A Novel Therapeutic Treatment Utilizing Cordycepin and Cladribine

INTERNSHIP ARTICLE
A Novel Therapeutic Treatment Utilizing
Cordycepin and Cladribine Synergy to Decrease
Adverse Treatment Effects in Various Cancer Cell
Lines
Jason Cui1*, Robert Culbertson2, Zebin Mao3, and Beverly Mock4
Student1, Teacher2: Langley High School, 6520 Georgetown Pike, McLean, VA 22101
Mentor3: Peking University School of Basic Medical Sciences, 38 Xue Yuan Road, Beijing, China
Mentor4: National Institutes of Health, 37 Convent Drive, MSC4258, Bethesda, MD 20892-4258
*Corresponding author: [email protected]
Abstract
Modern methods of cancer treatment like chemotherapy are
effective in eradicating cancer cells; however, the following
immunosuppression and cellular damage that occur are
often strenuous on the human body. This study sought to
design a novel therapeutic treatment model that could not
only reduce adverse effects experienced by patients, but also
be applied to multiple forms of cancer and their respective
treatment regimes. Studies with the adenosine analog
3’-deoxyadenosine, extracted from Cordyceps sinensis, have
proven its ability to induce cancer cell apoptosis through
polyadenylation interference while preventing damage
to healthy cells. However, Cordycepin meets intrinsic
resistance in the body from adenosine deaminase (ADA).
Therefore, Cordycepin coupled with an ADA inhibitor can
potentially form a novel therapy that expresses a decrease
in cytotoxicity toward healthy cells. The chemo-drug/
ADA inhibitor Cladribine (2-chlorodeoxyadenosine), a
synthetic analog of 3’-deoxyadenosine, used in synergy was
hypothesized to work in two ways: 1) Cladribine, as an ADA
inhibitor, induces synergism in its apoptotic anti-cancer
effect on cancer cells, and 2) the enhanced efficacy of the
combined treatment allows for the reduction of the toxic
chemo-drug, alleviating Cladribine’s toxicity on healthy
cells while maintaining treatment efficacy. The novel
treatment was assessed through cell proliferation assays,
morphological observation, and confirmed through flow
cytometric analysis. Results indicated that Cordycepin/
Cladribine synergy experienced similar levels of cancer cell
inhibition as Cladribine alone, but exhibited a significant
increase in healthy cells left unharmed. The combined
treatment also effectively models the potential to expand
Cordycepin to additional chemo-drugs/ADA inhibitors
and better patient outlooks for respective treatments.
Introduction
Traditionally to combat cancer, most patients receive chemotherapy,
radiation, and/or surgery to reduce cancerous tumor cells.
However, these methods involve destroying healthy cells and
lead to plethora of side effects including immunosuppression,
myelosuppression, and gastrointestinal distress1. The purpose
of this study in its entirety was to research and develop a novel
cancer therapy that could potentially reduce toxic treatment side
effects to healthy cells without sacrificing treatment efficacy.
Cordyceps sinensis is a species of endoparasitical fungi of
the genus Cordyceps, also referred to as the Chinese Caterpillar
Fungus, or “Dong Chong Xia Cao”2. As its name suggests,
Cordyceps sinensis is a fungus, but what differentiates it from
the latter is its unique way of imbedding itself on a host tissue.
Caterpillar fungi are the result of a parasitical relationship between
the larva of the ghost moth and the fungus, both of which are
located in the Tibetan plateau. The fungus imbeds itself into and
mummifies the larva, and then grows from the body insect.
Cordyceps sinensis has had an extremely long history of use,
the first by the Chinese Qing Dynasty dating back to 1757 AD. The
documented uses back then were as lung and kidney supplements
to improve health. However, the caterpillar fungus has now been
called an overall tonic that can “improve the general well being of
a human”3. Cordyceps sinensis has been acknowledged to treat a
wide variety of conditions like respiration problems, pulmonary
diseases, renal, liver, and cardiovascular diseases, low sex drive,
and immune disorders4. It is also considered to be a remedy
for fatigue, being used by multiple athletes in the 2008 Summer
Olympic Games. Studies have also shown that Cordyceps sinensis
is able to increase cellular ATP levels3.
