University of Massachusetts Amherst

Up-regulating Apoptosis in Cancerous Cells using the Bcl-2 Family of Proteins and Apoptosis

Biology 499T/P Honors Thesis

Natalie L. Saloio5/3/2016

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ABSTRACT

The Bcl-2 family of proteins plays a crucial role in negatively and positively regulating

apoptosis. Cancerous phenotypes create a cellular environment where apoptosis is negatively

regulated, which makes manipulations of the Bcl-2 family of proteins an appropriate method for

cancer therapy. This paper addresses potential manipulations which can be done to the pro-

apoptotic and anti-apoptotic Bcl-2 family of proteins which will increase the level of intrinsic

apoptosis in tumor cells, and act as cancer therapies. Scientists, through their literature, have

reviewed drugs which mimic the actions of the Bcl-2 family of proteins to either increase or

decrease levels of intrinsic apoptosis in cells and many therapies have been produced which

trigger the extrinsic pathway of apoptosis, but none of the processes involved manipulating the

actual Bcl-2 family of proteins and very few involve triggering the intrinsic apoptotic pathway.

This thesis proposes increasing expression of the Bcl-2 BH3-only protein, and inhibiting the BH4

domain of anti-apoptotic Bcl-2 proteins using recombinant protein expression, small molecule

inhibitors, and gene therapy. These therapeutic methods adhere to targeted cancer therapy

which allows consumption of higher doses without damaging the rest of the patient’s cells by

directing the drug to target only tumor cells, making it a more effective drug with a higher

efficacy and less side effects

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INTRODUCTION

Cellular Mechanisms of Apoptosis

Cell death is an existential component of living organisms. When our cells absorb too

many ultraviolet rays, radiation, toxins, and free radicals, DNA damage occurs, and our cells use

DNA repair mechanisms to mend the damage. If the damage is not repaired, the cell will be

signaled to self-destruct through pathways of programmed cell death, such as apoptosis. If the

cell’s pathways of programmed cell death are inhibited, damaged cells may accrue mutations that

lead to transformation of the cell, which will lead to tumor formation, possibly cancer, and

eventually, death of the organism.

Cell death can be regulated and accidental (Kroemer et al. 2009). Oncosis is an accidental

type of cell death, characterized by swelling of the nucleus and cytoplasm after a lethal injury

(Kroemer et al. 2009). Programmed cell death is a form of regulated cell death that is mediated

by an intracellular process (Kroemer et al. 2009). There are many forms of programmed cell

death, such as apoptosis, autophagic cell death, necroptosis, and pryoptosis, classified by their

morphological appearance, enzymological criteria, functional aspects, or immunological

characteristics (Kroemer et al. 2009). Apoptosis is a very common process of programmed cell

death that occurs in multicellular organisms (Kroemer et al. 2009).

Apoptosis is characterized by its specific morphological features, responsible for

rounding-up of the cell, retraction of pseudopodes, reduction of cellular and nuclear volume,

nuclear fragmentation, minor modification of cytoplasmic organelles, plasma membrane

blebbing, and engulfment by resident phagocytes (Kroemer et al. 2009). The three important

biochemical features of apoptosis include protein cleavage, DNA breakdown, and phagocytic

recognition. Apoptosis is a necessary function of cells throughout development and aging of

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organisms as a balancing mechanism to maintain cell populations in tissues (Ouyang et al. 2012).

For example, apoptosis is the process responsible for creating the space between our fingers and

toes as we are developing as human embryos. It is also required for proper functioning of the

immune system, such as eliminating activated or auto-aggressive immune cells during

maturation in the central lymphoid organs, or in peripheral tissues (Ouyang et al. 2012). To

accommodate for the different functions of apoptosis, there are two pathways it encounters.

