Received: 11 November 2019 | Revised: 15 January 2020 | Accepted: 9 February 2020

DOI: 10.1111/jcmm.15156

ORIGINAL ARTICLE

CDK4/6 inhibition suppresses tumour growth and enhances

the effect of temozolomide in glioma cells

Yingxiao Cao1 | Xin Li2 | Shiqi Kong1 | Shuling Shang3 | Yanhui Qi4

1
Department of Neurosurgery, Xingtai
People’s Hospital, Xingtai, China
2
Department of Neurosurgery, The First
People's Hospital of Shenyang, Shenyang,
China
3
Department of Operating Room, Xingtai
People’s Hospital, Xingtai, China
4
Department of Intensive Care Unit, Xingtai
People’s Hospital, Xingtai, China
Correspondence
Yingxiao Cao, Department of Neurosurgery,
Xingtai People’s Hospital, Xingtai, Hebei
054031, China.
Email: caoyingxiao2019@163.com
Abstract
In adults, glioma is the most commonly occurring and invasive brain tumour. For ma-
lignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide).
However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to
an urgent need for more efficient treatment choices. Here, we report that cyclin-
dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell
lines and tissues. CDK4 overexpression enhances colony formation and proliferation
of glioma cells and extends resistance to inhibition of TMZ-mediated cell proliferation
and induction of apoptosis. However, CDK4 knockdown impedes colony formation
and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective
inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induc-
tion. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ.
We further showed the possible role of RB phosphorylation mediated by CDK4 for
its oncogenic function in glioma. The growth of glioma xenografts was inhibited in
vivo, through combination treatment, and corresponded to enhanced p-RB levels,
reduced staining of Ki-67 and enhanced activation of caspase 3. Therefore, CDK4
inhibition may be a favourable strategy for glioma treatment and overcomes TMZ
resistance.

KEYWORDS

CDK4/6, drug resistance, glioma, p-RB, temozolomide

1 | I NTRO D U C TI O N in treatment of glioma, the overall survival (OS) of glioma patients is

An important category of human brain tumours, gliomas, are the most
invasive and fatal brain tumour of adulthood that are well-differenti-
ated astrocytomas of low-grade, anaplastic astrocytomas and GBM
1
(glioblastoma multiforme). The most aggressive subtype among the
gliomas is GBM with the poorest rates of survival.2 The commonly ap-
plied treatments are chemotherapy, surgical resection and radiother-
apy.3,4 Although the past few years have seen significant advancement
only 1-1.25 years.5
Temozolomide (TMZ), an alkylating agent, is currently used as
a first-line drug for treatment of glioma including GBM.6 TMZ leads
to guanine methylation in the DNA at position O6, causing incorrect
pairing of O6-methylguanine with thymine, activating the mismatch
repair system.7 This results in a break in the double-strand genome
that causes cell cycle arrest and apoptotic induction.6,8 TMZ resistance
is induced by MGMT (O6-methylguanine-DNA methyltransferase)

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J Cell Mol Med. 2020;24:5135–5145.  wileyonlinelibrary.com/journal/jcmm | 5135