Recent research, however, has investigated the possible antitumor or anti-cancer ability of Cordyceps sinensis with the basis
that it can prove to be a therapeutic alternative to traditional
methods of treatment. Studies have been done with mice that have
cervical cell carcinoma and Lewis lung cancer in which extracts
from Cordyceps sinensis were added to mice showed significant
reductions in tumor size5. In a different experiment, extracts from
the genus Cordyceps was applied in a dose of 0.5 mg/kg to a
mouse with a resulting tumor inhibition rate of 98.7%6.
It has been found that the key factor contributing to
Cordyceps sinensis’ anti-cancer ability is a chemical compound
found in Cordyceps sinensis called Cordycepin. Cordycepin
(3’-deoxyadenosine) is a nucleoside analog and a derivative
of the nucleoside adenosine, differing from the latter by the
absence of oxygen in its 3’-position2. Cordycepin is a known
polyadenylation inhibitor with a large spectrum of biological
activities, including anti-proliferative, pro-apoptotic and antiinflammatory effects2. Polyadenylation is a necessary step for
the synthesis and maintenance of functional RNA transcripts
catalyzed by poly(A) polymerase. Polyadenylation involves the
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Jason Cui, Robert Culbertson, Zebin Mao, and Beverly Mock
addition of a poly(A) tail near the end of the RNA transcription
process and before RNA translation. The addition of a 3’-end
poly(A) tail to eukaryotic mRNAs is required for the transport of
RNA from the nucleus to the cytoplasm, translation efficiency,
and the regulation of mRNA degradation.
The poly(A) tail consists of multiple adenosine
monophosphates; in other words, it is a stretch of RNA that
has only adenine bases. Because Cordycepin is so similar to
adenosine, it is able to interfere with poly(A) polymerase and
disrupt the polyadenylation process, allowing for its substitution
into the poly(A) chain. The resulting disruption terminates
RNA transcription/translation, leading to the termination of
protein synthesis and subsequent cell proliferation. Cordycepin,
as a bona-fide PAP inhibitor with the potential to interfere with
RNA synthesis therefore has great potential as a possible anticancer/chemotherapeutic agent7. Previous experimentation with
Cordycepin on HeLa cervical cancer cells and healthy PBMC cells
has indicated that Cordycepin is able to effectively inhibit cancer
cell growth while leaving healthy cells unharmed.
Although presenting itself as an anti-cancer agent, Cordycepin
exhibits significantly lower levels of cancer cell inhibition when
compared to traditional methods of treatment like chemotherapy.
To be considered as a possible therapeutic alternative to
chemotherapy, the anti-cancer effects of Cordycepin need to be
enhanced in a way to promote its effectiveness in reducing cancer
cells without increasing toxicity toward healthy cells.
Cordycepin’s effectiveness is reduced due to intrinsic resistance
in the body from adenosine deaminase. Adenosine deaminase
(ADA) is a key enzyme in purine metabolism and catalyzes the
irreversible hydrolytic deamination of active adenosine (or
2’-deoxy-adenosine), which yields the inactive metabolite inosine1.
It is present is nearly all mammalian cells, occurring in many forms.
However, due to Cordycepin’s nature as an adenosine analogue,
adenosine deaminase irreversibly deaminates Cordycepin thereby
reducing its effectiveness (Figure1).
Once deaminated, Cordycepin is unable to incorporate into
the poly(A) tail of the mRNA, allowing for normal cancer cell
proliferation to continue. To increase the anti-cancer effect of
Page 2 of 5
(2-chlorodeoxyadenosine) is a purine analogue used mainly to
treat hairy cell leukemia (HCL, leukemic reticuloendotheliosis),
multiple sclerosis, and other forms of lymphomas. As a purine
analog, it is a synthetic anti-cancer agent with varying side effects,
including suppression of the immune system. Its methods of
cancer cell inhibition are DNA-linked, but involve methods
similar to that of Cordycepin due to their similarities as purine
analogues. The unique ability that Cladribine possesses is its
significant ability to inhibit adenosine deaminase, the enzyme
responsible for the deamination of adenosine (or in this case
Cordycepin8). Over a 48-hour period, Cladribine was shown to
inhibit both DNA synthesis and adenosine deaminase activity
by 80-90%8. Cladribine, like the majority of chemotherapy
drugs used in treatment, is plagued with side effects that include
immunosuppression and fever in treated patients.
