Current Research Updates @ the University of Miami
University of Miami / Miller School of Medicine
Department of Neurological Surgery
Childhood Cancer Research Lab
young Researchers like our own Frederic Vallejo who continue to give
Hope to kids battling Childhood Cancer. Today we Honor and thank this
inspiring young man for his passion and dedication to finding a cure for
children battling this devastating disease.
"My name is
Frederic Vallejo and I have been a research volunteer in the lab working
with Dr. Graham since May of 2016. My younger brot
Joey was diagnosed with Stage IV Neuroblastoma on his first birthday.
My family’s devastating entanglement with Neuroblastoma has fueled a
passion in me to help other children and families suffering from this
disease. Since joining the lab, my projects have been primarily focused
on killing cancer cells by exploiting their strange metabolic
tendencies. Research thus far has shown that many cancers heavily
upregulate the glycolytic pathway while forgoing the Krebs cycle and
electron transport chain almost completely. I have been using different
glycolytic inhibitors which enter the cancer cell, back up glycolytic
machinery, and force the malignant cells to stress out and ultimately
die while leaving normal cells unharmed. I have also been heavily
investigating exogenous ketone body acetoacetate and
beta-hydroxybutyrate treatment which has shown extremely promising
anti-cancer results thus far. I was recently awarded the Beyond the Book
Summer Scholarship at the University of Miami. One student is chosen
out of a pool of applicants hoping to conduct research over the summer.
This research grant of $4000 allows me to fund a self-designed
comprehensive Neuro-Oncologic research project during the Summer of
2018. I am eager to continue developing my own hypotheses for combatting
malignant tumors while instructing and leading a team of undergraduates
to execute my proposed experiment."
By Dr. Regina Graham
Congratulations to our medical student volunteers Gregor
Rodriguez and Zach Gersey for their recent publications. Gregor published a review
article entitled: “Investigating the therapeutic role and molecular biology
of curcumin as a treatment for glioblastoma” in Therapeutic Advances in Medical Oncology. This publication sets the stage for our work examining the effects of curcumin on
glioblastoma stem cells. Zach’s original research article “Curcumin
decreases malignant characteristics of glioblastoma stem cells via induction of
reactive oxygen species was recently published in BMC Cancer. Zach
with the help of other members in the lab demonstrated that curcumin
significantly reduces cancer stem cell replication and promotes cell death. One
caveat with using curcumin is the difficultly in maintaining concentrations
high enough in the body to kill the cancer cells. To address this issue we are
collaborating with Dr. Roger LeBlanc, Chairman of the UM Chemistry Department
and Dr. Eduardo Véliz to develop more bioavailable forms of
curcumin. We have now tested a series of
curcumin analogs and have found several that are significantly more potent than
the natural form of curcumin. We are currently writing this manuscript and hope
to submit for publication soon. In addition, Dr. Véliz is working on generating
new novel compounds designed specifically as potential anti-cancer molecules. This
research is based upon a new concept in drug design and development which is to
produce hybrid-drugs which combine the active regions of 2 or more known
anti-cancer molecules with the hope of improved drug effectiveness compared to each
We are also collaborating with the LeBlanc laboratory, in the development
of a carbon-dot nanoparticle based chemotherapeutic for the treatment of
pediatric brain tumors. We recently published our work entitled “Transferrin
conjugated nontoxic carbon dots for doxorubicin delivery to target pediatric
brain tumor cells” in the journal Nanoscale. Our volunteer and Nicklaus Children’s
Hospital chief resident Scott Raskin contributed significantly to this
publication by performing many of the cell viability assays on our pediatric
brain tumor cell lines. Given the positive results from our initial carbon dot studies,
we are working with the LeBlanc’s lab to generate multidrug and multi-ligand
(able to bind to and enter cancer cell by multiple mechanisms) conjugated
Congratulations to Megan Marlow and Sumedh Shah for their recent
publication “Treatment of adult and pediatric high-grade gliomas with
Withaferin A: antitumor mechanisms and future perspectives” which was
published the Journal of Natural Medicines. In this paper Megan and
Sumedh examine the anti-cancer mechanisms of Withaferin A and discuss the
effects on high-grade gliomas.
A belated congratulation goes to our medical student volunteers Zachary
Gersey and Sumedh Shah who tied for best abstract/poster at the 2016
Florida Brain Tumor Biomedical Technology Summithosted by the
University of Miami Brain Tumor Initiative. We would also like to congratulate
Zach Gersey who was accepted into the Neurosurgery residency program at the
University of Pittsburg, which is ranked among the top five neurosurgical
residency programs in the country!
We welcome our new volunteers; Dr. Maria Carter, Frederic (Freddy) A.
Vallejo, Denis A Ortega Ioni, Wanda Gonzalez, Katrina Kostenko and Ingrid
Torrens. Maria is a resident at Nicklaus Children’s Hospital and is working on
examining the potential of Resperidone and Quetiapine, both atypical
anti-psychotic drugs, as an adjuvant therapy for medulloblastoma treatment. She
is focusing on the molecular subgroup, Group 3 which is more common in infants
and children but rare in adults. This subgroup is MYC amplified and currently
has the worst prognosis. Maria has found that clinically relevant
concentrations significantly reduce medulloblastoma cell proliferation. She is
presenting her findings at the American Society of Hematology and Oncology
(ASPHO) annual meeting in April.
Freddy along with Nico De Cordoba have been examining ways to target
the metabolic pathway in cancer. They are looking at combining glycolytic inhibitors
with the anti-diabetic drug Metformin. Metformin is known to lower blood
glucose but may also have direct anti-cancer on the tumor cell. Additionally
they are exploring the potential of a ketogenic diet. Originally developed for
children with epilepsy, this high fat, and very low carbohydrate diet forces
the body to burn fat instead of glucose to generate energy. Cancer cells prefer
to use glucose to generate energy. The use of glucose by cancer cells also
prevents oxidative damage by detoxifying reactive oxygen species. Ketogenic
diets, therefore, may act as a cancer therapy by severely limiting glucose
availability and by inducing oxidative stress within the cancer cell. The ketone bodies acetoacetate (AA) and b-hydroxy buterate
(bHB) are produced by the liver from fatty acids of an individual on a
ketogenic diet and may have direct anti-cancer effects. Freddy and Nico have
been evaluating their effects on brain tumor and neuroblastoma cells. Freddy
presented our results on brain tumor cells at the 2017 Miami Winter Symposium
in January and at the International Pediatric Neuro-Oncology / Florida Brain
Tumor Summit in March. Nico will present our findings on neuroblastoma at
Denis, Wanda, Ingrid and Katrina are Miami-Dade College students. Denis
and Wanda started in the lab last summer and have been working on the curcumin
analog project and will be determining the cytotoxic effects of the novel
hybrid compounds developed by Dr. Velez. Ingrid and Katrina just started in the
laboratory and will be working on targeting tumor cell metabolism. Freddy,
Denis, Wanda, Ingrid and Katrina will be presenting their data at the Life
Sciences South Florida STEM Undergraduate Research Symposium at Palm Beach
State College in April.
