Aveneu Park, Starling, Australia

DETERMINATION knowledge of risk factors and good funding

DETERMINATION OF STATIN ACTIVITY AGAINST PANCREATIC CANCER
CELL LINES IN 2D AND 3D CULTURES

1.0 INTRODUCTION

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Pancreatic cancer (PC) continues to have one of the poorest
prognoses among all cancers. PC accounts for about 3% of all cancers in the US
and about 7% of all cancer deaths (American Cancer Society, 2017). Cancer death rates trend has
continued to go down for other types of cancers such as breast and prostate
cancers, this is due to the availability
of early detection biomarkers, improved treatments, better knowledge of risk
factors and good funding and awareness programs (Stewart et al., 2016). However the cause of
pancreatic cancer still remain unknown, Cancer status is usually determined
when the disease is well beyond established and intervention
at this period only through surgery, and remission works only in few cases. The reason behind being luck of screening or diagnostics tests (Herreros-Villanueva & Bujanda, 2016).

1.1 SIGNIFICANCE OF THIS RESEARCH STUDY

Pancreatic cancer burden to world health population is a
major concern. Pancreatic cancer contributes to loss to an individual utility
to the quality of life through its debilitating
nature and compromising economic objectives of a person. Currently, there is no effective mode of
treatment for pancreatic cancer, and long-term survival of cancer patients
remains poor. A lot of research is focused on finding better treatments for
pancreatic cancer. Many clinical trials are testing new combinations of
chemotherapy drugs for pancreatic cancer. This study will validate the
effectiveness of statins against pancreatic cancer cell lines.

2.0 RESEARCH BACKGROUND

Pancreatic ductal adenocarcinoma (PDAC) comprise 80% of
pancreatic cancer cases (Ying et al., 2016). A number of predisposing
factors have been attributed to etiology
of PDAC and some of the factors include genetic predisposition, environmental
exposure and chronic inflammation (Knudsen et al., 2017). PDAC of particular concern
has shown to be resistant to all currently
available treatment regimens such as chemotherapy, radiotherapy, and immunotherapy.  PDAC common feature is the growth of dense fibrous or connective tissue
called desmoplasia. Desmoplasia is a reaction secondary to an insult where
overactive tumor-associated fibroblasts
deposit excessive extracellular matrix (ECM), stellate cells as well as immune
cells, which play a significant role in tumor progression and resistance to
therapy (Nielsen, Mortensen, & Detlefsen, 2016). 

Pancreatic cancer poor prognosis was believed to be due to
quick progression to metastatic disease. However, recent sequencing data from
excised tumor and metastatic tissue shows that there is a period of over ten
years between initiating mutation and formation of the tumor mass. Metastasis occurs five years later with death following
after two years (Roe et al., 2017). Therefore, proving this
theory in the general population, and if biomarkers for early diagnosis can be achieved,
there is ample time to treat pancreatic cancer before the metastatic disease.

2.1 EVALUATION OF DRUG IN 3D CULTURE MODEL

3D in vitro tumor model has been adopted as a preclinical
tool for evaluating anticancer therapeutic candidates (Fang & Eglen, 2017). Research shows that 3D
culture accurately reflects in vivo drug responses and mechanisms of
chemotherapy than 2D monolayer based cultures (Adcock, Trivedi, Edmondson, & Yang, 2015). It has been reported cells
grown in 3D cultures show high resistance to anticancer drugs than cells grown
in monolayer culture. This corresponds to massive resistance to anticancer
agents in vivo shown in clinical settings (Breslin & O’Driscoll, 2016). The increased resistance to
chemotherapeutic agents has been attributed to intrinsic mechanism in tumor
microenvironment which includes factors
such as increased cell to cell contact, pro-cell
survival signaling, general 3D cellular architecture, lowered proliferation
rate, and oxygen and drug diffusion gradient of site-specific
non cancer cells (Senthebane et al., 2017).