For this reason, the connection was established between the
inherent weakness of Cordycepin due to its deamination and the
toxic side effects of Cladribine – a connection that became the
focus of this study. It was hypothesized that a synergy between
the two treatments would be able to form a more effective cancer
therapy by reducing the amount of adverse effects the holistic
treatment creates. The synergy is rationalized to operate in two
ways: First, Cladribine, as an adenosine deaminase inhibitor,
is effectively able to induce synergism in its apoptotic anticancer effect on cancer cells. By effectively inhibiting adenosine
deaminase, it is theorized that this action will translate into an
increased effectiveness in Cordycepin’s anti-cancer activity.
Secondly, the increased effectiveness of Cordycepin in the
combined treatment has the potential to alleviate Cladribine’s
toxicity toward healthy cells. Due to the potential increase in
Cordycepin’s anti-cancer effect, the dosage of Cladribine can be
significantly reduced, with the end goal being to achieve similar
levels of cancer cell inhibition with the synergy treatment.
With the reduction of a toxic chemotherapy substance and
the supplementation of Cordycepins and Cladribine, the new
synergy treatment is hypothesized to reduce damage to healthy
cells while maintaining similar cancer cell inhibition as traditional
treatment alone. This new combination therapy would then prove
to be more efficient than chemotherapy alone in providing a
treatment with minimized distress toward the patient. Previous
research has independently confirmed the efficacy of Cordycepin
and Cladribine, and also adenosine deaminase’s activity against
Cordycepin. The focus of this particular study is therefore to
implement Cordycepin and Cladribine into a novel therapy in
which significant synergism is induced.
Materials and Methods
Figure 1. The deamination of Cordycepin. Cordycepin is
naturally deaminated by adenosine deaminase (ADA) due to its
similarity to adenosine.
Cordycepin over a longer period of time, the use of a co-drug
in synergy with Cordycepin is required; more specifically, an
adenosine deaminase inhibitor.
A vast variety of potential co-drugs were considered
to be utilized in synergy with Cordycepin. Cladribine
Cell Line: Throughout the experiment, HeLa cervical cancer
cells, Human dermal fibroblast (HDF) cells, and U266 multiple
myeloma cells were utilized to gauge the effects of Cordycepin
and Cladribine. HeLa and HDF cell lines were obtained from Dr.
Zebin Mao (Peking University, Beijing, PRC). U266 cancer cells
were obtained from the National Cancer Institute (Bethesda, MD,
USA) courtesy of Dr. Beverly Mock. Cells were cultured in vitro
using DMEM (Invitrogen, Carlsbad, CA, USA) supplemented
with 10% fetal bovine serum and were stored in a 37°C incubator
with 5% CO2. Cells were cultured using proper sterile techniques
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Jason Cui, Robert Culbertson, Zebin Mao, and Beverly Mock
Page 3 of 5
and conformed to safety guidelines. Drugs and Chemicals: Cordycepin and Cladribine were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Rapamycin, MS-275, Adriamycin, Bortezomib, and Vincristine were all obtained from the National Cancer Institute (Bethesda, MD,
USA) courtesy of Dr. Beverly Mock. Morphology Observation: Preliminary morphology observation was completed to gauge the effects of
Cordycepin, Cladribine, and Cladribine synergy on HeLa cervical cancer cells. HeLa cells (6 x 105) were seeded in 6-cm Petri dishes (Techno
Plastic Products AG, Transadingen, Switzerland) with 2mL DMEM medium. Petri dishes were then treated with variables including a
control, 1 mM Cordycepin, 10 µM Cladribine, and a synergy between the two. Previous dose-response curve analysis indicated the optimum
dosage of Cladribine to be used in synergy with Cordycepin while retaining maximum anti-cancer effective to be around 5 µM. Therefore,
5 µM of Cladribine was used in synergy with 1 mM of Cordycepin and tested. After 24 hours, cell morphology was observed and recorded
under light microscopy (Olympus, CK 40). The goal was to seek out signs of cellular apoptosis, characterized by plasma membrane
blebbing, condensation of the nucleus and cytoplasm, and loss of cellular contact with the matrix. Annexin V-FITC Apoptosis Detection: After
using morphological observations to provide qualitative data, Annexin V-FITC was used to quantify the apoptotic abilities of Cordycepin,
Cladribine, and their synergy. Cellular apoptosis is characterized by certain morphologic features, including loss of contact of the plasma
membrane, condensation of the cytoplasm and nucleus, and internal cleavage of DNA. The loss of the plasma membrane is one of the
earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is transferred from the inner to the outer leaflet
of the plasma membrane, exposing PS to the external cellular environment. Annexin V is a phospholipid-binding protein that has a high
affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes (fluorescent markers) including FITC.
