SYDNEY MEDICAL SCHOOL

Anti-tumour activity of Omega -3 Epoxy Fatty Acid analogue in human cancer

cell lines: a comparative study

Supervisor: Professor Michael MurrayPharmacogenomics & Drug Development GroupDiscipline of Pharmacology

Master of Philosophy CandidateWilliam Sudarmana

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POLYUNSATURATED FATTY ACIDS (PUFA)

› Polyunsaturated fatty acids (PUFAs) such as omega-3 and omega-6 are essential and vital for the human body to function.

› Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the 2 main components of omega-3 PUFA, are mainly found in cold-water fish such as salmon and tuna. Alpha linolenic acid (ALA), another omega-3 fatty acid, is found in plant sources such as nuts and seeds.

› Omega-6 typically comes as linoleic acid from plant oils such as corn oil, soybean oil, & sunflower oil, as well as from nuts & seeds.

› Epidemiological studies observed that

- Omega-3 PUFA have been shown to impede tumour growth.

- Omega-6 PUFA have been shown to promote tumour growth.

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6ω

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BIOTRANSFORMATION OF AA AND EPA

• Recent evidence indicates that epoxyeicosatrienoic acids (EETs) stimulate tumour cell growth.

• EETs promote the neoplastic phenotype of carcinoma cells and increase the proliferation of tumour cells.

• EETs may also enhance tumour growth by stimulating angiogenesis, which increases delivery of nutrients and oxygen to tumours and promotes metastatic disease.

MEMBRANE – PHOSPHOLIPIDS

Phospholipase A2

EPAAA

CYTOCHROME P450

11,12-epoxyeicosatrienoic acid (11,12-EET)

17,18-epoxyeicosatetraenoic acid (17,18-epoxy-EPA)

2018

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The double bonds seen are able to undergo oxidation to form various omega-3 or omega-6 epoxides

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PREVIOUS STUDY IN THE GROUP

› Our group have synthesised 17,18-epoxyeicosatetraenoic acid (17,18-epoxy-EPA)

› It has been shown to impair cell proliferation by- Down-regulating cyclin D1, preventing the

progression to G2-phase of cells that are already in the cell cycle

- Preventing cell cycle progression through S-phase

- Activating apoptosis

› These actions were specific to 17,18-epoxy-EPA because the regioisomeric epoxides formed at the alternate olefinic bonds in omega-3 EPA stimulated proliferation and did not activate apoptosis.

› The group then sought to develop molecules that capture the antiproliferative and pro-apoptotic actions of omega-3 17,18-epoxy-EPA while minimising the potential prostimulatory effects of the regioisomeric epoxides by eliminating the double bonds as shown on the right

16-(3-ethyl-2-oxiranyl) hexadecanoic acid (C20 epoxide)

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11,12-EET

17,18-epoxy-EPA

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8 9 11 12 14 15

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AIM AND METHOD

RATIONALE› This project follows the work completed by the previous PhD graduate, Herryawan Dyari. He has

proposed a mechanism of action for the C20 epoxide on MDA-MB-231 breast adenocarcinoma. The Pharmacogenomics and Drug Development Group is now interested in understanding the critical parameters present in the pathways of C20 epoxide in more responsive cell lines. This will enhance the Group’s understanding of C20 epoxide’s strengths and limitations for future in-vivo testing.

AIM› The objective of this study is to investigate the effects of 16-(3-ethyl-2-oxiranyl) hexadecanoic acid (C20

epoxide) on six different human cancer cell lines- A549 lung carcinoma- BeWo placenta choriocarcinoma- DU145 prostate carcinoma- HeLa cervical adenocarcinoma- HEPG2 hepatocellular carcinoma- MDA-MB-468 breast adenocarcinoma

METHOD› The experiments that I will conduct include:

- MTT assay – to measure the viability of cells and antiproliferative response to the compound of interest- Propidium iodide (PI) staining by flow cytometry – for cell cycle analysis

16-(3-ethyl-2-oxiranyl) hexadecanoic acid (C20 epoxide)

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MTT ASSAY

The absorbance at 570 nm is measured using a spectrophotometer

Media were aspirated and the purple formazan dissolved with dimethyl sulfoxide (DMSO)

25 μL of MTT (2.5 mg/mL) are added into each well

Cells were treated with C20 epoxide at 3 different concentrations: 10 μM, 20 μM, 40 μM + control (DMSO)

Cells were starved with serum free medium (DMEM / F12K + P/S)

Cells (1x104/well) were seeded in 96-well plates in complete growth medium (DMEM / F12K + FCS 10% + P/S)

24 h

24 h

48 h

2 h

30 min

EFFECT OF C20 EPOXIDE ON DU145 CELL VIABILITY

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A 96-well of DU145 after an MTT assay. Increasing amounts of cells resulted in increased purple colouring.