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5136
by eliminating methylation from guanine at O6 position.9 Therefore,
patients with methylated MGMT promoter and silenced MGMT ex-
pression showed a better median and 2-year survival through com-
bined radiotherapy and TMZ chemotherapy.10 In patients with GBM
resistant to TMZ, resistance was anticipated to be inhibited by MGMT
pseudo-substrates through MGMT depletion, although no significant
restoration of TMZ sensitivity was observed in clinical trials.11 Thus,
new strategies are needed to sensitize patients to the efficiency of
chemotherapy using TMZ.
The common features in several types of cancer, including mela-
noma, include constitutive CDK (cyclin-dependent kinases) activation
and of cell cycle deregulation.12,13 In 38% of cancer, there is a deletion
of a tumour suppressor and a negative regulator of CDK4, P16INK4a.14
Furthermore, there are CDK4 gene amplification and mutations in ger-
mline in melanoma, causing unhindered CDK4 activity and enhanced
cell proliferation.15 CDK6 is regulated by cyclin, more specifically by cy-
clin D protein and CDK inhibitor protein. CDK6 is overexpressed in can-
cers including leukaemia, melanoma, medulloblastoma and lymphoma,
which associated with chromosomal rearrangements. Generally, the
cyclins control the cell cycle entry in proliferating adult mammalian cells
by binding and activating CDK4 and CDK6 and facilitation of retinoblas-
toma (RB) phosphorylation and G1 to S transition.16 As a result, CDK4
activity deregulation correlated with tumorigenesis, although, there
are only rare reports of studies on the function of CDK4 in glioma.17
Palbociclib (PD0332991), a specific CDK4/6 inhibitor, was developed to
18
arrest the cell cycle progression in proliferating tumour cells. It proved
to be beneficial especially in tumours lacking p16INK4a or overexpress-
ing cyclin D, such as bladder and gastric cancers, while those without
functional RB1 have been refractory to palbociclib treatment.19 Upon
promising results in preclinical models of various cancers including
GBM, palbociclib was tested in several clinical trials in phase I/II and
has been approved by the FDA in combination with anti-oestrogen
therapies against hormone receptor-positive breast cancers.20,21 These
clinical studies indicate that as a single agent, palbociclib fails to provide
durable responses, potentially due at least in part to tumour adaptation,
and suggesting a need of combination with other agents.22 Previously
study has shown that pharmacologic inhibition of cyclin-dependent ki-
nases 4 and 6 arrests the growth of glioblastoma multiforme intracranial
xenografts.23 In addition, palbociclib significantly prolongs survival in a
genetically engineered mouse model of brainstem glioma.24 Previously
studies also have shown that combining CDK4/6 and mTOR inhibitors
offers increased benefits against glioma through a number of mech-
anisms, including blockade of compensatory signalling mechanisms,
improved brain penetration of palbociclib, enhanced metabolic effects
and cytostatic to cytotoxic conversion.25 Although modifications in
several cell cycle regulators, such as activation of CDK2, amplification
of cyclin D and loss of p21CIP1 or p27KIP1, may contribute to tumour
adaptation, the main resistance to palbociclib treatment is mediated by
RB1 inactivation.26 We therefore sought a combination regimen using
CDK4/6 inhibitors that inhibits overall cell cycle progression.
In this study, we report the overexpression of CDK4 in glioma tis-
sues and cell lines, promoting proliferation of glioma and formation
of spheroid and bestow TMZ resistance. The inhibition of activity
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CAO et al.
of CDK4 deacetylase by specific CDK4 inhibitors impedes prolifer-
ation and enhances apoptosis in glioma cell lines. The glioma cells
can be sensitized to TMZ by inhibitors of CDK4. Our outcome here
indicates that CDK4 inhibition may be a possible treatment strategy
for glioma. In this study, we evaluated the effect of CDK4 overex-
pression and its inhibition in glioma cells in vitro and in vivo, and
determined the characteristics of glioma cell such as apoptosis and
associated protein markers.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Collection of samples from patients
Between 2015 and 2018, tissues from normal brain (n = 6) and gli-
oma tissues of high grade (n = 12) were collected during surgery from
Xingtai People's Hospital. The samples were frozen immediately in
liquid nitrogen till used further. The ethics committee of Xingtai
People's Hospital gave consent to the project.
2.2 | Cell culture
The ATCC (American Type Culture Collection) provided us with the
cell lines for human glioma (U87, U251, H4 and A172). NHAs (Normal
human astrocytes) were procured from Lonza (Switzerland) and cul-
tured as per supplied instructions. These cell lines were maintained in
DMEM (HyClone) containing FBS (10%) and penicillin-streptomycin
(1%).
2.3 | Assay to detect colony formation
Seeding of cells was done in 6-well plates at of the rate of 500 cells
in each well. Post-adhesion, treatment was done with TMZ with or
without CDK inhibitors. Every three days, renewal of the culture
medium was done. After two weeks, the cells were crystal violet
stained and imaged.
2.4 | CCK-8 assay
The cells cultured in 96-well plates were treated with the specified
compounds, or transfected with indicated siRNA or plasmids, and at
indicated times, measurement of cell proliferation was done using
CCK-8 kit (Sigma).
2.5 | Detection of apoptosis
The apoptosis assay was performed as previously study. 27,28
Briefly, cultured cells in six-well plates were treated with the speci-
fied compounds, collected after trypsinization, given PBS wash

