Download Lactoferrin and its impact on breast cancer cell lines

Carolyn Alvarez
Hamid Hussani
Ejike Okoye
Jennie Williams, PhD
The evidence of various macromolecules as factors inhibiting the progression of chronic
diseases has promoted the study of cancer chemoprevention. The mechanisms of action of
these agents are not fully understood. However, their use has been shown to be effective in
cancer prevention and therapy. Glycoproteins, such as lactoferrin, are presently being
studied for their anticancer effect. In this study, the effect of human lactoferrin on human
breast cancer cells (MDA-MB-231 and MCF-7) was investigated. Cells were treated with
lactoferrin at concentrations ranging from 0µM to 500µM. Lactoferrin decreased cell
viability of MDA-MB-321 and MCF-7. The LC50 of MCF-7, as determined by presto blue
cell viability assay, was 224±17µM at 24 hrs. Apoptosis, as determined by the comet assay,
was induced in both cells lines. In addition, a scratch test analysis demonstrated a decrease
in cell migration upon treatment of lactoferrin. The ability of lactoferrin to induce cell cycle
arrest was examined; however, at the concentrations examined no arrest was detected. The
results collected from this study indicated that lactoferrin inhibits several steps involved in
cancer development.
Breast Cancer is currently the second leading cause of mortality in the world. It is a
tragic disease that affects millions of people worldwide; with an estimated 1.6 million
persons to be diagnosed with cancer in 2012 (National Cancer Institute). As of today,
cancer is one of the most researched topics in science; resulting in many breakthroughs of
discovery. The disruption of normal cellular process and the aggressive proliferative
attributes make studies to eradicate this disease an important focus of research. Over the
past years, clinical studies have demonstrated the existence of a link between cancer,
environmental factors and nutrition. Studies have shown that natural nutritional products
have an effect on either the acquisition of or the prevention of chronic diseases.
Consumption of certain foods we eat can serve as promising chemopreventive agents. In
fact, these evaluations are currently being studied clinically (Baveye et al., 1999). The
term chemoprevention is defined as a chemical that can alter, interrupt, block and/or
prevent development of diseases such as cancer. Since cancer itself is a multistage
disease, there is a period of time in which development of the malignant tumor can be
arrested or inhibited.
The human body is made of trillions of cells that undergo mitosis and apoptosis in
an orderly fashion. Cell proliferation and differentiation is a normal process of constantly
dividing cells. The normal function of cells undergoing mitosis is to grow, to copy and
synthesize DNA and to replicate. However, this process is not always successful. Proteins
involved in the normal check and repair mechanism of the cell incur mutations and is
aberrantly expressed. When this occurs, cells that have mistakes in their DNA continue to
replicate and grow uncontrollably. Although there are many checkpoints in place to evade
DNA damage or mutations, cells sometimes lose the ability to stop the proliferation of
damaged DNA. In order for cells to replicate, proliferate, and differentiate an available
iron supply is needed. Cell proliferation and most DNA synthesis require iron; thus,
rapidly dividing cells like cancer cells utilize iron to maintain their existence.
Lactorferrin, a transferrin family of proteins, is an iron-binding glycoprotein. LF regulates
the amount of iron transported into cell. The chelation of iron can promote fast
proliferating cells such as cancer cells to undergo cell cycle arrest. Therefore, as the
internal concentration of lactoferrin increases an increase of iron is chelated by this
glycoprotein; thus, leading to an inhibition of proliferation and eventually an induction of
apoptosis.
Evidence demonstrates that Lactoferrin has anti-inflammatory, anti-helminitic and
antioxidant abilities as well as the ability to induce apoptosis of malignant cells and
disrupt cellular proliferation. Apoptosis and its link to cancer have been extensively
studied. An important aspect of cancer chemoprevention is the discovery of agents that
can induce apoptosis in rapidly dividing cells. Of pertinent, studies examining the effect
of LF have demonstrated a significant inhibition of metastasis of cancer cell; specifically,
in breast cancer cell lines. Breast cancer primarily metastasizes to nearby lymph nodes,
liver, lungs and local bones. Human LF has shown, in vivo, to inhibit distant metastasis in
mice models.
Here, we show that breast cancer cells treated with lactoferrin has an effect on the
metastasis of cancer.
Scratch test: MCF-7 cells were treated with
different concentrations of lactoferrin and
observed at 24 hour increments under 250x
magnification. Migration and metastasis of cells is
shown.
Morphology: MCF-7 cells were treated with different concentrations of
lactoferrin. The morphology of the cells was recorded at 24 hour
increments under 250x magnification.
Flow Cytometry: Fixed MCF-7 cells
were stained with PI, which stains
nuclear material. Peaks in the graphs
show location where cell cycle arrest
has occurred. Treatment of lactoferrin
had no effect on cell count at different
points of cell cycle.
•Cell Morphology
Treatment with lactoferrin had an effect on the morphology of the MCF-7 cells; 24
hours post treatment.
Treatment with lactoferrin had no effect on the morphology of the MCF-7 cells; 48
and 72 hours post treatment. This may be attributed to cellular rebound after the drug
is exhausted from the medium.
•FLOWCYTOMETRY W/ PI STAINING
Treatment of MCF-7 cells with lactoferrin had no effect on the progression of cells
through the cell cycle.
Induction of apoptosis in MCF-7 cells treated with lactoferrin could not be detected
(G0-G1) via this method.
•SCRATCH TEST
Migration or wound healing of MCF-7 cell line treated with lactoferrin was retarded
in a concentration and time dependent manner.
After 72 hours (last time point examined), complete wound healing was seen in the
control well and minimal migration was seen in the well with the highest concentration
(500 μM).
•COMMET ASSAY
DNA fragmentation of MDA-MB-231 and MCF-7 cancer cells was induced by
treatment of lactoferrin in a concentration dependent manner.
Distinct comet “tails” in both breast cancer cell lines, indicating apoptosis in the cells,
was observed.
•FUTURE PLANS: Replication of present study and additional in vivo and in vitro
analysis to define the mechanism of action of lactoferrin .
Comet Assay: Both MCF- and MDA-MB231 cells were treated with different
concentrations of lactoferrin in order to
observe the apoptotic ability of cells. The
“tail” of the comet increases with increasing
apoptosis, showing the greatest amount of
apoptosis in the highest concentration of
treatment.
MCF-7 0μM
Comet Length: 201 px
Dr. Daniel Moloney
Dr. Jennie Williams
Dr. David Bynum
Ms. Judy Nimmo
MCF-7 500 μM
Comet Length: 303 px
MDA_MB_231 0 μM
Comet Length: 86 px
MDA-MB-231 500 μM
Comet Length: 142 px
Ms. Kristen LaMagna
Dr. Farah Daccueil
Mr. Delon Callender
Ms. Debbie Pelio
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