Lastly we would like to acknowledge our collaborators, Dr. Roger
LeBlanc, Dr. Zhili Peng and Dr. Eduardo Véliz from the department of Chemistry at UM. As
well as Dr. Brian Marples, Professor of Radiation Oncology at UM. Dr. Marples
has been collaborating with us on a project centered on radio sensitizing
cancer stem cells. We thank Dr. Guillermo De Angulo and Winston Walters for
their contribution to our efforts. We are forever grateful to our
generous supporters who provide the much needed funding critical for our
By, Dr. Regina Graham
There have been many exciting
things happening in the research laboratory that I want to share with you. First of all, a belated congratulations to
our Nicklaus Children’s Hospital volunteer Claudia Zapata who won for best
original research abstract at the 7th Annual Residents and Fellows’
Scholarship Day last June. Claudia also
matched at Children’s Hospital of Philadelphia (CHOP). She will be starting her
pediatric hematology/oncology fellowship in July and will be working with Dr.
John Maris who specializes in treating children with neuroblastoma. We would
also like to congratulate our new volunteer Amelia Bahamonde who won first
place at the Life Sciences South Florida STEM Undergraduate Research
Symposium! Amelia is a University of Miami (UM) undergraduate who has been
working in the lab since last summer. In addition, Amelia was one of only six UM
students selected to attend the Annual Atlantic Coast Conference Meeting of the
Minds in Syracuse, NY. Congratulations to our second year medical student,
Gregor Rodriguez who won an honorable mention at the
Eastern-Atlantic Student Research Forum (ESRF)! The ESRF is an international
meeting held each year at the University of Miami were selected medical,
graduate, M.D./Ph.D. students, and resident physicians present original basic
science and clinical research in multiple biomedical fields. Claudia, Gregor
and Amelia all work on projects focused on determining the efficacy and the molecular
mechanisms of Withaferin A, the main constituent of the plant Withania
somnifera, for neuroblastoma treatment. We have found that withaferin A
promotes neuroblastoma cell differentiation by down-regulating genes important
for cancer stem-cell self-renewal. Currently retinoids (13-cis-retinoic acid) are
used for cancer cell differentiation therapy, however their use can be limited
due to toxicity and acquired resistance.
We found that withaferin A not only promotes cancer cell differentiation
but potentiates the effects of the retinoids indicating that perhaps lower doses
of retinoids can be used in conjunction with withaferin A.
Congratulations to our first year
medical student, Sumedh Shah for winning the Alpha Omega Alpha medical honor
society 2016 Carolyn L. Kuckein Student Research Fellowship! In an
ongoing collaboration with Drs. Roger LeBlanc and Shanghao Li in the department
of chemistry at the University of Miami (UM), we have been examining the
potential of using novel transferrin conjugated chemotherapies. Brain tumors are
notoriously difficult to treat in part due to the lack of effective agents able
to cross the blood brain barrier (BBB). The BBB is a cellular barrier between
the blood and the brain environment that regulates the entry of substances into
the brain tissue, such as glucose or amino acids and serves to protect the
brain from pathogens and other potential neurotoxins. However, this “barrier” also
prevents many anti-cancer drugs from entering the brain from the blood. One
method to help drugs cross the BBB is to take advantage of the transferrin
receptors present on the cells that constitute the BBB. Transferrin is
iron-binding proteins that modulate the free iron in the blood. When a chemotherapy
such as doxorubicin (also know as Adriamycin) is attached to the transferrin
(TF) molecule, it can pass through the BBB to reach the tumor. Since brain
tumor cells have increased levels of transferrin receptors, the
chemotherapy-transferrin molecule is more likely to enact its effects on the
cancerous cells. Dr. Li has added a carbon dot (C-Dot) to the doxorubicin-transferrin
molecule (Dox-C-Dot-TF). C-Dots are novel non-toxic nano-particles that can be
made from any carbon source such as bread or orange juice, for example. The addition
of the C-dot would allow a wider range of drugs to be conjugated to
transferrin, and because c-dots are fluorescent we can visualize transferrin
conjugate inside cells. Dr. Scott Raskin, a resident at Nicklaus Children’s
Hospital who is volunteering in the laboratory, demonstrated that treatment of
pediatric brain tumor cell lines (medulloblastoma, ATRT, and glioblastoma) with
the Dox-C-Dot-TF conjugate was just as effective as the doxorubicin alone
indicating that doxorubicin is still effective when conjugated to the C-Dot and
transferrin molecule. Scott will be presenting these findings at the American
Society of Pediatric Hematology Oncology (ASPHO) 2016 annual meeting. Scott was
selected to give an oral-platform
presentation on our exciting findings! Sumedh and Scott will now be
examining the potential of combining multiple chemotherapeutic agents to the
C-Dot-transferrin molecule. Combing agents that work together could
significantly increase the efficacy.
In addition to Amelia Bahamonde and
Dr. Scott Raskin we welcome Anthony Sanchez, Sarah Samuals and Beatriz Hawkins.
Anthony is a UM graduate who has been helping out on multiple projects
including examining the effect of curcumin on brain tumor stem cells. Anthony
has also been instrumental in teaching the student volunteers. Beatriz is an
undergraduate at Florida International University and has also contributed to
multiple ongoing research projects. Sarah has been examining the effects of
atypical antipsychotics on brain tumor cells. Currently there is a push to use
FDA approved drugs in new ways. Since antipsychotics already cross the BBB, and
certain antipsychotics can ease brain tumor patient’s symptoms, we sought to
determine if any of these might also have direct anti-cancer effects. Sarah will be presenting her findings at the Annual
Zubrod Memorial Lecture & Cancer Research Poster Session at UM. Sarah,
along with medical students Zachary Gersey and Gregor Rodriguez will be
presenting their work at the 2016 Florida Brain Tumor Biomedical Technology
Summit hosted by the University of Miami Brain Tumor klInitiative.
We have recently published our work
in the journal Experimental and Molecular Medicine. Below is the research
summary from the journal website and the link to the full publication.
Cancer: Combined therapy destroys neuroblastoma cells
Combined treatment with a sugar molecule and a chemical
found in red wine helps destroy cancer cells originating from immature nerve
cells. Found primarily in young children, neuroblastoma is an aggressive
disease in need of better treatment options. Most cancer cells use glucose as
their main source of energy, but the glucose analogue 2-deoxy-D-glucose (2-DG)
can block energy production. A team led by Regina M. Graham from the University
of Miami Miller School of Medicine in Florida, USA, tested whether resveratrol,
a compound found in grape skin, would augment the tumor-killing power of 2-DG
in cell culture. They found that the combined treatment was more potent than
2-DG alone at killing cells from human neuroblastoma. Resveratrol decreased the
activity of a key survival enzyme called Akt, thereby increasing 2-DG induced
cellular stress pathways, which ultimately led to cell death. Link to
We would like to thank Dr.
Guillermo De Angulo, Dr. John Thompson, Dr. Ricardo Komotar, Winston Walters, Dr.
Michael Ivan and Nadia Myrthil for their contribution to our research efforts.
We would also like to thank our collaborators; Dr. Roger LeBlanc, Dr. Shanghao Li,
Dr. Eduardo A. Véliz and Lorenzo Sansalone at the UM department of Chemistry and
Dr. Jeffrey Prince at the UM department of Biology.
All of this work has been made
possible from the generous research grants from the Mystic Force Foundation. We would like to thank everyone who has
donated to the Mystic Force Foundation. Your generous gifts bring us one step
closer to achieving our goal of finding more effective, less toxic therapies
for children with cancer.