CAO et al.
and suspended again in binding buffer. These were then stained
for 15 minutes with Annexin V/PI as per instructions from Becton
Dickinson. A Becton Dickinson flow cytometer was used to detect
fluorescence.
2.6 | Plasmids and transfection
The wild-type CDK4 (CDK4-WT) and CDK4-DN expressing plasmids
29
were procured from Addgene. Transfection of plasmids was done
into cells along with DNA transfection agent TurboFect as per in-
structions (Thermo Scientific). At 20%-30% confluence of cells, 2 μg
plasmids were transfected per well of the six-well plate and 0.1 μg
plasmids in each well of a plate with 96-wells. The siRNAs target-
ing human CDK4 and control siRNA were procured from RiboBio.
Transfection of siRNA was done along with Lipofectamine 3000
from Invitrogen into the cells as per the protocol of the manufac-
turer. At 20%-30% confluence of cells, siRNA at 50 pmol was trans-
fected in each well of a six-well plate and at 2.5 pmol in each well of
a 96-well plate.
2.7 | Quantitative real-time PCR
For two days, the cells were treated and then extraction of RNA was
done using TRIzol reagent (Sigma). Using PrimeScript™ RT reagent
Kit (Perfect Real Time), reverse transcription was carried out fol-
lowed by quantitative RT-PCR in a CFX96 Real-Time PCR Detection
System from Bio-Rad using SYBR® Premix Ex Taq™ II master mix (Tli
RNaseH Plus) from Takara, China. Synthesis of the primers was done
at Sangon Biotech Co., Ltd. Per experiment was done independently
in triplicates. The expression of β-actin was used for normalization
of relative gene expression which was determined through the 2-ΔΔCt
method.
2.8 | Western blotting
Western blotting was performed as previously research.30,31 The
lysis of cells was done in lysis buffer. The samples were resolved by
SDS-PAGE, transferred to PVDF (polyvinylidene fluoride, Roche)
membrane and blocked with skim milk (5%) and kept along with the
specified primary antibody. Then, incubation with the appropriate
secondary antibody conjugated with HRP (horseradish peroxidase)
was done. The bands that appeared were observed with Western
Bright ECL HRP substrate and Kodak films were used to develop
them.
2.9 | Xenograft studies
The right back of BALB/c nude mice (5-week-old female) were in-
jected subcutaneously with U251 cells (2 × 10 6 cells). On reaching
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5137
nearly 100 mm3 tumour volume, the mice were arbitrarily grouped
into four categories (6 mice/group). Treatment of mice per group
was done daily through oral gavage with vehicle, abemaciclib
(150 mg/kg/d), TMZ (50 mg/kg/d) or their combination for three
weeks. Tumours were measured Every two days, tumours were
measured through a Vernier caliper, and their volume was deter-
mined by the formula: V = (L × W2)/2 (W, width; L, Length). The
experiments were carried out as per the Regulations for the
Administration of Affairs Concerning Experimental Animals and
consented by the Experimental Animal Ethics Committee of Xingtai
People's Hospital.
2.10 | Immunohistochemical (IHC) staining
Immunohistochemical was performed as previously study.32,33
Tissue sections (4 μm) were first fixed with 1:1 ratio of
Acetone:Methanol for 10 minutes. Peroxidase inhibitor (CM1) was
used to block the endogenous peroxidases for 8 minutes prior to
the incubation of the section with primary antibody at 1:100 di-
lution overnight at 4°C. The DISCOVERY OmniMap AntiRb HRP
detection system and DISCOVERY ChromoMap DAB Kit (Ventana
Co.) were used to detect antigen-antibody complex. Then, each
slide was counterstained with haematoxylin, dehydrated, cleared
and assessed after mounting.
2.11 | Analysis of statistical data
Data are presented as the mean ± SD (standard deviation).
Student's t test was applied to carry out all statistical assess-
ments and realized through GraphPad Prism VI statistical soft-
ware. A difference representing P < .05 was deemed significant
statistically.
3 | R E S U LT S
3.1 | Glioma tissues and cell lines overexpress CDK4
Up-regulation of CDK4 has been observed in several human cancers,
but its role in glioma tumorigenesis unknown to a large extent. To
ascertain CDK4 level in glioma, the comparison of CDK4 mRNA levels
of normal brain tissues from humans and glioma tissues from pub-
lished gene profiling studies (TCGA data). CDK4 expression was ob-
served higher compared to that in normal control tissues (Figure 1A).
Then, through IHC a Western blot assay, the expression of CDK4
in glioma tissues and normal human brain tissues was done. In ac-
cordance with mRNA data, high expression of CDK4 was confirmed
in glioma specimens when compared to nontumour brain tissues
(Figure 1B,C). A similar outcome was observed when the expression
of CDK4 in glioma cell lines and normal human brain tissue were com-
pared (Figure 1D).
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5138 CAO et al.