2.2 USE OF STATINS AS ANTICANCER AGENTS

Statins work by targeting rate-limiting enzyme of the
mevalonate pathway, 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase (Mullen, Yu, Longo, Archer, & Penn, 2016). The products of mevalonate
pathway: Sterols, such as cholesterol are involved in membrane integrity and
steroid production. Ubiquinone (coenzyme Q) is involved in electron transport
and cell respiration. Farnesyl and geranylgeranyl isoprenoids involved in
covalent binding of proteins such as the Ras family to membranes. Dolichol is
required for glycoprotein synthesis. Isopentenyl adenine is essential for
certain tRNA function and protein synthesis (Tricarico, Crovella, & Celsi, 2015). Importantly statins
inhibition of HMG-CoA is applied in clinical settings to treat
hypercholesterolemia and as prophylaxis for cardiovascular disease (Yang et al., 2017). As an anticancer
agent, statins inhibit HMG-CoA
reductase, which results in a reduction
of isoprenoids synthesis, geranylgeranyl, and
farnesyl (Likus et al., 2016). These substances bind to Ras
protein, which is involved in signaling pathways essential for cell growth,
proliferation, differentiation, and cancer development.  Lack of isoprenoids leads to Ras protein inactivation,
blockade of cell signaling, and tumor regression (Jonckheere, Vasseur, & Van Seuningen, 2017). Based on recent studies, we will
analyze the activity of four lipophilic statins from different classes (I
class: lovastatin (LOVA), mevastatin (MEVA), and simvastatin (SIMVA); and II
class: pitavastatin (PITA)) in 2D and 3D human pancreatic cancer cell (BxPC-3,
MIA PaCa-2, and PANC-1) cultures

3.0 HYPOTHESIS

Inhibition of
mevalonate will result in depletion of downstream products needed for cell proliferation,
which will result in the arrest of cell
growth and eventual cell death.  HMG-CoA
reductase is the major rate-limiting enzyme of the mevalonate pathway. Inhibition
of HMG-CoA reductase prevents the conversion of HMG-CoA to mevalonate, and thereby
reduce levels of mevalonate and its downstream products. Many products of the
mevalonate pathway are necessary for critical cellular functions such as
membrane integrity, cell signaling, protein synthesis, and cell cycle
progression. Disruptions of these processes result
in cell death.

4.0 STUDY AIMS

·        
To determine the activity of statin against pancreatic
cancer cell lines in 2D and 3D cultures.

·        
To compare drug interaction in traditional 2D
cell monolayer and 3D culture model.

5.0 MATERIALS AND METHODS

5. 1 STUDY DESIGN

Reproducibility of results study will be conducted.  All data will be obtained by performing experiments.
All experiments will be done in professor’s Chi-Tsai laboratory at Kaohsiung
Medical University, Kaohsiung, Taiwan.

5.2 STUDY METHODS

METHOD

AIM

Cell culture

To grow and maintain pancreatic
cancer cell lines in appropriate media until further analysis

Cell viability assay (MTT)

To determine effectiveness of a
therapeutic agent to inhibit or kill pancreatic cancer cells

spheroid 3d culture

To determine drug interaction
against pancreatic cancer cell lines in 3D tissue-mimicking environment

Cell colony formation

To determine proliferative
ability of cells and long-term survival
after treatment  with cytotoxic drug

Cell death assay

To evaluate type of cell death
whether due to apoptosis or necrosis after statins treatment to cells

Statistical analysis

To determine the significance of data obtained through experiments.

5.3 Materials

All materials including culture media, reagents, consumables
and equipment will be provided by professor Chi-Tsai laboratory.

6.0 BIOMEDICAL TECHNIQUES IN THIS RESEARCH

In this project will use the following biomedical
techniques: cell culture, cell viability test, spheroid growth test, cell
colony formation, cell death and statistical analysis.

6.1 CELL CULTURES

Aseptic technique and good cell culture practice should be
practiced all the time to ensure all cell culture procedures are performed to a
standard that will prevent contamination from bacteria, fungi and mycoplasma
and cross contamination with other cell lines. Human pancreatic cancer cell
lines: BxPC-3, MIA PaCa-2, and PANC-1 will be obtained from the American Type
Culture Collection (ATCC, Taiwan R.O.C). Human foreskin fibroblast cells
CRL-4001 labeled with green fluorescent protein (HF-GFP) will be obtained from
ATCC, Taiwan R.O.C. BxPC-3 cells will be grown in Roswell Park Memorial
Institute 1640 GlutaMAX medium, MIA PaCa-2 and PANC-1 cell lines will be
cultured in Dulbecco’s Modified Eagle’s Medium GlutaMAX medium. Both media will
be supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 10%
fetal bovine serum. HF-GFP cells will be grown in Medium 106 with Low Serum
Growth Supplement. Media and supplements will be purchased from Gibco
(Carlsbad, CA, USA). Cells will be maintained in a humidified atmosphere
containing 5% CO2 at 37°C. The cells will be quickly thawed in warm media to
stop DSMO activity.  The cells will then
be cultured in respective media as mentioned above until 80 – 85% confluence of
growth is reached. Subculture or passage of cells by removing all the old
media, and wash with pre-warmed PBS to remove FBS. Trypsinization to break
adhesion and free up cells. Check under inverted
microscope and tap the dish against the base of microscope to ensure all the
cells are released. Stop the action of trypsin by adding medium containing 10%
FBS. Pipette the cells up and down to ensure all the cells are resuspended.
Perform cell count and subculture as required.

