Download Linking Cataracts to Cancer

Linking Cataracts to Cancer: The Diagnosis of a Novel Oncoprotein to Predict
Premature Basal-Like Breast Cancer
Gregory Konar
Massachusetts Academy of Mathematics and Science
Abstract
Alpha-basic crystallin and its associated gene CRYAB were studied for potential
oncogenic activity in MDA-MB-231 basal like breast cancer cells under conditions of elevated
stress with the inclusion of Rhodiola crenulata root extract. It was hypothesized that the protein
will exhibit oncogenic activity in the stressed breast cancer cells and not in the normal
immortalized cell line control, and the Rhodiola crenulata extract will suppress the protein. A
Western Blot Assay was used for the testing with SDS-PAGE gel electrophoresis and protein
exposure using chemoluminescense caused by Luminal. The proteins were lysed from the cell
lines, and then they were separated and transferred onto a gel blot using the electrophoresis
chamber. The proteins were blocked using the αb-crystallin antibody conjugated to HRP, and
they were exposed using a GBox Exposure Machine. The protein was shown to be specific to
only the breast cancer cells, and it had a 390% higher concentration in the MDA-MB-231 cells
than in the normal cells. Rhodiola crenulata root extract showed marginal suppressive abilities,
with the proteins being suppressed approximately 24% in both cell lines. The elevated expression
of the alpha-basic crystallin in basal like breast cancer cells but not in normal breast cells proved
that the protein is a feasible target for oncogenic activity in malignant diseases such as cancer,
and that it should be looked at with association to poor clinical outcomes in the medical society.
Introduction
The primary objective of a significant amount of scientific research performed today is to
discover a method to cure cancer; however, nobody has succeeded in fully curing it yet. An
important part of the process of eliminating cancer cells and increasing survival chances is via
early detection. Recent studies have produced increasingly positive results in accurately
detecting premature cancer through protein expression. Proteins are ubiquitous molecules that
are used in the function of both normal and malignant cells. In cancer cells, the genes that make
up these proteins, referred to as oncogenes, play the greatest role in cell division through a
process called the cell cycle.
The cell cycle comprises four primary phases, which include S phase, M phase, G1 phase,
and G2 phase. In a normal instance, the proteins would ensure that the DNA of the cell is copied
during M phase, and that two daughter cells are produced during S phase. But when carcinogenic
cells perform cellular division, mutated oncogenes target the growth regulators in the cell at G1
phase, and this causes the cell to stay in the division cycle and produce multitudes of cells in a
shorter period of time than normal (Sherr, 1996). Because these oncogenes are found only in
newly mutated carcinogenic cells, they have become a primary target in research on how cancer
proliferates and metastasizes inside of the body. Targeting these anti-apoptotic or proliferator
enhancers is achieved in multiple different ways, which range from examining active receptor
kinases and growth factor regulators to detecting the expression of the oncoproteins associated
with the oncogene. If scientists are able to detect all of the oncogenes and oncoproteins
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associated with the different types of cancer, then they will learn to predict premature cancer
through simply observing the proteins and genes in an individual’s body.
The protein that is the primary focus of this project is referred to as alpha-basic crystallin
(αb- crystallin). Αb- crystallin is part of the crystallin protein family, and even though crystallin
proteins are ubiquitous, αb- crystallin is found mostly in the eye lens or in some tissues
throughout the body. Detection of small heat shock type proteins like αb- crystallin can be
achieved through a variety of different methods, but for this testing the process that will be used
is the Western Blot Assay. This means that the protein concentrations of the cell lines need to be
found, and then gel electrophoresis needs to be performed in order to expose the proteins. The
gel will then be transferred to a PVDF membrane and exposed to an antibody for 30 minutes, and
then exposures will be taken of the proteins using an exposure machine. The exposures of this
transfer blot should be analyzed at the 20 kilo-Dalton range in order to locate and detect the
protein accurately. The phosphorylation status of the protein will also play a huge role in protein
detection and activation. Phosphorylated proteins have a phosphate group added to certain amino
acid locations found in the molecule. This additional phosphate group can either turn the protein
on or off, which controls whether it will be expressed when exposed to its conjugate antibody.
The amino acids that are associated with phosphorylation are serine, threonine, and tyrosine; in
this testing the αb-crystallin protein will be phosphorylated at serine-59.
Literature Review
Basal Type Carcinoma of the Breast
Ongoing concern in the area of breast cancer has led to not only increased awareness of
this malignancy, but also to more research that focuses on different types of breast cancer.
Cancer comprises four main groups of malignancies, carcinoma, sarcoma, lymphoma, and
leukemia. All four of those groups affect different parts of the body, whether it is the blood, skin,
or organs of an individual. Carcinoma is the most common form of cancerous malignancy that
occurs in the body; it occurs when tissue cells become mutated and begin to form tumor cells.
Common forms of carcinoma include most lung cancers, basal cell carcinomas,
adenocarcinomas, and squamous cell carcinomas (Berman, 2011). Sarcomas are very rare strains
of cancer; they form when cells that originate from bone, cartilage, muscle, or fat mutate and
form malignant tumors. Sarcomas are the most difficult type of cancer to detect early because
most patients feel little to no pain or discomfort in the afflicted area(s) (Bueckler, 2005).
Lymphoma is also known as cancer of the lymphocytes, which are cells that work to form the
immune system of an organism. Lymphoma, like almost all cancers, is considered to be an
incurable disease, meaning that even the most aggressive forms of chemotherapy or radiation
therapy will only alleviate the symptoms and not rid the body completely of the cancer.
Leukemia is cancer of the blood or bone marrow, but is not just limited to cancerous
malignancies. It covers an entire group of diseases that affect the blood, bone marrow, or
lymphoid system referred to as hematological neoplasms. The biomarker that doctors use to
determine whether leukemia is present in the body is by looking at the white blood cell count in
an organism. If the number of cells is elevated greatly, then that signals that a malignancy is
present.
Basal cell carcinoma is classified under the carcinoma subtype and is the most common
form of skin cancer in the United States. Over 2.8 million people in the United States are
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diagnosed with it every year. Basal cell carcinoma isn’t a highly fatal cancer with approximately
70,000 deaths occurring each year. It is a very slow growing cancer and can be highly mutilating
if not treated immediately. Basal cell carcinoma is a nonmelanoma type of skin cancer, meaning
that it is induced by overexposure to sunlight or x-rays; often this overexposure is caused by
tanning beds or sun lamps (“Melanoma/Skin Cancer”, 2010). There are three main types of basal
cell carcinoma, all of which are characterized by the lack of gene expression in a specific area,
hormone receptor (HER2), estrogen receptor (ER), and progesterone receptor (PR). The people
who are at the greatest risk of developing basal cell carcinoma are those with relatives who have
had the disease, those who receive a heightened amount of sun exposure either from work or
from tanning, and those who have many moles (because the cancer is apt to start growing from
that area). The symptoms of basal cell carcinoma are subtle, painless, and usually resemble sores
on the surface of the skin (Figure 1) that are discolored and will not heal.
Figure 1. Morphology and cross section of basal cell carcinoma. The
carcinoma at first looks like a mole or a sore, but it will not heal over time,
because the cancer is developing under the basal skin cell layer (Berman,
2011).
Because basal cell carcinoma is an anti-metastatic cancer, it will almost never spread to other
parts of the body; instead it will stay in one primary location if detected and treated early.
Neglecting to take steps in removing the cancerous area opens up the possibility for the cancer to
metastasize to local parts of the body, most often the nose, eyes, and ears which will result in the
disfiguration of those body parts (Berman, 2011). Basal cell carcinoma of the breast is a
cancerous skin growth that is detected on the surface of the breast of an individual. There are
numerous methods available today to remove the cancerous growth; these include excision,
cryosurgery, electrodessication, and photodynamic therapy. All of these are non-invasive
procedures and depending on how quickly the cancer was diagnosed, should permanently
remove the cancer. Most basal cell carcinomas do not return once they are removed by surgery
or therapy. In the present study performed the basal like cell line MDA-MB-231--- will be used to
simulate basal cell carcinoma of the breast.
Linking Cataracts to Cancer 3
MDA-MB-231- - - (Figure 2) is a basal-like breast cancer cell line available for purchase
at the American Type Culture Collection (ATCC). It is an epithelial based cell line that is found
in the mammary gland, or breast of humans.
