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Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models OPEN

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Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.
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Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
Intravenous Formulation of
HET0016 Decreased Human
Glioblastoma Growth and
Implicated Survival Benet in Rat
Xenograft Models
Meenu Jain1, Nipuni-Dhanesha H. Gamage2, Meshal Alsulami1, Adarsh Shankar1,
Bhagelu R. Achyut1, Kartik Angara1, Mohammad H. Rashid1, Asm Iskander1, Thaiz F. Borin1,
Zhi Wenbo3, Roxan Ara1, Meser M. Ali2, Iryna Lebedyeva4, Wilson B. Chwang2, Austin Guo5,
Hassan Bagher-Ebadian2 & Ali S. Arbab1
Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective
CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to
explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016
with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single
IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at
60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered
vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in
syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed
prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016
and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased
neovascularization (laminin and αSMA), in addition to inammation and angiogenesis markers in the
treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is eective in inhibiting tumor
growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016
signicantly prolonged survival in PDX GBM811 model.
Glioblastoma (GBM) is a hypervascular malignant tumor with poor prognosis1,2. Because of hypervascularity,
anti-angiogenic therapies (AATs) targeting the vascular endothelial growth factor (VEGF) and VEGF receptor
(VEGFR) axis have been attempted in clinical trials, but the results have not been encouraging3. Moreover, our pre-
clinical studies in a rat model of human GBM also showed resistance to the treatment of receptor tyrosine kinase
inhibitors (RTKIs) and resulted in paradoxical enhancement of neovascularization and tumor growth4,5. erefore,
we need an agent that will decrease tumor growth and neovascularization with reduced resistance to therapy.
Recently, studies have shown the role of N-hydroxy-N’-(4-butyl-2 methylphenyl) formamidine (HET0016), a
highly selective inhibitor of 20-hydroxy arachidonic acid (20-HETE) synthesis involving enzymes of the CYP4A
and CYP4F families, in inhibiting tumor angiogenesis, proliferation, migration, and regulation of CD133+ /
CD34+ EPCs6–8. HET0016 was able to inhibit angiogenic responses to several growth factors as well as angio-
genesis in gliosarcoma and in the cornea induced by implanted human U251 GBM cells9,10. Our previous study
in breast cancer also showed decreased tumor growth aer treatment with HET00168. In previous studies of
GBM, HET0016 was prepared in cremophor and DMSO that was administered either orally or intraperitoneally
1Tumor Angiogenesis Laboratory, Georgia Cancer Center, Augusta University, Augusta, GA, USA. 2Cellular and
Molecular Imaging Laboratory, Henry Ford Health System, Detroit, MI, USA. 3Center for Biotechnology and Genomic
Medicine, Augusta University, Augusta, GA, USA. 4Department of Chemistry and Physics, Augusta University,
Augusta, GA, USA. 5Department of Pharmacology, New York Medical College, Valhalla, NY, USA. Correspondence
and requests for materials should be addressed to M.J. (email:
Received: 22 July 2016
Accepted: 28 December 2016
Published: 31 January 2017
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
(IP). However, there was limited success in controlling the glioma due to low bioavailability11. In the present
study, we optimized a condition to make IV formulation of HET0016 with 2-Hydroxypropyl Beta Cyclodextrin
(HPßCD) to improve bioavailability and deliver an eective dose of the drug to the tumor site, especially in the
hypervascular and hypoxic areas of GBM. HPßCD is a derivative of β -cyclodextrin that has been extensively used
as a drug delivery vehicle and recently, FDA has approved the use of HPßCD as a treatment for Niemann Pick
Type C disease12,13. e exploitation of HPßCD as delivery vehicle in GBM may be benecial due to the porosity
of the tumor vasculature, an enhanced permeability and retention (EPR) eect and reduced o target eects in
the tumor neovasculature14. Moreover, due to smaller size (< 1 nm) of the HPßCD encased drug, excess drug can
be cleared rapidly through kidneys. erefore, we believe that the use of HPßCD as a drug delivery system for
delivering HET0016 in GBM will not have any detrimental eect.
In the current rat xenogra model of GBM, we have performed magnetic resonance imaging (MRI) studies,
which help in analysis of changes in tumor vascular physiology, tumor size, backward transfer constant (kep),
tumor plasma volume (vp), vascular permeability (forward transfer constant, Ktrans ), extravascular and extracel-
lular space volume interstitial space volume fraction (ve)15. In addition to MRI, we have also evaluated changes in
histological parameters, eect on survival and signaling pathways following HET0016 treatment. ese parame-
ters may allow for a better understanding of the physiological characteristics of the regional tumor environment
and vascularity aer HET0016 treatment.
e main purposes of the study are (1) to determine whether HPßCD can be complexed with HET0016 within
reasonable period of time (practical kitchen chemistry) to obtain a water soluble formulation that can be admin-
istered intravenously, (2) to investigate the eects of IV administration of HET0016 on human and syngeneic
GBM tumor growth, and survival in patient derived GBM animal models, vascular parameters, histology, and (3)
to identify critical signaling pathways inhibited by HPßCD-HET0016, when administered through IV in the rat
GBM xenogra model.
IV formulation of HET0016 with 30% cyclodextrin. IV formulation of HET0016 using 30% HPβ CD
(in solution with water) was stable both at 4 °C and room temperature for an extended time without any precip-
itation of HET0016 (Fig.1a shows the structure of HET0016 with cyclodextrin). IV administration of 5-day old
complex solution did not cause any immediate or late detrimental eect on health of the animals. ere was no
change in the mass-spectrometric prole between the naked and HPβ CD-HET0016 complex alone or mixed with
plasma or cell lysate (Fig.1b). All proles showed the molecular weight of HET0016 (complex or naked) between
206 and 207 kDa with retention and elution time of 2.9 minutes.
Figure 1. Preparation of HPßCD-HET0016 complex (a) Structure of HET0016 and HPßCD is shown. HPßCD
resembles a shell and it can encase any deliverable drug. To prepare the IV formulation of HET0016, it was rst
dissolved in DMSO and added to 30% HPßCD prepared in water. e bucket like structure of HPßCD with
hydrophilic exterior helps encase HET0016 and delivers to the target area. (b) Mass spectrometry of HET0016
in DMSO (le panel), HET0016 complex in plasma (middle panel). HPßCD-HET0016 complex in tissue lysate
(right panel). Plasma and tissue lysate was collected from the rats bearing glioma and injected with single dose
of HPßCD-HET0016 for evaluation of pharmacokinetics.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
HET0016 levels in plasma and tumor tissue. Rats were orthotopically implanted with U251 cells and
allowed to form the tumor. HET0016 was administered to rats IV and IP as a single dose and sacriced for phar-
macokinetics at dierent time points. A total of 12 rats were serially euthanized for the measurement of HET0016
levels in plasma and brain tumor tissue at 0, 5, 30, 60, 180 min and 24 h (1440 min) aer single IV and IP HET0016
administration (10 mg/kg) (n = 2–4 per time point) and analyzed by LC-MS/MS as described in pharmacokinet-
ics section in material and methods. e level of HET0016 was 7-fold higher in plasma in the group of IV route at
5 min and 60 min time point (105042 and 36880.18 ng/ml) as compared to the IP group (15938.1 and 4773.0 ng/
ml) (p < 0.005) (Fig.2a). e plasma concentration suggests there was rapid elimination of HET0016 with a
half-life of approximately 45 min for both the IV and IP groups. e concentration of HET0016 in tumor tissue in
the IV group was much higher than that of the IP group at 60 min (9251 ng/g vs 325 ng/g) and 24 h (42.7 ng/g vs
7.4 ng/g) (p < 0.01) (Fig.2b). ere was no signicant dierence at middle time point at 180 min.
