachchy1, Mahendran Mahadevan2 and Jay Rayaprolu1 1Department of Food Science, University of Arkansas, Fayetteville, AR 72704 2 Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Bioactive peptides derived from food sources with anti-proliferative properties against cancer have drawn more attention in recent years. A pentapeptide derived from rice bran has shown anti-proliferative property on human breast cancer cells. The objective of this study was to investigate the apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell models (MCF-7 and MDA-MB-231). The growth inhibition activity of pentapeptide was evaluated by phenazine methosulfate - 3 - (4, 5 - dimethylthiazole - 2 - yl)] - 2, 5 -diphenyl tetrazolium bromide (MTS) assay in a dose-dependent manner. The levels of molecular targets (TNF-α, Bax, Bcl-2, Fas, and erbB-2) were evaluated by ELISA kits. Pentapeptide showed growth inhibition activity on MCF-7 and MDA-MB-231 in a dose-dependent pattern. Significant (p < 0.05) decreases in the levels of Bcl-2 and erbB-2 and increases in the levels of TNF-α and Bax were detected after pentapeptide treatment from 72 to 96 h. The results suggest that pentapeptide inhibits growth of human breast cancer cells by introducing apoptosis through a caspase-dependent pathway. ABSTRACT METHODS RESULT HIGHLIGHTS AND DISCUSSION Breast cancer cell lines (MCF-7 and MDA-B231) Anti-proliferative effects of pentapeptide using MTS assay: A dose-dependent growth inhibition pattern was observed with pentaptpeide on MCF-7 and MDA-MB-231 cells (Figure 1). Significant increases in caspases -8 and -9 was observed in pentapeptide treated MCF-7 and MDA-MB-231 cells for after 72 and 96 hours (Figs. 2 and 3). The activated caspase-3/7 cleaved other cellular proteins leading to the apoptosis. Bcl-2 blocks cell death while Bax accelerates the apoptosis in cancer cells by binding to inactivate Bcl-2. Significant increases in Bax/Bcl-2 ratios were observed in pentapeptide treated MCF-7 cells for 72 and 96 hours compared to the control (absence of pentapeptide). TNF and Fas are considered as members of the death domain receptor family, they activate caspases through their death receptor-activator complexes. Pentapeptide treatment induced increases in levels of TNF-α, Fas, and Caspase-8 in MCF-7 and MDA-MB-231 cells. These data suggested that the pentapeptide stimulated apoptosis by triggering death receptor signaling involving the activation of TNF-α and Fas followed by the caspase cascade by caspase-8. The results of MCF-7 and MDA-MB-231 cells treated with pentapeptide suggested that pentapeptide may enhance the apoptotic signal by suppressing the expression of ErbB-2 Pentapeptide Treatment Apoptosis Molecular Targets Effect/Features TNF-α, Fas, Bcl-2, Bax, and ErbB-2 Anti-proliferative property Activation of Caspases Caspases Glo®8 and 9 kits ELISA assay (Abcam, Enzo 2012) MTS assay (Kannan et al. 2010) Data Collected using multi-well plate reader RESULTS a b c c d e f g INTRODUCTION h Breast cancer is one of the leading causes of cancer related deaths and illnesses in the United States (American Cancer Society 2013). Apoptosis, also named programmed cell death (PCD), is a genetically regulated process initiated by physiological and pathological stimuli (Broker et al. 2005). The apoptosis is characterized by caspases cascade, morphological changes, and DNA fragmentation (Earnshaw et al 1999). Molecular targets are individual genes or molecules can affect any other molecular events that are relevant to the process of carcinogenesis (Milner et al. 2001), such as TNF-α, Bax, Bcl-2, Fas, and erbB-2. Kannan et al. (2010) prepared a novel pentapeptide from rice bran with a sequence Glu-Gln-Arg-Pro-Arg and showed nearly 80% of growth inhibition activity on human breast cancer cell (MCF-7). However, the apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell lines remains to be investigated. i i i The results demonstrated that there are two apoptotic pathways involved in pentapeptide-induced apoptosis in human breast cancer cell lines. In one pathway, the apoptosis is activated after the increased Bax/Bcl-2 ratio leading to the release of cytochrome c in mitochondria-mediated pathway. In the second pathway, pentapeptide induces apoptosis via death receptor-mediated pathway by stimulating the expression of TNF-α, followed by forming the death-inducing signaling complex (DISC) through Fas expression and the activation of caspase-8. The pentapeptide may amplify apoptotic signals by down-regulating the expression of ErbB-2. CONCLUSION Figure 1. Growth inhibition effects of pentapeptide at various concentrations on human breast cancer cell lines (MCF-7 and MDA-MB-231) after 96 hours incubation. The cells treated with various dosages of pentapeptide. The cells cultured with media (Eagle's Minimum Essential Medium) alone and genistein (400 ug/mL) are negative control and positive control. Values with the same letter are not significantly different(P < 0.05). Values are means ± standard deviation of three determinations. The standard deviation are shown by the error bars. Figure 2. The relative intensity of activated caspase-8 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. a b a c c c b d d d d e e f f g g g g g g h h h Figure 3. The relative intensity of activated caspase-9 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. Figure 4. The levels of TNF-α in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. This research/work was supported by a grant from the Arkansas Breast Cancer Research Program and the University of Arkansas for Medical Sciences Translational Research Institute (CTSA Grant Award # UL1TR000039). ACKNOWLEDGEMENT a a a a b b c d Determine in vitro apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell model (MCF-7 and MDA-MB-231) OBJECTIVES d d b e c e c c c e f f d e f g American Cancer Society. 2013. Bröker LE, Kruyt FE, Giaccone G. 2005. Cell death independent of caspases: a review. Clinical Cancer Res 11: 3155–62. Milner J, McDonald SS, Anderson DE, Greenwald P. 2001. Molecular targets for nutrients involved with cancer prevention. Nutrition Cancer 41: 1–16. Kannan A, Hettiarachchy NS, Lay JO, Liyanage R. 2010. Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran. Peptides 31: 1629–34. Abcam and Enzo, 2012. Company protocols. REFERENCES Figure 5. The levels of Bcl-2 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. Figure 6. The levels of Bax in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. MATERIALS Human breast cancer cell line (MCF-7 and MDA-MB-231), Leibovitz's L-15 Medium, and Eagle's Minimum Essential Medium were purchased from ATCC. Trypsin-EDTA solution was purchased from Sigma. The human ELISA kits for Bcl-2 and ErbB-2 were supplied by Abcam Plc. (Cambridge, MA, USA). The TNF-α, Bax, and Fas human ELISA kits were from Thermo Fisher Scientific Inc. (Waltham, MA, USA), Enzo Life Science Inc. (Farmingdale, NY, USA), and RayBiotech, Inc. (Norcross, GA, USA), respectively. a a b c b d c c e e c d e e f e d e f g f h h h Figure 7. The levels of ErbB-2 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars. Figure 8. The levels of Fas in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