Figure 2. MDA-MB-231 cell line. This is
a basal type carcinogenic cell line that
will have proteins isolated from it for
further analysis. (own photo)
MDA-MB-231--- is based on a disease called adenocarcinoma and is a derivative of the pleural
effusion of a 51 year old Caucasian female in 1974. This cell line can be applied as a transfection
host, and expresses both an epidermal growth factor (EGF) and a transforming growth factor
alpha (TGF alpha). These are both very important growth factors for a cell line to have because
EGF stimulates cellular growth and proliferation in cells, and TGF alpha is upregulated in cancer
and stimulates proliferative cells in the body. MDA-MB-231--- is tumorigenic meaning that the
cell line is active and readily forms tumors under any laboratory conditions. The cell line itself is
categorized as aneuploid female, with a modal number of 64 and a range from 52-68. The
chromosomes N8 and N15 are absent in this cell line, but there are still 11 stable marker
chromosomes, some unassignable chromosomes, and a majority of autosomes found in this cell
line. MDA-MB-231- - - cells are shown to express the WNT7B oncogene (and also the CRYAB
oncogene). The cells are shipped frozen in Leibovitz’s L-15 Medium, and it is advised to
produce a new 10% fetal bovine serum so that the cells can thaw properly (“MDA-MB-231”,
n.d.). In order to ensure that the cell line is ready for use, the cells need to have the culture
medium removed and then need a rinse with a trypsin solution so that latent trypsin inhibitor is
removed. New growth media should be pipetted onto the cells and they need to be incubated at
37o centigrade under 5% CO2 conditions until the cells are needed for testing. Not following
these instructions to subculture the cells will lead to sources of error in the data because of the
trypsin inhibitor.
MDA-MB-231--- cells have been used in scientific testing multiple times before, but not
specifically for the target protein from this experiment. Triple negative basal like breast cancer
lines are normally used so that scientists can acquire a better look at the BRCA1 gene, which is
upregulated in the cell line. This means that some of the carcinogenic activity of the cell line
comes as a result of BRCA1 gene mutations coupled with other genetic variations. Alpha-basic
crystallin is a very recent discovery inside of the cell line; therefore, not much research has been
performed on its role in the cell line. MDA-MB-231--- cells are shown to be the second most
aggressive basal like breast cancer phenotype when tested in vitro. Other studies performed with
the cell line concluded that alpha- basic crystallin expressions in basal like cell lines are
associated with poor clinical outcome in breast cancer. This results because the protein induces
anchorage independent growth in the cell line, leading to increased cell migration and invasion.
Linking Cataracts to Cancer 4
(Moyano, 2006). In order to test that specific qualities occur in only the basal like cell line,
another cell line is tested to compare the results of the experimentation.
MCF-10A is an immortalized breast cancer cell line acquired from the American Type
Culture Collection (ATCC). It is an epithelial based cell line that is found in the mammary gland
of the breast of humans.
Figure 3. MCF-10A cell line. This immortalized breast
cancer cell line is incapable of forming tumors on its own,
meaning that scientists use this cell line to look at natural
agents that tumorigenic (Debnath, 2003).
The cell line derives from a fibrocystic disease isolated in 1984 from a 36 year old Caucasian
female. The cell line is also applied as a transfection host and is non-tumorigenic, meaning that
the cell line will not readily form tumors unless forced to with the addition of a chemical reagent.
This means that the cell line is important when the goal of the testing is researching potential
tumorigenic agents. The cell line is produced by long term culture in serum free medium with a
low Ca++ concentration. MCF-10A cells are derived from the adherent cells of the culture and
the other nonadherent cells are found in different cell lines available from the ATCC (i.e. MCF10F). The cells test positive for epithelial sialomucins, cytokeratins, and milk fat globule antigen.
The cell line shows no signs that it is terminally different or that it senesces. The stimuli that the
cells respond to are insulin, glucocorticoids, cholera enterotoxin, and epidermal growth factor
(EGF). EGF is common to both cell lines used in the testing. Electron microscopy reveals that
these cells display characteristics similar to those of ductal luminal cells and not of myoepithelial
cells. Reactions tested with monoclonal antibodies of MFA-Breast and MC-5 proteins revealed
an expression of breast specific antigens, which is a positive sign for scientists (“MCF-10A”,
n.d.). The non-tumorigenic nature of the cell line is important for this testing because the cell line
being compared to the basal type breast cancer line needs to be a non-cancerous line. This is
because it doesn’t form tumors or add any unnecessary proteins to the testing conditions.
Although this cell line is used for a variety of purposes, it is not normally used in comparisons
with other cell lines.
The primary usage of MCF-10A cell lines, along with similar immortalized cell lines, is
to look at different potential tumorigenic agents. These cell lines are considered immortal
because they can proliferate indefinitely under proper lab conditions without interruption from
other cellular properties. Usually the indefinite proliferation is caused by a mutation or the
introduction of some type of oncogene. MCF-10A cell lines have been previously used in
laboratory testing to look at such processes as mutagenesis, morphogenesis, and oncogenesis.
Linking Cataracts to Cancer 5
Mutagenesis is the origin and development of a mutation inside of a cell line and MCF-10A cells
aid in the study of this process by allowing the cell line to be cultured in culture media that
contains a frame shift-inducing agent. This biological agent mutates an aspect of the cell line
which the researchers can study as the cell line proliferates. Morphogenesis and oncogenesis are
commonly tested for together in laboratory testing, where morphological growth agents are
cultured into the cell line and then the cell line is exposed to oncogenes in order to disrupt the
growth process of these agents (Debnath, 2003). The MCF-10A cell line is extremely versatile
and can be used in a variety of ways.
Oncoproteins and Oncogenes
As discovered through decades of research, oncoproteins play a pivotal role not only in
causing a person to develop cancer, but also in helping doctors detect cancer. Oncoproteins are
comprised of oncogenes, which are genes that have the potential to cause cancer. Oncogenes are
often expressed at high or elevated levels in cancer cells or in tissue cells that have the potential
to develop tumors. The term oncogene was coined in 1969 by oncologists Robert Huebner and
George Todaro, but the first oncogene itself was not discovered until 1970 when the oncogene
src was concluded to be a part of chicken retrovirus. After post-experimentation confirmed the
conclusion, the discovery of both new oncogenes and proto-oncogenes (activated oncogenes)
lead to current research studies performed all over the world (Mukherjee, 2011).
Figure 4. Oncogenetic growth. Oncogenes form from normal cells that contain a
proto-oncogene that mutates in some way that causes the cells to undergo neoplastic
transformations that change them into cancerous cells (“Oncogene”, 2012).
Proto-oncogenes, the basic form of an oncogene, are commonly found in all types of cells in the
body and are proven to regulate the promotion and differentiation of the cells. There are multiple
proto-oncogenes found in the genomes of cells where they are involved in different steps of the
cell’s growth cycle. If there are any alterations in the gene sequence of the cell (caused by the
insertion and removal of a retrovirus) or there is an overexpression of a specific protein in that
cell, then the proto-oncogenes will cause the host cell to undergo neoplastic transformation
(Figure 4) and convert to carcinogenic cells with uncontrolled growth and proliferation. When
observing a normal gene found inside the human body, one is also looking at multiple protooncogenes in the DNA of that gene which could mutate at any time. All normal genes have this
mutational ability, and more and more retroviruses are causing mutations in the genomes of
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genes. This leads to neoplastic transformation and the end result of the development of a cancer
cell. There are five other mutations that lead to the transformation of the cells, they are point
mutations (hyperactive gene product and transcription proliferator), chromosomal translocation
(new location, gene fusion), and gene amplification (Chial, 2008). All of these potential
mutations cause the proto-oncogene to transform the host cell.
The expression levels of oncogenes and oncoproteins in cancer cells are always found to
be much higher than levels found in normal cells. As explained above, when the proto-oncogenes
are expressed at elevated levels, they mutate and form tumorigenic and carcinogenic cells. A low
expression level of the oncogene means there is no visible damage or harm done to the cells,
more so it means that the cell is completely normal and functioning properly (Chial, 2008). Any
mutation that leads to the transformation of a cell must first initiate with an abrupt
overexpression and activation of a specific oncogene which produces excessive amounts of the
coded oncoprotein. The overabundance of the oncoprotein will lead to the stimulation of the cells
that signal for the tissue cells to proliferate uncontrollably, this leads to tumor growth and
metastasis of the newly formed cancer cells. Without the overexpression of the oncogene there
would not be a transformation of the cell, and the proto-oncogenes would stay in a down
regulated state, meaning that there is little to no activity shown.
There are two states that a gene or a protein can be in at any given time when not at
normal levels: up-regulated and down-regulated. When a gene is up-regulated, there are either
external or internal stimuli present and they cause the gene to become expressed at a higher level
than normal. This occurs in a cell when it is deficient in some receptor, and receptor genes
become up-regulated, leading to the formation of more receptor proteins. These proteins then
regulate the sensitivity of the cell, and the gene returns to its normal level. The opposite of this
process is down-regulation where a gene is decreased in expression by a natural force, and its
corresponding protein expression is also decreased. If a cell is overstimulated with a certain
neurotransmitter or hormone for an elongated period of time, the receptor genes become downregulated (the corresponding protein expression would also decrease), and the cell stimulation
would return to normal levels. When an oncoprotein is down-regulated, there will be a lower
expression of that protein. This also means that the gene/protein combination is not as potent, or
it expresses the same power in controlling cellular processes as it would if it were at normal
levels. The hope for alpha basic crystallin is that the set experimentation parameters will provide
the ideal environment for up-regulation of the protein.
