Department of Biochemistry Faculty of Medicine, KKU

Anticancer effect of on cholangiocarcinoma cell

Mr. Bundit Promraksa Ph.D. candidate Medical biochemistry and molecular biology program Department of Biochemistry, Faculty of Medicine, KKU

1 Contents

• Introduction • Cholangiocarcinoma • Coniferyl alcohol • Metabolomics • Materials and Methods • Results • Discussion

2 Introduction Cholangiocarcinoma: combating a silent killer

Treatment of cholangiocarcinoma  Operative therapy  Radiation  Chemotherapy  Chemoradiation treatment

Table 1 The toxicity of chemotherapy in advanced CCA patients

Figure 1 Epidemiology of CCA in Thailand Blood cell production

Figure 2 Opisthorchis viverrini metacercaria Electrolyte imbalance and their host (cyprinoid fish)

Petney et al., Parasitol Int; 2011 3 Anderson et al., Oncologist; 2004 Butthongkomvong et al., Asian Pac J Cancer Prev; 2013 The effective treatment with less toxicity of CCA is still needed Introduction

Lignin as anticancer potential against cholangiocarcinoma cell (1) • Generally, found in cell wall of wood and bark. • are cross-linked phenolic polymers. (2) • Three monomers are precursors including (3) (1) paracoumaryl alcohol (PA) (2) (SA) Figure 1 monolignol monomers (3) coniferyl alcohol (CA) - 50-60% • Lignin macromolecules are formed by the dehydrogenative polymerization of three with carbohydrates • 50-60% of lignin’s interunit linkage is β-O-4 unit • The pharmacological studies of lignin have been limited.

4

Lundquist, K. (1992). Introduction Lignin as anticancer potential against cholangiocarcinoma cell

Sample preparation NMR acquisition Multivariate statistical analysis Generation of the datasets Transform variables Plant extracts

C. halicacabum G. celosioides S. dulcis %Cell viability ECH EGC ESD Biological testing

Plant extracts

2D-NMR for structure Candidate compound confirmation Identification of active metabolites

Lignin 5 Promraksa et al. PLOS One; 2019 Introduction Metabolomics

• Metabolomics is the simultaneous (multiparallel) systematic identification and measurement of many cellular metabolites of a biological system at a specific point in time. It is a high-throughput analysis of metabolites. • Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are often used for metabolome profiling as they produce rapid and reproducible results and samples can easily be prepared. • NMR focuses on the metabolic profiling of all of metabolites (“fingerprint”) in a sample.

Untargeted metabolomics

6 1.Nature 2. Phytochem Rev (2008) 7:525–537 Research questions

• Can coniferyl alcohol be inhibit the CCA cell viability?

• What are the key metabolites change after treatment with coniferyl alcohol that induced apoptosis in CCA cell line?

7 Materials and Methods

Cytotoxicity test on cholangiocarcinoma cells

Flow cytometric analysis

Coniferyl alcohol KKU-213 cell Cultured in 10 cm3 (In house synthesis) dishes and treatment with or without coniferyl alcohol Western blotting 110 V Collected the cancer cells

NMR acquisition

KKU-213 cell Cultured in 15 cm3 Collected the cancer cells for dishes and treatment with or 15x106 and extracted with without coniferyl alcohol chloroform/methanol (1:2 v/v) 8 Results Coniferyl alcohol (CA) induced apoptosis

100 48 hrs 72 hrs 75

50

Cell viability (%) viability Cell 25

0 0 100 200 300 400 500 Coniferyl alcolhol concentration (g/mL)

Figure 1 Viability of CA on KKU-213 Figure 2 Flow cytometric analysis of apoptotic Figure 3 Effect of coniferyl alcohol on proapoptotic for 48 and 72 hrs KKU-213 cells stained with PI and FITC- BAX and Bcl-2 protein in KKU-213 cells Annexin V after treatment with CA for 48 hrs

CA can inhibit KKU-213 in dose dependent manner by inducing apoptosis 9 Results

Metabolic profiling of coniferyl alcohol induced KKU-213 cell apoptosis

Carnitine

Methanol

Phosphocholine

Lactate

Gluthatione

Creatine

Guanine

inositol

-

Lactate

Creatine

Glycine

Myo

Choline

Gluthatione

Alanine

inositol

-

acid

Taurine

Gluthatione

Betaine Betaine aldehyde

Adenine

Succinate

Myo

Gluthatione

Acetate

Oxaloacetate

Inosine

Formate

Isoleucine

Ethanol

ATP

Leucine

Proline

Valine

Valine

Gluthatione

NAD+

Homocysteine

ATP

Fumarate

DHAP

Tyrosine

Uracil

Tyrosine

NAD+

Guanosine

Glutamate

Uracil

Isoverarate

NAD+

Uridine

Dimethylmalonic

Phenylalanine

Uridine Dimethylamine

10 Figure 4 H1 NMR spectra and metabolite identification of KKU-213 cell Results

0 µg/mL CA 50 µg/mL CA 100 µg/mL CA 200 µg/mL CA

R2 = 0.773 Q2 = 0.637

Figure 5 Principal component analysis of Intracellular metabolites of KKU-213 after treatment with or without CA 11 Results

