Expression of HIF-1α and MDR1/P-glycoprotein in refractory mesial temporal lobe epilepsy patients...
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ORIGINAL ARTICLE
Expression of HIF-1a and MDR1/P-glycoprotein in refractorymesial temporal lobe epilepsy patients and pharmacoresistanttemporal lobe epilepsy rat model kindled by coriaria lactone
Yaohua Li • Jianbin Chen • Tianfang Zeng •
Ding Lei • Lei Chen • Dong Zhou
Received: 2 December 2013 / Accepted: 11 February 2014
� Springer-Verlag Italia 2014
Abstract Hypoxia-inducible factor-1a (HIF-1a) is
thought to mediate pharmacoresistance in tumor by inducing
Pgp overexpression. We aimed to investigate the expression
of HIF-1a and MDR1/P-glycoprotein in refractory epilepsy,
to explore the correlation of HIF-1a with epilepsy multidrug
resistance. We collected hippocampus and mesial temporal
lobe (MTL) cortex of refractory mesial temporal lobe epi-
lepsy (mTLE) patients that underwent surgery, and estab-
lished a pharmacoresistant TLE rat model kindled by coriaria
lactone. We used real-time quantitative PCR (RQ-PCR) and
western blot to investigate expression of HIF-1a and MDR1
in hippocampus and MTL/entorhinal cortex. We found that
the expression of HIF-1a and MDR1, at both mRNA and
protein levels, were up-regulated in hippocampus and MTL
cortex of mTLE patients compared with the control cortex
(all P \ 0.05), and increased in hippocampus and entorhinal
cortex of kindled rat model versus the control group (all
P \ 0.05). These results demonstrated the overexpression of
HIF-1a and MDR1/Pgp in hippocampus and MTL/entorhi-
nal cortex of mTLE patients and the pharmacoresistant TLE
rat model. HIF-1a may have a regulatory effect on MDR1
expression in refractory epilepsy, which is probably con-
sistent with MDR mechanism in tumor.
Keywords HIF-1a � Pgp � Multidrug resistance � mTLE �Epilepsy rat model � Coriaria lactone
Introduction
Resistance to multiple anti-epileptic drugs (AEDs) has
been an important clinical challenge in refractory epilepsy
therapy for neurologists. Numerous studies have found that
overexpression of multidrug transporters, such as the
multidrug resistance gene 1 (MDR1) product P-glycopro-
tein (Pgp), in the blood–brain barrier (BBB) restricted
anticonvulsant effect by promoting AEDs efflux [1–4].
Numerous studies have revealed that overexpression of
MDR1/Pgp conferred multidrug resistance in cancer [5, 6],
and hypoxia-inducible factor-1 (HIF-1) may be a primary
factor regulating the expression of MDR1/Pgp [7, 8]. HIF-1
consists of a heterodimer of HIF-1a and HIF-1b, in which
HIF-1a determines the biological activity associated with
hypoxic adaptation and pathological response [9]. A study
revealed that the MDR1 gene-promoter contains a func-
tional HIF-1a binding site known as classical hypoxia
response element (HRE) [10]. Recurrent seizures and fre-
quent epileptic discharges may also cause ambient hypoxia,
resulting in HIF-1a accumulation to adapt to hypoxic
environments.
Considering those evidences, we hypothesized that HIF-
1a has a regulatory function in MDR1 expression in
refractory epilepsy resembling in tumors. Temporal lobe
epilepsy (TLE) accounts for the largest proportion of
refractory epilepsy, and hippocampal sclerosis (HS) is the
most frequent pathological finding in TLE [11]. Therefore,
Lei Chen and Dong Zhou, as the co-corresponding authors,
contributed equally to this study.
Y. Li � T. Zeng � L. Chen (&) � D. Zhou (&)
Department of Neurology, West China Hospital, Sichuan
University, No. 37 Wainan Guoxue Road,
Chengdu 610041, Sichuan, China
e-mail: [email protected]
D. Zhou
e-mail: [email protected]
J. Chen � D. Lei
Department of Neurosurgery, West China Hospital, Sichuan
University, No. 37 Wainan Guoxue Road,
Chengdu 610041, Sichuan, China
123
Neurol Sci
DOI 10.1007/s10072-014-1681-0
we designed this study to investigate the expressions of
HIF-1a and MDR1 in refractory mesial temporal lobe
epilepsy (mTLE) and in a pharmacoresistant TLE Spra-
gue–Dawley (SD) rat model kindled by coriaria lactone
(CL), using real-time quantitative PCR (RQ-PCR), and
western blot (WB) for analysis.