Alpha- Basic Crystallin
Recently, researchers have begun to express great interest in a small heat shock protein
called alpha-basic crystallin (αb- crystallin) because of it molecular chaperone like ability when
it is exposed to elevated levels of stress. Alpha-Basic crystallin is a member of the crystallin
protein family, which are structural proteins that are water soluble and are most commonly found
on the surface of the eye lens. These proteins are responsible for maintaining the transparency of
the lens and working on nerve regeneration in the eye lens due to injury. Crystallin proteins in
the eye lens also work to increase the refractive index of the eye lens while not hindering light
progression to the optic nerve. This allows for the individual to perceive objects with a clearer,
more defined picture. However, these proteins do not only exist in the eye lens and are referred
to as ubiquitous because of their ability to exist in different areas of the body. Areas outside of
the eye lens that commonly express crystallin proteins are the heart and the breast, but in the
Linking Cataracts to Cancer 7
breast the proteins are often associated with aggressive breast cancer tumors. Under normal
physiological conditions, these proteins look and act like normal structural proteins; however,
when the protein is exposed to elevated conditions of heat or stress, it begins to resemble
molecules called chaperone proteins.
Chaperone proteins prevent newly formed polypeptide chains or subunits from
aggregating in one specific area of the body. When chaperone proteins are denatured by either
elevated heat or cellular stresses, they lose the ability to prevent the aggregation of cellular
material. If denaturing occurs, then the chaperone proteins attract other soluble proteins and
cause them to aggregate and clump; this aggregation can lead to the development of oxidative
nuclear cataracts. Crystallin proteins are split into two primary groups, alpha crystallin and
beta/gamma crystallin. While both groups of proteins are ubiquitous in nature and are found in
almost all vertebrate organisms, they serve different purposes in the body. Alpha crystallin is
further broken up into two subgroups, acidic and basic. Both of the subgroups are found in large
aggregates, contribute to cataract formation, and are one of the major chaperone proteins.
Beta/Gamma crystallin is usually associated with several metabolic or regulatory factors in the
body and does not play a role in cataract formation in the eye.
Alpha crystallin, the more molecular chaperone like protein, is also classified as a heat
shock proteins, which is transcriptionally controlled and is up-regulated under conditions of heat
or stress. When the body receives a heat related stress signal, it undergoes heat shock response
which leads to an increased proliferation of those proteins. Each heat shock protein is named for
its molecular weight (in kDa or kilo-Daltons), and because the molecular weight of alpha
crystallin is approximately 20 kDa, it is referred to as Hsp-20. Heat shock proteins play
important roles in cellular maintenance where they can aid in protein folding and intracellular
transport (the transportation of other proteins across the cellular membrane). Different heat shock
proteins aid the body in different ways; for instance, certain proteins are important
phosphoproteins in the heart and aid in smooth muscle relaxation. Other heat shock proteins
work in the immune system by binding to specific antigens and help present them to the immune
system for removal or acceptance.
CRYAB, the oncogene associated with alpha-basic crystallin, is a member of the small
heat shock protein (sHsp or Hsp20) family. While alpha basic crystallin (CRYAB) and alpha
acidic crystallin (CRYAA) are both chaperone like and heat shock proteins, they are expressed
differently and are found in different areas of the body. CRYAB is more ubiquitous then
CRYAA, and is found throughout the body of any vertebrate. CRYAA, which is expressed in a
3:1 ratio with CRYAB, is limited to the function of the eye. In basal like breast cancer and
metaplastic tumors where there is an elevated level of stress placed on the cells, CRYAB levels
are shown to be an average of 83% higher when compared to the normal levels of the gene
(Sitterding, 2008). This confirmed the hypothesis that CRYAB is a small heat shock protein,
because the elevated cellular stress levels lead to up-regulation in the oncogene and subsequently
lead to the up-regulation of the alpha basic crystallin protein. When the gene is exposed to
elevated stress or heat levels, whether radiation induced or physiologically changed, it will
undergo both up-regulation and denaturation. These changes cause the protein to become
expressed at a much higher level than normal. The heightened denatured expression of CRYAB
leads to the aggregation of soluble proteins and eventually to the activation of proto-oncogenes,
which signals the transformation of the host cell into a cancer cell.
Linking Cataracts to Cancer 8
Figure 5. Alpha basic
crystallin. This small heat
shock protein could be the
potential link between
cataract formation and breast
cancer (“HSPb2”, 2009).
Another method for looking at CRYAB and alpha basic crystallin is through
phosphorylation, where the addition of aT covalently bonded phosphate group to an amino acid
residue location on the protein causes the protein kinase to either become active or inactive,
aract amino acid residues that phosphorylation is acts on
turning the protein on or off. There are three
in proteins: serine, tyrosine, and threonine. Phosphorylation at different amino acid residues
leads to different levels of up-regulation or down-regulation in proteins. The most common areas
for phosphorylation in CRYAB are on the serine residue points 45 or 59.This phosphorylation is
supposed to increase the expression of the alpha basic crystallin in lab tests, although some
experts disagree with that theory. Serine-59 phosphorylation of alpha basic crystallin is
associated with a down-regulation in the anti-apoptotic effects of the protein, meaning that it
would ameliorate the aggressive phenotype associated with the basal-like breast cancer tumors
(J. Moyano, personal interview, November 29, 2012). This phosphorylation has no associated
effects on the expression levels of the alpha basic crystallin in the breast cancer, those levels
remain the same regardless of the phosphorylation location.
Western Blot Assay
The need to figure out protein concentrations and expressions in cells has been so great in
recent years that Western Blot Assays (Westerns) have become a popular method of screening
for proteins. Western Blots, otherwise referred to as protein immunoblots, are a method of
screening for proteins in a sample of tissue or cells. Westerns use gel electrophoresis, PVDF
membranes, and antibodies to separate the proteins based on peptide length and then analyze
them using exposures. In order to run a Western, the first step is to lyse the cell lines/tissues with
a lysis buffer. The lysed cells are run through SDS-PAGE (sodium doddecylsulfatepolyacrylamide gel electrophoresis) using a polyacrylamide based gel. Then gel electrophoresis
is run on the SDS soaked (and negatively charged) cells. After the proteins in the lysed cells have
separated based on molecular weight (kDa), the gel membranes are then transferred to PVDF
(polyvinylidene difluoride) membranes through a process of either electroblotting or manual
transfer. Manual transfer uses filter paper, transfer buffers, and capillary action to transfer the
proteins from gel to membrane, but this method is too lengthy (close to two hours) for it to be
considered practical in most laboratory cases. Electroblotting is the use of a transfer machine (i.e.
iBlot Transfer System) to transfer the proteins from the gel to the PVDF membrane. This method
takes approximately seven minutes depending on the machine, and it is more commonly used in
laboratories compared to the manual method. Once the transfers are complete the protein blots
Linking Cataracts to Cancer 9
are blocked overnight in a solution of TBST (TRIS Buffered Saline + Tween) and diluted
antibody. After the overnight blocking is complete, the blots are rinsed with TBST and blocked
with a secondary antibody, which is typically conjugated to HRP (horseradish peroxidase). Two
more TBST rinses follow that, and then the blots are looked at with an exposure machine to see
if the proteins are expressed. The overall Western Assay process can take as few as three days or
as much as one week to complete.
Antibodies, otherwise known as immunoglobulin, are Y shaped proteins used in the
immune system to identify and neutralize any foreign objects that are present. These foreign
objects range from harmless bacteria to lethal diseases. The antibody detects the foreign object
from its antigen, a unique area that is specialized in every different bacteria or disease, and the
paratope of the antibody binds to the epitope of the antigen seamlessly. This causes the antigen
to be tagged for destruction by other cells in the immune system. In a Western Assay, the
antibody is used to bind to the antigen that it has a similar paratope. An alpha basic crystallin
antibody has a paratope that is specifically meant to bind with the epitope of the alpha basic
crystallin protein antigen. The binding between the antibody and the antigen means that the
protein becomes blocked into the blot while it rotates overnight in a cold room. Once the bound
protein is blocked into the blot, subsequent TBST washes can be performed without the worry of
the target protein washing off the blot. This explains when the blot is taken to a machine that
performs exposures, only the location of that specific protein shows up on the exposure. The
antibody is reacting to the chemical reagent looked for during the exposure cycle, and signals
that there are bound antigens of proteins at that definite spot on the protein ladder. From the
exposed site, scientists can conclude if the protein is actually expressed there. If the protein is upregulated or down-regulated, this means that the protein concentration shown by the antibodyantigen binding is either higher or lower respectively.
Gel electrophoresis is a key step in the success of a Western Blot Assay. In gel
electrophoresis, cell or tissue samples are lysed for proteins using a lysis buffer and the samples
are then loaded into polyacrylamide gel chambers which are submerged in a buffering agent and
electricity is used to force the proteins to run down the gel. The proteins, in running down the
gel, become separated by their molecular weight (in kilo-Daltons). Smaller proteins (like alpha
basic crystallin) will stop running down the gel sooner, while larger proteins will run much
further down the gel. The distance that the proteins run down the gel is proportional to the length
of time that the electricity is running. The separation of the proteins is critical for a successful
Western Blot Assay because if the proteins are not separated by any means, then there would be
no easy way to determine which protein is actually being expressed in the assay.