The two pathways of apoptosis are intrinsic and extrinsic. These pathways are

differentiated by the apoptotic signals received from inside and outside the cell. The intrinsic

pathway of apoptosis is a response to signaling molecules originating from inside the cell, i.e.,

the cell kills itself because it senses stress. The signaling molecules originating inside the cell can

be triggered by severe cell stress, such as, DNA damage, chromosome rearrangement, cytotoxic

stimuli, etc. (Ouyang et al. 2012). In contrast, the extrinsic pathway of apoptosis, is a response to

signaling molecules originating from outside the cell (Ouyang et al. 2012). In extrinsic apoptosis,

receptor-mediated signals stimulate cell death, i.e., receptors receive extracellular signaling

molecules which direct the cell to kill itself.  Receptors which receive the extracellular signals

and initiate extrinsic apoptosis are the tumor necrosis factor (TNF) receptor and the first

apoptosis signal (FAS) receptor. A process which would trigger receptor-mediated signals to

initiate the external pathway of apoptosis is modulation of immune function, e.g., extrinsic

apoptosis is used by white blood cells to kill viral infections (Ouyang et al. 2012). Another

process the extrinsic apoptotic pathway is used for is the separation of human toes and fingers

during development (Ouyang et al. 2012). Both intrinsic and extrinsic pathways are required for

proper organism function, especially when there are malfunctions in cellular pathways.

The Relationship Between Apoptosis and Cancer

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Cells with malfunctioning apoptotic pathways lack the ability to activate programmed

cell death processes, such as apoptosis: one defining characteristic of a cancerous cell phenotype

(Ouyang et al. 2012). In signal transduction pathways which promote cell survival and growth,

such as the Protein Kinase B (AKT) pathway, pro-apoptotic proteins are always active unless an

extracellular survival/growth signal binds to the survival/growth receptor (Zheng et al. 2015).

When the survival/growth signal binds to the  survival/growth receptor, an anti-apoptotic protein

will inhibit apoptosis (Zheng et al. 2015). An example of a mutation which could cause cancer in

the AKT signaling pathway is if the pro-apoptotic protein, in charge of initiating apoptosis, had a

loss-of-function mutation, where it no longer initiated apoptosis: this would lead to non-stop cell

reproduction (an over proliferation of cells). Another example of a mutation in the AKT

signaling pathway which could cause cancer is a gain-of-function mutation in the anti-apoptotic

protein, where it is constitutively active, inhibiting apoptosis: this would also lead to an over

1proliferation of cells. Although the two mutations affect two different proteins in the pathway,

the end result for both was the cell’s inability to initiate apoptosis. The pro-apoptotic and anti-

apoptotic proteins in the AKT pathway are both a part of the Bcl2-family of proteins. After

gaining an understanding of how a lack of apoptosis can lead to cancer, one can propose

potential cancer therapy methods by finding mechanisms to ensure that apoptosis is activated in

cancerous cells using the Bcl-2 family of proteins.

Bcl-2 Family of Proteins

The B-cell lymphoma 2 (Bcl-2) family of intracellular proteins located in the cytoplasm

and mitochondria are one of the many proteins which regulate intrinsic apoptosis (Shamas-Din et

al. 2011). There are two sub-families of Bcl-2 proteins, categorized by whether they positively

(pro-apoptotic Bcl-2) or negatively (anti-apoptotic Bcl-2) regulate apoptosis (Shamas-Din et al.

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2011). The anti-apoptotic Bcl2 proteins which negatively regulate apoptosis have domains 1,2, 3,

and 4 (BH1,2,3,4). The  pro-apoptotic Bcl-2 proteins that positively regulate apoptosis have two

sub-families of proteins, differentiated by their domains: BH3-only and BH1,2,3 (Shamas-Din et

al. 2011). Bcl-2 anti-apoptotic proteins have one domain which the Bcl-2 pro-apoptotic proteins

lack; the BH4 domain (Shamas-Din et al. 2011). Since the BH4 domain is the only domain

differentiating pro-apoptotic from anti-apoptotic activity in the Bcl-2 family of proteins,

scientists have inquired whether it was a key player in inhibiting apoptosis. It was discovered

that the BH4 domain was both necessary and sufficient to terminate the process of intrinsic

apoptosis in the cell (Monaco et al. 2015). It was also found that the BH3 domain on the pro-

apoptotic BH3-only protein was found necessary and sufficient to inhibit anti-apoptotic proteins

(Shamas-Din et al. 2011). When the intrinsic pathway for apoptosis is initiated, the pro-apoptotic

Bcl-2 proteins are one of the first proteins to become activated to start the apoptotic process.

Figure 1: Categorization of the Bcl-2 proteins by the domains they possess and their

ability to initiate or terminate cell death. Adapted from BH3-only proteins in apoptosis and

beyond: an Overview by Lomonosova E and Chinnaduri G 2010.