Rice Bran Derived Pentapeptide-induced Apoptosis in Human Breast Cancer Cell Models (MCF-7 and MDA-MB-231) Ruiqi Li1, Navam Hettiarachchy1, Mahendran Mahadevan 2 and Jay Rayaprolu1 1Department of Food Science, University of Arkansas, Fayetteville, AR 72704 2 Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Bioactive peptides derived from food sources with anti-proliferative properties against cancer have drawn more attention in recent years. A pentapeptide derived from rice bran has shown anti-proliferative property on human breast cancer cells. The objective of this study was to investigate the apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell models (MCF-7 and MDA-MB-231). The growth inhibition activity of pentapeptide was evaluated by phenazine methosulfate - 3 - (4, 5 - dimethylthiazole - 2 - yl)] - 2, 5 -diphenyl tetrazolium bromide (MTS) assay in a dose-dependent manner. The levels of molecular targets (TNF-α, Bax, Bcl-2, Fas, and erbB-2) were evaluated by ELISA kits. Pentapeptide showed growth inhibition activity on MCF-7 and MDA-MB-231 in a dose-dependent pattern. Significant (p < 0.05) decreases in the levels of Bcl-2 and erbB-2 and increases in the levels of TNF-α and Bax were detected after pentapeptide treatment from 72 to 96 h. The results suggest that pentapeptide inhibits growth of human breast cancer cells by introducing apoptosis through a caspase-dependent pathway. ABSTRACT

INTRODUCTION OBJECTIVES MATERIALS Breast cancer is one of the leading causes of cancer related deaths and illnesses in the United States (American Cancer Society 2013). Apoptosis, also named programmed cell death (PCD), is a genetically regulated process initiated by physiological and pathological stimuli (Broker et al. 2005). The apoptosis is characterized by caspases cascade, morphological changes, and DNA fragmentation (Earnshaw et al 1999). Molecular targets are individual genes or molecules can affect any other molecular events that are relevant to the process of carcinogenesis (Milner et al. 2001), such as TNF-α, Bax, Bcl-2, Fas, and erbB-2. Kannan et al. (2010) prepared a novel pentapeptide from rice bran with a sequence Glu-Gln-Arg-Pro-Arg and showed nearly 80% of growth inhibition activity on human breast cancer cell (MCF-7). However, the apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell lines remains to be investigated. Determine in vitro apoptotic pathways of pentapeptide-induced apoptosis in breast cancer cell model (MCF-7 and MDA-MB-231) OBJECTIVES MATERIALS Human breast cancer cell line (MCF-7 and MDA-MB-231), Leibovitz's L-15 Medium, and Eagle's Minimum Essential Medium were purchased from ATCC. Trypsin-EDTA solution was purchased from Sigma. The human ELISA kits for Bcl-2 and ErbB-2 were supplied by Abcam Plc. (Cambridge, MA, USA). The TNF-α, Bax, and Fas human ELISA kits were from Thermo Fisher Scientific Inc. (Waltham, MA, USA), Enzo Life Science Inc. (Farmingdale, NY, USA), and RayBiotech, Inc. (Norcross, GA, USA), respectively.