Rhodiola
Rhodiola is a genus of the family Crassaluceae, which is a family of hardy plants that all
store water in succulent leaves. The range for Crassaluceae extends from the northern extremes
of the northern hemisphere, to areas in southern Africa where water is scarce.
Linking Cataracts to Cancer 10
Figure 6. Rhodiola rosea plant. Rhodiola is only
found in high altitudes and in colder regions, and
its roots can be used for significant medicinal
purposes (“What is Rhodiola?”, 2009).
There are 33 genera in the family Crassaluceae, and most of these plants possess no real
agricultural significance. The more common uses for these plants are in either herbal medicine or
horticulture, where the primary usage for the plants is either as herbal supplements or garden
plants. The most common genera of Crassaluceae are Crassula ovata (jade tree), Rhodiola rosea
(Rhodiola), and Kalanchoe blossfeldia (Kalanchoe). Rhodiola are often similar in appearance to
the related genus Sedum, the stone crops. Often, many authors will merge Rhodiola with Sedum
into one genus under the name Sedum.
Rhodiola comprises as many as 200 different species, all of which grow in high altitude
regions of Asia or Europe. They are often referred to as stonecrops (Sedum) for their ability to
grow in even the smallest amount of soil. The most common species of Rhodiola that are used in
medicine are R. rosea, R. imbricata, R. crenulata, R. sacra, and R. kirilowii. These plants are the
most frequently found and grown species in the wild, and they possess the most desirable sets of
natural compounds that can be isolated and used in medicine today. Each species of Rhodiola has
its own unique set of phytochemicals that are used in different ways in a laboratory setting. The
way to synthesize the nutrients and compounds from the Rhodiola plant is to harvest the root of
the plant at the peak growth time (late fall) and then grind it up into a powder. The powder is
then suspended in either an aqueous or hydro alcoholic solution (typically of ethyl alcohol) and
then stored in a cool place for up to three months or until needed. Another method of working
with Rhodiola is to freeze dry the roots of the plant until they are needed to be ground up for use.
This allows for the water content of the roots to go down, and therefore they will have a higher
concentration of nutrients left over in the root. This method allows for optimal compound
synthesis in the lab. Ancient Asian cultures also used Rhodiola for medicinal tea; this method of
ingesting Rhodiola is not as nutrient rich, but is still commonly used today in Russia. The other
way to ingest Rhodiola is by taking capsules of powdered Rhodiola, which are available
commercially.
The medicinal qualities of Rhodiola are so numerous and plentiful, that it can be
classified as an adaptogen, which is a plant that possesses the innate ability to withstand
biological, physical, and physiological stressors. There are currently only seven known
Linking Cataracts to Cancer 11
adaptogens in the world which are all found in high mountainous regions in either Tibet or
China. Some examples of adaptogens are Panax ginseng (Panax quinquefolius), Siberian ginseng
(Eleutherococcus senticosus), and licorice (Glycyrrhiza glabra). Adaptogens are shown to not
only help the body fight stress, but also combat other problem areas in the body. Each one of
these abilities is controlled by specific compounds found inside of these plants. It is a
combination of multiple of these compounds that contribute to the anti-carcinogenic nameplate
that they bear. In Rhodiola, the phytochemicals rosin, salidroside, beta-sitorserol, gallic acid, and
kaempferol are the main compounds that have anti-carcinogenic implications. These five
compounds have also been shown to control the anti-anxiety, anti-viral, anti-fungal, and antistressing abilities of the plant. When a combination of these compounds are exposed to cancer
cells, multiple different smaller compounds work at the molecular level to perform tasks such as
preventing metastasis, and increasing radiation based death in the cancer cells.
The phytochemistry of Rhodiola has revealed that the successfulness of Rhodiola is
because of specific sets of compounds. All of the compounds have been grouped and classified
under six specific groups: phenylpropanoids, phenylethanol derivatives, monoterpenes,
triterpenes, flavanoids, and phenolic acids. Phenylpropanoids are organic compounds that are
synthesized from the amino acid phenylalanine. In Rhodiola, an example of a phenylpropanoid is
rosavin, which in itself is made up of rosin and rosarin. The presence of rosavin in an extract of
Rhodiola has been used to make the determination of whether that extract is genetically pure,
with 3% concentrations of rosavin being the standard for a naturally pure extract. Phenylethanol
derivatives, like salidroside and tyrosol, are (like the name suggests) derivatives of phenylethanol
or phenylethyl alcohol, which is an organic compound that is commonly found in the essential
oils of the plant. These derivatives have large antioxidant properties and are the most important
compounds for antioxidant therapeutic usage. Monoterpenes and triterpenes are both based on
terpenes. The difference between monoterpenes and triterpenes is that the former are made up of
two isoprene units in the chemical symbol C10H16, and the latter are made up of six isoprene
units in the chemical symbol C30H48. They are both naturally occurring organic extracts that are
synthesized from plants. Examples of monoterpenes in Rhodiola are rosiridol and rosaridin,
while examples of triterpenes are daucosterol and beta-sitosterol.
Flavanoids are ubiquitous in plants and are the most commonly found group of
polyphenolic compounds in the human diet. In Rhodiola, flavonoid examples include
kaempferol, rodiolin, and tricin. The primary role that flavonoids play in Rhodiola is to act as a
response modifier to certain allergens and carcinogens, meaning that they allow for the body to
respond in a different, sometimes better, way to a foreign object. Phenolic acids are the sixth
group of compounds commonly synthesized from Rhodiola. Gallic acid is the primary phenolic
acid synthesized from Rhodiola, and the primary function of it is to serve as an anti-fungal and
anti-viral compound. All six of those groups can be synthesized from Rhodiola by a means of
fractionation, where scientists take a primary compound, and separates it based on composition
of a certain gradient (Khanum, 2006).
Research Plan
Researchable Question:
What is the effect of Rhodiola crenulata root extract on the biosuppression of alpha-basiccrystallin, a novel oncoprotein that serves as a biomarker for predicting premature basal-like
breast cancer?
Linking Cataracts to Cancer 12
Hypothesis:
The protein alpha-basic-crystallin will signal premature basal-like breast cancer, and Rhodiola
crenulata root extract will suppress the expression of the protein.
Procedure: The cell lines used in the testing were received from the ATCC, cultured into new
tissue culture plates, and placed into an incubator. The cell lines were then lysed of their
proteins, and a BCA Protein Assay was run in order to determine relative protein concentrations.
Gel electrophoresis was then performed to separate the proteins, and the separated proteins were
transferred onto a PVDF membrane. The membrane was then blocked in an antibody, and the
membranes were coated in a Luminal bath. The membranes were then exposed in a standard
laboratory imager scanning for chemoluminescense. The exposures were then analyzed for the
protein content using band normalization and pixel density.
Methodology:
BCA Protein Assay
1.1)
1.2)
1.3)
In a laboratory setting, two frozen cell lines were received for testing, MDA-MB-231--(HTB-26, ATCC) and MCF-10A (CRL-10317, ATCC). The cell lines were thawed in a
Styrofoam box (12 in x 12in x 12in) filled completely with ice for one hour until the
provided culture media defrosted. Inside a standard tissue culture hood (Thermo
Scientific) the cell lines were then plated into eight sterile cell culture plates (clear
polystyrene, 60mm x 15mm, BD Falcon) for each cell line and were fed with 4mL of
primary growth media (Advanced DMEM, Gibco). The plated cell lines were then taken
to a cell culture incubator (150L, LEEC GA2000) and were exposed to 37 degrees
centigrade temperatures and 5% carbon dioxide levels for 3 days. Every day the cell
plates were removed from the incubator, placed into the same tissue culture hood, and fed
with 4mL of new growth media (Advanced DMEM, Gibco).
Every day prior to the testing of the cells, the cell lines were looked at under a
microscope (BA410 model) to ensure that the cell lines were still alive. Using a black
indelible marker (Sharpie, fine tip), the plates that contain the MCF10A cells were
labeled as follows (there were 2 of each label): 10A-IR-C (Irradiated Control cells), 10ANoIR-C (Non-irradiated Control cells), 10A-IR-Rh (Irradiated Rhodiola cells), and 10ANoIR-Rh (Non-irradiated Rhodiola cells). The process was repeated starting at step 1.2
using the MDA-MB-231--- cell line except on the labels, the 10A was replaced with 231.
A 10% ethanol solution was made by diluting ethanol (Fisher, 100%) with 1X Phosphate
Buffer Saline, otherwise known as PBS-1X (Cell Signaling Technology) at a dilution rate
of 1:10. A 1% solution of Rhodiola crenulata root extract was made by diluting Rhodiola
crenulata root extract (1μg/mL, Barrington Nutritionals) in the 10% ethanol solution so
that the final concentration of Rhodiola was 100μg/mL. In the same tissue culture hood,
plates with the “R” tag in the label received 20μL of the diluted Rhodiola extract, which
was pipetted onto each of the plates and then subsequently swirled and mixed by hand.