By, Dr. Regina Graham
In our continued collaboration
with Nicklaus Children's Hospital (formerly Miami Children’s Hospital MCH), Claudia Zapata, a pediatric resident at NCH has been working on elucidating the molecular mechanism of withaferin A induced
neuroblastoma cell differentiation. Differentiation therapy aims to mature the
cancer cells into a more differentiated phenotype with less growth potential.
Currently, retinoids are used to promote neuroblastoma cell differentiation,
however inherent resistance and toxicities hinder their effectiveness. We have found that withaferin A can promote
neuroblastoma cell differentiation, inhibit cell proliferation and limit
neuroblastoma stem cell renewal. Claudia will be presenting our findings on the
effects of withaferin A treatment on neuroblastoma at the 28
annual American Society of Pediatric Hematology/Oncology (ASPHO) meeting taking
place in Phoenix AZ, in May.
Cosme-Blanco, another NCH
resident, who is working in the laboratory, is currently examining the
antitumor effects of activating or inhibiting neuroendocrine signaling in
neuroblastoma. Neuroblastoma is a neuroendocrine tumor and as such may be
sensitive to neuroendocrine regulators. It has previously been demonstrated that gastrin-releasing
peptide, luteinizing hormone-releasing hormone, growth hormone releasing hormone, and somatostatin
can regulate tumor growth. Wilfredo has found that affecting neuroendocrine
signaling can significantly reduce neuroblastoma cell proliferation.
Congratulations to University of Miami medical student Eric
Barbarite who won Best Poster Award at the Miami Winter Symposium: Towards
Personalized Cancer Medicine (January 18-21, 2015). Eric presented our findings
on the radio- sensitization of glioblastoma stem cells via autophagy inhibition.
The cellular process known as autophagy, meaning self-digestion can be a
survival mechanism and can direct cell fate in response to stresses such as
radiotherapy. Eric found that quinacrine, also known as Mepacrine, effectively
inhibited autophagy and increased glioblastoma stem cell death in response to
radiation. Radiation remains a mainstay for the treatment of solid tumors, such
as brain tumors. Understanding how the brain tumor stem cells survive
radiotherapy provides us with therapeutic targets in order to increase the effectiveness
of radiation therapy. Eric will also present our work at the Young Neurosurgeon's Forum
for the American Association of Neurological Surgeons (AANS) annual meeting in
Washington DC this May.
also congratulate Sumedh Shah for being selected to represent the
University of Miami at the 9 annual Atlantic Coast Conference
(ACC) Meeting of the Minds Research Conference at NC State University (April
10-12, 2015). Sumedh is currently in the University of Miami Honors program in
Medicine and has been contributing to multiple ongoing research projects by
investigating the ultrastructure of brain tumor stem cells in response to
different therapies by electron microscopy under the direction of Dr. Jeffery
Prince, an associate professor in the department of Biology. Using electron
microscopy we can analyze the structural diversity and dynamics of cancer
cells. Sumedh is now examining the potential of inhibiting the glycolytic
pathway to eliminate the brain tumor stem cells. Sumedh examined multiple
patient-derived glioblastoma stem cell lines and found that the mitochondria,
sometimes referred to as “the powerhouse of the cell” were abnormal suggesting
that these cells may rely primarily on the use of glucose to generate the
energy for the cell. Sumedh has presented his finding at the Research, Creativity, and Innovation Forum
at the University of Miami in March and will also present our research at the
AAC meeting in April.
We would also like
to welcome our new volunteers. Gregor Rodriguez, a first year University of
Miami medical student has been coming to the laboratory regularly and has
contributed to multiple ongoing projects. Gregor has found that treating brain
tumor stem cells with low doses of curcumin, isolated from the spice turmeric,
significantly reduces cell proliferation and neurosphere formation, an
indicator of ‘stemness”. Nicolas (Nico) de Cordoba, MCH patient and a childhood cancer survivor, is an undergraduate at
Springhill College. Nico is looking at ways to target nuclear factor
erythroid 2 [NF-E2]-related factor 2 (nrf2). Nrf2 is increased in many cancers
and correlates with a poor prognosis. In addition, it contributes to
chemotherapeutic and radiation resistance by promoting the expression of antioxidant
and detoxification genes. Inhibiting nrf2 may potentially increase the efficacy
of current cancer treatments.
We recently submitted our research on targeting the glycolytic
in neuroblastoma for publication. One way to contribute to
cancer research is to present your data at local, national and internationally
attended scientific meetings. Another way is to publish your findings in
peer-reviewed journals. Lastly, we want to thank our continuing volunteers,
Nadia Myrthil and Zach Gersey for all their hard work. We thank Winston
Walters, Dr. Amade Bregy, Dr. Ricardo Komotar, and Dr. Guillermo De Angulo, for
their contribution to our research efforts.
By Dr. Regina Graham
Congratulations to Dr. Branko
Cuglievan who won first prize in
the 6 Annual Miami Children’s Hospital residents and fellows
research day. His research focuses upon targeting Nicotinamide adenine dinucleotide (NAD), which
is a coenzyme
found in all living cells.
NAD is synthesized in the cell by two pathways, from either amino acids (de novo pathway) or metabolites (salvage
pathway). Studies have shown that cancer cells rely heavily on the salvage
pathway of NAD formation. A key enzyme in this pathway is nicotinamide phosphorribosyl
transferase (NAMPT). NAMPT inhibitors are in development and have been used in
clinical trials. NAD is an important cofactor for the glycolytic pathway, which
is increased in cancer, the activation of PARP (DNA repair protein) and
sirtuins or SIRTS (cellular stress response proteins). Work by Dr. Fiorela Hernandez,
a former volunteer from Miami Children’s hospital, and others in the lab have
demonstrated that SIRTs play an important role in mediating cancer cell
resistance to chemotherapeutic agents. We hypothesize that inhibiting NAMPT may
impact multiple cellular processes important for cancer cell survival and
resistance to therapies. We have recently received a grant from Cancer Free
Kids to help fund these studies examining the role of NAMPT in mediating
chemoresistance in neuroblastoma.
We welcome our new volunteer Dr. Wilfredo
Cosme-Blanco, currently a resident at Miami Children’s Hospital. In addition to
having his MD degree, Wilfredo also has a Ph.D. from the University of Texas
where he studied the role of telomere dysfunction, DNA damage response and p53 mutations
in cancer as well as aging.
We would like to thank our summer volunteers for
all their hard work. Daniel Benito, a second year medical student from Drexel
University who examined the potential of inhibiting autophagy in order to
sensitize cancer cells to chemotherapy and radiation therapy. Autophagy, from the Greekauto-, "self" and phagein, "to eat",
the process whereby cells degrade unnecessary or damaged cellular components. Autophagy is now
recognized as a cellular process that plays an important role in maintaining
cellular homeostasis in response to nutrient deprivation, chemotherapy and
ionizing radiation therapy (radiotherapy). Studies have shown that inhibiting
autophagy can greatly increase the effectiveness of therapeutic agents. Daniel
found that the potent anti-malarial drug quinocrine was more effective than
chloroquine, an agent currently used in clinical trials to inhibit autophagy. Alex
undergraduate from Brandeis University who worked on developing techniques used
to assess the potential of withaferin A, a natural compound isolated from Withania somnifera, as a differentiating agent in neuroblastoma.
