Dr. Gautam Sethi Associate Professor Dept of Pharmacology Yong Loo Lin School of Medicine

1. Potential application of gamma-tocotrienol as a novel chemosensitizer in gastric cancer Dr. Gautam Sethi Associate Professor Dept of Pharmacology Yong Loo Lin School of Medicine National University of Singapore Singapore

2. Global Variation in Cancer Incidence and Mortality 50 New York Times

3. BAD LUCK AND CANCER CONNECTION!

4. Cancer is a Preventable Disease That Requires Major Lifestyle Changes Pharmaceutical Research, 2008; 25, 2097-2116; Clin Cancer Res. 2009 Jan 15;15(2):425-30.

6. Different faces of inflammation and its role in tumorigenesis Environmental pollutants (Cigarette smoke, Diesel) Stress (Chemical, physical,and psychological) Viruses (HTLV1, HPV, HCV, HBV, EBV ) Bacteria (e.g; Helicobacter pylori ) Food Factors (Grill, Fried, red meat) Reactive oxygen species Tumor necrosis factor Interleukin-1 Interleukin-6 Interleukin-8 Interleukin-18 Nuclear Factor-B STAT3 AP-1 Hypoxia-inducible factor Cyclooxygenase-2 PI3K/AKT Inducible nitric oxide- synthase Matrix metallo- proteinase-9 Chemokines Chronic inflammation Acute inflammation • Tumor cell survival • Tumor cell proliferation • Tumor cell invasion • Tumor angiogenesis • Tumor metastasis • Tumor chemoresistance • Tumor radioresistance • Innate Immunity • Humoral immunity • Immune surveillance Therapeutic inflammation Pathological inflammation Biochem Pharmacol. 2006 Nov 30;72(11):1605-21.

7. Gastric cancer is the second leading cause of cancer death in both sexes worldwide (736,000 deaths, 9.7% of the total). The highest mortality rates are estimated in Eastern Asia (28.1 per 100,000 in men, 13.0 per 100,000 in women), the lowest in Northern America (2.8 and 1.5 respectively). High mortality rates are also present in both sexes in Central and Eastern Europe, and in Central and South America. There are marked geographic variations in gastric cancer incidence, with the highest rates in Japan, China and South America 7

8. Etiological factors for Gastric Cancer 8

9. Treatment options SURGERY: The most common postoperative complication is tumor recurrence. Many patients present with distant metastases or direct invasion of organs, obviating the possibility of complete resection. Surgical procedures such as wide local excision, total gastrectomy, or gastrointestinal bypass may be performed to allow oral intake of food and alleviate pain. CHEMOTHERAPY: Not very successful due to number of toxic side effects/chemoresistance. One study revealed recurrence rates of up to 80 percent in patients undergoing surgical resection alone, suggesting a need to continue investigation of adjuvant therapies to chemotherapy. RADIOTHERAPY: Only a modest survival advantage has been shown. The adverse effects caused by radiation therapy include gastrointestinal toxicity from dose-limiting structures surrounding the stomach. 9

10. What is NF-kB? p65 p50 IkBa Activator Ubiquitination & Phosphorylation Nuclear import p50 p65 p50 p65 IkBa kB enhancer Degradation IkBa Nucleus Cytoplasm Trends Pharmacol Sci. 2009;30:313-321.

11. NF-κB in Gastric Cancer 11

12. NF-κB is constitutively activated in advanced gastric carcinoma patients and correlated to the survival time of patients after chemotherapy. FOLFOX: Folinic acid + 5-FU+ Oxaliplatin

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14. Trends Pharmacol Sci. 2009;30:313-321. 14

15. Identification of novel pharmacological agents that can overcome chemoresistance

16. Vitamin E Family Vitamin E Tocotrienol (T3) Tocopherol (TP) α- TP β- TP γ- TP δ- TP α- T3 β- T3 γ- T3 δ- T3 Tocopherols are reported to: • Neutralize metabolic and UV-induced reactive oxygen species (ROS) • Protect neuron from death due to ROS • Maintain skin cell membranes intact from ROS Tocotrienols are reported to: • Anti-Metabolic: Lower cholesterol : Lower triglyceride • Anti-Cancers: Inhibit of cancer cell cycle, Inflammation Induce apoptosis in cancer cells Inhibit proliferation of cancer stem cells to delay cancer relapse