F I G U R E 1 CDK4 is up-regulated
in glioblastoma tissues and cell lines.
A, Analysis of TCGA datasets showing
high expression of CDK4 mRNA in
glioblastoma specimens compared with
nontumour brain tissues. B, IHC analysis
of CDK4 expression in glioblastoma
specimens and normal brain tissues. Scale
bar, 100 μm. C, Western blotting of CDK4
expression in glioblastoma specimens
and normal brain tissues. D, Comparison
of CDK4 expression between NHAs and
glioblastoma cell lines by western blotting

F I G U R E 2 CDK4 promotes the

proliferation and spheroid formation
of glioblastoma cells. A, U87 cells were
transfected with the indicated plasmids
and cell proliferation was monitored
by a CCK-8 assay. B, U251 cells were
transfected with the indicated plasmids,
and cell proliferation was monitored
by a CCK-8 assay. C, U87 cells were
transfected with the indicated siRNA,
and cell proliferation was monitored
by a CCK-8 assay. D, U251 cells were
transfected with the indicated siRNA,
and cell proliferation was monitored
by a CCK-8 assay. E, U87 cells were
cultured in a nanoculture plate and then
transfected with the indicated plasmids.
Spheroid formation was monitored
under a microscope. F, U87 cells were
cultured in a nanoculture plate and then
transfected with the indicated siRNA.
Spheroid formation was monitored under
a microscope