Materials and methods
mTLE patients and control group
We planned to collect the hippocampus and MTL cortex of
mTLE patients. Patients recruited in this study were diag-
nosed with refractory epilepsy by neurologists according to
the definition of pharmacoresistant epilepsy [12]. The 24-h
EEG monitoring indicated that epilepsy-like waves origi-
nated from unilateral temporal lobe. In addition, there was
ipsilateral hippocampal sclerosis identified by MRI or low
metabolism in ipsilateral mesial temporal lobe identified by
PET, without other pathological changes. Surgery was
determined with neurologist and neurosurgery specialist
consultation. In operation, cortical electrode monitoring
confirmed that epilepsy-like waves originated from the
interior temporal lobe, thus anterior temporal lobe resection
was performed. HS was confirmed by frozen pathological
examination. It was infeasible to obtain brain tissues of
normal hippocampus and mesial temporal lobe (MTL) cor-
tex, so that normal temporal cortex tissues were used as
negative control. All patients in control group did not have
history of epilepsy and other systematic diseases.
The study was approved by the Ethics Committee of
West China Hospital. Informed consents were obtained
from the patients and their legal guardians on the use of
their brain tissues in this research. Finally, we collected
five refractory mTLE patients with the hippocampus and
MTL cortex and five cases with normal temporal lobe
cortex as control group. The brain tissues were separated as
needed and immediately preserved in liquid nitrogen. The
clinical data are shown in Table 1.
Pharmacoresistant TLE rat model
The lack of normal hippocampus as control compromised
the validity of study in patients. As supplement, the study
was also performed in a refractory epilepsy rat model. In
our previous study, a kindled Sprague–Dawley (SD) rat
model induced by CL was confirmed as a refractory TLE
model [13]. This epilepsy model is similar to human mTLE
with MTL epileptic genesis and pharmacoresistant prop-
erties [13]. We also have received a Chinese patent for this
epilepsy animal model.
The animal study was approved by the Experimental
Animal Management Institute of Sichuan University and
strictly performed in compliance with the ‘‘Laboratory
Animal Welfare Protection Law of China’’. Fifteen healthy
male SD rats aged 6–8 weeks and weighing 100–120 g were
used. The rats were acclimated under laboratory conditions
Table 1 Clinical data of mTLE patients group and control group
(a) Clinical data of mTLE patients group
Case Gender Age, y Seizure type Duration, y EEG, sp ori MRI/PET
1 M 20 CPS, SGS 6 L-T L-HS/–
2 M 20 CPS, SGS 11 L-T L-HS/–
3 M 19 CPS, SGS 8 L-T L-HS/–
4 F 36 CPS, SGS 15 R-T R-HS/–
5 M 24 CPS, SGS 9 R-T N/R-Ta
(b) Clinical data of control group
Case Gender Age, y Tissue sources
1 M 49 Operative route of benign neoplasm in deep area of brain
2 F 29 Adjacent normal cortex in surgical evacuation of IH
3 F 47 Adjacent normal cortex in surgical evacuation of IH
4 M 36 Adjacent normal cortex in surgical evacuation of IH
5 M 39 Operative route of benign neoplasm in deep area of brain
mTLE mesial temporal lobe epilepsy, M male, F female, y year, CPS complex partial seizure, SGS secondarily generalized seizure, sp ori spikes
origin, L left, R right, T temporal lobe, HS hippocampal sclerosis, N normal, IH intracerebral hematoma
– indicates no PETa Indicates PET show low metabolism
Neurol Sci
123
for 1 week before the start of the experiment. CL is an epi-
leptogenic agent extracted from a traditional Chinese herb
coriaria [14], containing tutin (C15H19O6) and coriamyrtin
(C15H18O5) at a concentration of 5 mg/ml (tutin[50 %).