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The Process of Intrinsic Apoptosis

Bcl-2 Pro-apoptotic Protein Dimerization and Mitochondrial Outer Membrane Permeabilization

When internal cellular stress triggers the initiation of the intrinsic pathway of apoptosis,

the BH3-only pro-apoptotic proteins inhibit the anti-apoptotic proteins. BH3-only proteins

essentially inhibit the inhibitors, by binding the anti-apoptotic Bcl-2 proteins and forming a

sequestration complex. After the BH3-only proteins inhibit the inhibitors, the pro-apoptotic Bcl-

2 proteins are set free in the cytoplasm and their activity is encouraged (Tait and Green 2010).

Two of the same pro-apoptotic Bcl-2 proteins, Bcl-2 antagonist killer (BAK), dimerize through

their BH3 domains, producing a homodimer. One BAK is located in the cytoplasm and the other

on the outer membrane of the mitochondria.  The dimerization of the two BAK Bcl-2 pro-

apoptotic proteins, initiates mitochondrial outer membrane permeabilization (Tait and Green

2010).  The BAK protein localized in the cytoplasm is controlled by anti-apoptotic Bcl-2

proteins when apoptosis is not taking place. When one of the BAK proteins is activated its BH3

domain is exposed, and is inserted into the hydrophobic groove of another BAK molecule (Tait

and Green 2010). When BAK in the cytoplasm and BAK on the outer membrane of the

mitochondria are not dimerizing, and apoptosis is not taking place, an anti-apoptotic Bcl-2

protein is inhibiting BAK in the cytoplasm. When the cell is signaled to apoptosis, a Bcl-2 BH-3-

only protein localized in the cytoplasm inhibits the anti-apoptotic Bcl-2 protein, allowing BAK

in the cytoplasm to dimerize with BAK on the outer mitochondrial membrane.

Mitochondria contain an outer and inner phospholipid bilayer membrane separated by the

intermembrane space differentiated by their protein and lipid compositions (Tait and Green

2010). The inner membrane is composed of about twenty percent lipids and eighty percent

proteins, making it only permeable to oxygen, carbon dioxide, and water (Tait and Green 2010).

In contrast, the outer membrane is composed of fifty percent lipids and fifty percent proteins

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allowing the membrane to be highly permeable to small proteins like cytochrome c (Tait and

Green 2010). The cells are committed to apoptosis when intermembrane space proteins, such as

cytochrome c, are released into the cytoplasm, during mitochondrial outer membrane

permeabilization (MOMP).

The Release of Cytochrome C

Cytochrome c is an apoptosis-dependent protein released from the mitochondrial

membrane, which initiates a cascade of events that leads to the activation of the apoptosome,

caspase proteases, and other proteins required for the completion of cell demolition. Cytochrome

c does this by binding to an apoptotic protease activating factor (APAF), and releasing the

caspase recruitment domain (CARD)(Martinou and Youle 2011). Many APAF proteins

aggregate together through their CARD domains. This leads to the formation of an apoptosome,

a large quaternary structure which recruits and activates an initiator caspase, leading to a positive

feedback event of caspases required to complete apoptosis (Tait and Green 2010).

The Activation of Caspases

Most apoptosis pathways are caspase-dependent, and therefore require the activation of

these enzymes to perform proteolysis, the breakdown of proteins (Czabotar et al. 2014).

Caspases initiate cell shrinkage, nuclear fragmentation, chromatin condensation and membrane

blebbing, completing the process of apoptosis (Tait and Green 2010). The caspase family of

proteins is a family of twelve enzymes (Ouyang et al. 2012). They are known as zygomens,

meaning they are always inactive until a biochemical change in the cell, such as internal damage

or death factors, causes their activation (Brentnall et al. 2013). This family of enzymes has

cysteines and histidine residues in their active sites, which act by stabilizing the cleavage of

apoptosis-targeted proteins at specific aspartic acids, a characteristic which makes them very

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selective (Ouyang et al. 2012). The initiator caspase 9 plays an important role in initiating the

proteolytic cascade of the other 12 caspases required to completely demolish the cell (Tait and

Green 2010). This is because caspase 9 is the only one which has a CARD domain which allows

it to bind to the CARD domain on the apoptosome (Tait and Green 2010).