Eect of HET0016 on tumor growth and vascular parameters. Rats were orthotopically implanted
with U251 cells and allowed to form tumor for 8 days. Aer day 8, animals were treated with HET0016 and fol-
lowed up for 21 days. A detailed schema of treatment schedule is shown in Fig.3. Treatments on the same day and
seven-day waiting period were chosen to mimic the post-surgical cases and post diagnosis of GBM, respectively,
as described previously4. Tumor growth was measured by in vivo MRI. IV administration of HET0016 signi-
cantly reduced the tumor growth (in delayed [day 8–21] treatment) compared to that of vehicle treated animals
(p < 0.001) (Fig.4a) and there was reduced growth with early (day 0–21) treatment although signicant dierence
was not achieved. When compared to the IP administered treatment, tumor growth was reduced only with early
IP treatment, although signicant dierence was not achieved (Fig.4a). However, when all groups were com-
pared, signicant (p < 0.005) reduction of tumor volume was found for delayed IV treatment group compared to
the vehicle and delayed IP HET0016 treated groups (Fig.4a).
Vascular parameters were evaluated to gain information about the vascular kinetics of the tumor environment.
Delayed IV HET0016-treated animals showed signicantly increased blood ow, which may indicate normali-
zation of blood vessels causing enhanced ow although there was no signicant dierence in blood ow (rCBF)
among the IP and early IV treated groups (Fig.4b). As expected, late IP or IV HET0016 treated animals showed
signicantly lower vp (blood plasma pool), ve (extracellular space or interistial volume), Ktrans (forward permea-
Figure 2. Pharmacokinetics of HPßCD-HET0016 in plasma and tissue aer IV and IP dose administration
in rat glioblastoma model. e concentrations of HET0016 in plasma or tissue versus time post dose
administration in rat model are shown. Blood samples were obtained for 0–24 hrs aer dose administration.
Plasma and tissue HET0016 concentrations were determined by LC-MS/MS aer liquid phase extraction. Each
data point is presented as the mean concentration ± SEM (n = 2–4). Concentration of HET0016 in plasma
(a) and tumor tissue (b) aer a single HET0016 (10 mg/kg) dose through IV and IP route. Signicant values
from t Student test and Mann-Whitney’s test are represented by *p < 0.05 in comparison to IV and IP doses in
the same time point.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
bility transfer constant) compared to that of corresponding vehicle treated groups (p < 0.01) (Figs4c and 5a–c).
Both early IP and early IV groups also showed signicantly lower vp compared to that of corresponding vehicle
treated groups (Fig.4c). As rCBF analysis showed higher blood ow (Fig.4b), it is expected that normalized ves-
sels will have less Ktrans (Fig.5a) compared to that of neovessels. erefore, the blood plasma pool (vp) would be
reduced in tumors with normalized vessels (Fig.4c). When the extravascular extracellular space volume (ve) was
compared, both early and delayed IV HET0016- treated groups showed signicantly decreased ve compared to
that of vehicle and IP-treated groups (Fig.5c). ere was no dierence in kep (backow transfer) between the IP
and IV group of animals (Fig.5b). We also evaluated the blood chemistry to determine the toxicity following 0–21
days of treatment with either vehicle, IV formulated or IP HET0016 in GBM xenogras. We did not observe sig-
nicant changes on liver function, renal function, important enzymes, pancreas, and lipid prole aer HET0016
treatment in IP and IV groups (SupplementaryTable1).
Proliferation (Ki67), Micro Vessel Density (MVD), Smooth muscle Actin (αSMA), extracellular
and extravascular space (EES). Brains were removed from the rats (U251 xenogra model) following
euthanasia and prepared for immunohistochemistry as described in material and methods. Tumor section in the
early and delayed IV or IP treatment and vehicle groups were stained for Ki67, which is a marker of cellular prolif-
eration. Ki67 positive and negative tumor cells were counted within the tumor areas. Ki67 positive cells along the
endothelial lining were omitted during counting and calculation of the proliferative cells. In IP treatment groups,
only delayed treatment showed reduced proliferation but not signicant (Fig.6a,c). However, in IV treatment
groups, signicant decreased proliferation was observed in both the early and late treatment groups as compared
to corresponding vehicle groups (Fig.6b–d).
Tumor tissues were also stained for laminin to analyze for microvessel density (MVD). Laminin, a basement
membrane glycoprotein, is found under the endothelium, encasing the pericytes and smooth muscle cells in the
vessel wall and has been shown to be an excellent marker for blood vessels in the brain. Tumor sections were
stained with laminin antibody and images from the positive area were taken at 10x and 20x and counted for
a number of vessels. We found no signicant dierence in expression of laminin among the IP-treated groups
(Fig.7a–c, top le panel). However, there was signicant reduced expression of laminin with lower number of
vessels (3–50 fold less) in the IV treatment group especially around the rim of tumor as compared to vehicle
(p < 0.01) (Fig.7d–f top right panel). We also performed α SMA immunostaining to demonstrate the eect on
vessels in pericyte area around tumor periphery and found there was a signicantly smaller number of ves-
sels with reduced expression of α SMA in the IV treatment group as compared to vehicle (p < 0.02) (Fig.7e,f).
However, no such dierences were detected in the early and delayed IP-treatment groups (Fig.7b,c).
Dierences in the area of extracellular and extravascular space were also analyzed by using H&E stained
images from the IP and IV groups using the color thresholding method as described earlier5. ere was no dif-
ference observed among IP treatment groups as compared to vehicle (SupplementaryFigure2a,b top panel).
However, we found a threefold reduction in the extracellular and extravascular space in the IV-treated groups
(both early and delayed) as compared to vehicle (p < 0.01) (SupplementaryFigure2c,d bottom panel).
Reduced expression of cell proliferation, inammatory and angiogenic proteins were detected
in glioma treated with HET0016. A protein array was performed using tissue lysates for angiogenic
related growth factors using a membrane based protein array kit. SupplementaryFigure3 shows the expres-
sion of important proteins related to angiogenesis among the control, early and delayed IV treatment groups.
Pro-angiogenic proteins such as VE-cadherin (vascular endothelial cadherin-vasculogenesis), bFGF (basic
brobalst growth factor), IL-8 (chemokine CXCL8) and MCP-1 (a CCL2 ligand) were signicantly downregu-
lated in the delayed IV treatment group while expression of anti-angiogenic proteins such as Tie-2, angiostatin
and angiopoietin-2 was increased in the delayed treatment group (day 8–21; SupplementaryFigure3a) as com-
pared to control (p < 0.03). In the early treatment group, expression of proteins, such as MCP-1, angiostatin
and angiopoietin-2 followed a similar trend, indicating a signicant eect of the treatment, particularly on the
factors secreted or expressed by endothelial and inammatory cells. Western blot was performed to determine
the expression of proteins related to cellular proliferation (p-ERK), survival (p-AKT), inammation (COX-1/2),
arachidonic acid metabolism (CYP4A11), signal transducers and activators of transcription (p-STAT1). We also
Figure 3. Schematic representation of treatment schedule. U251 glioma cells were implanted orthotopically
in rat’s brain and treated with HPßCD-HET0016 as described in material and methods.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
Figure 4. HPßCD-HET0016 reduces tumor growth and vascular parameters in rat glioblastoma model.