SD rats were randomly divided to experimental (n = 10)
and control (n = 5) groups. The rats in experimental group
were intramuscularly injected with CL 0.4 ml/kg every 72 h,
while in control group with same dose normal sodium. Sei-
zures were graded according to Racine’s five-stage scale
(1972) [15]. The rats were considered as completely kindled
if they had five or more consecutive stage 4 or 5 seizures with
generalized high-amplitude epileptiform discharges on EEG
[13]. After a maximum of 18 times of CL injections, five rats
in experimental group were successfully kindled. In control
group there was no unexpected deaths. Brain tissues of the
kindled group and control group were removed immediately
after deep anesthesia with 6 % chloral hydrate by peritoneal
injection. The hippocampus and entorhinal cortex were
quickly dissected out and preserved in liquid nitrogen.
RQ-PCR
Total RNA was extracted according to Trizol method
(Invitrogen, USA). RNA integrity was analyzed by 1 %
agarose gel electrophoresis. The b-actin was taken as an
internal control. PCR primers and TaqMan probe were
designed and synthesized by Shanghai Shenggong (China).
The sequences are shown in Table 2. RQ-PCR was per-
formed with the FTC2000 PCR system (Funglyn, Canada).
The following conditions were used for amplification:
94 �C for 2 min; 94 �C for 20 s; 54 �C (MDR1, b-actin)/
50 �C (HIF-1a) for 20 s; and 60 �C for 30 s for 45 cycles.
PCR-amplified products qualitatively detected by 2.0 %
agarose gel electrophoresis. The expression rate between
groups was calculated according to the equation derived by
Livak and Schmittgen [16]: expression rate (R) = 2-DDCT.
CT denotes the number of cycles at which the fluorescent
signals in the reaction system are detected by the thermal
cycler, DCT = CT (target gene) - CT (b-actin), and
DDCT = DCT (trial group) - DCT (control group).
WB
Tissues samples (100 mg) were homogenized and centri-
fuged. The protein concentration was determined by BCA
protein assay kit (Pierce, USA) and adjusted to 2 lg/ll. Each
protein sample (10 ll) was resolved by SDS-PAGE (Amer-
sham 80-6418-77, USA) and wet transferred to PVDF mem-
brane (Amersham TE22, USA). The samples were blocked
and then incubated with the primary antibody diluted at
1:1,000 (mouse anti-HIF-1a monoclonal antibody, Novus,
USA; rabbit anti-MDR1 polyclonal antibody, Boaosen, China)
at 4 �C overnight. The membranes were washed and then
incubated with HRP-labeled goat anti-mouse (for HIF-1a) or
anti-rabbit (for MDR1) secondary antibody (1:10,000, Pierce,
USA) at room temperature for 1 h, separately. The bands were
detected using a chemiluminescent substrate (Millipore,
USA). Theb-actin was used as an internal control. Gray values
were measured using ImageJ Analysis Software (NIH). The
relative expressions of samples in different duplications were
standardized by a same sample of control group.
Statistical analysis
Data were processed by SPSS16.0 and expressed as
mean ± standard deviation (SD). The results were com-
pared by one-way analysis of variance (ANOVA), followed
by least-significant difference (LSD) test for multiple
intergroup comparisons as needed. All tests were two
sided, and P \ 0.05 was considered statistically significant.
Results
RQ-PCR
In the human brain tissue samples, the R value of HIF-1amRNA was 2.91 in the hippocampus and 3.39 in the MTL
cortex versus the control cortex, with significant differ-
ences, respectively (both P \ 0.05). The R value of MDR1
mRNA in the hippocampus and MTL cortex were 2.71 and
2.87 versus the normal cortex, respectively (both
P \ 0.05). No significant difference of HIF-1a mRNA, as
well as MDR1 mRNA, was obtained between hippocampus
and MTL cortex (both P [ 0.05). See in Fig. 1.