Therapy Proposals

For this project, I seek to uncover methods which could act as cancer therapies by

enabling or disabling the functions of proteins involved in the intrinsic pathway of apoptosis. I

will be analyzing and reviewing scientific literature to research the Bcl-2 family of proteins as

well as other proteins which regulate the intrinsic pathway of apoptosis. It is my assertion that

the proteins and pathways involved in intrinsic apoptosis can be exploited to create an increase in

apoptotic activity in cancerous cells.

There are many ways in which the intrinsic apoptotic pathway and the proteins involved

can be manipulated and exploited to increase the frequency of programmed cell death in cells

containing malfunctioning pathways which have lost the ability to activate apoptosis. After

analyzing this pathway, I hypothesize that increasing the expression of the pro-apoptotic Bcl-2

BH3-only proteins and inhibiting the BH4 domain on the anti-apoptotic Bcl-2 proteins  are both

manipulations, that individually, could increase the levels of apoptosis and prevent tumor

growth. Since the goal of this therapy is to induce apoptosis, it will need to be a targeted event,

which kills only cancer cells. Correctly targeting cancer cells is required for the efficacy of the

treatment.

STRATEGIES FOR THERAPY

Inhibiting the BH4 Domain of Anti-apoptotic Bcl-2 Proteins

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The BH4 domain on the anti-apoptotic Bcl-2 proteins binds to BAK in the cytoplasm,

and inhibits it from dimerizing with BAK on the outer mitochondrial membrane. The anti-

apoptotic Bcl-2 protein binds to the VDAC1 channel and inhibits its activity, inhibiting the

influx of calcium ions which prevents MOMP (Monaco et al. 2015). The dimerization of BAK

proteins are required for MOMP, which is required for the release of cytochrome c and the

completion of apoptosis. Inhibiting the BH4 domain on the anti-apoptotic Bcl-2 proteins will

increase the level of apoptosis in the cell, and eventually kill the tumor. The inhibition of the

anti-apoptotic Bcl-2 protein BH4 domain can be completed through small molecule inhibitors.

To use a small molecule inhibitor against the anti-apoptotic Bcl-2 BH4 domain, a target

residue must be selected and surface binding  pockets must be detected in the BH4 domain,

favorable for small molecule binding (Johnson and Karanicolas 2015). Random ligand selectivity

must be studied through the Bcl-2 BH4 domain, and the ligand protein interaction can be

detected through screening (Ishima 2015). The small molecule inhibitor will be transported into

the cancer cells using lipid nanoparticles (Rostami et al. 2014).

Small molecule inhibitors force the inhibitor-bound protein to adopt a confirmation that is

opposite or distinct from its unbound conformation. Therefore, inhibiting the BH4 domain of the

anti-apoptotic Bcl-2 protein is expected to force the anti-apoptotic Bcl-2 proteins to gain pro-

apoptotic activity. The BH4 domain of Bcl-2 (a specific anti-apoptotic Bcl-2 protein) is required

for the protein’s anti-apoptotic function (Chen and Deng 2015). Knowing this, the BH4 domain

could be responsible for the anti-apoptotic characteristics of all the anti-apoptotic Bcl-2 proteins.

Chen and Xingming found that when the BH4 domain is cleaved or removed from the anti-

apoptotic Bcl-2 proteins, they act as pro-apoptotic Bcl-2 proteins, promoting apoptosis (Chen

and Deng 2015). The BH4 domain on the anti-apoptotic Bcl-2 proteins inhibits the BH3-only

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Bcl2 pro-apoptotic proteins from dimerizing on the outer mitochondrial membrane, which

prevents MOMP, the release of cytochrome c, the activation of caspases, and the completion of

apoptosis. The BH4 domain also inhibits the influx of calcium ions required for the release of

cytochrome c, and therefore the completion of apoptosis.

Increasing Expression of Bcl-2 BH3-only Protein

Increasing the expression of the pro-apoptotic Bcl-2 BH3-only proteins will allow the

anti-apoptotic Bcl-2 proteins to be inhibited, releasing BAK located in the cytoplasm, allowing it

to dimerize with BAK on the outer mitochondrial membrane, leading to a constantly

permeabilized mitochondria outer membrane, allowing the continuous release of cytochrome c.