(a) Representative post contrast T1-weighted images from in vivo imaging of rat with glioma show tumor size
from dierent groups of animals. Semi-quantitative analysis shows signicantly reduced tumor volume in
animals that received HET0016 from day 8 and continued for 2 weeks (n = 5 to 8). (b) Relative cerebral blood
ow maps created from arterial spin labeling techniques. Semi-quantitative analysis (bar graphs) indicates
higher ow in IV HET0016-treated groups (started on day 8) compared to that of vehicle-treated group
(n = 5 to 8). (c) Tumor plasma volume maps and semi quantitative analysis are shown. Both IV and IP
treatment with HET0016 caused a signicant decrease in tumor plasma volume (vp) compared to vehicle and
corresponding IP-treated groups. Each group is represented as the mean of the total measurable MRI sections
from animals of each group (n = 5–8 animals but 7–16 sections). Signicant values from ANOVA test followed
by Fisher’s exact test are represented by *p < 0.05 compared to the corresponding vehicle group. An outlier data
point (from a single MRI section) was removed as described in our material and methods.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
Figure 5. HPßCD-HET0016 inuences vascular parameters in rat glioblastoma model. MRI images from
in vivo rat imaging show signicant changes in vessel permeability (Kt rans) (a), and ve (interstitial space volume)
(C) but no changes in kep (backow rate constant) (b) in early and delayed treatment in IV groups. Signicant
values from ANOVA test followed by Fisher’s exact test are represented by * and #p < 0.05 compared to (*)
vehicle 8–21 vs IV HET 8–21 and vehicle 0–21 vs IV HET 0–21, and (#) IP HET 8–21 vs IV HET 8–21 and IP
HET 0–21 vs IV HET 0–21. Each group is represented as the mean of the total measurable MRI sections from
animals of each group (n = 5–8 animals but 7–16 sections), analyzed by MRI at 0.5 μ m thickness in a range up to
7–16 viewed slices of each animal.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
determined the expression of, HIF-1α , EGFR, VEGF, MMP-2 (angiogenesis) and p-NFKB (inammation), to
investigate the eects of IV administered HET0016 on the rat model of GBM. We observed reduced levels of
COX-1, CYP4A11, p-ERK, p-AKT, p-STAT1, HIF1α , EGFR, VEGF, MMP-2 and p-NFKB proteins in tumors
obtained from both IV treatment groups as compared to vehicle (SupplementaryFigure3b), which conrms the
inuence of HPβ CD-HET0016 on cell proliferation, migration, and inammation pathways.
Eect of HET0016 on survival in GBM811 and HF2303. We evaluated the eect of HET0016, TMZ
alone or in combination with radiation on survival in PDX derived tumor model of GBM811 and HF2303. First,
we tested the eect of treatment in vitro 3D culture model (HF2303) and found that the treatment with HET0016
or TMZ alone or combination showed the inhibition of the growth of neurospheres Fig.8a). Similar studies were
planned in vivo model and we observed that HET0016 treatment alone or in combination with TMZ in irradiated
tumor bearing animals resulted in reduced tumor growth and prolonged survival in both GBM811 (p < 0.004)
and HF2303 PDX models (p = 0.18) (Fig.8b). e overall survival in GBM811 model was prolonged to 26 weeks
aer the treatment with HET0016 plus TMZ and radiation, while control animals (supercontrol and irradiated
only control) survived only for 10 weeks similar to the clinical setting (Fig.8c). e cumulative survival was 100%
for HET0016 plus TMZ combination group as compared to 66% for TMZ alone, 33% for HET0016 alone at 26
weeks until the end of the study without demonstrating any evidence of recurrence (Table in Fig.8d). erefore,
the current study provides evidence that combination therapy (HET0016 + TMZ) with irradiation can improve
survival in GBM. In the second PDX model of HF2303, the HET0016 treatment alone and in combination with
TMZ, resulted in reduced tumor volume and prolonged survival of 26 weeks vs. only 17 weeks of irradiated con-
trol (p < 0.10). e cumulative survival was 25% for both HET0016 plus TMZ combination group and HET0016
alone group at 26 weeks as compared to 0% in radiation only group (p < 0.18). us, data indicates combination
treatment with HET0016 plus TMZ and irradiation resulted in better survival benet as compared to radiation
alone. Overall, the response was better in GBM811 model than HF2303 model and may be attributed to dierence
in growth and genetic characteristics. We also measured the presence of 20-HETE and MGMT in dierent glioma
cells (GL261, U251, HF2303 and GBM811) and GBM811 showed comparatively lower amount of 20-HETE and
methylguanine DNA methyltransferase (MGMT) levels (SupplementaryFigure5and6), which may indicate
better treatment results following HET0016, radiation and TMZ in GBM811 PDX model16,17.
Figure 6. HPßCD-HET0016 treatment results in reduced proliferation, migration in rat glioblastoma
model. (a,b) Ki-67 immunohistochemical staining was done as a marker of proliferation in tumor tissues.
Rats treated with HET0016 starting on day 8 showed signicantly fewer proliferative cells in the tumor in the
IV group compared to the IP group. (c,d) Bar graphs represent the proportion of Ki67 positive tumor cells
compared to total number of tumor cells in tumor area of each group. Images were taken in 40x magnication.
e brown nuclei color shows the positive labelling cells. Statistically signicant dierences were veried by
ANOVA followed by Bonferroni’s test. *p < 0.05 in comparison to the respective vehicle group.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
Eect of IV HET0016 on GL261 syngeneic GBM. We also assessed the eect of IV HET0016 on tumor
growth in syngeneic model of GBM using GL261 cell line. All animals underwent MRI and tumor volume
was measured from the MRI images. ere was signicantly reduced tumor volume in animals treated with
IV HET0016 (54.33 ± 12.37 mm3) compared to that of vehicle treated animals (131.70 ± 19.45 mm3) (p < 0.007)
Despite the availability of current chemotherapy and targeted therapies in GBM (e.g. temozolomide and bevaci-
zumab), not many therapeutic benets have been achieved due to target developing drug resistance18. Most ther-
apies have produced a decrease in tumor growth during early stages of treatment followed by aggressive tumor
recurrence. Mu et al. previously developed water-soluble IV formulation of HET0016 which rapidly penetrated in
the rat normal brain and inhibited the formation of 20-HETE aer cerebral ischemia19. Here, we have developed
an improved IV formulation of HPßCD-HET0016 that dissolves in 2–3 min compared to the 48 hrs as reported
by Mu et al. IV administration of HPßCD-HET0016 in a rat model of human GBM signicantly reduced tumor
growth in developing or developed tumors as compared to IP HET0016 treatment. It appears that the IV formu-
lation facilitated increased delivery of HET0016 to the hypervascular and hypoxic tumor sites, due to EPR eects.
Moreover, the eect of IV HET0016 was not cell specic and showed similar reduced tumor growth in syngeneic
Figure 7. HPßCD-HET0016 treatment results in reduced neovascularization in rat glioblastoma
model. Laminin and α SMA immunohistochemistry staining was done in tumor tissues to determine the
neovascularization (a,b,d and e) Representative images from brain tumor tissues are shown at 10x and 20x
from the IP (le panel) and IV (right panel) groups. Red arrows indicate blood vessels. Four areas on the tissue
section were selected and the number of vessels counted. (c and f) Laminin and α SMA quancation. Each
bar represents an average of four areas and was estimated in multiple samples from each group (n = 2–4).
Signicant dierence is indicated by *p < 0.05 compared to the respective vehicle group.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
tumor (GL261) models. e distribution prole of HPßCD-HET0016 formulation showed better bioavailability
and retention in tissue with a higher brain to plasma ratio in the IV treatment group as compared to the treatment
with IP preparation. e ndings suggest that the IV formulation or encapsulation of HET0016 in a non-toxic
delivery system may have the benets of increased half-life (protect the drug from degradation during circulation
and early clearance), lower toxicity, enhanced EPR eect over the IP preparation and improved the therapeutic
index14. Furthermore, enhanced accumulation of macromolecular drugs in tumor tissues can occur as compared
to normal tissue due to impaired lymphatic drainage and porous blood vessels in GBM with abnormal molec-
ular and uid transport dynamics20,21. e eect of enhanced delivery of IV HET0016 is supported by vascular
parametric analysis in MRI studies. Vascular parametric analysis supported the eect of the drugs, where the IV
formulation decreased vascular permeability (Ktrans), tumor blood volume (vp), and extracellular extravascular or
interstitial space volume (ve) but increased the blood ow to the delayed treated tumors in GBM.