These specific plates were then moved from the tissue culture hood into the same cell
culture incubator (at the same temperature and carbon dioxide specifications) and were
incubated for fifteen hours. The plates that had the “C” tag in the label received 20μL of
Linking Cataracts to Cancer 13
1.4)
1.5)
the 10% ethanol solution, and were also swirled by hand. These cell plates were then
moved to the same cell culture incubator and were incubated for fifteen hours. After the
fifteen hours had elapsed, the plates that had the “IR” tag in the label were removed from
the incubator and were taken to a Cesium-137 irradiator (details withheld per FBI
measure) and were exposed to 1 Gray of radiation for 20 seconds which served as a
simulation of natural stress in the body. After the exposures, the plates were placed back
into the same incubator and incubated for an extra three hours. The plates with the
“NoIR” tag in the label remained in the incubator for the three hours.
After the three hours had elapsed, a clean, used Styrofoam box (12 in x 8 in x 8 in) was
filled entirely with shaved ice from a standard ice machine. The cell plates were removed
from the incubator and immediately placed on top of the ice (but not into the ice) so that
the cells would stay below room temperature throughout the duration of the step. The
growth media that remained on the cells was aspirated using a standard laboratory grade
vacuum aspirator (AMETEK). PBS-1X was then added in varying amounts (enough to
cover the bottom of the cell culture plate) to the plates. The PBS-1X was the swirled
using hands, and then subsequently aspirated using the same device as before. This step
was repeated twice to ensure that all of the loose cells were properly taken care of. Then
0.5 mL of a RIPA Buffer (cell lysing agent, Cell Signaling Technology, #9806) was
added to the cells using a standard micropipettor (200μL) and was left to coat the cells for
five minutes. After the five minutes had elapsed, a cell scraper (sterile, BD Falcon
353085) was used to scrape the bottom of the plate so that the cells lysed off of the plate
and into the lysing agent. The lysed cells were then transferred into separate sterile
microcentrifuge tubes (1.5mL, USA Scientific) kept slightly embedded in the ice using
the same micropipettor; the tip of it was changed after every complete transferal. The
sixteen microcentrifuge tubes were then brought into a temperature controlled four
degree centigrade room, and were placed into a standard microcentrifuge (sixteen tube
capacity, Sorvall Micro 17) which was set to 14,000 revolutions per minute for duration
ten minutes. After the ten minutes had elapsed, the tubes were placed into a standard
centrifuge tube holding rack and were placed into a -80o Celsius freezer (22 cubic feet,
U85-22 commercial model) until needed further on in the testing.
A BCA Protein Assay Kit (Pierce) was used for this part of the experiment. Solid Bovine
Serum Albumin, also known as BSA (sc-2323, Santa Cruz) was diluted into nine specific
standards in accordance to the standard test tube protocol instructions included with the
kit. In order to figure out the total volume of the working reagent needed for testing, the
equation WR= (Standards + Unknowns)*(Replicates)*(Volume of WR per sample) was
used. The working reagent was then created by adding 50 parts of Reagent A and one
part of Reagent B to a standard 50mL conical tube and then the tube was vortexed using a
vortex machine (Corning LSE) for approximately three seconds; the solution turned a
green color. Using a 100μL version of the same brand of micropipettor used before, 25μL
of all the standards and unknowns were pipetted into three separate wells (none were in
the same well) of an eight by twelve sterile well plate (BD Biosciences), the
micropipettor tips were changed after every transferal. Then, 200μL of the working
reagent was added into each of the wells and the wells were covered and mixed by hand
for thirty seconds. Each of the wells that contained either a standard or an unknown
mixed with the working reagent was subsequently given 1% TRIS Buffered Saline
+tween, referred to as TBST (Cell Signaling Technology, #9997) so that it was a 1:3
Linking Cataracts to Cancer 14
dilution of standard/working reagent to TBST. The well plate was then covered and
placed in the same incubator for 30 minutes. After the 30 minutes, the plate was removed
from the incubator and allowed to cool to room temperature, at which time the plate was
taken to a Microplate Reader (96 well reader, SpectraMax Plus384) and the absorbances
of the solutions on the plate were read at 562 nm. The quantitative values that were
produced by the machine were copied down onto paper using a standard black pen, and
the values were transferred into a Microsoft Excel file. The absorbance values for the
standards only were graphed versus the respective concentration of the standard. The
values of absorbance were graphed on the x axis, while the concentration (in μg/mL) was
graphed on the y axis. A linear trendline (otherwise known as the standard curve) was
then calculated using Excel software, and the equation and R2 values were shown on the
graph. The equation that was received was then used to determine the protein
concentrations of the unknowns. The average absorbance value for the unknowns was
given by the Microplate Reader, so for each of the unknowns, the absorbance value was
substituted into the equation, and the concentration results were acquired; these results
were multiplied by three to take into consideration the fact that the original unknowns
were diluted by a factor of three. These values were then converted from μg/mL to μg/μL
so that they would compatible with the stacking gel that was made in the next step. In
preparation for making the gels, a 10% ammonium Persulfate solution was made using
1mL of PBS-1X mixed with 100μg of solid ammonia (NH3).
Western Blot Assay
1.6)
1.7)
The stacking gel was made in a different (but same company as before) standard 50mL
conical tube using a recipe of 8.2mL of distilled water, 10.0mL of acrylamide (100%,
Fisher Scientific), 6.3mL of TRIS (1.0M, 8.8pH, Fisher Scientific), 0.25mL of the 10%
ammonium Persulfate solution made earlier, 0.25mL of Sodiumdoddecylsulfate (SDS,
10%, Fisher Scientific), and 0.01mL of N, N, N’, N’- Tetramethylethalinediamine
(TEMED, 100%, Thermo Scientific). The same standard 10mL serological pipette as
before was used for the liquid volumes above 1mL, except the tip of the pipette was
changed after each liquid was transferred. For liquid values less than 1mL, the same
micropipettor was used except the tips were exchanged for each liquid. Extreme caution
was taken when the acrylamide was used, because it is a neurotoxin. The solution was
vortexed for five seconds and then was taken to a gel chamber (Enduro Vertical PAGE
System) where the gel was transferred into the chamber using a standard plastic dropper.
The gel was transferred into the chamber until it reached a point that was approximately
one centimeter below where the teeth of the 1.5 mm gel combs (that went with the gel
chamber) would reach to. In order to prevent the stacking gel from drying out, distilled
water was carefully layered on top of the gel using a different standard dropper. Care was
taken to perform this step slowly so that the water did not combine with the recently
poured gel. Standard paper towels were then soaked with faucet water and rung out.
These paper towels were then laid over the top of the chamber, and Saran wrap (Stretchtite) was laid over the top of the paper towels. The gel was then left overnight so that it
could properly polymerize (this could be done in three hours minimum).
The standing gel was made in a similar 15mL conical tube using the recipe 5.5mL
distilled water, 1.3mL acrylamide (100%, Fisher Scientific), 1.0mL TRIS (1.0M, 6.8pH,
Linking Cataracts to Cancer 15
Fisher Scientific), 0.08mL Sodiumdoddecylsulfate (SDS, 100%, Fisher Scientific),
0.08mL ammonium
Persulfate, and
0.008mL of
N,
N, N’, N’Tetramethylethalinediamine (TEMED, 100%, Thermo Scientific). The same standard
10mL serological pipette as before was used for the transferal of liquid volumes above
1mL, except the tip of the pipette was changed after each liquid was transferred. For
liquid values less than 1mL, the same micropipettor was used except the tips were
exchanged for each liquid. Extreme caution was taken when the acrylamide was used,
because it is a neurotoxin. The saran wrap and paper towels that covered the gel chamber
were removed and the gel chamber was rinsed with distilled water from a standard squirt
bottle so that any excess acrylamide washed away. Any remaining moisture left in the
chamber was then wicked out using the edge of a paper towel. The standing gel was then
poured on top of the separating gel until the level of gel reached the top of the chamber.
Two 1.5 mm gel combs were then inserted into the standing gel in set spots on the
chamber. The gel was left alone to polymerize for two hours, afterwards the gel combs
were removed.
1.8) The cells that were lysed at the end of section 1.4 were removed from the -80o centigrade
freezer and were immediately placed into a Styrofoam bucket filled with shaved ice.
Once the cells had warmed up to just below room temperature, micropipettors were used
to transfer ¼ parts of the cells to new microcentrifuge tubes. The amount varied from cell
line to cell line because of the calculations that were done based off of the equation
results concluded in section 1.5. Once the cells were transferred, the new tubes were
labeled appropriately with the same labels as before. 2μL of dye (4X, purple, Thermo
Scientific) was transferred into each of the tubes using a micropipettor. Two tubes that
would serve as markers for the rest of the tubes were made using 5μL of marking agent
(New England Bio Labs) and 2μL of the 4X dye in each.
