Aconitine Induces Cell Apoptosis in Human

Pancreatic Cancer via NF-κB Signaling Pathway

Journal from: European Review for Medical and Pharmacological Sciences

2016; 20: 4955-4964

By: B.-L. JI, L.-P. XIA, F.-X. ZHOU, G.-Z. MAO, L.-X. XU
Department of Laboratory Medicine, Hospital of Traditional Chinese
Medicine of Lishui City, Lishui, Zhejiang, China

PRESENTER: Widya Yanti Sihotang

ADVISOR : Prof. Jwu-lai Yeh, PhD
2017/10/12
 Introduction
Pancreatic cancer is one of the leading causes of cancer-related deaths in the world.

National Cancer Institute, 2017

Figure 1. Incidence of Pancreatic Cancer
2
Pancreas
The pancreas is an organ found
deep in the body, behind the
stomach. It is shaped a little bit like
a fish. It is about 6 inches long and
less than 2 inches wide. It goes
across the belly (abdomen).

There are 2 diffrent kinds of glands:

1. Exocrine
2. Endocrine Figure 2. Pancreas
https://www.cancer.gov/images/cdr/live/CDR636528.jpg

3
Function of Pancreas
• The endocrine gland
consists of the islets of
Endocrine Langerhans and
secretes hormones into
function the bloodstream
• Release of insulin and
glucagon

• Acini (cluster of
secretory cells)
Exocrine digestive enzymes
• Secretes pancreatic
function juice which breaks
down all categories of
foodstuff
Figure 3. Exocrine and Endocrine Pancreas
https://www.cancer.org/cancer/pancreaticcancer/about/what-
is-pancreatic-cancer.html
4
Diseases of Pancreas

 Diabetes mellitus
• Type 1 Pancreas failure to produce enough insulin
• Type 2 Insulin resistance
 Pancreatitis Inflamation of the pancreas
• Acute pancreatitis

• Chronic pancreatitis

 Exocrine Pancreatic Insufficiency inability to break down and digest food properly

 Pancreatic cancer

5
https://en.wikipedia.org/wiki/Pancreas
Figure 4. Hallmarks of cancer 6
APOPTOSIS
Apoptosis is a tighty regulated form of cell death, also called the programmed cell death.

Figure 5. Apoptosis Pathway 7

 Normal Cell vs Cancer Cell

Normal Cell division

Cancer Cell division Uncontrolled

growth

8
APOPTOSIS: PATHWAY
“Extrinsic Pathway”
Death Ligands Death Iniciator
Receptors Caspase 8

Effector Cell
Caspase 3 Death
“Intrinsic Pathway”
DNA damage Mitochondria/ Iniciator
p53 Cytochrome C Caspase 9
9
 • a sequence spesific transcription factor
that is involved in many cellular activities
Nuclear Factor including the inflammatory and immune
response
of kappa B • NF-B also inhibits apoptosis by
(NF-B) activation of several anti-apoptosis
proteins

10
 NF-B Pathway
Canonical Signaling Pathway Non-Canonical Signaling Pathway

Figure 6. NF-B Pathway http://www.abcam.com/research-areas/overview-of-nf-kb-signaling

11
 The Role of NF-B in Apoptosis

https://www.ncbi.nlm.nih.gov/pubmed/25120954 12
Figure 7. The Rule of NF-B in Apoptosis
 ACONITINE（烏頭鹼）
Aconitine is a major bioactive diterpenoid
alkaloid with high content derived from herbal
aconitum plants

In Asia, aconite poisoning is much more

common because of the continuing use of Figure 8. Structure of Aconitine
aconite roots in traditional medicine as https://pubchem.ncbi.nlm.nih.gov/compound/aconitine
analgesic, anti-inflammatory, and cardiotonic
agents 科別 毛茛科 Ranunculaceae
屬名 烏頭屬 Aconitum
Despite its narrow therapeutic index, aconitine 中文學名 烏頭
is used as a traditional medicine in China
拉丁學名 Aconitum carmichaelii Debx.
because of its analgesic and anti-inflammatory
activities and currently has limited applications 英文名稱 Aconite
in herbal medicine 中文俗名 苦烏喙、奚毒、雞毒、莨、毒公、獨白
Chan, T. Y. (2009). "Aconite poisoning." Clinical Toxicology 47(4): 279-285.
草、土附子、草烏、斷腸
http://tcm-toxic.kmu.edu.tw/index.php/%E7%83%8F%E9%A0%AD 13
Although thousands of studies have been conducted, the specific
mechanism involved remain unknown. Therefore, obtaining an in-
dept understanding of pancreatic cancer and finding a novel
therapeutic strategy to diagnose and treat this disease are
necessary.