TMZ is widely used alkylating agent for the treatment of primary as well as recurrent GBM. Eect of TMZ
requires functional DNA mismatch repair (MMR), low levels of methylguanine DNA methyltransferase (MGMT)
and DNA repair genes16,22,23. Unfortunately, majority of the primary and recurrent GBMs have unmethylated
or active high level of MGMT, which make TMZ treatment unresponsive17,24,25. We evaluated the synergistic
eect of HET0016 alone or in combination with TMZ on the survival of animals treated with 30 Gy irradiation
in patient-derived xenogra models (PDX) (GBM811, HF2303) (treatment schedule shown in Supplementary
scheme). Aer Administration of HET0016 alone and in combination with TMZ and radiation in GBM811 and
HF2303 models for 6 weeks resulted in good response and tumor did not relapse until 6 months (endpoint of
the study). erefore, HET0016 plus TMZ combination may prolong survival and reduce therapy resistance. e
synergistic eect may be due to the role of HET0016 in sensitizing the action of TMZ and irradiation by reducing
DNA repair mechanisms. Our preliminary study indicated that HET0016 administration resulted in down reg-
ulation of DNA repair genes (unpublished preliminary data, SupplementaryFig.4). e continuous exposure of
Figure 8. Treatment with HET0016 and TMZ prolongs survival in PDX models. (a) Treatment with
HET0016 and TMZ inhibit neurospheres growth in vitro: HF2303 was treated with HET0016 and TMZ alone
(100 μ M), in combination with TMZ and followed up for 14 days. (b) HF2303 and (c) GBM811 show the
eect of HET0016 and TMZ on survival rate of the mice in groups 1, 2, 3, 4 and 5 of treatment schedules (as
described in Material and Methods) were evaluated from the rst day of treatment until death. X-axis represents
cumulative survival time in weeks. Table in Fig. 8d summarize the details of duration in weeks and % survival.
Athymic nude rats were implanted orthotopic with HF2303 and GBM811. Six to ten weeks aer implantation,
rats were randomized into ve treatment groups receiving PBS, radiation, HET0016, TMZ, HET0016 plus TMZ
for another 6 wks, as described in Materials and Methods. #e animals were not included due to the technical
diculty. Signicant dierence is indicated by *p < 0.05 vs irradiation and super-control, $p-value was 0.18 vs
irradiation control, achieved by Kaplan Meier analysis and Log-rank test.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
cellular DNA to potentially harmful environmental and internal insults necessitates redundant and overlapping
DNA repair mechanisms26. erefore, successful destruction of GBM tumors may require a combined approach
utilizing standard treatments in combination with inhibition of DNA repair pathways.
e anti-tumor eect of HPßCD-HET0016 was supported by decreased tumor cell proliferation, migration,
and neovascularization. ere was a clear reduction in the number of Ki67 + cells in the IV treatment groups as
compared to IP groups. e reason for observing the signicant reduced proliferation in IV treatment groups
may be that HET0016 is eective in preventing the regrowth of established tumor due to increased bioavailability
following IV administration. We also believe established tumor has developed-neovascularization and leaky ves-
sels, which allow EPR eect to be more pronounced in groups treated with IV formulated HET0016. In addition,
we suggest that the eect of HET0016 on tumor growth may be attributed to the reduced basic broblast growth
factor expression (bFGF or FGF-2) aer early and delayed treatment. bFGF is a potent mitogen that maintains
cancer cell stemness and leads to drug resistance by enhancing the blood-brain-barrier function of endothelial
cells27,28. ese results are supported by an earlier study of Guo et al.29 which showed chronic in vivo admin-
istration of HET0016 (IP, 10 mg/kg/day) reduced the volume of 9 L gliosarcoma, accompanied by mitotic and
vascularization reduction.
GBM tumor vessels are tortuous, disorganized, highly permeable, decreased pericyte coverage and a solid
basement membrane structure30,31. We found overall fewer vessels in both early and delayed IV treatment group,
with reduced EES, laminin, and α SMA staining, especially at the invasive margin of the tumor indicating the role
of HET0016 in inhibiting the growth of new blood vessels. ese observations were supported by a published
report that suggested that the pericyte line around new endothelial cells sprouts from tumor vessels, play a role
in blood vessel growth, and is suggested to be a potential target in AAT therapy32. In addition, this is further
supported by a high expression of angiopoietin-2, Tie-2 molecules and reduced VE-cadherin expression, which
is associated with pericyte endothelium suggesting an eect of HET0016 on vascular development and stabil-
ity33. We suggest that HPßCD-HET0016 has a primary eect on blood vessels, which by normalization increased
the bioavailability of drug to the hypoxic tumor sites. MRI vascular parametric analysis also correlates with
immunohistochemistry; especially the normalization of vessels in the IV delayed treatment groups that resulted
in decreased permeability (Ktrans) and overall reduced plasma volume fraction (vp). e treatment also caused
decreased EES volume indicated by decreased ve, which is also validated in H&E histological analysis. Similarly,
Guo et al also showed normalization of vessels in 9 L tumor following HET0016 treatment10.
In our study, we observed that HET0016 reduced expression of the pro-angiogenic proteins but increased the
expression of anti-angiogenic proteins expression to achieve equilibrium to reduce tumor growth. Expression
of pro-angiogenic factors such as IL-8, MCP-1, VEGF and SDF-1α were decreased, while expression of inhibi-
tors of the angiogenic process such as angiostatin, angiopoietin-2/Tie-1, and Tie-2 proteins were increased aer
HET0016 treatment. Recently, a study in triple negative breast cancer showed a similar eect of IP HET0016 on
pro-angiogenic factors8. We observed that HPßCD-HET0016 reduced IL-8 expression. IL-8 has recently been
shown to be a critical factor in regulating cancer cell stemness and invasion, and higher expression was associated
with poor survival and therapy resistance in glioma34. Moreover, HET0016 treatment reduces the expression of
MCP-1 and SDF-1α , key chemokines responsible for tracking and activation of monocytes/macrophages and
have been involved in inammation and angiogenesis35. MCP-1-induced angiogenesis has been reported to be
mediated through up-regulation of HIF-1α and subsequent activation of VEGF35. erefore, HPßCD-HET0016
may be acting as an inhibitor of inammation and angiogenesis growth responses in GBM tumor cells through
regulating HIF-1α and VEGF. Interestingly, there were less hypoxic areas in HPßCD-HET0016-treated tumors as
shown by reduced HIF-1α protein expression. Reduced HIF-1α expression also resulted in low VEGF expression
aer HPßCD-HET0016 treatment. VEGF signaling and angiogenesis and highly aected by HIF-1α levels and
regulates endothelial cell proliferation, migration, and permeability of blood vessels. Studies have shown high
expression is correlated with increased metastases, vasculature, and tumor recurrence28. ese ndings suggest
that HPßCD-HET0016 regulated the expression of pro-and anti-angiogenic factors to achieve the balance in the
maintenance of the tumor microenvironment8.
In the current study, we studied eect of HPßCD-HET0016 treatment in the arachidonic acid metabolism
pathway. Previously, HET0016 has been shown to have an eect on expression of CYP4A enzymes. In present
study, we found HET0016 was able to reduce the protein expression of CYP4A11 and COX-1, thereby inuencing
inammation, angiogenesis, and MAPK signaling in GBM36,37. Previous studies have also documented a similar
role of HET0016 in glioma, gliosarcoma, lung, and breast cancers10,29,38. A study in colon cancer suggested com-
bination therapy of rofecoxib and HET0016 might be a new treatment that can improve the anti-tumor ecacy of
rofecoxib alone39. In addition, we also investigated role of p-NFκ B as marker of inammation and proliferation.