6.2 CELL VIABILITY TEST (MTT ASSY)

To determine the effect of drug on cell viability, 3-(4, 5-dimethylthiazol-2-yr)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St
Louis, MO, USA) assay will be performed. BxPC-3, MIA PaCa-2, and PANC-1 cells
will be seeded in 96-well plates in a volume of 100 ?L (5,000 cells/well).
After 24 h pre-incubation, the cells will be treated with 100 ?L of different
concentrations of statins. Only medium without cells will be used as a positive
control, and the medium with 0.5% DMSO (Sigma-Aldrich Co.) will serve as a
negative control. After 24, 48, and 72 hours the cells will be incubated for 3
h with the MTT solution (Sigma-Aldrich Co.). The absorbance will be measured at
a wavelength of 570 and 630 nm.

6.3 SPHEROID GROWTH

Spheroids growth will be performed on BxPC-3, MIA PaCa-2,
and PANC-1 cells by 3D Bioprinting method (Tseng et al., 2015).  The cancer cells will be mixed with human
fibroblasts (1:1), to better represent the tumor microenvironment. The cells
will be incubated with nanoparticles NanoShuttle
(Nano3D Biosciences Inc., Houston, TX, USA) for 8–10 h. Then cells will be
resuspended and seeded into ultra-low attachment 96-well plates in a volume of
100 ?L (2,000 pancreatic cancer cells and 2,000 human fibroblasts per well). The
plate will be placed on a magnetic drive, and will be incubated in humidified
atmosphere containing 5% CO2 at 37°C until spheroids formation. Photos of
spheroids will be taken after 2 days of incubation. The medium will be replaced
by new medium containing 5, 10, and 20 ?M of statins. Photos will be taken
every 48 h, and medium will be replaced every 96 hours of incubation. The
effect of statins in 3D pancreatic cancer cell cultures will be examined by
measuring the size change of spheroids using ImageJ software (National
Institutes of Health).

6.4 CELL COLONY FORMATION

BxPC-3, MIA PaCa-2, and PANC-1 cells will be seeded in
12-well plates in a volume of 1 mL (100 cells/well) and will be treated with
100 ?L of 10 and 90% half maximal effective concentration (EC50) of statin
solutions. Cells will be incubated in a humidified atmosphere containing 5% CO2
at 37°C. The cells will be incubated for 12 days, after which they will be
rinsed with phosphate-buffered saline (PBS, Gibco) and then fixed with 4% paraformaldehyde
(Thermo Scientific, Waltham, MA, USA) solution in PBS for 15 min. Then the
cells will be rinsed with PBS, incubated with 0.1% aqueous crystal violet
solution for 15 minutes. Finally, the cells will be washed with sterile
deionized water. Pictures will be taken using G:BOX gel documentation system
(Syngene International Ltd, Bengaluru, India) and Genesys software (Syngene
International Ltd). The number and percentage area of colonies will be
calculated.

6.5 TYPE OF CELL DEATH

Pancreatic cancer cells will be seeded in 24-well plates in
a volume of 0.5 mL (1,500 cells/well), and incubated in a humidified atmosphere
containing 5% CO2 at 37°C for 24 hours. After incubation, cells will be treated
with 10, 50, and 90% EC50 of statins. After 72 hours, cells will be rinsed with
PBS and the medium will be replaced with fresh one. 3 ?L of 10 mg/mL Hoechst
33342 aqueous solution and 1 ?L of 1 mg/mL propidium iodide will be added into
each well and cells will be incubated for 10 min. The type of cell death will
be examined (apoptotic and necrotic cells) by fluorescence microscopy, and the
number and percentage of cells will be calculated.

6.6 STATISTICAL ANALYSIS

Statistical analysis will be performed using Microsoft
Office Excel 2016 software (Microsoft Corporation, Redmond, WA, USA). All the
experiments will be done in triplicate independent measurements and the
obtained values were reported as mean ± standard deviation. Student’s t-test
will be used and p-values will be calculated. A value of p

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