With the FITC marker, Annexin V is able to retain its affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells
that are undergoing apoptosis. Annexin V-FITC staining is completed before the loss of PS contact, which accompanies the latest stages
of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with V-FITC was done with the dye propidium
iodide (PI) in order to identify early apoptotic cells (PI negative, FITC Annexin V positive). Similar to previous tests, HeLa cervical cancer
cells were applied with constant treatments of a control, 1 mM Cordycepin, 10 µM Cladribine, and a synergy between 1mM Cordycepin
and 5 µM Cladribine. HeLa cells were then washed twice with cold phosphate buffered saline (PBS) and suspended in Annexin V binding
buffer (0.1 M Hepes/NaOH [pH 7.4], 1.4 M NaCl, 25 mM CaCl2) with a concentration of 1 x 106 cells/mL. 100 µL of each solution (1 x
105 cells) was then transferred to 5 mL culture tubes. Annexin V-FITC and PI were then both added at concentrations of 5 µL. The three
controls included: unstained HeLa cells, cells with Annexin V and no PI, and cells with PI and no Annexin V. Cells were then gently rocked
and stored at room temperature (25˚C). 400 µl binding buffer was re-added to each tube, and flow cytometry was completed within one
hour. MTT Proliferation Assay Analysis: Methylthiazoletetrazolium (MTT) assay was used to determine cell viability of both cancerous HeLa
cells and healthy human dermal fibroblast (HDF) cells with the treatment of Cordycepin, Cladribine, and their synergy. Both HeLa cells
and HDF cells were seeded in a 96-well plate with each well containing 100 μl of growth medium. 40 wells were allotted each for HeLa
and HDF cells, while 16 wells acted as blanks for the negative control. Both HeLa cells and HDF cells were then treated with 0.1 mM
Cordycepin, 0.5 mM Cordycepin, 1 mM Cordycepin, 10 μM Cladribine, and Cordycepin in synergy with Cladribine for 48 hours. MTT
was added to each well for a final concentration of 0.5 mg/mL and incubated at 37°C for 4 hours. Finally, the medium was removed and
dimethyl sulfoxide (DMSO) was added to each well. The OD value of each well was then determined with automated spectrophotometry.
Further Study: Further study was completed with Cordycepin, in which MTT assays, reciprocated to those above, were performed on
Cordycepin in synergy with Rapamycin, MS-275, Adriamycin, Bortezomib, and Vincristine on U266 multiple myeloma cells. Cells were
cultured for 48 hours and the OD value was determined through automated spectrophotometry.
Results
Morphology Observation
Under light microscopy, it was clear that HeLa cells without
Cordycepin (control) had polygonal shapes with healthy appearances
and firm attachment, characteristics associated with healthy cells
(Figure 2).
Cells with 1mM Cordycepin were rounded but still adherent to
the ground matrix, with the total cell count having decreased. Cells
applied with 10 μM Cladribine experienced heavy levels of apoptosis,
with membrane blebbing and a condensed cytoplasm/nucleus in
individual cells. Cordycepin/Cladribine synergy experienced similar
apoptotic effects, especially with the compact cytoplasm/nucleus
existent in cells.
Annexin V-FITC Apoptosis Detection
Apoptosis induction was observed after 24 hours of drug
applications. At 1mM of Cordycepin treatment, approximately 7% of
cells underwent apoptosis after 24 hours, including early and late stage
apoptosis (Figure 3).
Cladribine, which was applied at a concentration of 10 μM,
exhibited a much higher level of cytotoxicity toward HeLa cells,
Figure 2. Morphology observation under various treatments.
Cells applied with Cordycepin/Cladribine synergy experienced
similar apoptotic affects as Cladribine alone, especially in
cytoplasm and nuclei reduction in cells.
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Jason Cui, Robert Culbertson, Zebin Mao, and Beverly Mock
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inhibiting cellular growth to 84% of the control. However, Cladribine/
Cordycepin synergy displayed a similar level of inhibition to 86% of
the control. All data was the result of three independent experiments.