CAO et al.
3.2 | CDK4 promotes the proliferation and spheroid
formation of glioma cells
Next, we examined the possible regulation of glioma cell prolif-
eration by CDK4 as result of its overexpression in glioma cells. To
assess this, wild-type CDK4 (CDK4-WT) and CDK4-DN overex-
pression plasmids were transfected in U87 and U251 cells, and
CCK-8 assay was done to assess cell proliferation. A significant
rise in the rate of cell proliferation was observed on up-regulation
of CDK4 (Figure 2A,B). The CDK4-DN–transfected group exhib-
ited similar rate of proliferation as the control group (Figure 2C,D).
Furthermore, CDK4 overexpression mainly facilitated the sphe-
roid formation of glioma cells (Figure 2E), although CDK4-DN
overexpression exhibited no evident effect of glioma cells sphe-
roid formation. The ability of glioma to form a spheroid was nearly
abolished as a result of CDK4 knockdown (Figure 2F). Thus, CDK4
promotes spheroid formation and cell proliferation ability of
glioma.
3.3 | The TMZ-mediated killing of glioma cells is
resisted by CDK4
A standard therapy for glioma is surgical resection and radio-
therapy combined with TMZ. 5 However, the effectiveness of this
modality is limited by acquired chemoresistance of glioma cells.
Thus, it is imperative to study TMZ resistance and we conjecture
that CDK4 overexpression in glioma might bestow resistance to
TMZ. Figure 3A,B and E shows that CDK4 overexpression signifi-
cantly enhanced the percentage of living cells post-TMZ treatment.
Furthermore, after TMZ treatment, nearly the same percentage of
living cells was observed in the group transfected with CDK4-DN.
As anticipated, CDK4 knockdown made the glioma more sensitive to
TMZ (Figure 3C,D and F). We evaluated the role of CDK4 in regulat-
ing TMZ-mediated cell apoptosis in glioma cells because at higher
concentrations, TMZ causes apoptotic cell death. TMZ-induced ap-
optosis is remarkably reduced as a result of CDK4 overexpression
(Figure 3G,H), while CDK4 knockdown sensitized glioma cells to
TMZ-mediated apoptosis (Figure 3I-L). Our results thus indicate that
CDK4 overexpression in glioma may possibly be an innate process
that grants TMZ resistance.
3.4 | Selective inhibitors of CDK4/6 inhibit
proliferation and induce apoptosis of glioma cells
From the data mentioned above, CDK4 was found to have a
crucial part in controlling the proliferation of glioma cell, to cor-
roborate this, tried to inhibit CDK4 activity using three selective
inhibitors abemaciclib, palbociclib and ribociclib and assessed
growth inhibition in three human glioma cell lines: A172, U87 and
U251. For all three chemicals tested, a decline in cell prolifera-
tion dependent on dose was observed (Figure 4A-C). Like a few
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5139
anticancer drugs, CDK4 inhibitors led to increase in apoptosis in
glioma U87 cells (Figure 4D,F). Moreover, inhibition of apoptosis
by pan-caspase inhibitor Z-VAD-FMK in combination with inhibi-
tors of CDK4 improved the cell viability percentage remarkably in
comparison with CDK4 inhibitors alone (Figure 4G-I). To further
verify the above results, we carried out Western blot to evalu-
ate the changes in apoptosis-related proteins (cleaved caspase-3,
Bax and Bcl-2) in U87 and U251 cells after 24 hours of treatment
with abemaciclib. Our results indicated abemaciclib enhance Bax
and decrease Bcl-2 levels, and increased caspase 3 activation
(Figure 4J-K).
3.5 | CDK4 inhibition resensitize glioma cells
to TMZ
Overexpression CDK4 confers resistance to TMZ; thus, we speculated
that CDK4 inhibition might aid in an enhanced TMZ efficiency in glioma.
To assess any cooperation between TMZ and inhibitors of CDK4 in the
chemotherapy of glioma, we treated glioma cells with TMZ and abe-
maciclib either singly or in combination. Accordingly, the cotreatment
with TMZ and abemaciclib significantly lowered the cell viability when
compared to treatment with TMZ alone (Figure 5A,B). In addition, the
combination index (CI) values of abemaciclib and TMZ were evaluated,
and abemaciclib showed strong synergistic effects with TMZ in U87
and U251 cells (Figure 5C,D). After 2 weeks, we assessed long-term
survival by staining through crystal violet and observed that the com-
bination nearly fully eliminated U87 and U251 cell clonogenic survival
(Figure 5E,F). Furthermore, an increase in apoptotic rate was also ob-
served after treatment with the combination, as an increase in Annexin
V-positive cell number was found (Figure 5G,H). Thus, the combination
treatment of TMZ and CDK4 inhibitor is therapeutically effective on
glioma.
3.6 | The sensitivity of glioma cells to TMZ is
enhanced by abemaciclib through p-RB repression
We then analysed whether the inhibitory effect of TMZ and abe-
maciclib treatment is mediated by the p-RB suppression. For this,
we evaluated the effect of the dosage of TMZ or abemaciclib on
p-RB, by treating U87 glioma cells for 48 hours and observed
up-regulation of p-RB (Figure 6A). However, when the cells were
treated with abemaciclib alone, down-regulation of p-RB was ob-
served (Figure 6B). To assess the interaction of abemaciclib to TMZ,
studies were conducted in parallel where U87 cells were treated
with TMZ and abemaciclib for one full day and the Western blot re-
sults showed that the drug combination resulted in more decline in
RB phosphorylation (Figure 6C). Considering these results, RB acti-
vation is induced by TMZ treatment, leading to induction of apopto-
sis in glioma cells. Nevertheless, p-RB induced by TMZ is repressed
by abemaciclib, and a possible cause of enhanced sensitivity of cells
to TMZ.
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5140 CAO et al.