17. Sources of Vitamin E TOCOTRIENOLS TOCOPHEROLS Source : 2010 Soya & Oilseed Bluebook

18. Science of Tocotrienol & Chronic Diseases [J Atheroscler Thromb. 2010 Oct 27] Obesity Cholesterols [Chem Biol Inter. 2010 Dec] [Am J Cardiol. 2006 Sep ] Diabetes NeuroProtection Skin Whitening Tocotrienols [Pig. Cells 2010 Oct] [Stroke. 2011 Aug] Liver Cancer Pancreatic Cancer Prostate Cancer Breast Cancer [BJP. 2011 Jul-Aug] [Mol Cancer Ther. 2011 Oct 4] [Int J Cancer. 2011 Mar 11] [Cell Prolif. 2011 Oct 4]

19. Hypothesis! • Whether γ-tocotrienol can overcome chemoresistance in gastric cancer through modulation of pro-inflammatory NF-κB signaling cascade.

20. In Silico Proteomics Analysis The predictive analysis was run by Cellworks, (California) using information available in the public domain. To Cellworks’ best knowledge, information and belief, no proprietary or confidential information of any third party has been used in running the studies. The predictive data were generated using Cellworks’ proprietary Oncology technology asset and is shared for discussion purposes.

21. NF-κ Inhibition Study Data

22. Effect of NF-κB Inhibition on key biomarkers associated with GC progression

23. γ-Tocotrienol inhibited the proliferation of different gastric cancer cells in a dose and time dependent manner

24. γ-Tocotrienol induced apoptosis in gastric cancer SNU16 cells in a dose-dependent manner 45 * 40 35 30 25 0μM 10μM 20 15 10 5 0 0 10 25 50 (24 h) * = p<0.05 25µM 50μM

25. γ-Tocotrienol potentiates the apoptotic effects of capecitabine in gastric cancer cells 60 50 40 30 20 10 0 Cape γ-Toco+ Cape γ-Toco Control Untreated γ-Toco γ-Toco Untreated Capecitabine γ-Toco+ Cape Capecitabine γ-Toco+ Capecitabine 60 * * 50 % Sub G1 40 30 % Inhibition of esterase activity 20 10 0 γ-Toco+ Cape Cape γ-Toco Control

26. 0.4 0.3 Optical density (450 nm) p65 DNA binding activity 0.2 0.1 0 AGS A293 SNU16 MKN45 Constitutive NF-κB activation in different gastric cancer cell lines * * * = p<0.05 (TransAMNF-kB p65 transcription factor assay kit (ActiveMotif, Carlsbad, CA, USA)

27. γ-Tocotrienol inhibited constitutive and capecitabine-induced NF-κB activation in gastric cancer cells in a dose-dependent manner 0.4 SNU16 0.3 0.2 0.1 0 0 10 25 50 γ-Tocotrienol (µM)/12 h 0.4 0.3 0.2 0.1 0 MKN45 * * Optical density (450 nm) p65 DNA binding activity * * * * - + - 10 25 50 (γ-Toco, µM) - - + + + + (Cape, 25 µM)

28. γ-Tocotrienol suppresses various anti-apoptotic, metastatic and angiogenic gene products in gastric cancer cell lines

29. -Tocotrienol potentiates the antitumor effects of capecitabine in vivo using athymic (nu/nu)mouse model γ γ

30. -Tocotrienol potentiates the effects of capecitabine to inhibit the growth of gastric cancer in nude mice

31. 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Effects of -tocotrienol and capecitabine on NF-κB activation in gastric tumor tissue samples * * Optical density (450 nm) p65 DNA binding activity VC Cape γ-Toco γ-Toco + Cape