The continuous release of cytochrome c will ensure the activation of the apoptosome and the

initiation of the cascade of caspase activation completing the process of apoptosis. Increasing the

expression of the pro-apoptotic Bcl-2 BH3-only protein can be accomplished through

recombinant protein expression and gene therapy.

To obtain recombinant protein expression, the gene of interest which codes for the

protein of interest will be cloned in vector, transformed into the host, induced and purified

(Rosano and Ceccarelli 2014). For this therapy, multiple copies of the pro-apoptotic BH3-only

Bcl-2 protein are desired. The first step in making a recombinant protein is isolating the gene of

interest which codes for the protein of interest (Rosano and Ceccarelli 2014). Therefore,

restriction endonuclease (a restriction enzyme) will be used to cleave and isolate the BH3-only

gene from the genome. This gene will then be added to a vector, making a construct (Rosano and

Ceccarelli 2014). The construct will then be inserted into cancer cells. To successfully deliver the

construct to cancer cells only, gene therapy will be used (Rosano and Ceccarelli 2014).

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Vectors used for recombinant proteins can be viruses or bacteria (Duffy et al. 2013). A

popular bacterial vector is plasmid from E. coli (Duffy et al. 2013). There are many popular viral

vectors used for recombinant proteins such as adenovirus, baculovirus, herpes virus, retrovirus,

lentivirus, etc. (Rosano and Ceccarelli 2014). Viral vectors can both integrate their genome into

the cell’s chromosome, so it can self-replicate, or they can be transient and replicate once

(requiring readministration) (Duffy et al. 2013). Viral vectors must include an origin of

replication, promoter, selection marker, and a cloning site (Rosano and Ceccarelli 2014). The

promoter on the viral vector must be specific to cancer cells (Duffy et al. 2013). The viral vector

will act as the gene delivery vehicle in the host (Westphal et al. 2013).

A common virus used as a viral vector in cancer gene therapy is the adenovirus

(Westphal et al. 2013).  Adenovirus is a vehicle which could transport therapeutic genes into

hosts in gene therapy (Westphal et al. 2013). Since human contact is very common with

adenovirus it is very likely that the patient of this gene therapy has developed antibodies against

it, which means the patient could reject the therapy more easily than if the expression vector was

a virus which doesn’t come into human contact very much (Wold and Toth 2013). Adenovirus

serves as a good virus vector because its genome is very well known and can be easily modified,

it efficiently delivers the viral genome to target cells, and it can be produced in large quantities

(Westphal et al. 2013). Adenoviruses will need to be readministered because the extra genes are

not replicated when the cell undergoes cell replication, i.e., it is a transient vector (Westphal et

al. 2013). Using a viral vector, such as adenovirus, which needs to be readministered, is a safe

approach to the therapy because if something went wrong and the adenovirus vector was

targeting the wrong cells, the problem could be fixed before it did too much damage. This is in

contrast to using a viral vector which inserts its genome into the targeted cell’s genome and

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essentially self-replicates, such as a lentivirus. Self-replicating vectors which inflict cell death

could do a lot of damage in a short amount of time if the vector was targeting the wrong cells.

Multiple conditions will be tested to obtain the desired protein. A western blot analysis

will be performed to detect the presence of the protein in the cancerous cell. To direct this

protein into cancerous cells it will be required to use receptor-mediated gene transfer with

receptor-ligand interactions where the ligand is coupled to the Bcl-2 BH3-only DNA complex

(Giacca and Zacchigna 2012). This will ensure proper cellular targeting and delivery (Giacca and

Zacchigna 2012). Receptor-mediated gene transfer will convey the recombinant Bcl-2 BH3-only

protein from the site of administration to the surface of cancerous cells, and facilitate endocytosis

(Giacca and Zacchigna 2012).

DISCUSSION

Both therapeutic mechanisms are dependent on a normal functioning intrinsic

mitochondrial apoptotic pathway. If there is a mutation in this apoptotic pathway, such as a loss-

of-function mutation, both of these mechanisms will not work. Some examples of mutations in

this apoptotic pathway which could negate both therapeutic mechanisms is; a mutation which

prevents BAK dimerization even after a small molecule inhibited the BH4 domain and BAK in

the cytoplasm was released; a mutation which prevents MOMP even after BAK dimerization; a

mutation which prevents the release of cytochrome c into the cytoplasm even after MOMP, etc.