F I G U R E 3 Overexpression of CDK4 confers resistance to TMZ-mediated cell proliferation inhibition. A, U87 cells were transfected
with the indicated plasmids. Then, 24 h after transfection, the cells were treated with the indicated concentrations of TMZ. After another
48 h, cell viability was measured by a CCK-8 assay. B, U251 cells were transfected with the indicated siRNA. Then, 24 h after transfection,
the cells were treated with the indicated concentrations of TMZ. After another 48 h, cell viability was measured by a CCK-8 assay. C, U87
cells were transfected with the indicated plasmids. Then, 24 h after transfection, the cells were treated with the indicated concentrations
of TMZ. After another 48 h, cell viability was measured by a CCK-8 assay. D, U251 cells were transfected with the indicated siRNA. Then,
24 h after transfection, the cells were treated with the indicated concentrations of TMZ. After another 48 h, cell viability was measured by a
CCK-8 assay. E, Colony formation of U87 cells transfected with vector, CDK4 or CDK4-DN, was treated with TMZ for two weeks. F, Colony
formation of U87 cells transfected with siRNA against CDK4 was treated with TMZ for two weeks. G, U87 cells were transfected with
the indicated plasmids. Then, 24 h after transfection, the cells were treated with the indicated concentrations of TMZ. After another 48 h,
cell apoptosis was analysed. H, U251 cells were transfected with the indicated siRNA. Then, 24 h after transfection, the cells were treated
with the indicated concentrations of TMZ. After another 48 h, cell apoptosis was analysed. I, U87 cells were transfected with the indicated
plasmids. Then, 24 h after transfection, the cells were treated with the indicated concentrations of TMZ. After another 48 h, cell apoptosis
was analysed. J, U251 cells were transfected with the indicated siRNA. Then, 24 h after transfection, the cells were treated with the
indicated concentrations of TMZ. After another 48 h, cell apoptosis was analysed. K, U87 cells were transfected with the indicated plasmids.
Then, 24 h after transfection, the cells were treated with the indicated concentrations of TMZ. After another 48 h, cleaved caspase 3 was
analysed by Western blotting. L, U87 cells were transfected with the indicated siRNA. Then, 24 h after transfection, the cells were treated
with the indicated concentrations of TMZ. After another 48 h, cleaved caspase 3 was analysed by Western blotting

3.7 | TMZ and abemaciclib combination treatment
improved antitumour efficacy in a xenograft model
On observing the in vitro results, we assessed the efficiency of com-
bination treatment in xenografts. The combination therapy led to
significantly reduced tumour volume compared to the controls or
after monotherapy at end of three weeks (Figure 7A,B). Therefore,
like in vitro outcome, combination therapy markedly repressed RB
dephosphorylation (Figure 7C). Likewise, IHC staining for Ki-67 re-
vealed a decline in proliferation, and IF staining for cleaved caspase
3 revealed increase in apoptosis (Figure 7D,E). Therefore, a combi-
nation of abemaciclib and TMZ exhibits a synergy in the therapeutic
effect in vivo.
4 | D I S CU S S I O N
A type of primary tumour of the brain, glioma, is the most common
and the most aggressive subtype is GBM.1,34 Currently, the common
treatment option, chemotherapy, is largely ineffective because of
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CAO et al. 5141