32. Combination of -tocotrienol and capecitabine was effective in downregulating the overexpression of various gene products involved in proliferation, survival, and invasion in gastric cancer tumor tissues

33. γ-tocotrienol in combination with capecitabine inhibited the expression of proliferative biomarker Ki67 in GC tumor tissues

34. γ-tocotrienol in combination with capecitabine inhibited the expression of angiogenic biomarker CD31 in GC tumor tissues

35. First evidence that gamma-tocotrienol inhibits the growth of human gastric cancer and chemosensitizes it to capecitabine in a xenograft mouse model through the modulation of NF-κB pathway Manu KA, Shanmugam MK, Ramachandran L, Li F, Fong CW, Kumar AP, Tan P, Sethi G Clinical Cancer Research, 2012; 18: 2220-2229

36. Angiogenesis • Angiogenesis is the formation of new blood vessels from pre-existing vessels. • It is a normal process in growth and development, as well as in wound healing. • However, it is also a fundamental step in the transition of tumors from a dormant state to a malignant state. • Once a tumor has grown to ~3mm diameter, it requires its own blood supply to stay alive and grow. • Tumor angiogenesis is essential for • supplying nutrients and oxygen and removing waste products • dissemination of the tumour cells to distant sites • growth of metastatic colony at new site

37. Angiogenesis is tightly controlled by the balance of two sets of counteracting factors; angiogenic activators and inhibitors. The 4 major steps of endothelial cells in angiogenesis: • Breaking through of the basal lamina that envelopes existing blood vessels • Migration toward a source signal • Proliferation • Formation of tubes Menzel-Severing, Eye (2012) 26, 2–12

38. Effect of γ-tocotrienol on VEGF induced migration of HUVECs VEGF VEGF + γ-tocotrienol + + VEGF - + γ-tocotrienol HUVECs (70 µl; 5×105 cells/ml) were added into the two reservoirs of the culture insert (IBIDI GmbH). After 12 hours, the insert was gently removed creating a gap of ~500 µm. The cells were treated with 50 μM γ-tocotrienol for 12 h before being exposed to 10ng/mL VEGF for 24 h. Width of wound was measured at time zero and 24h of incubation. The representative photographs showed the same area at time zero and after 24 h of incubation. Columns, mean; bars, SD. *, p < 0.05.

39. Effect γ-tocotrienol on VEGF induced invasion of HUVECs VEGF + γ-tocotrienol VEGF VEGF + γ-tocotrienol VEGF HUVECs (5×104) were seeded in the top-chamber of the Matrigel coated polycarbonate membrane containing inserts. After pre-incubation with or without 50 µM γ-tocotrienol for 12 h, transwell chambers were placed into the wells of a 24-well plate, in which we had added Medium 200 containing 10 ng/mL VEGF. After incubation for 24h, cell invasion was analyzed. Columns, mean; bars, SD. *, p < 0.05.

40. Effect on tube formation of HUVECs VEGF Control VEGF + γ-tocotrienol (25 μM) VEGF + γ-tocotrienol (50 μM) HUVECs were pretreated with various dilutions of γ-tocotrienol for 12 h and then seeded onto the Matrigel layer in 24-well plates at a density of 5 × 104 cells in Medium 200 with or without VEGF. After 6 h, tubular structure of endothelial cells was photographed using an inverted microscope.

41. Effect on VEGF-induced microvessel sprouting from rat thoracic aorta VEGF Control VEGF + γ-tocotrienol (25μM) VEGF + γ-tocotrienol (50μM) Aortas isolated from Sprague-Dawley rats were cut into 1 mm rings, randomized into Growth Factor Reduced Matrigel-coated wells and further sealed with 100 μl of Matrigel. Medium 200 with and without VEGF along with different dilutions of γ-tocotrienol was added to the wells and incubated for 6 days. Columns, mean; bars, SD. *, p < 0.05.