Cells applied with Cordycepin/Cladribine synergy showed similar
levels of inhibition as cells with Cladribine alone, exhibiting the
treatment efficacy of the novel therapy. Due to the short period of
time after application, it can be expected that HeLa cells treated with
varying treatments for 48 and 72 hours will experience even higher
levels of apoptosis.
MTT Proliferation Assay Analysis
The morphological changes suggested the involvement of cell
death induced by Cordycepin and Cordycepin/Cladribine synergy,
and the apoptotic effects were confirmed through Annexin V-FITC
Apoptosis Detection. The MTT viability assay was therefore used to
investigate the effect of Cordycepin/Cladribine synergy. MTT tests
showed that Cordycepin exhibited a significant anti-cancer effect,
with the optimal dosage of 1 mM reducing HeLa cells to 77% of the
control. Cladribine, which exhibited a much greater cytotoxic effect,
inhibited HeLa cells to 46% of the control (Figure 4).
Cordycepin/Cladribine synergy however inhibited HeLa cells
to 47% of the control, exhibiting a 1.93% increase in efficacy over Figure 3. The efficacy of various treatments over 48 hours.
Cladribine alone. When applied to healthy PBMC cells, Cordycepin Notably, the application of Cordycepin and Cladribine synergy
alone decreased cell viability to 99% of the control, exhibiting little (1mM Cordycepin & 10 µM Cladribine) produced similar levels
to no toxic side effects to healthy cells. Cladribine, when applied, of cellular apoptosis as Cladribine alone.
decreased cell viability to 55% of the control. However, Cordycepin/
Cladribine synergy decreased cell viability to 66% of the control, exhibiting a 10.73% increase in healthy PBMC cells left unharmed. All
data was taken from three independent trials.
Further experimentation proved this same model to be effective with a wide variety of different treatments, specifically those used in
the treatment of Multiple Myeloma (MM), a cancer of the plasma cells (Figure 5).
Figure 4. MTT assay analysis. Cordycepin and Cladribine
synergy exhibited a 47% inhibition, with a 1.93% increase in
efficacy over Cladribine alone.
Figure 5. Further MTT Assay Analysis on a variety of
treatments for Multiple Myeloma (MM). U266 Multiple
Myeloma cells were treated for 48 hours. Cordycepin synergy
proved to be more effective in most cases.
Discussion
Through experimentation the hypothesis was tested and confirmed. The goal was to research and design a novel cancer therapy that could
retain its effectiveness toward cancer cells, but reduce the amount of toxic side effects toward healthy cells. Morphological observation
results indicated that both Cladribine and Cordycepin/Cladribine synergy induced rounded-up cells with blebbed membranes, hallmark
signs of apoptosis. Although only tested on HeLa cancer cells, the new synergy treatment showed an increase in treatment efficacy over
Cordycepin alone. Annexin V-FITC indicated that Cordycepin/Cladribine synergy and Cladribine both induced significant amounts of
apoptosis in HeLa cells. The synergy treatment showed an increase in effectiveness over Cordycepin alone, and also showed similar levels
of cancer cell inhibition as the chemo-drug while increasing healthy cell viability.
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Jason Cui, Robert Culbertson, Zebin Mao, and Beverly Mock
Due to the novel treatment’s ability to decrease adverse
treatments in healthy cells, numerous possibilities exist for its
application: Cordycepin/Cladribine therapy is able to significantly
decrease side effects experienced during chemotherapy
treatment, including immunosuppression, myelosuppression, and
gastrointestinal distress. This creates a better outlook for cancer
patients and increases the rate of survival from chemotherapy
side effect-related deaths. Similarly, due to Cordycepin’s beneficial
impact on cells, novel treatment regimens can be developed to
allow for less time between drug administrations.
A wealth of opportunities for future research exists as well.
Investigations of an adenosine deaminase-resistant strain of
Cordycepin would greater reduce toxic side effects to healthy
cells, producing an even more effective treatment. Finally,
the experimentation of Cordycepin with a wider variety of
chemotherapy drugs would provide a better understanding of
Cordycepin as a polyadenylation inhibitor and its effectiveness on
a wider variety of cells and treatments.
Page 5 of 5
Acknowledgements
The author would like to thank Dr. Zebin Mao and Dr. Beverly
Mock for respective lab spaces and materials, along with the
encouragement and inspiration to pursue research. The author
would also like to thank Mr. John Simmons for providing guidance
and help with lab procedures, and Mr. Robert Culbertson for
support and help throughout the paper-writing process.
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