A High in treatment group

R2X = 0.709 Q2Y = 0.599 P-value = 0.670

High in control group

B High in treatment group

R2X = 0.812 Q2Y = 0.804 P-value = 0.120

High in control group

C High in treatment group

R2X = 0.885 Q2Y = 0.795 P-value = 0.007

High in control group

Figure 6 O-PLS corresponding coefficient loading plots displaying significant 12 metabolites after treatment with or without CA Results

Taurine Phosphocholine Glutathione Myo-inositol Choline p=0.004 p=0.011 p=0.045 p=0.010 p=0.020 p=0.027 1000 2500 p=0.038

500 1500 800

M)

M)

M)

M)

M)

  800 2000 400 600 1000 300 600 1500 400 200 400 1000 500 200

100 200 500

Absolute concentration ( concentration Absolute

Absolute concentration ( concentration Absolute

Absolute concentration ( concentration Absolute Absolute concentration ( concentration Absolute Absolute concentration ( concentration Absolute 0 0 0 0 0 0 0 0 0 0 50 50 50 50 50 100 200 100 200 100 200 100 200 100 200 CA concentration (g/mL) CA concentration (g/mL) CA concentration (g/mL) CA concentration (g/mL) CA concentration (g/mL)

Succinate ATP Inosine p=0.002 Adenine Uracil p=0.006 p=0.004 p=0.040 p=0.017 p=0.038 400 p=0.031 p=0.006 p=0.002 150

300 M) 1000

250 M)

M)

M)

M)

  200 300 800 200 100 150 600 200 100 400 100 50 100

50 200

Absolute concentration ( concentration Absolute

Absolute concentration ( concentration Absolute Absolute concentration ( concentration Absolute 0 ( concentration Absolute 0 0 ( concentration Absolute 0 0 0 0 50 50 0 0 0 100 200 100 200 50 50 50 100 200 100 200 100 200 CA concentration (g/mL) CA concentration ( g/mL)  CA concentration (g/mL) CA concentration (g/mL) CA concentration (g/mL)

Figure 7 Comparison of significantly metabolites concentration 13 Discussion

➢ CA induced apoptosis in the cellular ratio of BAX/Bcl-2 that observed by western blot analysis and flow cytometry.

➢ The upregulation of BAX and downregulation of Bcl-2 protein results in the release of apoptogenic factors such as cytochrome c. In our results, ATP significantly increased which plays a critical role in early apoptosis as its interacts with apoptosis protease activating factor-1 (Apaf-1) before activation of caspase cascade pathway. At 200 ug/mL of CA treatment, KKU-213 cell partly underwent necrosis as observed in annexin V/PI staining (Tsujimoto Y, 1997).

➢ Moreover, Glutathione depletion has important in cellular defense against reactive oxygen species (ROS) especially in apoptosis indicated that KKU-213 underwent the activation of the apoptotic signaling cascade (Circu ML and Aw TY, 2012).

➢ Previous study reported that choline-containing metabolites, taurine and glutathione were significantly decreased after treatment with doxorubicin which was in similarly to our results (Opstad et al., 2009).

➢ Soares and coworker (2015) reported that inosine induces melanoma cell proliferation through phosphoinositide 3-kinase (PI3K) pathways. Therefore, inosine is associated with proliferation of melanoma cell proliferation. In our results, the lower of inosine content was present in CA treatment group compared to non-treatment group. It might be a potential marker for detection of CCA cell proliferation. 14 Reference

• Promraksa B, Phetcharaburanin J, Namwat N, et al. Evaluation of anticancer potential of Thai medicinal herb extracts against cholangiocarcinoma cell lines. PLoS One. 2019;14(5):e0216721. • Circu ML and Aw TY. Glutathione and modulation of cell apoptosis. Biochim Biophys Acta. 2012;1823(10):1767-77. • Tsujimoto Y. Apoptosis and necrosis: Intracellular ATP level as a determinant for cell death modes. Cell Death Differ. 1997.4; 429–434. • Opstad KS, Bell BA, Griffiths JR, et al. Taurine: a potential marker of apoptosis in gliomas. Br J Cancer. 2009 Mar 10;100(5):789-94.

15 Acknowledgements

Overseas advisor:

Advisor: Assoc. Prof. Dr. Watcharin Loilome Dr. Jia Li Assoc. Prof. Dr. Nisana Namwat Faculty of Medicine, Dr. Anchalee Techasen Department of Surgery & Cancer Dr. Jutarop Phetcharuburanin

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