F I G U R E 4 CDK4/6-selective inhibitors impair the proliferation of glioblastoma cells. A, U87 cells were plated in 96-well cell culture
plates, and one day later, they were treated with the increasing dose of CDK4/6 inhibitors. After 72 h of treatment, cell viability was
measured by a CCK-8 assay. B, U251 cells were plated in 96-well cell culture plates, and one day later, they were treated with the increasing
dose of CDK4/6 inhibitors. After 72 h of treatment, cell viability was measured by a CCK-8 assay. C, A172 cells were plated in 96-well cell
culture plates, and one day later, they were treated with the increasing dose of CDK4/6 inhibitors. After 72 h of treatment, cell viability was
measured by a CCK-8 assay. D, U87 cells were treated with abemaciclib for 48 h, cell apoptosis was measured by flow cytometry. E, U87
cells were treated with palbociclib for 48 h, cell apoptosis was measured by flow cytometry. F, U87 cells were treated with ribociclib for 48 h,
cell apoptosis was measured by flow cytometry. G, U87 cells were treated with abemaciclib with or without 10 μmol/L Z-VAD-FMK for 48 h,
cell apoptosis was measured by flow cytometry. H, U87 cells were treated with palbociclib with or without 10 μmol/L Z-VAD-FMK for 48 h,
cell apoptosis was measured by flow cytometry. I, U87 cells were treated with ribociclib with or without 10 μmol/L Z-VAD-FMK for 48 h,
cell apoptosis was measured by flow cytometry. J, U87 cells were treated with abemaciclib at indicated concentration, and indicated protein
level was analysed by Western blotting. K, U251 cells were treated with abemaciclib at indicated concentration, and indicated protein level
was analysed by Western blotting

chemoresistance, leading to a recurrence of cancer.35,36 Anti-TMZ
resistance, as a form of anti-chemoresistance, is a potentially prom-
ising option for glioma treatment.37 Abemaciclib exhibits favour-
able therapeutic properties and potential anticancer efficacy.38
Therefore, we assessed the in vitro activity and in vivo activity of
abemaciclib against glioma, as well as the synergy between abemaci-
clib and TMZ. Indeed, abemaciclib significantly induce apoptosis in
glioma cells in vitro, therefore, its repressed cell proliferation and
survival. Further, this pro-apoptotic effect was found to occur via
RB pathway, in addition to a decline in Bcl-2 level and activation of
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5142 CAO et al.

F I G U R E 5 CDK4/6 inhibitors sensitize

glioblastoma cells to TMZ. A, U87 cells
were treated with the given doses of
TMZ with abemaciclib. Cell viability was
measured by a CCK-8 assay. B, U251
cells were treated with the given doses
of TMZ with abemaciclib. Cell viability
was measured by a CCK-8 assay. C, Drug
combination results (CI) for abemaciclib
and TMZ examined in U87 cells. D, Drug
combination results (CI) for abemaciclib
and TMZ examined in U251 cells. E,
U87 cells were treated with 200 μmol/L
TMZ, 0.1 μmol/L abemaciclib or their
combination. Long-term survival was
visualized after 14 d by crystal violet
staining. F, U87 cells were treated with
50 μmol/L TMZ, 0.1 μmol/L abemaciclib
or their combination. Long-term survival
was visualized after 14 d by crystal
violet staining. G, U87 cells were treated
with 200 μmol/L TMZ, 0.1 μmol/L
abemaciclib or their combination for
48 h. Cell apoptosis was measured by
flow cytometry. H, U251 cells were
treated with 50 μmol/L TMZ, 0.1 μmol/L
abemaciclib or their combination for 48 h.
Cell apoptosis was measured by flow
cytometry

F I G U R E 6 CDK4 inhibition suppressed RB phosphorylation. A, U87 cells were treated with indicated concentration of TMZ. Indicated
protein levels were analysed by Western blotting. B, U87 cells were treated with indicated concentration of abemaciclib. Indicated protein
levels were analysed by Western blotting. C, U87 cells were treated with indicated concentration of TMZ and abemaciclib. Indicated protein
levels were analysed by Western blotting

caspase-3 and Bax in glioma cell lines. A preferred drug for GBM The acquired or inherent resistance to TMZ is considerable, and, the
treatment is TMZ, but it is not curative and, thus, more efficient resistance of glioma cells primarily involves the MGMT DNA-repair en-
treatment options are needed. zyme.39 MGMT, a 22 kD protein, repairs TMZ-induced lesions directly
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15824934, 2020, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.15156 by Turkey Cochrane Evidence Aid, Wiley Online Library on [10/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CAO et al. 5143