1.9) The microcentrifuge tubes were brought over to the gel chamber, which had two sub
chambers with ten loading chambers in each sub chamber. Each sub chamber was set up
in the order of (from left to right): Marker, Blank Chamber, 231CN, 231CI, 231RN,
231RI, 10ACN, 10ACI, 10ARN, and 10ARI. The tag “C” meant it was a control, “R”
meant Rhodiola, “I” meant irradiation, “N” meant no irradiation, “231” meant MDAMB-231 cell line, and “10A” meant MCF-10A cell line. The respective proteins were
transferred from their tubes to their corresponding chambers using micropipettors, where
the tips were exchanged after every protein was transferred. The gel chambers were
placed into a larger gel electrophoresis chamber (Enduro Vertical PAGE System), which
was then filled completely with an electrophoresis buffering agent (10X, Sigma-Aldrich,
B6185) The top was then placed onto the chamber, and the anode/cathode wires were
attached from the top of the chamber to an electrical box. The electrical box was then set
to 100V and it was left to run for 90 minutes. The chamber worked because of the
presence of bubbles in the buffering agent.
1.10) Once the 90 minutes of run time had elapsed, the electricity was turned off and the
electrophoresis chamber was opened up. The gel chambers were removed from the
electrophoresis chamber and the combination of the two gels were removed from the gel
chambers. Using a standard sterilized Exacto knife, the standing gel (the gel found on top
of the stacking gel, there was a definitive line between the two gels) was cut away and the
stacking gel was slid off of the gel chamber. The electrophoresis worked if the proteins,
more specifically the markers, have visibly run down the gel. Two separate standard
Linking Cataracts to Cancer 16
containers (the tops to standard micropipettor tip boxes) were filled with enough distilled
water to coat the bottom of it, and one gel was submerged into either container. The
electrophoresis chamber and all other objects that were associated with the
electrophoresis process were rinsed with distilled water and left to dry on a drying rack
until they were needed to be used again in the next section (1.11).
1.11) The diluted transfer buffer was made in a 500mL standard graduated cylinder using a
recipe of seven parts distilled water, two parts methanol, and one part Transfer Buffer
(20X, Life Technologies). Proper safety equipment was an absolute necessity for this step
because methanol is a caustic chemical and can be harmful to skin. The diluted transfer
buffer was then poured into the rinsed and dried electrophoresis chamber, and the
chamber was set aside for later use. On the lab table next to the electrophoresis chamber,
eight pieces of standard filter paper (2 in by 4 in) and two PVDF membranes (2 in by 4
in, WestTran) were wet with distilled water, and were set aside until the next step. Then,
a gel holder cassette (supplied with the electrophoresis chamber) was opened and was
layered with a foam sponge, two pieces of wet filter paper, one PVDF membrane, one of
the pieces of gel that has the proteins on it, two more pieces of wet filter paper, another
foam sponge, and then the cassette was closed and sealed. The loaded cassette was then
placed into the electrophoresis chamber. The second gel holder cassette was then loaded,
using the same methodology as the first one, with the other piece of gel. Once this
cassette was loaded, it was also placed into the electrophoresis chamber. The
electrophoresis chamber was then placed on top of a standard hot plate (Yellowline MAG
HS 7, Laboratory Analysis Unlimited) that had a magnet underneath it. A magnetic
stirring rod (40mm by 8mm, SPI Supplies, 01405-AF) was then placed into the
electrophoresis chamber, and the hot plate was turned on so that the magnetic stirrer spun
but the hot plate did not produce any heat. The cover was then placed onto the chamber,
and the anode/cathode wires were connected to the electrical box. The box was then set to
80V of electricity for duration of one hour. After the hour had elapsed, the electricity and
magnetic stirrer were both turned off, and the chamber was removed from the hot plate.
The top of the chamber was removed and both of the gel holder cassettes were removed
from it and were opened up. The transferal of the proteins was successful if the proteins
have visibly transferred from the gel to the PVDF membrane. The two membranes were
then placed into separate containers filled with a blocking buffer (5% BSA TBST) and
were allowed to block for 30 minutes at room temperature.
1.12) Two 50mL conical tubes were set up by filling them both with 25mL of a buffering agent
(BSA Blocking Buffer, Thermo Scientific). The alpha- basic crystallin antibody (Santa
Cruz Biotechnology) was then added to the solution with a micropipettor so that the
antibody was at a dilution rating of 1 part antibody to 400 parts buffering agent. Each of
the conical tubes were then vortexed for five seconds each, and were placed into a tube
holding rack until they were needed later on. The two PVDF membranes that held the
transferred proteins were transferred to separate conical tubes (that were prepared in the
last step) using standard metal tweezers. The conical tubes were then capped, and then
were taken to a temperature controlled 4o centigrade room, where they were placed
horizontally onto a rotating machine (360 degree rotation, PTR-35, Grant Industries) that
kept them moving constantly. The conical tubes were left overnight to rotate in the room.
1.13) The next morning, the tubes were removed from the rotating machine and removed from
the temperature controlled room. * Two containers (same two containers used before in
Linking Cataracts to Cancer 17
section 1.10) were filled with lab made 1% TRIS Buffered Saline (TBST), but only
enough was used just to coat the bottom of the containers. The membranes with the
proteins were removed from the conical tubes using standard metal tweezers and were
placed into separate containers of TBST. The containers were then covered with a top,
and then were taken to a rocking machine (Labnet ProBlot 25 XL, Spectra Services)
where they were placed on the top of the machine, and then rocked at a setting of four for
ten minutes. After the ten minutes elapsed, the TBST in the containers was poured out
into the sink (the membranes were not removed) and new TBST was poured onto the
membranes in the container. The containers were then rocked (at the same setting) for
another ten minutes, where the process was repeated once more. This means that the
TBST rinse and rock was done a total of three times. After the third TBST wash, the
containers were filled with exactly 15mL of TBST, and an anti-rabbit antibody
conjugated to HRP (Santa Cruz Biotechnology) was added with a micropipettor so that
the dilution rate was 1 part antibody to 3000 parts TBST. Both of the containers were
then placed onto the rocking machine for 30 minutes at a setting of 2. This was done to
provide gentle motion and to expose the antibody to all parts of the membrane. After the
30 minutes of rocking had finished, the TBST antibody mixture was poured off into the
sink, and the TBST rinse process repeated itself for another three times. After the TBST
rinses were finished, the protein blots (membranes) were rinsed with 1X PBS in order to
remove any remaining bits of extra antibody or residue that might become a source of
error. In two separate standard plastic weighing trays, equal parts of Luminal Reagents A
and B (Santa Cruz Biotechnology) were transferred onto them using a serological pipette.
The amount of the reagent is negligible, just be sure to cover the bottom of the weighing
tray. Each of the weighing trays had one of the protein blots placed into them and the
trays were placed onto the rocking machine at setting 3, where they were rocked for two
minutes. While the proteins were being rocked, a piece of plastic wrap (Saran Wrap) was
placed flat on the top of the lab bench. Once the protein blots were done rocking, the
blots were moved from the weighing trays to the plastic wrap, where they were stacked
from bottom (length wise) to top facing in the same direction as each other. The plastic
wrap was then folded over the tops of the protein blots. The blots were then taken to an
exposure machine (GBox Scanning Machine), and the machine was set up so that there
was a plain white board underneath the blots, and it was scanning for chemoluminescense
caused by Luminal. The focus and lighting were corrected so that the exposures would
look the best. The exposure times were set for 30 seconds, 1 minute, and 10 minutes.
After that, the machine was left to run for the allotted amount of time specified by the
exposure times. Once the exposures complete, make sure to save the exposure files to a
safe place, and print out copies of each exposure using a printer. These images that were
received are where the results were based from, but another test had to be run using an
actin antibody to help determine the location of the protein that was just exposed.
1.14) The protein blots were taken out of the plastic wrap and were transferred using tweezers
to separate containers that were filled with the same type of buffering agent as used
before. The only difference here was that the antibody used was an actin antibody diluted
1:400. The containers were placed onto the rocking machine at a setting of 4 where they
rocked for a total of one hour. After 30 minutes, the blots were flipped over. Then the
exact same methodology was used starting at the asterisk (*) found in the beginning of
section 1.13. After the actin antibody exposures were completed, the exposures were
Linking Cataracts to Cancer 18
compared to each other, and a protein ladder was used to determine the exact location (in
kilo Daltons) of the protein along with the activity under certain conditions.
Results
Western assays showed that the concentration levels of the alpha-basic crystallin protein
increased in the MDA-MB-231--- cell line when compared to the MCF-10A cell line. The assay
also showed that the Rhodiola crenulata root extract had a negative effect on the alpha-basic
crystallin concentration levels, which is a positive result. When the Rhodiola crenulata root
extract was added to the cells, the concentration level of the protein decreased regardless of the
cell line. While the greater portion of the testing produced qualitative results in the form of
exposure pictures, quantitative results were extracted from the exposures using a process called
band normalization through pixel density. The data was arranged into tables separated by the two
different blots, even though later the data would be appended into a final full data table. Each
table included the initial pixel density value for the CRYAB target protein, the pixel density
value for the Actin protein, the ratio of the proteins, and the normalized CRYAB concentration.