Table 2 Sequences of RQ-PCR primers and probes for HIF-1a,
MDR1, and b-actin
Gene Sequences
HIF-1a
Primer
Forward 50-TGCTGATTTGTGAACCCATT-30
Reverse 50-CCAAAGCATGATAATATTCAT-30
TaqMan probe 50-CTCAGTCGACACAGCCTC-30
MDR1
Primer
Forward 50-GCCGAAAACATTCGCTATG-30
Reverse 50-TCTCACCAACCAGGGTGT-30
TaqMan probe 50-CTGTCAAGGAAGCCAATGCC-30
b-Actin
Primer
Forward 50-AAGGCCAACCGCGAGAA-30
Reverse 50-CCTCGTAGATGGGCACA-30
TaqMan probe 50-CTGCACCACCAACTGCTTAGC-30
Neurol Sci
123
In the rat samples, the expression rates of HIF-1amRNA in the kindled group were 1.74 in hippocampus
and 1.39 in entorhinal cortex compared with that in the
control group (both P \ 0.05). The mdr1 mRNA
expression rate values were 2.30 and 1.47 in hippocampus
and entorhinal cortex, respectively (both P \ 0.05). See in
Fig. 2.
WB analysis
For the human brain tissue samples, significant differences
were observed in terms of HIF-1a protein expression in the
hippocampus (1.55 ± 0.12) and MTL cortex (1.46 ± 0.06)
versus the control cortex (1.08 ± 0.18) (both P \ 0.05).
Expressions of Pgp in the hippocampus (1.56 ± 0.18) and
Fig. 1 DCT values of HIF-1a(a) and MDR1 (b) mRNA in
human brain tissue samples.
Higher mRNA expression was
indicated by lower DCT.
Asterisk indicates a significant
difference
Fig. 2 DCT values of HIF-1a(a) and mdr1 (b) mRNA in rat
brain samples. Higher mRNA
expression was indicated by
lower DCT. Asterisk indicates a
significant difference
Fig. 3 Relative expression of
HIF-1a protein (a) and Pgp
(b) in human brain samples. The
representative immunoblot
bands are shown below the
histograms. Asterisk indicates a
significant difference
Neurol Sci
123
MTL cortex (1.51 ± 0.12) versus control cortex
(1.08 ± 0.14), both increased significantly (P \ 0.05), which
was consistent with the up-regulation of HIF-1a. There were
no statistical difference between the hippocampus and MTL
cortex for HIF-1a and Pgp expressions. See in Fig. 3.
In the rat brain samples, the expression of HIF-1a in
hippocampus was significantly higher in kindled group
(1.56 ± 0.11) compared with that in control group
(0.97 ± 0.10; P \ 0.05), and in entorhinal cortex was
1.40 ± 0.07 in kindled group versus 1.00 ± 0.11 in control
group (P \ 0.05). Consistent with HIF-1a, in hippocampus
Pgp overexpressed in kindled group (1.75 ± 0.19) com-
pared with control group (0.99 ± 0.12; P \ 0.05), and in
entorhinal cortex kindled group scored at 1.48 ± 0.07
compared with control group scored at 0.99 ± 0.11
(P \ 0.05). See in Fig. 4.
Discussion
Transporter theory commends that transport protein, such as
Pgp, overexpression and over-activity can hinder AEDs
reaching an effective therapeutic concentration in the epi-
leptogenic focus [17]. On the other hand, numerous studies,
in the tumor pharmacoresistance mechanism, have dem-
onstrated that MDR1 expression is modulated by HIF-1a[10, 18]. In the present study, we found that expression of
HIF-1a and MDR1, at mRNA and protein levels, up-regu-
lated in hippocampus and MTL cortex/entorhinal cortex.
The expression of MDR1 in both rat and human are posi-
tively correlated to HIF-1a, at mRNA and notably at protein
levels, indicating the potential relationship between them.
This study was the first, to our knowledge, to report
expression of HIF-1a in refractory epilepsy patients and the
possible relationship between HIF-1a and MDR1. These
findings suggested that HIF-1a may potentially induce
multidrug resistance in epilepsy by up-regulating MDR1,
which was consistent with the MDR mechanism in tumor.