Title: Aconitine Induces Cell Apoptosis in Human

Pancreatic Cancer via NF-B Signaling Pathway

14
How if aconitine induces cell apoptosis in human
pancreatic cancer via NF-κB signaling pathway?

The aim: To explore the effects of aconitine in human

pancreatic cancer

15
 MATERIALS AND METHODS

In vitro study

Cells: Cell viability assay MTT

Miapaca-2 Colony formulation assay Microscope

Cell apoptosis assay Hoechst-33258

stain; Annexin V-
FITC Apoptosis
Western Blot analysis
Detection Kits
With different concentration
PANC-1
Caspase activity detection Caspase-3, -8, -9
activity kits

16
 MATERIAL AND METHOD
In vivo study
Three groups: each n=10
Xenograf Mouse Model I. Vehicle (methanol)
𝟏𝐱𝟏𝟎𝟔
Miapaca-2 4 weeks
II. 50 mg/kg
cells were s.c. once a day
injected

4 weeks old, male III. 100 mg/kg

athymic nude mice once a day
Molecule Structure of Aconitine

Immunohistochemistry
(IHC) Analysis

Tumor size : once/ 4 days

Statistical Analysis : Presented as the means ± SD and use
t-test to compare the difference between group
Weight was obtained at the end 17
Results

19
 Aconitine Inhibits Cell Viability in Cultured
Human Pancreatic Cancer Cell Lines

Figure 9 20
Aconitine Suppresses Cell Proliferation in Miapaca-2 and PANC-1 Cells

Figure 10 21
 Aconitine Increases Cell Apoptosis in a Dose-dependent Manner
in Pancreatic Cancer Cells

0 15 30 60

0 15 30
60

Figure 11
22
Aconitine Reduces the Number of Survival and Increases Cell Apoptosis in a Dose-dependent
Manner in Pancreatic Cancer Cells

Figure 12 23
Aconitine-induced Pancreatic Cancer Apoptosis Through NF-B Signaling Pathway

NF-B
Bax(cyto.)
Bax (mito.)
Bcl-2
Cl-caspase-9
Cl-caspase-3
Cl-PARP
Cyto.C (cyto.)
-atcin
Miapaca-2 PANC-1

24
Figure 13
Aconitine Suppress Tumor Growth and Induced Cell Apoptosis in a Xenograft
Mouse Model of Pancreatic Cancer

Figure 14 25
Discussion
 This research is the first to explore the potensial role of aconitine in
pancreatic cancer cell proliferation.
 In xenograft mouse model, aconitine administration was shown to
inhibit the grownth of pancreatic cancer and induce cell apoptosis.
 Found through Hoechst staining and cell cytometry analysis that
aconitine administration could remarcably increase cell apoptosis in
both of Miapaca-2 and PANC-1 cell lines.
 The extrinsic pathway begin outside a certain cell when stimulated by
oxidant stress or chemical attack.

26
Discussion
 Stress induced apoptosis involves changing the mitocondrial
permeability, the subsequent cyto.
 Multiply proteins are involved in this process, including Bcl-2-
antagonist of cell death, and cleaved PARP.
 The expressions of Bax, Bcl-2 and NF-B decreased as the aconitine
concentration increased.
 The protein levels of Bax, cl-caspase-9, cl-caspase-3, cl-PARP, and
cyto.C were remarcably upregulated after aconitine administration,
which were coincident with the process of cell apoptosis.
27
Conclusion

In vitro In vivo
Miacapa-2 PANC-1 Athymic nude mice

Cells viability
Colony cells Volume of Tumor
Apoptosis Weight of Tumor
NF-B
Bax (cyto.) Expression
Bax (mito.)
Bcl-2
Cl-caspase-9
Cl-caspase-3
Cl-PARP
Cyto.C (cyto.)

28
 This study validated the inhibitory effects
of aconitine on pancreatic cancer, which
was demonstrated to be associated with
Conclusion cell apoptosis.
This findings may provide novel evidence
of aconitine as a potential therapeutic
reagent against pancreatic cancer.

29
THANK YOU