42. Photograph Effect on angiogenesis in Matrigel plugs implanted in mice VEGF + γ-tocotrienol (10 μg) VEGF + γ-tocotrienol (20 μg) Control VEGF H & E stain Matrigel (0.5 mL) containing 100 ng VEGF and 20 units of heparin with or without 10 or 20 μg of γ-tocotrienol were injected subcutaneously into the ventral area of C57/BL/6 mice (n=5 per group). After 6 days, the mice were euthanized and intact Matrigel plugs from all groups of mice were removed. The matigel plugs were photographed and then fixed with 10% neutral buffered formalin and paraffin sections were prepared and used for H&E staining to identify the formation and infiltration of new microvessels.

43. A. B. VEGF (50ng/ml) VEGF (50ng/ml) 0 0 1 2 4 6 h (γ-Tocotrienol) 0 0 1 2 4 6 h (γ-Tocotrienol) ◄ pTyr1175-VEGFR2 ◄ pSer473-AKT ◄ β-actin ◄ AKT γ-Tocotrienol suppressed the activation of VEGFR2, and the AKT/mTOR signaling pathway in HUVECs ◄ pSer2448-mTOR ◄ mTOR ◄ pThr389 p70S6K1 ◄ p70S6K1 ◄ pThr389/Ser424 -p70S6K1 ◄ p70S6K1 ◄ β-actin A, γ-tocotrienol suppressed the activation of VEGFR2 induced by VEGF in a time dependent manner. The activation status of VEGFR2 was tested by western blot analysis and probed with anti–phosphorylated VEGFR2 antibody. The same blot was stripped and reprobed with β-actin antibody to verify equal protein loading. B, γ-tocotrienol inhibited the activation of AKT/mTOR signaling pathway in endothelial cells. Proteins from different treatments were probed with phospho-specific antibodies.

44. A. C. 0 1 2 4 6 h (γ-Tocotrienol) ◄ pSer473-AKT ◄ AKT ◄ pSer2448-mTOR ◄ mTOR γ-Tocotrienol induced apoptosis and inhibited the AKT/mTOR pathway in HCC cells ◄ pThr389 p70S6K1 ◄ p70S6K1 0 6 12 24 h (γ-Tocotrienol) B. ◄ PARP ◄ Cleaved PARP ◄ pThr389/Ser424 p70S6K1 ◄ S6K1 ◄ β-actin ◄ β-actin A, γ-Tocotrienol reduced the viability of HCCLM3 cells in a dose dependent manner. Cell viability was quantified by MTT assay. Columns, mean; bars, SD. *, p < 0.05. B, HCCLM3 cells were treated with 50 μM γ-tocotrienol for indicated times, whole-cell extracts were prepared, subjected to western blot analysis against PARP antibody. C, γ-tocotrienol suppressed the phosphorylation of mTOR signaling pathway kinases in HCCLM3 cell. HCCLM3 cells were treated with γ-tocotrienol for the indicated times, and whole-cell extracts were prepared, and subjected to western blot analysis against phospho-specific antibodies.

49. Conclusion! Pro-inflammatory transcription factors Can be considered As Ideal Targets to Discover Therapeutics for Treatment of cancer

50. Acknowledgment(s) Cancer Pharmacology Members Dr. Muthu K Shanmugam (Post-doc) Dr. Wang Hong(Post-doc) Dr. Siveen KS (Research Assistant) Mr. Li Feng (Graduate student) Ms. Radhamani Kannaiyan (Graduate student) Ms. Lalitha Ramachandran (Graduate student) Ms. Alamelu Nachiyappan (Graduate student) Ms. Jingwen Zhang (Graduate student) Ms. Xiaoyun Dai (Graduate student) Ms. Fan Lu (Lab manager) Collaborators Prof. Kam Man Hui (NCCS) Prof. Patrick Tan (DUKE-NUS) Dr. Alan Prem Kumar (CSI) Prof. John Luk (China) Grant Support National Medical Research Council, Singapore Academic Research Fund Tier 1 and 2, Ministry of Education, Singapore