Our study demonstrated that, in mTLE patients and TLE
model, mRNA and protein of HIF-1a up-regulated in
hippocampus and MTL/entorhinal cortex. HIF-1a is
induced in hypoxic condition but rapidly degraded under
normoxic conditions by the ubiquitin–proteasome system
[19]. Recurrent seizures and frequent subclinical electro-
graphic seizure discharges originated from MTL can lead
to enhanced local oxygen consumption and oxygen desat-
urations in whole body [20, 21], resulting in HIF-1aaccumulation especially in MTL. In addition, some studies
have demonstrated that in patients with mTLE, interictal
and periictal hypoperfusion of MTL was found with
SPECT [22, 23], and interictal hypometabolism was
exhibited in MTL with PET [24, 25], which may explain
overexpression and accumulation of HIF-1a in MTL.
Other studies in animals have also demonstrated that
some downstream target genes of HIF-1, such as EPO and
VEGF, participated in epileptic neurogenesis [26, 27]. As
in ischemic/hypoxic encephalopathy studies HIF-1 mani-
fests dual nature of apoptosis and adaptive [28]. Consid-
ering HIF-1a overexpressed in hippocampus, we
speculated that HIF-1a may involve hippocampal neuronal
apoptosis, secondary glial cell proliferation, and incorrect
connection of the neural network. Therefore, it suggested
that HIF-1a may be a core factor involving occurrence and
development of HS.
A limitation of our study is that optimal control brain
tissues of human, i.e., normal hippocampus and normal
MTL cortex, were infeasible to gain. Thus, we adopted a
pharmacoresistant TLE rat model as supplement. More-
over, the correlation of HIF-1a and MDR1/Pgp should be
further detected. In further research, the co-expression of
HIF-1a and Pgp in spatial distribution at cellular and
structural levels based on immunohistochemistry is needed,
and inhibiting HIF-1a expression using RNA-interference
technology in the rat model will be studied to verify the
possible regulation effect of HIF-1a on MDR1 in refractory
epilepsy.
Fig. 4 Relative expression of
HIF-1a protein (a) and Pgp
(b) in rat brain samples. The
representative immunoblot
bands are shown. Asterisk
indicates a significant difference
Neurol Sci
123
In summary, both HIF-1a and MDR1 up-regulated in
the hippocampus and MTL/entorhinal cortex in mTLE
patients and the pharmacoresistant TLE rat model. HIF-1amay have a regulatory effect on MDR1 expression in
pharmacoresistant epilepsy, which is similar to the multi-
drug resistance mechanism in tumor. The present research
is likely to provide new insights on pharmacoresistance
mechanism in refractory epilepsy.
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (no. 81371425), the scientific
research foundation of Sichuan University for outstanding young
scholars (no. 2082604164246), Sichuan Province basic research plan
project (no. 2013JY0168) and Chengdu City Science and Technology
Bureau fund (no. 12DXYB209JH-002).