Alex found that very low levels of withaferin A promoted neuroblastoma cell
differentiation and decreased tumor formation in an in vitro assay. Neal Patel,
a University of Miami (UM) undergraduate evaluated the levels of a novel
protein in brain tumor samples. Together with Dan Benito, he optimized a
protocol for immunostaining frozen tissue specimens. He found that the degree
of staining was higher in the high-grade astrocytoma compared to low-grade
astrocytoma. We are currently determining if this protein is a valid therapeutic
target for the treatment of high-grade brain tumors. Eric Barbarite, a second
year UM medical student, pioneered our research centered on understanding why
cancer stem cells are often resistant to radiotherapy and finding agents that
sensitize these cells to radiotherapy. Neil Soni, another UM undergraduate
student focused on targeting 3- phosphoinositide dependent protein kinase 1 (PDK1)
in cancer stem cells. PDK1, a master kinase, is often overexpressed in cancer. Eric
Barbarite, Neal Patel and Neil Soni will continue with us through the fall (and
hopefully longer). Lastly, we would like to welcome Aura Ordonez, a Florida International University undergraduate student.
We continue to present our findings
at local, national and international meetings. Branko Cuglievan presented our
findings on the role of sirtuins in chemoresistence in neuroblastoma at the 27
annual meeting of the American Society of Pediatric Hematology and Oncology
(ASPHO) meeting in Chicago (May 2014) and Fiorela Hernandez also presented the
finding at the Advances in Neuroblastoma Research meeting also in Cologne
Germany (May 2014). In addition we will be presenting additional research
findings at the AACR specialized meeting “Targeting the PI3K-mTOR Network in Cancer” in September, the Congress of Neurological Surgeons (CNS) 14 annual meeting
in October, and the 19th
Annual Scientific Meeting and Education Day of the Society for Neuro-Oncology
(SNO) in November.
we would like to thank our volunteers, Zach Gersey and Nadia Myrthil and UMBTI
fellows Dr. Baoyu Zhang and Dr. Ahmad Alshareef for their continued research
efforts. In particular we would like to thank Dr. Zhang for his help in
teaching the newer volunteers. We would also like to thank UMBTI members Dr.
Amade Bregy, Dr. Ashish Shah, Winston Walters and Dr. Ricardo Komotar for their
continued support. Finally we thank Dr. Guillermo De Angulo (Miami Children’s
Hospital) for his continued input into our research endeavors as well as Dr.
John Thompson for his intellectual input
and editorial support.
By, Dr. Regina Graham
our work on therapeutic targeting cancer stem cells. Cancer stem cells are the
driving force of the tumor and are often resistant to standard chemotherapy
agents. We are currently examining the effects of different therapeutics on
neuroblastoma, medulloblastoma and glioblastoma stem cells. We have found that Withaferin
A; a natural product derived from the medicinal plant Withania
Somnifera, is cytotoxic to cancer stem cells but not to normal cells. Withania Somnefera, also know as
ashwagandha or Indian ginseng, has been used in Ayurveda (one of the worlds
oldest medical systems originating in India) for centuries. We have found that Withaferin
A induces cellular stress pathways and programmed cell death. Furthermore Withaferin
A can potentiate the effects of some chemotherapeutic agents. Dr. Baoyu Zhang
recently presented these findings locally at the University of Miami Cancer Symposium and at the university
wide research symposium, Canesearch. We will also present our results at the
105 annual American
Association for Cancer Research (AACR)
meeting in April.
to Raisa Uddin! Raisa was chosen (as one of only ten students!) to represent
the University of Miami at the 9 annual Atlantic Coast Conference
(ACC) Meeting of the Minds in April. Raisa is a UM undergraduate and as part of
her honors thesis project has been doing research focusing on the AKT pathway
in glioblastoma. She will present our results on targeting the AKT pathway in
glioblastoma stem cells. Glioblastoma is a type of brain tumor that arises from the brain’s
supportive tissue (glial cells) and unfortunately the prognosis for children
(and adults) diagnosed with this disease is poor. These tumors are very
aggressive and even with surgery are difficult to treat. The AKT pathway is upregulated
in many cancers and often negatively influences prognosis. Recent evidence
suggests that AKT plays a key role in cancer stem cell biology. In cancer, AKT
can be activated by activation of receptors on the cell surface or within the
cell by activation of intracellular molecules. Using patient derived glioblastoma
stem cell lines generated in the lab, Raisa tested various inhibitors of the
AKT pathway targeting proteins both upstream and downstream of AKT to determine
at which point in the AKT pathway is most critical for glioblastoma stem cell
We would like to mention that Zach Gersey, a second
year UM medical student volunteer in the laboratory, will be featured in the
latest issue of Medical IBIS, the University of Miami Miller
School of Medicine's publication highlighting medical students and their
research. Zach has been working on determining how curcumin induces
glioblastoma stem cell death. Lastly we would like to thank all of our
volunteers for their had work as well as Dr. Amade Bregy and Dr. Ricardo
Komotar for their continued support of our research efforts.
By, Dr. Regina Graham
We have recently moved into our
new laboratory space and have joined forces with the University of Miami Brain
Tumor Initiative (UMBTI). The new laboratory is located in the Lois Pope Life Center,
which gives us access to state of the art equipment for conducting cutting edge
research. A goal of the UMBTI is to translate advances made in the laboratory into
treatments for brain tumor patients.
While we continue our efforts on neuroblastoma, we have expanded
research into other areas of childhood cancers including gliomas and
medulloblastoma. Brain tumors are the second most common cancer in children particularly
in young children. Blood cancers, such as leukemia, account for 1/3 of all
childhood cancers while brain tumors account for slightly more than 20%. We are
working towards being able to collect both brain and spinal cord tumors. Spinal
cord tumors can occur in children of any age but are more frequent in older
children. We will bank (store) these tumor specimens, perform molecular
analysis and grow tumor cell lines. Molecular analysis will provide insights on
potential therapeutic targets and the cell lines provide a vehicle to examine
the efficacy of novel, FDA approved and natural products in inducing cancer cell
research focus of the lab has been on tumor stem cells. Recent evidence
suggests tumors contain a small population of cells capable of driving tumor
growth termed tumor stem cells. The tumor stem cells are resistant to
chemotherapy and radiotherapy and are hypothesized to be responsible for tumor
regrowth following treatment and subsequent patient relapse. So while
conventional treatments aimed at targeting the bulk of the tumor may induce
tumor shrinkage, cancer stem targeted treatments will eliminate the driving force
of the tumor, ultimately leading to tumor demise.
say farewell to Dr. Fiorela Hernandez who is now in a fellowship program at the
University of Texas, MD Anderson Cancer Center. We would also like to wish her
belated congratulations on winning first place (again!) in the 5 Annual
Miami Children’s Hospital residents and fellows research day. Her work focused on sirtuins as a novel drug
target for neuroblastoma. Sirtuins are histone deacetylases, which have been
implicated in neurodegenerative diseases such as Alzheimer’s
and Huntington’s disease and in cancer cell survival. In neuroblastoma, we have
found that neuroblastoma cancer cells activate sirtuins in response to chemotherapy
leading to cancer cell protection. Blocking sirtuin activation was found to increase
the therapeutic potential of chemotherapy. We have presented our sirtuin findings
at several internationally attended scientific meetings including the American
Society of Pediatric Hematology/Oncology (ASPHO) 26 annual meeting
and the 104 annual American Association for Cancer Research (AACR)
welcome our newest volunteer, Dr. Branko Cuglievan who is
currently in the pediatric residency program at Miami Children’s Hospital. Dr.