Previous studies have shown a pro-survival role for p-NFκ B in glioma and an association with chemo resistance40.
We demonstrated reduced levels of p-NFκ B aer HET0016 treatment suggesting an anti-proliferative eect in
Previous study have shown that GBM has frequent overexpression of EGFR, which leads to activation of
the P13/Akt pathway, associated with adverse clinical outcome and has been suggested to be a therapeutic tar-
get41–43. We showed that HPßCD-HET0016 repressed the expression of EGFR, ERK, and Akt, a target of MEK.
We also determined expression of p-STAT1, a tumor suppressor protein involved in the p38/MAPK pathway in
response to IFN-α and stress. Higher expression of p-STAT1 was shown to be associated with poor prognosis
in glioma and depletion of IRF1/STAT1 signaling has been reported to increase the ecacy of anti-VEGF (bev-
acizumab) therapy in a glioma xenogra model44–46. We found that HPßCD-HET0016 treatment also reduced
the levels of pSTAT1, indicating HET0016 can improve overall prognosis in glioma. In summary, we have estab-
lished a highly soluble HPßCD-HET0016 complex for IV administration that reduced GBM growth, normal-
ized vasculatures, decreased permeability, and EES volume both in established and growing tumors through
enhanced bioavailability compared to conventional IP HET0016 preparation. Our results showed that HET0016
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
in combination with TMZ and radiation enhanced the anti-tumor ecacy and prolonged survival in GBM xen-
ogra models as compared to TMZ alone. Anti-tumor properties of HPßCD-HET0016 resulted in decreasing
proliferation, hypoxia, migration, stemness, and vasculatures in glioma by altering the balance of pro-angiogenic
and anti-angiogenic balance, PI3K/Akt, p38/MAPK and inammation pathways (Summary model in Fig.9).
erefore, HPßCD-HET0016 could be tested in clinics to explore combination therapies with TMZ or radiother-
apy for GBM.
Materials and Methods
Ethics statement. Animal experiments were performed according to the NIH guidelines and the experi-
mental protocol was approved by the Institutional Animal Care and Use Committee of Henry Ford Health System
and Augusta University (formerly Georgia Regents University) (Approval number: 2014–0625). All animals were
kept under pathogen-free conditions at room temperature (21 to 25 °C) with exposure to light for 12 hours and
12 hours in the dark. Food and water were oered ad libitum. Body weight was measured twice weekly as an
indicator of overall animal health. All surgeries were performed under ketamine - xylazine anesthesia, and eorts
were made to minimize suering. Euthanasia for the moribund animals was performed in a CO2 cha mber.
Chemicals. HPßCD (2-hydroxy Propyl-β -Cyclodextrin) was purchased from Sigma-Aldrich (St. Louis, MO),
cell culture media was from ermo Scientic (Waltham, MA), and fetal bovine serum was purchased from
Hyclone (Logan, Utah). HET0016 was obtained from Dr. JR Falck of UT Southwestern University, Texas, and
also synthesized by Dr. Iryna Lebedyeva in the department of Chemistry and Physics, Augusta University with a
purity of more than 97%. Cell culture grade DMSO was purchased from Fischer Scientic (PA). Blood chemistry
proles and electrolytes were determined Using third party vendor (Antech Diagnostic).
Tumor cells. Human glioma U251 cell line was obtained from Dr. Steve Brown of Henr y Ford Health System.
e cell line was authenticated in July 2014 using the STR proling method. GL261 syngeneic (C57BL/6 mouse
derived) GBM cell line was obtained from Dr. Ted Johnson (Augusta University) and was authenticated in 2016.
e cell line, U251 was grown in high glucose (4.5 g/L) Dulbecco’s modied eagles medium (DMEM) and GL261
in RPMI (Roswell Park Memorial Institute) (ermo Scientic), supplemented with 10% fetal bovine serum
(FBS), 2 mM glutamine and 100 U/ml penicillin and streptomycin at 5% CO2 at 37 °C in a humidied incubator.
Figure 9. Summary and hypothetical model. Tumors consist of an abnormal vasculature, composed mainly
of immature vessels with increased permeability. e less densely packed cells allow drugs or complexes
to accumulate in tumor tissue. Treatment with HPßCD-HET0016 leads to reduced expression of α SMA
and increased expression of angiopoietin-2 and Tie-2, which may lead to reduced tumor vasculature that is
inadequate to support tumor growth and may lead to tumor dormancy. Our results suggest that HET0016
reduces cancer cell growth, invasion, and vasculature by reducing the expression of signaling molecules in the
MAPK, PI3K/AKT, and inammation pathways.
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
Patient derived GBM cells (HF2303) was obtained from Dr. Tom Mikkelsen’ s lab at Henry Ford Hospital and
was grown in neurosphere medium (NM), composed of DMEM/F-12 supplemented with N2 (Gibco), 0.5 mg/ml
BSA (Sigma), 25 μ g/ml gentamicin (Gibco), 0.5% antibiotic/antimycotic (Invitrogen), 20 ng/ml basic broblast
growth factor, and 20 ng/ml EGF (Peprotech). Cells were maintained in culture for up to passage 10 (low passage).
Patient derived PDX GBM cells, GBM811 was obtained from North Western University and was propagated in
immunocompromised NOD-SCID mouse and tumor was disintegrated into cell suspension at the time of tumor
implantation in nude rats.
Intraperitoneal (IP) formulation of HET0016. For IP formulation, 2 mg of HET0016 was dissolved in
1 ml of solution containing 80% PBS, 10% of dimethyl sulfoxide (DMSO) (Sigma, St. Louise, MO, USA) and 10%
cremophor (Sigma, St. Louise, MO, USA). e mixture was vortexed and sonicated until HET0016 was dissolved.
Each rat received 10 mg/kg/day dose and the dose has been used according to previous publications10,11.
Intravenous (IV) formulation of HET0016 using HPßCD (2-hydroxy Propyl-B-Cyclodextrin).
Previous published methods have used 48 hours incubation with continuous rotation to make a complex of
HET0016 with HPßCD for the IV formulation19. Here, we describe a rapid and safe method to synthesize IV for-
mulation of HET0016 without using a sonicated water bath or long rotation. HPßCD has a bucket-like structure
with hydrophilic outer shell but hydrophobic inner cavity (Fig.1a). However, optimized conditions are needed
to insert the drug into the HPßCD cage since HET0016 is a heat sensitive compound and can be degraded with
long-term rotation at room temperature. HET0016 was dissolved in DMSO (2 mg/50 μ l) and was added slowly
to a 30% HPßCD solution in sterile water (950 μ l) with continuous vortex, and the turbid solution became clear
within 2–3 minutes. e nal concentration of DMSO was 5%. We have tested mass-spectrometric proles of
HET0016 complexed with HPßCD as well as HET0016 dissolved in DMSO (Fig.1b). e solution of HET0016
in DMSO showed a peak at 2.9 min retention elution time, and it did not change when HET0016 was complexed
or encapsulated with HPßCD.
HET0016 pharmacokinetics in plasma and tissue lysate. Twelve nude rats (RNU nu/nu) obtained
from Charles River Laboratory (Frederick, MD) were used for pharmacokinetics studies. A single IV injection of
HET0016 or vehicle (10 mg/kg) in 30% cyclodextrin or IP injection of HET0016 was administered to the animals
in the respective groups. Animals were euthanized at multiple time points, and brain tumor tissue was collected
(60, 180 min, 24 hrs). e details are provided in Supplementarymethods.