Small Molecule Inhibitors

A difficult aspect of producing small molecule inhibitors is finding the appropriate ligand

that will inhibit the specific domain (ligand selectivity) and for the purpose of this therapy;

finding a ligand that will inhibit the BH4 domain of anti-apoptotic Bcl-2 proteins. Scientists have

been combating this struggle by starting the process with an unbound protein structure and

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observing low-energy conformations that include deep-surface pockets, which enables inhibitors

to recognize low-lying excited states of the protein that are naturally occurring (Johnson and

Karanicolas 2015). Another solution could be viewing the interaction from the perspective of the

potential ligand, where an inhibitor is expected to act against a given protein, only if the protein

surface has a suitable pocket for the ligand to bind to under normal physiological conditions

(Johnson and Karanicolas 2015). The process of finding ligand selectivity can be completed with

the aid of computer software such as Rosetta (Johnson and Karanicolas 2015). Ligand selectivity

that is already known for certain domains in proteins that are very similar to the structure of the

proteins being used for therapy is very helpful in determining an effective ligand.

The Bcl2-family of proteins have posed great targets for creating small molecule

inhibitors to work against their anti-apoptotic activity (Johnson and Karanicolas 2015).

Therefore, selectivity of ligands has already been achieved towards some of the anti-apoptotic

Bcl-2 proteins (Johnson and Karanicolas 2015). This makes easier the search for an appropriate

ligand to bind to the BH4 domain of the anti-apoptotic Bcl-2 proteins.

Inhibiting the BH4 domain on the anti-apoptotic Bcl-2 proteins, through small molecule

inhibitors, and increasing the expression of the pro-apoptotic BH3-only Bcl-2 protein will

increase the level of apoptosis in the cell, and eventually kill the tumor, as long as the small

molecule inhibitor inhibits the BH4 domain of the anti-apoptotic Bcl-2 proteins. If the small

molecule inhibitor interacts with domains of proteins other than the BH4 domain of anti-

apoptotic Bcl-2 proteins, and inhibits the growth of non-cancerous, this could fatally harm the

organism.

Recombinant Protein Production

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Increasing the expression of the Bcl-2 BH3-only proteins through recombinant proteins

pose difficulties in some areas, including the potential for low protein activity and detection,

protein toxicity, and codon bias (Rosano and Ceccarelli 2014). If the protein imposes harmful

effects on the cell, its detection through western blot analysis could be very low or zero (Rosano

and Ceccarelli 2014). When introducing the recombinant proteins into the host cell it is

important to monitor the growth of the protein before it is induced due to the fact that it may be

toxic to the cell and perform unnecessary damaging functions which interfere with proliferation

and homeostasis (Rosano and Ceccarelli 2014). Codon biases can occur when the frequency of

synonymous codons in the foreign coding DNA is significantly different than the hosts which

could result in low levels of tRNAs and indirectly lead to low levels of expression of the

recombinant protein (Rosano and Ceccarelli 2014).

Recombinant protein production can be limiting when using retroviral vectors. Based on

the packaging constraints imposed by the viral proteins, vector genomes themselves have limited

space for insertion of foreign sequences (Rosano and Ceccarelli 2014). Along with the issue of

limited space comes the question of the stability of the vectors (Rosano and Ceccarelli 2014).

The natural processes which are required to proceed in a retrovirus vector, such as reverse

transcriptase, require a high level of rearrangement and instability which could pose problems in

the transduction and delivery of the vector into the cell (Rosano and Ceccarelli 2014). Due to

immune responses, sustaining long term gene expression of the therapeutic gene (in this case the

Bcl-2 BH3-only) using viral vectors is a challenge, especially amongst the other signals or drugs

administered to the host (Rosano and Ceccarelli 2014). To help avoid this, the use of a gene’s

own promoter, other than the viral promoter, can help stabilize long-term gene expression

(Rosano and Ceccarelli 2014). Another vector which could be used is a plasmid found in bacteria

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cells, such as E. coli. To conclude, integration of authentic genomic elements into a viral vector

can help avoid common problems by sustaining gene expression and preventing an immune

response (Rosano and Ceccarelli 2014).