Cuglievan will continue Dr. Hernandez’s work as well as examine ways to target
neuroblastoma and brain tumor stem cells. We also welcome UMBTI/MFF volunteers:
Zachary Gersey a University of Miami medical student (class of 2016), Kelechi
Ohaeto a Miami Dade College undergraduate and a Bridge to Baccalaureate
scholar, and Raisa Uddin and Nadia Myrthil both undergraduates. Zach is exploring
cell proliferation and survival signaling in tumor stem cells, Kelechi is
examining the potential of curcumin in targeting glioblastoma stem cells, Raisa
is determining the potential of targeting the PI3K/AKT cell survival pathway in
brain tumor stem cells, and Nadia is studying the role of aquaporins in brain
tumor pathophysiology. We have found that curcumin, a component of the spice
tumeric, is a potent inducer of glioblastoma stem cell death. Kelechi will
present these findings at the Annual Biomedical Research Conference for
Minority Students (ABRCMS) in November in Nashville, TN. Zach will present his
findings elucidating the molecular mechanism by which curcumin induces tumor
stem cell death at the 2014 Miami Winter Symposium, The Molecular Basis of Brain
Disorders in January.
We would also like to thank
UMBTI fellows Dr. Baoyu Zhang, Dr. Amad Alshareef, and Dr. Kyriakos
Papadmitriou for their technical assistance and intellectual input on ongoing
research projects. We also would like to thank Winston Walters for his
technical expertise, and lastly thanks to Dr. Ricardo Komotar and Dr. Amade
Bregy for their contributions to our research.
By, Dr. Regina Graham
March / 2013
Belated congratulations to out
volunteer Dr. Fiorela Hernandez who won first prize for the category of
Resident Abstract for Original Research in the 4th annual Miami
Childrens Hospital Research Day. Fiorela presented her research centered on
targeting metabolic and cell survival pathways in neuroblastoma.
One of our research projects is
focused on a novel drug target for neuroblastoma. We have found that neuroblastoma
cancer cells are sensitive to drugs that inhibit sirtuins. Sirtuins (or SIRTs)
are proteins capable of regulating the activity of other proteins as well as
gene expression. SIRTs belong to a family of proteins known as histone deacetylases
or HDACs. Together with histone acetyltransferases or HATs they regulate the
level of histone acetylation and the level of gene transcription. HDACs remove
acetyl groups from histones causing the DNA to wind tighter around the histone
and decrease gene expression. HATs do the opposite, they add acetyl groups to
histones causing the DNA to unwind and increase gene expression. Drugs that
target HDACs will alter gene transcription and therefore can affect multiple
aspects of the cells behavior including cell proliferation and cell survival. We
have found that blocking SIRT activity promotes neuroblastoma cell death and
enhances the efficacy of chemotherapeutic agents. Inhibiting SIRTs is emerging
as a promising anticancer strategy. We will be presenting our findings at the
American Society of Pediatric Hematology Oncology (ASPHO) and at the American
Association of Cancer (AACR) annual meetings.
have also established a new collaboration with Dr. Ricardo Komotar, Dr. Amade
Bregy and Ashish Shah of the Department of Neurosurgery in the development of
the University of Miami Brain Tumor Initiative. Dr. Komotar has spearheaded this
effort and we currently are able to bank patients brain tumors, grow patient
derived cell lines, perform molecular analysis of the tumors and test both
novel and FDA approved drugs on the patients cell lines. Identifying and
understanding the role of specific gene mutations or amplifications in a
patient’s tumor is the first step toward developing a more personalized
treatment for the patient. Identifying patients who will not respond to
standard therapy could allow them to be treated with alternative and/or
additional treatments. We are now working toward establishing collaboration
with Dr. Sanjiv Bhatia and Dr. John Ragheb at Miami Childrens Hospital to do
the same with medulloblastoma. Medulloblastoma is a fast growing high-grade brain
tumor primarily affecting young children. It is responsible for about 25% of
all pediatric brain cancers.
Lastly we would like to acknowledge
our volunteers: Jayanti Singh who spent several months with us and is now
working with Dr. Yanbin Zhang; Neal Patel, a University of Miami undergraduate who
spent the summer working in the lab, and Dr. Fiorela Hernandez, from Miami Children’s
Hospital, who has been with us almost two years now.
By, Dr. Regina Graham
Cancer remains the number one disease killer of children.
Although the overall 5-year survival rates have improved, one in four children
diagnosed with cancer will die and those that survive often suffer from the
damaging side effects of current treatment; including heart, lung, and liver
damage, secondary cancers, impairments in growth and development and
infertility. Previously we have focused solely on neuroblastoma but now we are
expanding our research efforts to include additional childhood cancers. Our
goal however, remains the same: find
more effective less toxic therapies for children diagnosed with cancer. By
indentifying therapeutic targets that are unique to tumor cells we can spare
normal cells and thereby reduce side effects. Our research currently focuses on
cancer cell metabolism, which differs fundamentally from that of non-cancer
cells. We have just presented our recent findings at the AACR (American
Association of Cancer Research) meeting in Chicago on April 3, 2012. The work presented
was very well received and we believe that cancer’s unique cellular metabolism
may very well be its’ achilles’ heel. We will publish our findings in cancer
research journals very soon.
In addition Dr. Guillermo De Angulo, a pediatric oncologist
at Miami Children’s Hospital and a part of the Mystic Force Foundation, is working
on joining a multicenter clinical trial using lenalidomide (Revlimid) for
patients with relapsed cancers. As well as having anti-blood vessel forming
properties lenalidomide is an immunomodulatory agent. Dr. De Angulo previously
found that higher absolute lymphocyte counts correlated with overall survival. A
lymphocyte is a type of white blood cell and includes T, B, and natural killer
(NK) cells. Lenalidomide has been shown to activate T cells and NK cells and
increase the levels of immune effectors. The hope is that lenalidomide will increase
immune function, tumor cell death and patient survival.
Another project in the laboratory is to examine the
potential of NK cells to kill tumor-initiating cells (also known as cancer stem
cells). It has been hypothesized that cancer relapse is due to remaining cancer
stem cells following therapy. We are examining ways to enhance NK cell mediated
cancer stem cell death. Dr. Dean Lee at MD Anderson Cancer Center, kindly
provided us with NK cells as well as protocols for the isolation and expansion
of NK cells from the peripheral blood.
We would like to thank our volunteers; Dr. Nataly Puerta MD,
Dr. Fiorela Hernandez MD, Sravana Paladugu and Ashima Madan. Nataly worked full
time with us last fall and now wants to continue her studies in the biomedical graduate program (PIBs) here at UM. Fiorela is in the
pediatric residency program at Miami Children’s Hospital and joined the laboratory
in order to gain research experience. Sravana and Ashima are undergraduate
students at the University of Miami; Sravana volunteered last fall and Ashima will
be joining the laboratory this spring. Lastly we thank Dr. Keith Webster for
use of lab space and equipment, and for his intellectual input into some of our
on going projects.