Animal model and treatment schedules. Forty-eight nude rats (RNU nu/nu) weighing 140–150 grams
obtained from Charles River Laboratory (Frederick, MD) were used in these experiments. Human glioma U251
cells (400 k in 5 μ L), were implanted orthotopically at 3 mm to the right and 1 mm anterior to bregma according to
published methods47. Following implantation, animals received treatment of HPßCD-HET0016 (IV, 10 mg/kg per
day); an equivalent dose of IV HPßCD alone as a vehicle; HET0016 (IP, 10 mgkg/day), or an equivalent dose of IP
vehicle alone for either two or three weeks. e details of animal model are described in Supplementarymethods.
In vivo MRI and measurement of vascular kinetics. All animals underwent MRI, vascular kinetic,
and tumor volume analyses as described previously on day 225 as depicted in Fig.3. e details on methods are
provided in Supplementarymethods section.
Tumor volume analysis. Post contrast T1-weighted images were used to determine the tumor volume. At
least two investigators, blinded to the various treatment groups, determined the volume by drawing irregular
region of interest (ROIs) for all slices containing tumor. To calculate the exact volume, investigators summed up
the number of slices and multiplied by the slice thickness.
Survival studies in PDX GBM models. Anti-tumor eects and survival studies were conducted in patient
derived xenogra GBM model (PDX) using HET0016 as an adjuvant to the current treatment strategies of GBM
(radiation and TMZ). Two dierent PDX models were developed using HF2303 (n = 11) and GBM811 (n = 13).
Cells were suspended in PBS and 2 × 105 cells in 5 μ l were injected orthotopically into brain of each, 5–7-week-old
nude rats, as described previously47–50. Prior to the start of treatment, animals underwent MRI at 6 weeks for
GBM811 and 10 weeks for HF2303 before the start of treatment to conrm the presence of a 3 mm3 t umo r.
ereaer, animals were randomly assigned to ve treatment groups and details of the treatment are shown
in Supplementary scheme. e ve groups of treatment in the present study were as follows: (1) supercontrol;
tumor bearing animals were treated with PBS (IV) twice weekly. (2) Irradiation control; tumor bearing animals
received a single dose of 30 Gy radiation encompassing the tumor. (3) Irradiation + HET0016; following single
dose of 30 Gy irradiation tumor bearing animals received IV HET0016 (10 mg/kg, 5 days/week, every other week
for 6 weeks. (4) Irradiation + TMZ; following single dose of 30 Gy irradiation tumor bearing animals received
oral TMZ (50 mg/kg, 2 days/week every other week for 6 weeks). (5) Irradiation + HET0016 + TMZ; follow-
ing single dose of 30 Gy irradiation tumor bearing animals received IV HET0016 plus oral TMZ for six weeks.
HET0016 was given on 1st, 3rd and 5th weeks and TMZ was given on 2nd 4th and 6th week following irradiation. All
animals (GBM 811 and HF2303) received 30 Gy of irradiation in a single fraction before the treatment of TMZ
and HET0016 alone or in combination except the super-control group that did not go through irradiation or any
other treatments. A single dose of 30 Gy radiation was given in an area encompassing the tumor using an X-ray
based image-guided micro small animal radiation research platform from Gulmay Medical Inc. (SARRPTM, an
Xstrahl company). All surviving animals underwent 2nd set of MRI following 6 weeks of treatments to determine
the eects on the tumor volume. 3rd set of MRI was obtained from all surviving animals 4–6 weeks aer the end
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
of the treatment. 4th set of MRI was also obtained from all surviving animals at the end of the studies (26 weeks
following tumor implantation). All animals were checked for weight gain or loss at least 2 times a week from the
day of tumor implantation. Any sign of morbidity was also noted. Survival was calculated from the day of tumor
implantation until death. One hundred and eighty-three days aer tumor implantation, the experiment was ter-
minated, and the surviving animals were sacriced. All rats were autopsied at death to examine the antitumor
ecacy of each treatment regimen. Our previous experience showed that HF2303 GBM bearing animals die by
16–20 weeks and GBM811 tumor bearing animals die in 12–16 weeks if not treated.
Histopathology. Tissues were stained for proliferation (anti-Ki 67 antibody, Millipore, USA), human spe-
cic MHC-1 marker (anti-MHC-1 antibody, now HLA-A, Abcam), anti-laminin antibody (for blood vessels), and
smooth muscle actin (SMA) for pericytes (anti-α SMA antibody abcam, USA) using standard immunohistochem-
ical procedure as described previously and included in Supplementarymethods8.
Statistical analysis. Comparison between drug and vehicle-treated groups was done by using Student’s
t-test. When more than two groups were analyzed, we used ANOVA followed by Bonferroni test for normal
distribution or Fisher’s exact test. All data were expressed as means ± SEM. P value of < 0.05 was considered sig-
nicant value in all tests. e criteria to exclude an outlier were the value outside of the range of mean ± twice the
standard deviation (if necessary). Body weight, tumor volume, and survival were compared among PBS-treated,
HET0016-treated, TMZ-treated, and HET0016 plus TMZ–treated groups. Kaplan Meier analysis was performed
to determine the survival of the animals bearing PDX derived GBM and log rank was used to compare the sur-
vival between the two groups.
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e authors thank the sta of the Georgia Cancer Center core facilities for their ecient assistance. e authors
also thank Rhea-Beth Markowitz, PhD for providing editing assistance in the manuscript and Ana de Carvalho
of Dr. Tom Mikkelsen’s group for providing HF2303 cells. is study is supported by the National Institutes of
Health (NIH) grants R01CA160216, R01CA172048 and Georgia Cancer Center startup funds.
Author Contributions
M.J., B.R.A. and A.S.A., designed research, performed the experiments, interpreted data and wrote manuscript.
M.A., A.I., T.F.B. conducted animal orthotopic injections and prepared paran blocks for histology. A.S., M.H.R.
and K.A., performed the immunohistochemistry and H & E staining. Z.W. conducted mass spectroscopy for
pharmacokinetic studies and R.A. conducted MRI and tumor volume analysis. M.M.A., N.H.G., designed the
composition of HET0016. I.L., A.G., performed the synthesis of HET0016. W.B.C. performed the MRI analysis
and H.B. helped in formulation of mathematical model for MRI analysis.
Additional Information
Supplementary information accompanies this paper at
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Jain, M. et al. Intravenous Formulation of HET0016 Decreased Human Glioblastoma
Growth and Implicated Survival Benet in Rat Xenogra Models. Sci. Rep. 7, 41809; doi: 10.1038/srep41809
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... When N-hydroxy-N'-(4-butyl-2methylphenyl) formamidine(HET0016), a highly selective inhibitor of 20-HETE synthesis, is used alone in tumor-bearing animals, tumor neovascularization is reduced (61,62). When the level of different pro-and anti-angiogenic factors in tumor lysates is detected, prominent changes occur after HET0016 treatment compared with placebo-treated tumors (63,64). When certain indicators, including extravascular cell space (EES), vascular parameters and tumor angiogenesis, are analyzed, HET0016 treatment reduces EES, tumor blood volume, permeability and tumor angiogenesis (63,64). ...
... When the level of different pro-and anti-angiogenic factors in tumor lysates is detected, prominent changes occur after HET0016 treatment compared with placebo-treated tumors (63,64). When certain indicators, including extravascular cell space (EES), vascular parameters and tumor angiogenesis, are analyzed, HET0016 treatment reduces EES, tumor blood volume, permeability and tumor angiogenesis (63,64). In the field of cancer, triple-negative breast cancer is not sensitive to chemotherapeutics. ...