The final data table included the addition of standard deviation values along with 95%
confidence interval values. Data tables from a preliminary assay performed early on in the
experimentation cycle is also included. The BCA Protein Assay was used in the experimental
process as a method of determining the amount of gel to make in order to perform successful gel
electrophoresis. The results of that assay were completely separate from the actual results of the
experiment, but they were pertinent for ensuring successful separation of proteins.
Data Tables
Table 1. Nomenclature of Tags used to define the different cell culture plates
used in the testing.
CN231
CI231
RN231
RI231
CN10A
CI10A
RN10A
RI10A
Ethanol
Yes
Yes
No
No
Yes
Yes
No
No
Rhodiola
crenulata
No
No
Yes
Yes
No
No
Yes
Yes
Irradiation
No
Yes
No
Yes
No
Yes
No
Yes
No
Irradiation
Yes
No
Yes
No
Yes
No
Yes
No
Linking Cataracts to Cancer 19
BCA Protein Assay
Table 2. BCA Protein Assay Standard Curve
Values used in the Figure 7 graph.
Concentration
2000
1500
1000
750
500
250
125
25
0
Absorbance
2.038
1.764
1.119
0.930
0.618
0.363
0.245
0.145
0.112
Protein Concentration
Concentration (mcg/mL)
2500
y = 987x - 120.96
R² = 0.9926
2000
1500
1000
500
0
0
0.5
1
1.5
2
Absorbance (at 542nm)
Figure 7. Protein Concentration as a function of Absorbance in
the BCA Protein Assay. This is the graph of the standard curve of
the concentration data, the equation shown in the graph is the
equation of best fit to determine the assay results.
2.5
Linking Cataracts to Cancer 20
Table 3: BCA Protein Assay Results that were used to determine the proper amount of Standing and Stacking Gel to
make for Gel Electrophoresis.
CN231
CI231
RN231
RI231
CN10A
CI10A
RN10A
RI10A
Concentration
603.00
486.54
465.81
372.05
378.96
508.75
345.40
496.90
3x Concentration
1809.01
1459.62
1397.43
1116.14
1136.87
1526.24
1036.19
1490.71
Average
0.734
0.616
0.595
0.500
0.507
0.638
0.473
0.626
Point 1
0.876
0.683
0.826
0.560
0.464
0.617
0.457
0.488
Point 2
0.591
0.548
0.363
0.439
0.549
0.659
0.488
0.764
Western Blot Assay
First Blot
Table 4. Blot One results with normalized CRYAB concentration values on
the right.
Lane
231-C-N
231-C-I
231-Rh-N
231-Rh-I
10A-C-N
10A-C-I
10A-Rh-N
10A-Rh-I
Average
CRYAB
69403
122763
74812
138910
46973
23507
52993
71803
75146
Actin
321169
520209
424323
447844
432316
338424
558339
400279
Ratio
0.216
0.236
0.176
0.310
0.109
0.069
0.095
0.179
Concentration
16239
17733
13249
23308
8165
5220
7132
13480
Ratio
0.017
0.021
0.021
0.027
0.026
0.020
0.029
0.020
30μg
16.584
20.553
21.468
26.878
26.388
19.656
28.952
20.125
Linking Cataracts to Cancer 21
Protein Normalization Ratios using Actin
0.5
0.4
0.3
0.2
0.1
0.0
231-C-N
231-C-I
231-Rh-N 231-Rh-I 10A-C-N 10A-C-I
Gel Membrane Lane Label
10A-Rh-N 10A-Rh-I
Figure 8. Protein Ratio values for Band
Normalization. These are the ratios of the
CRYAB concentration to the Actin
concentration; these values were used to
normalize the CRYAB values.
Alpha-Basic Crystallin Protein
Concentrations
35000
Protein Concentration (mg/mL)
CRYAB/Actin Ratio
0.6
30000
25000
20000
15000
10000
5000
0
231-C-N
231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I
Gel Lane on Membrane
Figure
9.
Protein
Concentrations
post
normalization for Blot One. Using the
CRYAB/Actin ratios, the average Blot One
CRYAB concentration was taken and normalized
to the values on the graph.
Linking Cataracts to Cancer 22
Second Blot
Table 4. Blot Two results with normalized CRYAB concentration values on
the right.
Lane
231-C-N
231-C-I
231-Rh-N
231-Rh-I
10A-C-N
10A-C-I
10A-Rh-N
10A-Rh-I
Average
CRYAB
120044
167168
73301
92564
21659
6465
19270
25588
65757
Actin
353653
344904
326762
363965
372278
274146
463582
376760
Ratio
0.339
0.485
0.224
0.254
0.058
0.024
0.042
0.068
Concentration
22321
31871
14751
16723
3826
1551
2733
4466
Protein Normalization Ratios using Actin
0.600
CRYAB/Actin Ratio
0.500
0.400
0.300
0.200
0.100
0.000
231-C-N
231-C-I
231-Rh-N 231-Rh-I 10A-C-N 10A-C-I
Gel Membrane Lane Label
Figure 10. Protein Ratios used for Band
Normalization in Blot Two. These are the
normalized ratios for the CRYAB in the
second blot.
10A-Rh-N 10A-Rh-I
Linking Cataracts to Cancer 23
Protein Concentration (mg/mL)
Alpha-Basic Crystallin Protein
Concentrations
35000
30000
25000
20000
15000
10000
5000
0
231-C-N
231-C-I
231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I
Gel Lane on Membrane
Figure 11: Protein Concentrations.
These are the final CRYAB
concentrations post normalization for
Blot 2.
Combined Blots
Table 5: CRYAB concentration results for both blots with standard deviation and 95% confidence values.
Lane
231-C-N
231-C-I
231-Rh-N
231-Rh-I
10A-C-N
10A-C-I
10A-Rh-N
10A-Rh-I
Concentration
(Blot 1)
16239
17733
13249
23308
8165
5220
7132
13480
Concentration
(Blot 2)
22321
31871
14751
16723
3826
1551
2733
4466
Average
Concentration
19280
24802
14000
20016
5995
3385
4933
8973
Standard
Deviation
4301
9997
1062
4656
3068
2594
3110
6374
95% Confidence
Interval
5960
13855
1472
6453
4252
3595
4311
8833
Linking Cataracts to Cancer 24
Protein Normalization Ratios using Actin
0.6
0.4
0.3
0.2
0.1
0.0
231-C-N
231-C-I
231-Rh-N 231-Rh-I 10A-C-N 10A-C-I
Gel Membrane Lane Label
10A-Rh-N 10A-Rh-I
Figure 12. Protein Ratios for Normalization
between both blots. These are the normalized ratios
for the CRYAB/Actin in both blots.
Alpha-Basic Crystallin Protein
Concentrations
35000
Protein Concentration (mg/mL)
CRYAB/Actin Ratio
0.5
30000
25000
20000
15000
10000
5000
0
231-C-N
231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I
Gel Lane on Membrane
Figure 13. Protein Concentrations for both of the blots.
These are the final CRYAB concentrations post band
normalization for both blots are shown on the same graph.
Linking Cataracts to Cancer 25
Average CRYAB Concentration
40000
CRYAB Concentration
35000
30000
25000
20000
15000
10000
5000
0
231-C-N
231-C-I
231-Rh-N 231-Rh-I
10A-C-N
10A-C-I
10A-Rh-N 10A-Rh-I
Variable Tested
Figure 14. Final average protein concentrations
for both blots. This graph includes the average
CRYAB concentration for both blots with error
bars.
% Change in Protein Concentration
Table 6. Percent Change Tables for the comparison of both cell lines.
Average Concentration
19280
24802
14000
20016
5995
3385
4933
8973
Test Variable
C-N
C-I
Rh-N
Rh-I
Average
% Change from 10A to 231
321.58
732.68
283.81
223.07
390.29
Linking Cataracts to Cancer 26
% Change in CRYAB concentration
% Change in CRYAB concentration from MCF10A to MDA-MB-231 cells
800
700
600
500
400
300
200
100
0
C-N
C-I
Rh-N
Rh-I
Variable Tested
Figure 15. Percent Change graph for both
blots. This graph shows the difference in the
CRYAB protein concentrations between the
10A and 231 cells.