By Dr. Regina Graham
We recently presented our findings
on the effects of 2-deoxy glucose (2-DG) on neuroblastoma cell viability at the
AACR 102nd Annual meeting 2011. More and more research today is focused
on understanding tumor cell metabolism and finding novel potential targets. It
is now recognized that the cancer cell’s reprogramming of energy metabolism is
an important step in tumor development. In the landmark paper D. Hanahan, &
R. A. Weinberg: (2000) The Hallmarks of Cancer, Cell, Vol. 100, pp. 57-70,
Hanahan and Weinberg described six fundamental attributes, which drive normal
cells to become cancerous. These include
insensitivity to growth arrest signals
of growth signals
to avoid apoptosis (cell death)
to create their own blood supply by inducing the formation of blood vessels
to invade and metastasize
Hanahan and Weinberg recently added
(2011), along with the cancer cells ability to avoid immune surveillance, the
reprogramming of cellular metabolism from one using oxygen (oxidative
phosphorylation) to one using glucose (glycolysis) in order to better support
the high replicative potential of cancer cells. Many talks at the meeting were
centered on dissecting the molecular pathways involved in tumor cell
metabolism and finding points for therapeutic intervention. The reliance on
glucose may have developed when the growing tumor outgrew its blood supply and
as such its oxygen supply. Cells using glucose were able to survive this hypoxic
microenvironment. Glucose not only provides ATP (energy) for the tumor cells
but carbon, and hydrogen which are necessary components along with nitrogen
derived from glutamine for the production of DNA, RNA and proteins. In addition
glycolysis leads to the production of lactic acid, which is excreted from the
cell and acidifies the extracellular microenvironment leading to increased drug
resistance and enhanced cancer cell invasion. We have found that 2-deoxy glucose induces neuroblastoma
cell cycle arrest and cell death. Neuroblastoma cells take up 2-DG but are
unable to fully metabolize it. Combining 2-DG with inhibitors of AKT can
potentiate neuroblastoma cell death. AKT is a protein kinase plays a key role
in multiple cellular processes including glucose metabolism as well as cell
proliferation, and the avoidance of apoptosis. We found that neuroblastoma
cells increase the activity of AKT in response to 2-DG treatment and inhibiting
AKT activity increases 2-DG-induced neuroblastoma cell death by an additional
50%. AKT inhibitors have shown promise for neuroblastoma treatment and are
currently in clinical trials.
have recently partnered with Antonello Podda, M.D., Director of Pediatric
Neuro-Oncology and Assistant Professor of Pediatrics here at the University of
Miami and Guillermo R. DeAngulo, M.D., Hematologist-Oncologist at Miami
Children’s Hospital and are currently discussing potential clinical trials for
neuroblastoma. We are thrilled and honored to have them on our Medical Advisory
Board for the Mystic Force Foundation and the fight against neuroblastoma. We
are currently determining which novel drug would be best for a neuroblastoma
clinical trial to be conducted here in Miami.
would like to congratulate our research assistant Constantinos Barth who was
recently accepted to Nova Southeatern University and will be starting a masters
program in biomedical sciences from which he will transition into their medical
school. We wish Constantinos all the best. Again we thank Dr. Keith Webster and
his lab members as well as Dr. John Thompson for all their help and support.
By, Dr. Regina Graham
Treatment for high-risk neuroblastoma remains a major
clinical problem. Despite aggressive multimodal therapy including multiple
rounds of chemotherapy, surgery and radiation, the prognosis for these children
remains very poor. We continue our efforts to find effective, less toxic
therapies for the treatment of neuroblastoma.
We have presented our research on cepharanthine at both
local meetings at the University of Miami, Miller School of Medicine as well as
internationally at the Advances in Neuroblastoma Research in Stockholm, Sweden.
At each of these venues our research was well received. We hope to publish our
findings in scientific journals very soon. We have found that clinically achievable concentrations of
cepharanthine enhanced the toxicity of several commonly used chemotherapy
agents by reversing a cancer survival mechanism called multidrug resistance. Multidrug
resistance refers to the ability of cancer cells to develop resistance to a
wide range of structurally and functionally unrelated chemotherapeutic drugs
and has been a major obstacle to the success of cancer chemotherapy. To better
understand the mechanisms of multidrug resistance in patients, we are
generating multidrug resistant neuroblastoma cell lines in the lab.
Understanding how the tumor cells develop resistance will allow for the finding
of additional therapeutic targets.
Another way to attack neuroblastoma cells is to target their
metabolism. We are currently investigating the potential of the glucose analog,
2-deoxyglucose. Cancer cells have higher rates of glycolysis than normal cells.
This observation was made by Otto Warburg who won the Nobel Prize in 1931 and
is known as the Warburg effect. The high rate of glucose metabolism
(glycolysis) by cancer cells is the basis for Positron Emission Tomography (PET) scan which uses a radiolabeled
glucose analog to image tumor burden in patients. This is also the basis for
the development of 2-deoxyglucose as a cancer therapy. Tumors take up the
2-deoxyglucose but are unable to metabolize it to get the energy needed to
grow. We are investigating ways to potentiate the effects of 2-deoxyglucose in
neuroblastoma. By understanding the cell signaling pathways induced by
2-deoxyglucose treatment, we can identify therapeutic targets. We have found
that cells exposed to 2-deoxyglucose upregulate several survival pathways and
by selectively inhibiting those pathways we can significantly increase
neuroblastoma cell death.
We are happy to announce that Constantinos Barth will be
joining the Neuroblastoma Research Group full time. Constantinos volunteered in
the lab this past summer and became quite skilled in the techniques commonly
used in the laboratory.
Finally, we would like to thank Dr. Keith Webster for
allowing us to continue to use space and equipment in his laboratory as well as
all the members of the Webster lab. We also thank Dr. John Thompson for his
continued intellectual input and technical support.
January 30, 2010
By Dr. Regina Graham
The primary goal of this research group is to find novel and effective treatments for neuroblastoma. We have a two-pronged approach to address this goal. One approach is to understand the molecular signaling pathways activated in neuroblastoma, which will allow the identification of possible therapeutic targets. The second approach is to address the immediate need for more effective neuroblastoma treatments. To this end we have amassed over 75 different drugs or reagents to test in our laboratory. These drugs include traditional chemotherapy agents used for neuroblastoma, as well as drugs developed for other diseases. In addition we are evaluating the potential of herbal medicines to supplement or potentiate the cytotoxic effect of traditional chemotherapy regimens.
The investigation of “off-label” use of various drugs in the treatment of cancer is not new. We are currently evaluating the potential of drugs approved for other cancers as a neuroblastoma treatment. For example, bone and bone marrow are common sites of metastasis in neuroblastoma; therefore, we are currently examining some of the drugs that are approved for multiple myeloma, a cancer of the bone marrow plasma cells. Besides similarity in the location, both myeloma and neuroblastoma cells can activate osteoclasts which can lead to bone lesions. Another off-label drug we have looked at are the proton pump inhibitors. Proton pump inhibitors are often used to reduce gastric acid production and are prescribed for gastroesophageal reflux disease (heartburn) and ulcers. However, these proton pump inhibitors can also inhibit a different type of proton pump, the vacuolar ATPase (V-ATPase). The V-ATPase is often upregulated in cancer cells and contributes to the maintenance of a neutral intracellular pH, especially in solid tumors. Inhibiting the V-ATPase can decrease intracellular pH and induce cancer cell death.