The androgen receptor (AR) and AR-driven genes are crucial in normal and neoplastic prostate tissue. Previous results showed a link between 20-hydroxyeicosatetraenoic acid (20-HETE) production and AR-driven prostate cancer (PCa) progression. This study aims to describe the contribution of GPR75, 20-HETE membrane receptor, in 20-HETE-mediated expression and transcriptional activity of AR in PCa. In LNCaP cells, 20-HETE increased AR expression, nuclear localization, and its transcriptional activity. Also, 20-HETE enhanced dihydrotestosterone (DHT) induced effects. All was abrogated by chemical antagonism of GPR75 (19-HEDE) or its transient knockdown. In human PCa, the expression of AR-driven genes correlated with GPR75. In LNCaP xenografts, tumors from castrated animals expressed higher levels of AR, this was impaired by inhibition of 20-HETE synthesis. These data suggest that 20-HETE, through the GPR75 receptor, regulates transcriptionally active AR in PCa cells, thus making 20-HETE/GRP75 potential targets to limit the expression of AR-driven phenotype in PCa cells.
Glioblastoma (GBM) is the most prevalent malignant tumor in the central nervous system. Owing to its hypervascular state and inevitable recurrence following standard therapies of surgical resection, radiation, and chemotherapies, clinicians as well as scientists have instituted antiangiogenic agents to counter the development of blood vessels in the recurrent and rapidly growing GBM. However, investigations from our group and from other laboratories, as well as from clinical data, indicate the development of therapy resistance and activation of alternate neovascularization in GBM following antiangiogenic therapies (AATs). Alternate neovascularization occurs in three distinct pathways, such as angiogenesis, vasculogenesis, and vascular mimicry. Beside angiogenesis, different mechanisms are postulated for the development of alternative neovascularization and therapy resistance in GBM following AAT including bone marrow-derived cell-mediated vasculogenesis, transdifferentiation of glioma stem cells to form vascular mimicry, and vasculogenic as well as angiogenic myeloid cell-mediated neovascularization. Specific agents that target bone marrow-derived cells, myeloid cells, and transdifferentiation of glioma stem cells could be added to current therapeutic strategies in GBM management to overcome the resistance.
Glioblastoma (GBM), the most common and aggressive brain tumor, can be treated using a multimodal treatment. Temozolomide (TMZ) has been the gold standard chemotherapeutic agent for newly diagnosed GBM for over a decade. However, almost all GBM patients eventually become resistant to chemotherapy including TMZ and develop recurrent tumors that are more aggressive than primary GBM. This chapter reviews the molecular mechanisms of innate and acquired TMZ resistance in GBM. It also summarizes potential treatment options and new target identification methods for TMZ-resistant GBMs.
Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs/EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450- (CYP) and lipoxygenase- (LOX) derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far has employed the use of enzyme inhibitors, HETE antagonists and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.
Ours and other previous studies have shown that CYP4Z1 is specifically and highly expressed in breast cancer, and acts as a promoter for the stemness of breast cancer cells. Here, we explored whether targeting CYP4Z1 could attenuate the stemness of breast cancer cells using HET0016, which has been confirmed to be an inhibitor of CYP4Z1 by us and others. Using the transcriptome‐sequencing analysis, we found that HET0016 suppressed the expression of cancer stem cell (CSC) markers and stem cell functions. Additionally, HET0016 indeed reduced the stemness of breast cancer cells, as evident by the decrease of stemness marker expression, CD44+/CD24− subpopulation with stemness, mammary‐spheroid formation, and tumor‐initiating ability. Moreover, HET0016 suppressed the metastatic capability through in vitro and in vivo experiments. Furthermore, we confirmed that HET0016 suppressed CYP4Z1 activity, and HET0016‐induced inhibition on the stemness and metastasis of breast cancer cells was rescued by CYP4Z1 overexpression. Thus, our results demonstrate that HET0016 can attenuate the stemness of breast cancer cells through targeting CYP4Z1.
Glioblastoma (GBM) is an aggressive form of central nervous system tumors that can occur in the brain or spinal cord. GBM treatment is challenging with limited options with traditional surgery, radiation, and chemotherapy due to mutational heterogeneity, hypervascularity, and predominant hypoxia throughout the tumors. When used as monotherapy or combination therapies, treatments involving angiogenesis pathway-based, mutation-based, or immune-based therapies have shown limited success in decreasing the GBM growth and improving survival, warranting urgent attention in both clinic and laboratory. We have been actively investigating the arachidonic acid (AA) metabolism pathways, where cytochrome P450 4 (CYP4) family of enzymes can produce 20-hydroxyeicosatetraenoic acid (20-HETE) metabolite, a critical signaling eicosanoid contributing to therapeutic resistance. We have shown that 20-HETE can activate several intracellular protein kinases, proinflammatory mediators, and chemokines in GBM. This chapter is focused on understanding the role of the AA metabolic pathway in GBM with an emphasis on the CYP4A-20-HETE axis as a novel therapeutic target. We have also discussed all the effective investigational mechanisms on how 20-HETE can promote GBM refractoriness to available therapies and how its inhibition could sensitize the tumor for improved outcomes.
Therapy resistance in glioblastoma (GBM), a hypervascular, hyperproliferative, and hypoxic neoplasia of the central nervous system with an extremely high mortality rate has always been an attractive topic of investigation. The transient benefits of adjuvant antiangiogenic therapies (AAT) in normalizing blood vessels, controlling abnormal vasculatures, and preventing recurrence coupled with higher rates of relapse are attributed to the AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms such as vascular mimicry (VM). An overactive IL-8-CXCR2 axis-driven VM contributed to AAT therapy resistance in preclinical models of GBM. Intervening the IL-8-CXCR2 axis with SB225002 (a CXCR2 antagonist) reduced (i) the GBM tumor burden, (ii) CXCR2 + and endothelial-like GBM subpopulations, and (iii) VM structures in the tumors. This chapter throws light on these novel findings and corroborates the therapeutic potential of a CXCR2 antagonist in combination with other antitumor agents in preclinical and clinical trials to reverse GBM therapy-induced resistance.
Almost 100% glioblastoma (GBM) recurs following extensive therapies. Current strategies of treatment of recurrent GBM varies from center to center and it depends on the site, size, and vascularity of the recurrent tumors. In this chapter, current views of managing recurrent GBM is discussed and also possible alternative intervention is pointed out.
We previously reported that deficiency in 20-HETE or CYP4A impaired the myogenic response and autoregulation of cerebral blood flow (CBF) in rats. The present study demonstrated that CYP4A was coexpressed with alpha-smooth muscle actin (α-SMA) in vascular smooth muscle cells (VSMCs) and most pericytes along parenchymal arteries (PAs) isolated from SD rats. Cell contractile capabilities of cerebral VSMCs and pericytes were reduced with a 20-HETE synthesis inhibitor, HET0016 but restored with 20-HETE analog WIT003. Similarly, intact myogenic responses of the middle cerebral artery and PA of SD rats decreased with HET0016 and were rescued by WIT003. The myogenic response of the PA was abolished in SS and was restored in SS.BN5 and SS.Cyp4a1 rats. HET0016 enhanced CBF and impaired its autoregulation in the surface and deep cortex of SD rats. These results demonstrate that 20-HETE has a direct effect on cerebral mural cell contractility that may play an essential role in controlling cerebral vascular function.
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Glioblastoma multiform (GBM) is the most common malignant glioma of all the brain tumors and currently effective treatment options are still lacking. GBM is frequently accompanied with overexpression and/or mutation of epidermal growth factor receptor (EGFR), which subsequently leads to activation of many downstream signal pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt/rapamycin-sensitive mTOR-complex (mTOR) pathway. Here we explored the reason why inhibition of the pathway may serve as a compelling therapeutic target for the disease, and provided an update data of EFGR and PI3K/Akt/mTOR inhibitors in clinical trials.