Data reported are mean ± SE of representative, N=3 experiments.*P < 0.05, ****P < 0.001 as compared with the DMSO control group

Control

10 μM

20 μM

40 μM

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EFFECT OF C20 EPOXIDE ON THE VIABILITY OF 6 HUMAN CANCER CELL LINES AT 48 HOURS (MTT ASSAY)

Dose-response graph of 6 human cancer cell lines treated with C20 epoxide for 48 hours. Cell survival / viability was quantified using the MTT assay, and values were expressed as percentages over those of control (DMSO). Data reported are mean ± SE of representative n=3 experiments.*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 as compared with the DMSO control group

53% 67% 67% 75% 31%

39%

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RESPONSIVENESS OF C20 EPOXIDE ON 6 HUMAN CANCER CELL LINES AT 48 HOURS (MTT ASSAY)

› C20 epoxide seemed more effective against these 4 cell lines:- A549 lung carcinoma

- BeWo placenta choriocarcinoma

- DU145 prostate carcinoma

- HeLa cervical adenocarcinoma

› C20 epoxide seemed less effective against these 2 cell lines:- HEPG2 hepatocellular carcinoma

- MDA-MB-468 breast adenocarcinoma

CELL PROLIFERATION AND REPLICATION ARE CONTROLLED BY THE CELL CYCLE

› The M phase – mitosis (cell nucleus splits in two)

› Followed by the G1 phase

- Interval between mitosis & initiation of DNA replication

› During G1, the cell is metabolically active & continuously grows but does not replicate its DNA

› G1 is followed by S phase

- DNA replication takes place

› The completion of DNA synthesis is followed by the G2 phase

- cell growth continues and proteins are synthesised in preparation for mitosis

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PROPIDIUM IODIDE STAINING AND ANALYSIS BY FLOW CYTOMETRY

Cells (1.5x105/well) were seeded in 6-well plates in complete growth medium (DMEM / F12K + FCS 10% +P/S)

Cells are starved with serum free medium (DMEM / F12K + P/S)

Cells were treated with the C20 epoxide at 3 different concentrations: 10 μM, 20 μM, 40 μM + control (DMSO)

Cells were fixed in 80% ethanol

Cells were resuspended in buffer containing 100μg/ml RNase A and 0.1% Nonidet NP40Stained with 12.5 μL of propidium iodide (50 μg/mL)

Samples were analysed by FC 500 flow cytometer and using the MXP software

24 h

24 h

48 h

24 h

1 h

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EFFECT OF C20 EPOXIDE ON CELL CYCLE OF HEPG2 CELLS

Control (DMSO) 10 μM 20 μM

40 μM

DNA content

DNA contentDNA content

DNA content

Num

ber o

f cel

ls

Num

ber o

f cel

ls

Num

ber o

f cel

ls

Num

ber o

f cel

ls

Sub-G1

G0/G1

S

G2/M

Significant in the proportion of cells in S-phase relative to control

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EFFECT OF C20 EPOXIDE ON CELL CYCLE DISTRIBUTION OVER 48 HOURS

60% 97%

10% 49% 45%

237%

4% 51% 39%

52%

17% 32% 22%

79% 103%

10% 6% 37%

Data reported are mean ± SE of representative n=3 experiments. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 as compared with the DMSO control group

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FURTHER WORK TO BE COMPLETED

› At the moment I have to finish the flow cytometry experiments for:

- BeWo

- HeLa

› The next direction in my project is to examine the effects of the C20 epoxide using:

- Caspase assay – to measure apoptosis

- Western blotting

- Cyclin D1

- Caspase 3 & 9

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ACKNOWLEDGEMENT

› I would like to thank:

- Professor Michael Murray for accepting me as an MPhil candidate

- The post-docs who have helped me gain the various skills

- Mai Tran

- Sarah Allison

- Julie Dwyer

- NHMRC for providing the grant to fund this project