METHODS Apoptosis Breast cancer cell lines (MCF-7 and MDA-B231) Pentapeptide Treatment Apoptosis Molecular Targets Effect/Features TNF-α, Fas, Bcl-2, Bax, and ErbB-2 Anti-proliferative property Activation of Caspases ELISA assay (Abcam, Enzo 2012) Caspases Glo®8 and 9 kits MTS assay (Kannan et al. 2010) Data Collected and Analyzed

RESULTS a a a a a a a a a a b b b b c c c c d e e e f f f g g Figure 1. Growth inhibition effects of pentapeptide at various concentrations on human breast cancer cell lines (MCF-7 and MDA-MB-231) after 96 hours incubation. The cells were treated with various dosages of pentapeptide. The normal human mammary epithelial cells (HMEC) were used to test the toxicity of pentapeptide. The cells cultured with media (Eagle's Minimum Essential Medium) alone and genistein (400 ug/mL) are negative control and positive control. Values with the same letter are not significantly different (P < 0.05). Values are means ± standard deviation of three determinations. The standard deviation are shown by the error bars. h i j

a a a a b b c d e e e e Figure 2. The relative intensity of activated caspase-8 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a b b b b c c c d d d d Figure 3. The relative intensity of activated caspase-9 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a b b c c d e e e e f f Figure 4. The levels of TNF-α in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hrs. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a b b c c c c d d d e e Figure 5. The levels of Bcl-2 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a a a b b b b b c d e e Figure 6. The levels of Bax in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a b b b b c c c c c c d Figure 7. The levels of ErbB-2 in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

a b c c d d d e f g g g Figure 8. The levels of Fas in pentapeptide (1000 µg/mL) treated MCF-7 and MDA-MB-231 cells after incubation for 72 and 96 hours. The cells cultured with media alone and genistein (400 µg/mL) are negative and positive control, respectively. Values are means ± standard deviation of three determinations. Values with the same letter are not significantly different (P>0.05). The standard deviations are shown by the error bars.

RESULTS HIGHLIGHTS AND DISCUSSION Anti-proliferative effects of pentapeptide using MTS assay: A dose-dependent growth inhibition pattern was observed with pentaptpeide on MCF-7 and MDA-MB-231 cells (Figure 1). Significant increases in caspases -8 and -9 was observed in pentapeptide treated MCF-7 and MDA-MB-231 cells for after 72 and 96 hours (Figure 2 and 3). The activated caspase-3/7 cleaved other cellular proteins leading to the apoptosis. Bcl-2 blocks cell death while Bax accelerates the apoptosis in cancer cells by binding to inactivate Bcl-2. Significant increases in Bax/Bcl-2 ratios were observed in pentapeptide treated MCF-7 cells for 72 and 96 hours compared to the control (absence of pentapeptide). TNF and Fas are considered as members of the death domain receptor family, they activate caspases through their death receptor-activator complexes. Pentapeptide treatment induced increases in levels of TNF-α, Fas, and Caspase-8 in MCF-7 and MDA-MB-231 cells. These data suggested that the pentapeptide stimulated apoptosis by triggering death receptor signaling involving the activation of TNF-α and Fas followed by the caspase cascade by caspase-8. The results of MCF-7 and MDA-MB-231 cells treated with pentapeptide suggested that pentapeptide may enhance the apoptotic signal by suppressing the expression of ErbB-2

The results demonstrated that there are two apoptotic pathways involved in pentapeptide-induced apoptosis in human breast cancer cell lines. In one pathway, the apoptosis is activated after the increased Bax/Bcl-2 ratio leads to the release of cytochrome c in mitochondria-mediated pathway. In the second pathway, pentapeptide induces apoptosis via death receptor-mediated pathway by stimulating the expression of TNF-α, followed by forming the death-inducing signaling complex (DISC) through Fas expression and the activation of caspase-8. The pentapeptide may amplify apoptotic signals by down-regulating the expression of ErbB-2. CONCLUSION

ACKNOWLEDGEMENT REFERENCES This research/work was supported by a grant from the Arkansas Breast Cancer Research Program and the University of Arkansas for Medical Sciences Translational Research Institute (CTSA Grant Award # UL1TR000039). ACKNOWLEDGEMENT American Cancer Society. 2013. Bröker LE, Kruyt FE, Giaccone G. 2005. Cell death independent of caspases: a review. Clinical Cancer Res 11: 3155–62. Milner J, McDonald SS, Anderson DE, Greenwald P. 2001. Molecular targets for nutrients involved with cancer prevention. Nutrition Cancer 41: 1–16. Kannan A, Hettiarachchy NS, Lay JO, Liyanage R. 2010. Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran. Peptides 31: 1629–34. Abcam and Enzo, 2012. Company protocols. REFERENCES