Data Analysis and Discussion
The overall trend of the data on the protein suggests that alpha-basic crystallin
concentration levels are upregulated in the presence of basal-like breast cancer cells. In normal
non-cancerous cells, the concentration of the alpha-basic crystallin has a pixel density of
approximately 5,800 pixels/mm3. In the presence of breast cancer, more specifically basal-like
breast cancer, the pixel density of the concentration of alpha-basic crystallin nearly quadruples to
a value of approximately 19,500 pixels/mm3. This suggests that the protein plays a crucial role in
the development of the cancer cells through its molecular chaperone like ability under the
stressful conditions posed by cancer. When the protein was exposed to elevated conditions of
stress through a radiation model, the data showed that the protein became upregulated and
therefore more active in the model. This reaffirms the prior knowledge that this protein has
behavior similar to that of a small heat shock protein. The addition of Rhodiola crenulata root
extract had a small effect on the data, suggesting that it may have minute clinical implications as
an anti-cancer agent. The presence of the alpha-basic crystallin protein most definitely has
implications as a potential cancer biomarker, especially because of the 390% change in
concentration values between the 231 cells and the 10A cells. As shown in Figure 15 below, the
exposure of the blots clearly show that the protein appears visibly in just the lanes that contain
the MDA-MB-231 cells, and not those that contain the MCF-10A cells. Not only has this protein
produced visual results suggesting that it is active in only cancer cells, but also it has
quantitatively backed up the visuals with data that shows the protein is much more present in the
Linking Cataracts to Cancer 27
breast cancer when compared to normal cells. Statistical analysis revealed that the null
hypothesis in two of the three cases could be rejected, which means that the concentration values
from the MDA-MB-231 cells are not statistically the same as the values of the MCF-10A cells,
meaning that they have an F value that that is greater than the F-critical value provided in the
Factorial ANOVA performed on the data. The addition of Rhodiola crenulata showed statistical
similarities, and the two blots were not statistically different.
Figure 15. Exposure photo looking at the alpha basic crystallin
concentrations. This is the final exposure photograph of the proteins. The 4
lanes on the far right are the 231 cells, where the 4 lanes on the left are 10A
cells.
Conclusions
The data that resulted from experimentation supported the hypothesis presented, but it did
not fully confirm it. The target oncoprotein, alpha-basic crystallin, was proved to be at elevated
concentration levels in MDA-MB-231 breast cancer cells when compared to the immortalized
MCF-10A cells. The addition of a stress model also succeeded in increasing the concentration
level of the protein. Additionally, the results of the western blot assay also proved that the
Rhodiola crenulata root extract had a slight preventative effect on alpha-basic crystallin
concentrations. However, unlike the results of the protein levels in different cell lines, the
Rhodiola induced cells did not undergo a great enough change in concentration level to be
considered significant by any medical means. The significance of the difference in alpha-basic
Linking Cataracts to Cancer 28
crystallin concentration levels just between the MCF-10A and MDA-MB-231 cells is something
of note to the medical community. Alpha-basic crystallin levels are a viable option to look at in
blood testing or urine testing to determine the presence of or stage of breast cancer. Although
there were only two data points taken for each testing condition, the trend of the data shows that
high levels of alpha-basic crystallin are associated with breast cancer, especially of the basal-like
nomenclature. This study is only at the pre-clinical level, but with access to different cell lines
and actual clinical patients, this study can be taken in vivo with live testing. Overall, the data
concludes that the protein alpha-basic crystallin and its relative oncogene CRYAB possess the
high potential for use in the prediction of basal-like breast cancer, a discovery that could
potentially save the lives of thousands.
Limitations and Assumptions
There were some significant limitations that were present in the project including time,
location, money, and resources. When it came to working in a lab with cell lines and numerous
different chemicals, the experimenter was limited to only the resources that the lab provided for
him to use in the testing. The cell lines and antibodies used in the experimentation cost
approximately $300-500 each, which meant that the testing was limited to only the materials
available without funding. The location of the labs also limited the amount of time spent working
in the labs, and the access to that place. The lab used for the testing is located 90 minutes away
from home and in order to test on consecutive days, the experimenter had to stay at the home of
the Qualified Scientist. This was normal for the experimenter because the QS is his aunt, and he
has stayed at her house before. These constraints really limited the scope of the project because
without these constraints multiple cell lines and proteins would have been examined instead of
just two cell lines and one protein.
The controls for testing included ambient air temperature, protein assay kit, incubator
temperature, radiation type/duration, freezer temperature, and sterilization technique in both the
flow hood and at the lab bench. The ambient air temperature in the lab setting was kept
consistent at 22˚ Celsius for the entire duration of the testing, while the incubator temperature
was kept at 37˚ Celsius at 5% CO2 concentration when the cells were incubating. In order to keep
the proteins from denaturing, it was necessary to use ice to bring down the temperature, and to
use a -80˚ Celsius freezer for extended storage. The BCA Protein Assay kit was kept constant
during the testing, as well as the radiation treatments for the cell lines that needed to receive
radiation. The radiation was 1 Gray of exposure for duration of 19.6 seconds, and a Cesium-137
ionizing irradiator was used for the testing. The control group for the testing was an
immortalized breast cell line referred to as MCF-10A. This cell line serves as the closest in vitro
simulation for regular breast cells that behave the way that was needed in a laboratory setting.
The control group used for the data analysis is the actin protein. This protein was used during the
quantification of the data because it is a ubiquitous protein and is accurately used to find ratios in
band normalization.
The entire project was based on the primary assumption supported by many smaller
suppositions. That primary belief was that the alpha-basic crystallin protein existed in the cell
lines that were tested. If this were not the case, then the project would have failed and ultimately
produced inconclusive results. It was assumed that the laboratory setting where the tests took
place was sterile and that proper laboratory sterilization technique was followed to ensure that
the data was not corrupted by outside sources. All of the machines that were used in the testing
Linking Cataracts to Cancer 29
were assumed to function properly and not produce rogue results. This includes the
hemocytometer, gel electrophoresis chamber, iBlot transfer machine, GBox exposure machine,
irradiator, freezer, incubator, and centrifuge. It was also assumed that if the machines did not
function properly, that a manual option was available to use and that it was just as effective as
the electronic method. This assumption was used when the electronic transfer machine broke,
and a manual method was used. It was assumed that throughout the course of the testing, that the
proteins were kept at ideal conditions and did not denature during testing. Denaturation of
proteins would have led to false results for the project. It was assumed that the choice of cell
lines and stress model were the most accurate to simulate actual in vivo situations. An
immortalized cell line was assumed to provide an accurate lab model of normal breast tissue as
compared to any other cell line, and a radiation based stress model was assumed to be more
accurate than a heat model. Finally, it was assumed that these results can accurately be
extrapolated when similar testing is performed on the same cell lines.
Sources of error were small because testing was performed in a laboratory setting under
flow hoods and incubators while using sterile technique. However, besides the human error that
is unavoidable in testing, other sources of error could include lapses in sterile technique leading
to outside contamination, uneven loading of the gel chambers leading to faulty plate readings,
and cutting short the amount of time the gel electrophoresis chamber was allowed to run. All of
these sources of error lead to minor alterations in the average expressions of the protein’s pixel
density, and ultimately to changes in the overall expression of the protein when compared to the
control group.
Applications and Future Experiments
Research is continually performed in the realm of oncology or cancer biology, with the
goal of it being to discover methods that could either prevent or treat cancer in people. While the
holy grail of all discoveries would be a cure for cancer, focusing in on early detection can be just
as effective in preventing fatalities from the disease all together. This is where looking at
proteins and protein signaling pathways plays a major role in science. The type of research that is
being performed in this project has a high potential for possessing positive implications in cancer
early detection and prevention.
While being largely attributed to cataract formation in the eye lens, small heat shock
proteins such as alpha-basic crystallin, with their ability to aggregate other proteins and then
denature them, are a novel target for cancer research. There are currently only a few (less than
10) published papers out about the implications of alpha-basic crystallin as an oncoprotein linked
to breast cancer and of the ten papers, only one of those papers pertains to basal like breast
cancer. That one paper looked at uses the same control cell line, but the focus of the paper is
completely off from the focus of the testing for this project. This means that the conclusions
drawn from this project are completely novel and can be used in studies conducted after this
testing. Alpha-basic crystallin levels can be found using ELISA Assays, and the specific
upregulated level that causes the neoplastic transformation of the cell can be determined. Once
this level is determined, it can be used in everyday oncological medicine as a biomarker for
premature/developing breast cancer.
Future extensions for this project include looking at other cell lines for alpha-basic
crystallin concentrations, using different models to simulate stress in vitro, using different testing
Linking Cataracts to Cancer 30
methods (for example, immunohistochemistry) to look at different aspects of the protein, and
performing in vivo studies in mice. Western Assays are the most basic type of test used to
determine the presence of a protein in a cell line, and there are more time consuming and
accurate testing methods that could be administered to test for the presence. Also, similar small
heat shock proteins can be scanned for to determine if the entire Hsp family has oncological
implications. Many other proteins can be discovered by using a genome-wide cDNA microarray,
and then those proteins can be looked at further. Anyway it is looked at, oncoproteins are
important molecules and should be taken seriously, because one day they could save millions of
peoples’ lives.
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Acknowledgements
The author wishes to thank many mentors for their help during the project. First, he
would like to thank all of the people at Pioneer Valley Life Sciences Institute in Springfield
Massachusetts for providing moral support and help when it was needed, and for their
generous donation of laboratory space and all of the cell lines, reagents, and materials that
were used in the project. He would also like to thank Dr. Sallie Smith Schneider for working
with the author throughout the course of the project, providing guidance and help when
necessary, and for allowing the author to stay at her house during the testing period. He
would like to thank his parents for their ongoing support of the topic, and their willingness to
provide transportation to and from school and the lab. Finally, he would like to thank Dr.
Judith Sumner and Mrs. Shari Weaver for their weekly help with the writing and paperwork
for the project.