References
1. Brandt C, Bethmann K, Gastens AM et al (2006) The multidrug
transporter hypothesis of drug resistance in epilepsy: proof-of-
principle in a rat model of temporal lobe epilepsy. Neurobiol Dis
24(1):202–211
2. Schinkel AH (1999) P-Glycoprotein, a gatekeeper in the blood-
brain barrier. Adv Drug Deliv Rev 36(2–3):179–194
3. Sisodiya SM, Lin WR, Harding BN et al (2002) Drug resistance
in epilepsy: expression of drug resistance proteins in common
causes of refractory epilepsy. Brain 125(Pt 1):22–31
4. Marchi N, Hallene KL, Kight KM et al (2004) Significance of
MDR1 and multiple drug resistance in refractory human epileptic
brain. BMC Med 2:37
5. Wu H, Hait WN, Yang JM (2003) Small interfering RNA-
induced suppression of MDR1 (P-glycoprotein) restores sensi-
tivity to multidrug-resistant cancer cells. Cancer Res
63(7):1515–1519
6. Gottesman MM (2002) Mechanisms of cancer drug resistance.
Annu Rev Med 53:615–627
7. Haar CP, Hebbar P, Wallace GCt et al (2012) Drug resistance in
glioblastoma: a mini review. Neurochem Res 37(6):1192–1200
8. Ding Z, Yang L, Xie X et al (2010) Expression and significance
of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in
human colon carcinoma tissue and cells. J Cancer Res Clin Oncol
136(11):1697–1707
9. Wang GL, Jiang BH, Rue EA et al (1995) Hypoxia-inducible
factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by
cellular O2 tension. Proc Natl Acad Sci USA 92(12):5510–5514
10. Comerford KM, Wallace TJ, Karhausen J et al (2002) Hypoxia-
inducible factor-1-dependent regulation of the multidrug resis-
tance (MDR1) gene. Cancer Res 62(12):3387–3394
11. Williamson PD, French JA, Thadani VM et al (1993) Charac-
teristics of medial temporal lobe epilepsy: II. Interictal and ictal
scalp electroencephalography, neuropsychological testing, neu-
roimaging, surgical results, and pathology. Ann Neurol
34(6):781–787
12. Kwan P, Arzimanoglou A, Berg AT et al (2010) Definition of
drug resistant epilepsy: consensus proposal by the ad hoc Task
Force of the ILAE Commission on Therapeutic Strategies. Epi-
lepsia 51(6):1069–1077
13. Wang Y, Zhou D, Wang B et al (2003) A kindling model of
pharmacoresistant temporal lobe epilepsy in Sprague–Dawley
rats induced by coriaria lactone and its possible mechanism.
Epilepsia 44(4):475–488
14. Zhou H, Tang YH, Zheng Y (2006) A new rat model of acute
seizures induced by tutin. Brain Res 1092(1):207–213
15. Racine RJ (1972) Modification of seizure activity by electrical
stimulation. II. Motor seizure. Electroencephalogr Clin Neuro-
physiol 32(3):281–294
16. Livak KJ, Schmittgen TD (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the 2(-Delta
Delta C(T)) Method. Methods 25(4):402–408
17. Feldmann M, Asselin M-C, Liu J, Wang S et al (2013) P-gly-
coprotein expression and function in patients with temporal lobe
epilepsy: a case-control study. Lancet Neurol 12(8):777–785
18. Song X, Liu X, Chi W et al (2006) Hypoxia-induced resistance to
cisplatin and doxorubicin in non-small cell lung cancer is
inhibited by silencing of HIF-1alpha gene. Cancer Chemother
Pharmacol 58(6):776–784
19. Huang LE, Gu J, Schau M et al (1998) Regulation of hypoxia-
inducible factor 1alpha is mediated by an O2-dependent degra-
dation domain via the ubiquitin–proteasome pathway. Proc Natl
Acad Sci USA 95(14):7987–7992
20. Maglajlija V, Walker MC, Kovac S (2012) Severe ictal hypox-
emia following focal, subclinical temporal electrographic scalp
seizure activity. Epilepsy Behav 24(1):143–145
21. Blum AS, Ives JR, Goldberger AL et al (2000) Oxygen desatu-
rations triggered by partial seizures: implications for cardiopul-
monary instability in epilepsy. Epilepsia 41(5):536–541
22. Tae WS, Joo EY, Kim JH et al (2005) Cerebral perfusion changes
in mesial temporal lobe epilepsy: SPM analysis of ictal and
interictal SPECT. Neuroimage 24(1):101–110
23. Oommen KJ, Saba S, Oommen JA et al (2004) The relative
localizing value of interictal and immediate postictal SPECT in
seizures of temporal lobe origin. J Nucl Med 45(12):2021–2025
24. Vielhaber S, Von Oertzen JH, Kudin AF et al (2003) Correlation
of hippocampal glucose oxidation capacity and interictal FDG-
PET in temporal lobe epilepsy. Epilepsia 44(2):193–199
25. Matheja P, Kuwert T, Ludemann P et al (2001) Temporal
hypometabolism at the onset of cryptogenic temporal lobe epi-
lepsy. Eur J Nucl Med 28(5):625–632
26. Eid T, Brines ML, Cerami A et al (2004) Increased expression of
erythropoietin receptor on blood vessels in the human epilepto-
genic hippocampus with sclerosis. J Neuropathol Exp Neurol
63(1):73–83
27. Rigau V, Morin M, Rousset MC et al (2007) Angiogenesis is
associated with blood–brain barrier permeability in temporal lobe
epilepsy. Brain 130(Pt 7):1942–1956
28. Chen W, Ostrowski RP, Obenaus A et al (2009) Prodeath or
prosurvival: two facets of hypoxia inducible factor-1 in perinatal
brain injury. Exp Neurol 216(1):7–15
Neurol Sci
123