Cepharanthine, which is isolated from the plant Stephania Cepharantha Hayata, has been widely used in Japan to treat both chronic and acute diseases for years without serious side effects. We are currently investigating this plant extract as a neuroblastoma treatment. We have found that at higher concentrations, cepharanthine induces neuroblastoma cell death, and at lower concentrations cepharanthine sensitizes neuroblastoma cell lines to vincristine and other chemotherapeutic agents by reversing multi-drug resistance. We are presenting these findings at the American Association of Cancer Research (AACR) annual meeting taking place in Washington DC in April 2010. Researchers around the world attend this meeting to learn about the latest breakthroughs in cancer research.
We now have six established neuroblastoma cell lines as well as a primary neuroblastoma cell line derived from a bone marrow aspirate of a neuroblastoma patient. We use these cell lines for our drug screening assays. In addition we have several non-neuroblastoma cancer cell lines, as well as non-cancerous cell lines used for comparison. The ultimate goal of our drug testing is to evaluate the potential neuroblastoma treatment in an animal model. One of the neuroblastoma cell lines expresses firefly luciferase, which can be used for metastasis studies. Using the firefly-luciferase expressing neuroblastoma cell lines in combination with non-invasive bioluminescent imaging we will be able to monitor neuroblastoma tumor growth and metastasis. We are currently setting up different metastasis models with these cells. In one model, the luciferase-expressing cells are injected directly in the bone marrow, the most common site of neuroblastoma metastasis. In the other models the cells are injected into the vasculature via the mouse-tail vein or into the heart. In these models the cells travel throughout the body and metastatic tumors develop at multiple locations. Lastly, we have successfully grown primary neuroblastoma cells derived from bone marrow aspirates in both NOD/Scid mice and the less immunocompromised Nude mice. We are currently planning experiments to test cepharanthine and other reagents that have shown promise in our cell culture experiments, in animal models.
Finally, we would like to express our continued gratitude to Dr. Keith Webster for providing laboratory space and use of the hypoxia chambers, and to Dr. Billy Thompson who regularly contributes to animal experiments and general technical support. We would also like to thank Dr. Sarah Woodrow who helped carry out the injections of neuroblastoma cells into the bone marrow.
By, Dr. James Guest
Since the last research report there have been several developments that have established our research efforts more effectively.
Firstly, Dr. Regina Graham, Ph.D was appointed as an assistant professor to the Department of Neurological Surgery at The University of Miami. Currently , Dr. Graham is working full-time on neuroblastoma projects. Dr. Graham has a strong background in cancer research and is a wonderful asset to our team.
We have purchased mice of a highly immune compromised strain for tumor testing and some animals have been injected with tumor cells.
For most tumor testing in animal models, the tumor injections are made under the skin of the flank. This is a preferred method because it is easy to monitor the growth and size of the tumors visibly and by palpation. However, it is not a very relevant location for neuroblastoma which usually starts within the body near the spine and then metastasizes to the bone marrow. It is the tumor in the bone marrow which is especially difficult to eradicate and we would like to be able test our therapies in an animal model of bone marrow neuroblastoma.
We have entered into several agreements and collaborations to share molecules for testing, covering a wide spectrum of drug mechanisms. Several drugs have been tested in the cultures of neuroblastoma. Some of these have shown potent cell killing.
A key issue to understand is that there must be a good correlation between the drug concentration tested in the cell culture and the drug concentration that can be obtained in a patient. Typically drug levels are limited by side effects. So, while it may be possible to kill cells “in a dish”, at a certain dose or concentration it may not be possible to achieve that same concentration in a patient without harmful toxicity. In that case the drug may be ineffective.
One way to get around this problem is that, sometimes, if you combine drugs, they will be effective at lower concentrations. Drug combinations have also been a cornerstone of cancer therapy because the combination can target multiple mechanisms promoting cell death. This also helps reduce the emergence of resistance.
The scientific method to conduct these studies is to work out a concentration- cell killing curve for a drug by itself with e.g. a line of neuroblastoma cells. Once this is known for the drugs independently, then they can be combined to see if more effective tumor cell killing occurs with lower concentrations.
In this way various drugs can be screened against a tumor cell line and the most promising are then tested in animals which bear tumors from the same cell line. Because it is much more costly to conduct the animal testing it is important to pare down the list of test agents to those which appear most promising.
The animal testing also must be conducted to show a strong effect. Ideally the drug(s) will eliminate an established tumor of considerable size, or at multiple sites. While a size reduction in the tumor is encouraging, this does not predict a true remission.
A separate important issue in the treatment of neuroblastoma concerns the role of “cancer stem cells”. Such cells have been identified in several types of human cancer. Stem cells have several survival mechanisms that allow them to survive within the human body under conditions that are lethal to other cells. If a stem cell becomes altered so that its growth is uncontrolled, it may become a cancer stem cell. It appears that if these cells are not killed tumors will recur. We are trying to understand why bone marrow may harbor neuroblastoma stem cells so effectively. As mentioned in previous reports we think the low oxygen tension of the marrow makes the cells more resistant to killing. Another possibility is that the neuroblastoma stem cells receive signals from the bone marrow cells that robustly promote their survival.
By, Dr. James Guest
The core of our research endeavor is to grow neuroblastoma cells in culture and transplant them into mice to form tumors. Both steps are necessary in order to test treatments. By having tumors growing in mice, and also by cryopreserving them, we are able to start the experiments over if we lose the cell cultures, which is always a risk. Because our cultures are long term, and in a specialized chamber, we are constantly battling with culture infections.
We have now grown neuroblastoma cells from 10 bone marrow specimens. There is an important difference between the early and later specimens. The most recent specimens are less differentiated. They grow under conditions that are used to cultivate cancer stem cells, and we are testing for markers of “stemness”, such as CD 133. We believe there is significant value in testing Sal's and other childrens cells. Because they are specific to each childs genetic makeup, the tumor cells may contain a unique pattern of mutations that affect their growth and response to different types of therapy.
We've included two established cell lines we have recently received from the University of Vermont and will soon begin including tumor cells from other children currently in treatment.
Transplants of bone marrow 5 have consistently yielded tumors in nude mice, some of which have been metastatic. This provides a good model of Sal’s disease. We have been able to recover these tumors back to cell culture and regrow them. Following transplant of several animals with bone marrow 8, tumors formed rapidly, being visible at 8 days. Then, unexpectedly they regressed. We are waiting to see if the tumors grow again. Among the reasons we have considered for this regression are that the tumor was rejected by the immune system. This is unexpected in nude mice which lack several immune system components. Another possible cause is that the extremely rapid growth outstripped the blood supply, and caused the tumors to die from lack of nutrients. It is always important to pay attention to the unexpected in science, since such observations may lead the way to important new knowledge.
We also submitted a letter of intent to a cancer foundation to request funding. The research work is time consuming and the materials are quite expensive. Sustaining the research effort in the long run is likely to require several sources of support . Dr. Gina Graham, PhD is now working full time on the project. I continue to work on the project about 12-15 hours per week and Dr. Billy Thompson, PhD also dedicates time to this effort. Dr.Keith Webster, Professor of Cell and Molecular pharmacology continues to permit our use of the hypoxia chamber which is a key aspect of deriving and testing cell cultures. We would benefit from a skilled technician with strong experience in cell culture and molecular biology techniques.