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Background: Due to the hypervascular nature of glioblastoma (GBM), antiangiogenic treatments, such as vatalanib, have been added as an adjuvant to control angiogenesis and tumor growth. However, evidence of progressive tumor growth and resistance to antiangiogenic treatment has been observed. To counter the unwanted effect of vatalanib on GBM growth, we have added a new agent known as N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine (HET0016), which is a selective inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis. The aims of the studies were to determine 1) whether the addition of HET0016 can attenuate the unwanted effect of vatalanib on tumor growth and 2) whether the treatment schedule would have a crucial impact on controlling GBM. Methods: U251 human glioma cells (4×10(5)) were implanted orthotopically. Two different treatment schedules were investigated. Treatment starting on day 8 (8-21 days treatment) of the tumor implantation was to mimic treatment following detection of tumor, where tumor would have hypoxic microenvironment and well-developed neovascularization. Drug treatment starting on the same day of tumor implantation (0-21 days treatment) was to mimic cases following radiation therapy or surgery. There were four different treatment groups: vehicle, vatalanib (oral treatment 50 mg/kg/d), HET0016 (intraperitoneal treatment 10 mg/kg/d), and combined (vatalanib and HET0016). Following scheduled treatments, all animals underwent magnetic resonance imaging on day 22, followed by euthanasia. Brain specimens were equally divided for immunohistochemistry and protein array analysis. Results: Our results demonstrated a trend that HET0016, alone or in combination with vatalanib, is capable of controlling the tumor growth compared with that of vatalanib alone, indicating attenuation of the unwanted effect of vatalanib. When both vatalanib and HET0016 were administered together on the day of the tumor implantation (0-21 days treatment), tumor volume, tumor blood volume, permeability, extravascular and extracellular space volume, tumor cell proliferation, and cell migration were decreased compared with that of the vehicle-treated group. Conclusion: HET0016 is capable of controlling tumor growth and migration, but these effects are dependent on the timing of drug administration. The addition of HET0016 to vatalanib may attenuate the unwanted effect of vatalanib.
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Malignant gliomas are aggressive brain tumors with limited therapeutic options, possibly because of highly tumorigenic subpopulations of glioma stem cells. These cells require specific microenvironments to maintain their “stemness,” described as perivascular and hypoxic niches. Each of those niches induces particular signatures in glioma stem cells (e.g., activation of Notch signaling, secretion of VEGF, bFGF, SDF1 for the vascular niche, activation of HIF2 α , and metabolic reprogramming for hypoxic niche). Recently, accumulated knowledge on tumor-associated macrophages, possibly delineating a third niche, has underlined the role of immune cells in glioma progression, via specific chemoattractant factors and cytokines, such as macrophage-colony stimulation factor (M-CSF). The local or myeloid origin of this new component of glioma stem cells niche is yet to be determined. Such niches are being increasingly recognized as key regulators involved in multiple stages of disease progression, therapy resistance, immune-escaping, and distant metastasis, thereby substantially impacting the future development of frontline interventions in clinical oncology. This review focuses on the microenvironment impact on the glioma stem cell biology, emphasizing GSCs cross talk with hypoxic, perivascular, and immune niches and their potential use as targeted therapy.
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Purpose: Antiangiogenic therapy is commonly being used for the treatment of glioblastoma. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. Determining the mechanism of treatment failure of the VEGF monoclonal antibody bevacizumab for malignant glioma would provide insight into approaches to overcome therapeutic resistance. Experimental design: In this study, we evaluated the effects of bevacizumab on the autophagy of glioma cells and determined target genes involving in the regulation of bevacizumab-induced autophagy. Results: We demonstrated that bevacizumab treatment increased expression of autophagy markers and autophagosome formation in cell culture experiments as well as in in vivo studies. Gene expression profile analysis performed on murine xenograft models of glioblastoma showed increased transcriptional levels of STAT1/IRF1 signaling in bevacizumab resistant tumors compared to control tumors. In vitro experiments showed that bevacizumab treatment increased IRF1 expression in a dose and time dependent manner, which was coincident with bevacizumab-mediated autophagy. Down regulation of IRF1 by shRNA blocked autophagy and increased AIF-dependent apoptosis in bevacizumab-treated glioma cells. Consistently, IRF1 depletion increased the efficacy of anti-VEGF therapy in a glioma xenograft model, which was due to less bevacizumab-promoted autophagy and increased apoptosis in tumors with down-regulated IRF1. Conclusions: These data suggest that IRF1 may regulate bevacizumab-induced autophagy, and may be one important mediator of glioblastoma resistant to bevacizumab.
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Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and it is associated with poor survival. The standard therapy for newly-diagnosed GBM is radiotherapy with concurrent temozolomide following maximal surgical resection. To improve the outcome of these patients, combinations of the standard therapy plus molecular-targeted agents have been tested in clinical trials. However, the addition of gefitinib to the standard therapy did not appear to improve clinical outcome, and the standard therapy plus bevacizumab showed no improvement in overall survival, although a 4-month improvement in progression-free survival (PFS) was observed. Phase II data have indicated the potential efficacy of talampanel combined with the standard therapy for patients with newly-diagnosed GBM, and these findings are awaiting validation in phase III trials. In addition, phase II trials have demonstrated that adjuvant immunotherapy is effective and tolerable for treatment of patients with GBM. In this article, we discuss topics in chemotherapy, molecular-targeted therapy, and immunotherapy for patients with newly-diagnosed GBM. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma.
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A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05). Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05) compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day’s data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.
In 2010, the National Institutes of Health (NIH) established the Therapeutics for Rare and Neglected Diseases (TRND) program within the National Center for Advancing Translational Sciences (NCATS), which was created to stimulate drug discovery and development for rare and neglected tropical diseases through a collaborative model between the NIH, academic scientists, nonprofit organizations, and pharmaceutical and biotechnology companies. This paper describes one of the first TRND programs, the development of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) for the treatment of Niemann-Pick disease type C1 (NPC1). NPC is a neurodegenerative, autosomal recessive rare disease caused by a mutation in either the NPC1 (about 95% of cases) or the NPC2 gene (about 5% of cases). These mutations affect the intracellular trafficking of cholesterol and other lipids, which leads to a progressive accumulation of unesterified cholesterol and glycosphingolipids in the CNS and visceral organs. Affected individuals typically exhibit ataxia, swallowing problems, seizures, and progressive impairment of motor and intellectual function in early childhood, and usually die in adolescence. There is no disease modifying therapy currently approved for NPC1 in the US. A collaborative drug development program has been established between TRND, public and private partners that has completed the pre-clinical development of HP-beta-CD through IND filing for the current Phase I clinical trial that is underway. Here we discuss how this collaborative effort helped to overcome scientific, clinical and financial challenges facing the development of new drug treatments for rare and neglected diseases, and how it will incentivize the commercialization of HP-beta-CD for the benefit of the NPC patient community.
Glioma is the most common form of primary malignant brain cancers. Tumor cell invasiveness is a critical challenge in the clinical management of glioma patients. The invasive biological feature of glioma cell is stimulated by both autocrine and paracrine factors including chemokine IL-8. In this study, we report that the production of IL-8 is higher in glioma tissues and cells than adjacent nontumor tissues (ANT) and normal glial cells. Autocrine IL-8 can increase the invasive ability of glioma cells by binding to CXCR1. In addition, high expression of IL-8 indicates poor prognosis of glioma patients. Furthermore, IL-8 is capable of modulating cell migration and invasion by regulating the activation of RAC1 which resulted in cytoskeletal reorganisation in an ELMO1 dependent manner. Finally, we found that IL-8 could enhance mesenchymal transition(MT) of glioma cells by activating ELMO1-NF-κB-Snail signaling. Our data indicate that IL-8 autocrine is responsible for the invasive phenotype of glioma and IL-8 may be a useful prognostic marker for glioma and novel therapeutic target for glioma invasion intervention.