F I G U R E 7 CDK inhibitor sensitized TMZ in vivo. A, Nude mice were injected s.c. with 5 × 106 U87 cells. After 1 wk, mice were treated
with TMZ, abemaciclib or their combination for 10 consecutive days. Tumour volume at indicated time-points after treatment was calculated
and plotted with P values, n = 6, in each group. B, Tumour weight was calculated at end of the experiments. C, The levels of indicated
proteins in randomly selected tumours were analysed by Western blotting. D, Ki-67 was analysed by IHC staining. E, Cleaved caspase 3 was
analysed by IF staining

by eliminating guanine site O6 methylation.39 Recently, GANT61, a
specific GLI (glioma-associated oncogene) inhibitor, was shown to in-
crease DNA damage, repress MGMT expression and recover the TMZ
sensitivity of glioma, implicating some association between MGMT
and the hedgehog signalling pathway.40 Likewise, in the primary glioma
tissues, the association of zinc finger protein Gli1 activity with MGMT,
with Gli1 binding to promoter region of the MGMT gene, implicating
MGMT to be a downstream target of HH/Gli1 pathway.41
Some CDKs have recently been conferred roles as immune re-
sponse and oncogenesis modulators.42 Particularly, genetic or phar-
macological inhibition of CDK4 and CDK6 could inhibit in vivo and
in vitro tumour growth and control tumour associated antigens ex-
43,44
pression. In the progression of cell cycle, CDK4 and CDK6, both
close homologs, interact with cyclin D and form heterodimers.45
One of the selective inhibitors of the CDK4/6-cyclin D complex is
P16, encoded by CDKN2A.45 CDK4 contributes to tumorigenesis in
several human cancers,46 and its inhibition can increase oncolytic
viral replication in glioma.47 Here, we showed that pharmacological
inhibition and genetic knockdown of CDK4 hinders growth of glioma
and TMZ resistance, via RB pathway regulation.
We report here that CDK4 enables glioma cell lines resistant
to TMZ, although the association between CDK4 and TMZ resis-
tance in terms of their levels in primary gliomas still remains to be
unravelled. Therefore, larger sample sizes are required to assess
the relationship between TMZ resistance and CDK4 levels. For
this, larger number samples that are resistant to TMZ are being
collected from our hospital, and the results will be presented in
our next manuscript.
Here, we focused on the synergism between CDK4/6 inhibi-
tors and TMZ, and report for the first time that abemaciclib and
TMZ combination is more effective in inhibition of tumour cell
proliferation and apoptotic induction in comparison with TMZ or
abemaciclib singly. In addition, the combination led to significantly
increased expression of apoptosis-related proteins (such as Bax,
Bcl-2 and cleaved caspase-3). To better understand the underlying
mechanism, we observed that p-RB levels up-regulated by TMZ
could be reversed by abemaciclib. The results were further cor-
roborated by our in vitro study which showed that combination
treatment extended median survival significantly in tumour-bear-
ing mice. In preclinical mouse models, abemaciclib shows promise
in controlling solid tumours and enhances sensitivity to gefitinib
and radiotherapy.48,49 To our knowledge, effects of abemaciclib
on the cytotoxicity of TMZ have not been reported in glioma cells.
While we show here the synergism between TMZ and abemaci-
clib in inhibiting glioma cells proliferation and bringing about the
much-enhanced rates of apoptosis, future studies need to be care-
fully conducted to assess the suitability of this drug combination
with TMZ.
To conclude, we report enhanced expression of CDK4 in glioma
and impaired proliferation of glioma as a result of CDK4 inhibition.
Further, the CDK4 inhibitors could sensitize glioma cells to TMZ-
induced inhibition of cell proliferation and induction of apoptosis.
Therefore, CDK4 inhibition may be a promising strategy for treating
glioma and surpassing TMZ resistance.
AC K N OW L E D G E M E N T S
None.
C O N FL I C T O F I N T E R E S T
The authors have no conflict of interest.
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15824934, 2020, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.15156 by Turkey Cochrane Evidence Aid, Wiley Online Library on [10/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
5144 CAO et al.

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How to cite this article: Cao Y, Li X, Kong S, Shang S, Qi Y.
CDK4/6 inhibition suppresses tumour growth and enhances
the effect of temozolomide in glioma cells. J Cell Mol Med.
2020;24:5135–5145. https://doi.org/10.1111/jcmm.15156