Recently, Gina began an extensive series of drug tests using assays that measure cell death and change in rate of cell division. She selected several currently used cancer drugs. Several of these showed a minimal or no significant effect on cells under the testing conditions. However, a drug called MG-132 was able to kill a very large percentage of these cells in 48-72 hours. Gina confirmed that the death mechanism involved “apoptosis” and we visualized numerous cells under the microscope showing apoptotic cell death. Mg-132 is a proteasome inhibitor. This means it blocks the protein complex within the cell that breaks down “used” proteins. Inhibition of this complex leads to accumulation of some proteins such as p53 that promote cell death. MG 132 is not approved for use in humans, although a similar drug called Velcade is approved for multiple myeloma. We have performed preliminary tests with Velcade at different doses on the cell cultures, and at doses of 1 micromolar the cells are heavily killed. Gina also tested drugs that function by some of the alternate mechanisms attributed to MG-132 and Velcade such as calpain inhibition, but these drugs did not have substantial effects.
The cell killing by Velcade and MG-132 is very impressive, however, it is essential to verify that the drugs will work to kill neuroblastoma cancers in mice. We will study this over the next several weeks.
Once you start growing cancers in mice and then recovering them to cell culture and retransplanting it is necessary to confirm that you are still studying a human-derived neuroblastoma as there is a small chance of causing a mouse tumor. For this reason we are having BM 5 cells tested for the presence of the Y chromosome, which should be absent in the mouse cells since they are female.
These results with proteosome inhibitors are very encouraging. To cure these children, it may be necessary to use a combination of drugs, or other treatment methods since it is possible their cells could develop resistance to the proteosome inhibitors. We are very interested to see if these cells contain cancer stem cells. There is some evidence that such stem cells underlie several forms of human cancer, and that they may be highly resistant to conventional therapies.
By, Dr. James Guest
December 6, 2008
Regina Graham will be joining us at a 50% time commitment. She will be supported by the Mystic Force Foundation with an appointment in Neurological Surgery at the University of Miami. She is a PhD with extensive post-doctoral experience in molecular biology and cancer biology. Recently she has been studying breast cancer using a flourescent luciferase model which allows imaging of human-derived tumors living inside mice. She has been performing this work under the supervision of Dr. Keith Webster, a well-known scientist in the Department of Cellular & Molecular Biology at the University of Miami. We hope to adapt this techinique to neuroblastoma studies.
Over the past few weeks we have successfuly grown tumors in mice using a bone marrow derived from the fifth marrow biopsy. The tumors have appeared as metastases in lymph nodes and the spleen. Although the cells appeared highly undifferentiated, when recovered to cell culture, they showed evidence of neuron ganglion cell differentiation. This is encouraging and indicates that they have retained the potential to differentiate. We will test if the cells further differentiate in response to retinoic acid, an important drug used to stabilize neuroblastoma minimal residual disease. It is thought that differentiated neuron cells are less aggressive and divide rarely.
We are also starting to test the bone marrow-derived cell lines for markers that stem cells are present. This will help us determine if a sub population of tumor cells have properties that may allow them to evade many current therapies.
This recent work has been conducted jointly between Dr. Webster and Dr. Guest's labs. Some diagnostic studies were provided free of charge by the UM Department of Pathology. Assistance of Dr. Billy Thompson is deeply appreciated.
By, Dr. Steven Vanni; Neurosurgeon, Researcher & Sal's Dad
It has been one year since Salvatore was first diagnosed with Neuroblastoma. Being a neurosurgeon this was a diagnoses I never wanted to hear. I have had patients in the past with this disease and their outcomes, to say the least, were not good. I felt we needed to do something more.
When our research started we first focused on evaluating the therapeutic effects of the various treatments on Salvatore's bone marrow. As simple as this sounds, it takes hours a day to extract the cells from the bone marrow. The Neuroblastoma cells are separated from the normal cells and then under a microscope we evaluate the cancer cells to see which are dying & which are growing. We try to characterize the response to figure out the effects of the various agents he's been exposed to. One of the first things we learned is that these cells don't grow well in a normal oxygen environment. Just to keep the cells alive we had to borrow a special chamber to keep the oxygen at at a lower than normal level. This was a very interesting fact that led us to the conclusion that one way to perhaps kill these cells is to figure out how to increase the oxygen concentration they are exposed to. This low level of oxygen unfortunately can lead to the cells being infected by bacteria that tend to flourish in this environment. This has caused us to have to repeat these experiments on numerous occasions. To date all of this has been done in house, by Dr. Jim Guest, my friend and colleague who has donated all of his time to this. We initially had employed a research fellow, Nicholas, who worked very hard on this but, due to visa issues, which are a nightmare since 911 had to return back to South America. Currently we are injecting Sal's cells into mice to try to reproduce the cancer to establish a working model of his disease. From there we can test various therapeutic agents in a living model. We are looking into purchasing a new chamber to eliminate the infection problems we've been experiencing. But even something as simple as this, that doesn't allow in outside air and can maintain an environment of low oxygen costs thousands of dollars. We need to hire a full time research fellow whose sole purpose is to test various therapeutic agents on Sal's as well as other childrens cancer cells to develop less toxic treatments for our children. Also to implement the studies we think are promising, to analyze the data from these experiments and then to publish the information so it can be shared with other researchers. These endeavors are not without significant cost and funds are needed to employ the help we desperately need.
Countless hours are spent reviewing abstracts and papers that have shown some promise and analyzing the data to understand the mechanism which the therapeutic intervention may have shown benefit.
We have contacted many of these researchers but most tell a very similar story, it is too hard to get government funding, and drug companies aren't interested in curing 600 kids a year, it is not profitable for them. A sad but realistic truth. But there are ways around this, that's were we can all make a difference. We have been asking for proposals from other researches both here and abroad whose early work has shown great promise. By funding their studies and guiding their research we can gather crucial information to further the fight. This can help us set up treatment protocols to help our children today.
Some of this research can be done in house at the University of Miami, but a lot of it needs to be spread out at various institutions. Why should we fund this, you may ask, versus a drug company. Our goal is not to make a profit. It is saving the lives of these innocent children.
A recent proposal which we are reviewing, is to further a study that was recently published that uses a polio virus that has been altered to infect neuroblastoma cells and may be able to kill them. You may be wondering, why a polio vaccine, interestingly polio attacks nerve cells. Neuroblastoma is a cancer that grows from nerve cells, so by being able to infect nerve cells but only selectively killing the abnormal ones, is a unique and promising concept. Early studies have shown a surprising response, but little available funding has halted this project, we would like to get it restarted. What does it take? Money, the 2 year cost just to further the testing in mice models is about 80,000 dollars a year. This is just one example. This doesn't include toxicology testing and the countless other studies needed to bring this to clinical use. Antibody treatments are also very promising similar to one that Sal is currently receiving at Sloan Kettering in New York. But these are made from mice which can only be used for a short time until the child becomes immune to it due to the portions made from mice. The estimated cost to make a humanized version is approximately 2 million dollars. It doesn't make financial sense for a drug company to make it.
We can make a difference, from the generous support by people like you. We can gain crucial information that can help us cure this deadly disease. We can support institutions which are showing promising results. We can initialize studies we think are promising and do these at the University of Miami.
Please help us help our children.
Mystic Force Foundation is a 501(c)(3) tax exempt organization