Gene Therapy

When executing gene therapy, there are many variables which must be considered

carefully in order for success, such as conquering intracellular and extracellular barriers (Giacca

and Zacchigna 2012). Common intracellular trafficking barriers occur when the transported

protein is unable to escape intracellular vesicles (Giacca and Zacchigna 2012). To help the

transported protein successfully escape the vesicle, vesicular releasing agents can be applied

when introducing the new gene to the host cell (Giacca and Zacchigna 2012). Vesicular releasing

agents include lysosomotropic agents, glycerol, virus particles, membrane disruptive peptides,

and photosynthesizing compounds (Giacca and Zacchigna 2012).

After the protein is released from the vesicle, it must avoid being degraded by the

cytoplasm and it must be successfully localized to the nucleus (Giacca and Zacchigna 2012). A

nuclear localization signal along with DNA carrier molecules will help ensure that the protein

enters the nucleus (Giacca and Zacchigna 2012).

Once the protein is located in the nucleus, it is at risk for DNA damage due to factors

such as immune responses, transcriptional shut off, inefficient nuclear trafficking, etc. (Giacca

and Zacchigna 2012). When the protein is in the nucleus, DNA damage can be prevented by

including specific sequences, derived from viruses or chromosomes, to the transferred DNA

which assure either integration of the DNA into the host chromosome, or extrachromosomal

replication of the transferred DNA with equal segregation to daughter cells (Wirth et al. 2013).

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Overcoming extracellular barriers include avoiding undesired interactions with plasma,

degradative enzymes, nontarget tissues, immune responses and allowing endocytosis to

successfully occur (Wirth et al. 2013). Some of the solutions to overcome these barriers include

studying the physicochemical structure of the protein and creating the best transfection

environment based on its features (Wirth et al. 2013). To help enable endocytosis, active

endogenous transport mechanisms can be used (Wirth et al. 2013). To avoid an immunological

response, polyplexes can be constructed in a specific manner (Wirth et al. 2013). Since the

positive charge DNA complexes possess attract the complement system, a solution could pose

coating the DNA positive charges with macromolecules, which could inhibit interactions with

the complement system of the host (Wirth et al. 2013). Viral delivery of gene therapy and

targeting cancer cells can be problematic with a viral vector. Using an adenovirus for gene

therapy can be risky because if the efficiency of infection is not %100, the therapy would not

work. Another option would be to use a targeted lipid nanoparticle delivery system.

Extrinsic Pathway of Apoptosis

The extrinsic pathway of apoptosis is a commonly manipulated pathway for cancer

therapies because it is initiated through the activation of death receptors which are always

present on the surface of the cells. Activating receptors which initiate apoptosis makes for a good

therapy because the receptor is always present. An example of a ligand which is a valuable target

for potential cancer therapies because it activates more than one death receptor, is the TNF-

related apoptosis-inducing ligand (TRAIL) (Sayers 2011). This ligand is studied heavily and

commonly manipulated for potential cancer therapies (Sayers 2011). Therapies include

administering TRIAL monoclonal antibodies (McGrath 2011). Another mechanism that could

work is using an adenoviral vectors to deliver TRIAL directly into tumor location (McGrath

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2011). Since the death receptor which TRIAL binds to is always present on the surface of the cell

this therapy would be more efficient than therapies involving intrinsic apoptosis.

CONCLUSION

Both proposed therapies in this paper share the commonality of targeting programmed

cell death pathways. Using small molecule inhibitors to interfere with the anti-apoptotic

properties that the BH4 domain usually expresses and changing its conformation to expresses

pro-apoptotic properties will interfere with its ability to inhibit apoptosis, and therefore

encourage apoptosis. Producing recombinant proteins and using gene therapy to increase the

expression of the BH3-only Bcl-2 protein, a protein which inhibits anti-apoptotic activity, will

also encourage a higher level of apoptosis. The two strategies proposed ensures that tumor cells

have the ability to initiate apoptosis.

Enabling cancer cells the ability to induce apoptosis is crucial since a common

occurrence in the development of tumor cells is the cell’s inability to activate it’s apoptotic

pathways. Enabling healthy cells which are normally signaled to proliferate, the ability to induce

apoptosis will kill the patient.  It is important to remember that for both therapies it is required

that they only target cancer cells. Producing a cancer therapy which only targets the cancer cells

is an enormous improvement from chemotherapy, radiation, and other non-targeted therapies,

because it enables patients to receive higher doses of a higher potency, with significantly less

side effects.

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