Nanomaterial-Facilitated Cyclin-Dependent
Kinase 7 Inhibition Suppresses Gallbladder
Cancer Progression via Targeting
Transcriptional Addiction
Chen-Song Huang,# Qiong-Cong Xu,# Chunlei Dai,# Liying Wang,# Yi-Chih Tien, Fuxi Li, Qiao Su,
Xi-Tai Huang, Jun Wu,* Wei Zhao,* and Xiao-Yu Yin*
Cite This: https://doi.org/10.1021/acsnano.1c04570 Read Online
ACCESS Metrics & More Article Recommendations *sı Supporting Information
ABSTRACT: Gallbladder cancer (GBC) is the most aggressive
malignancy of the biliary tract cancer, and there is a lack of
effective treatment. Here, we developed a nanoparticle platform
(8P4 NP) that can deliver THZ1, a cyclin-dependent kinase 7
(CDK7) inhibitor, to treat GBC. Analysis of datasets
demonstrated that CDK7 was positively correlated with poor
prognosis. CDK7 inhibition suppressed cell proliferation,
induced apoptosis, and caused cell cycle block in GBC cells.
THZ1 downregulated CDK7-mediated phosphorylation of RNA
polymerase II (RNAPII), resulting in a significant downregulation of transcriptional programs, with a preferential
repression of oncogenic transcription factors. To improve the
tumor targeting efficiency of THZ1, 8P4 NPs were prepared
and assembled with THZ1 to form THZ1@8P4 NPs. Compared with free THZ1, THZ1@8P4 NPs showed more advantages in
prolonging blood circulation, escaping from lysosomes and increasing cellular uptake. Importantly, THZ1@8P4 NPs
demonstrated a more significant inhibition effect on GBC cells than free THZ1 in vitro. In addition, THZ1@8P4 NPs could
efficiently deliver THZ1 to tumor sites in a patient-derived xenograft model of early recurrence, leading to tumor regression
and transcriptional inhibition with minimal toxicity. In summary, we conclude that THZ1@8P4 NPs provide a potent
therapeutic strategy that targets CDK7-mediated transcriptional addiction in GBC.
KEYWORDS: gallbladder cancer, nanoparticle, CDK7 inhibitor, THZ1 delivery, transcriptional addiction
INTRODUCTION
GBC is one of the most aggressive biliary tract malignancies,
ranking the fifth most common digestive tract malignancy.1
Complete resection of tumor is the main treatment that offers a
possibility of cure. However, the tumors of many GBC patients
are already very large and have invaded nearby tissues at the time
of diagnosis, making it difficult to completely remove them by
surgery.2 Although chemotherapy can prolong the survival of
patients with local advanced or distant metastasis, the regimen is
limited, because of insensitivity or the rapid development of
chemoresistance.3 Recent studies reveal a large number of
molecular pathogenesis involved in the development and
progression of GBC. So far, few are known to be useful for the
GBC treatment.4 Therefore, it is urgent to discover potential
therapeutic targets and treatment strategies.
Emerging evidence showed that a bewildering array of genetic
and epigenetic events resulted in dysregulated gene expression
programs in cancer cells, generating “addictions” that can be
targeted. But it remains an open question about what
transcriptional factors regulate the transcriptional addiction in
GBC. The high levels of oncogenic gene expression have high
demand for RNA polymerase II (RNAPII) and the basal
transcriptional machinery. Recent studies demonstrated that
“transcriptional therapy” with the drugs targeting the BRD4 is
Received: May 29, 2021
Accepted: August 13, 2021
Article
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effective in certain tumor types lacking targeted therapies.
CDK7, together with cyclin H and MAT1, phosphorylates the
RNAPII C-terminal domain (CTD) at gene promoters and
initiates transcription.5 Some small-molecule inhibitors of
CDK7, such as THZ1, have shown promise in the treatment
of various types of cancer.6−8 However, how CDK7 inhibitor
achieves cancer-specific regulation of gene expression is still
unknown. Furthermore, the effect of CDK7 inhibitor in GBC
has not yet been elucidated.
Many disadvantages of CDK7 inhibitor are exposed in the
treatment of cancers, such as strong hydrophobicity, low
bioavailability, and rapid metabolism in vivo. Nanosized drugdelivery system (DDS) can overcome current limitations of
THZ1 by sustained drug release in the circulation system.9 In
the meantime, the therapeutic effects of the CDK7 inhibitor can
be enhanced by the DDS through improving permeability and
attenuating systemic toxicity.10,11
In this study, we demonstrated that the upregulation of CDK7
expression was related to poor prognosis in GBC patients.
Moreover, CDK7 inhibition by small interfering RNAs or THZ1
could suppress cell proliferation, induce apoptosis, and cause cell
cycle block in GBC cells. By combining chromatin immunoprecipitation sequencing (ChIP-seq) assay with whole transcriptome sequencing (RNA-seq), we found that THZ1 played a
therapeutic role in GBC by suppressing JUN-/FOSL1-mediated
oncogenic transcriptions (Scheme 1). Importantly, we con-
firmed that nano-THZ1 (THZ1@8P4 NPs) have improved
pharmacokinetic and high GBC tumor accumulation properties.
Both in vitro and in vivo results with THZ1@8P4 NPs supported
the enhanced growth inhibition of GBC cells and GBC recurrent
PDX tumors with negligible toxicity.
RESULTS AND DISCUSSION
Effects of CDK7 Inhibition on GBC Cell Proliferation,
Apoptosis, and Cell Cycle. We analyzed the CDK7 level in
120 GBC tissues by immunohistochemical (IHC) staining and
found CDK7 was mainly localized in the nucleus of GBC cells
(Figure S2A in the Supporting Information). Analysis of clinical
characteristics revealed that CDK7 overexpression was closely
associated with increased TNM stage (P = 0.006) (Figure 1A)
and T stage (P = 0.043) (Figure S2B in the Supporting
Information and Table 1). To clarify the prognostic value of
CDK7 in GBC, the relationship between the expression of
CDK7 and survival period was investigated by Kaplan−Meier
survival analysis. We found that patients with higher CDK7
expression had shorter disease-free survival (DFS) (P = 0.0065)
and overall survival (OS) (P = 0.0006) (Figure 1B) in GBC,
indicating that high expression of CDK7 might be closely related
to rapid progression of GBC.
These findings reveal that CDK7 can be used as a good
biomarker of prognosis in GBC patients after surgical resection.
The abnormal activity of a variety of cell cycle regulators, such as
CDKs, often results in uncontrolled cell proliferation and
tumorigenesis.12 CDK7 has been found to be a key oncogene in
many cancers. Treatments targeting CDK7 have been confirmed
as an effective method in a variety of preclinical models of many
tumors. Lu et al. found that CDK7 inhibition is a promising
therapeutic strategy in pancreatic cancer by down-regulating a
series of gene transcription.8 Zhang et al. found that THZ1
effectively inhibits the viability of multiple myeloma cells by
down-regulating expression of MCL-1 and c-MYC.13 In our
previous study, we found that THZ1 showed antitumor activity
in intrahepatic cholangiocarcinoma by inhibiting the transcription of oncogenes and c-Met pathways involved in cell cycle
regulation.14
To further investigate the functional role of CDK7 in GBC
cells, we silenced CDK7 expression by the CDK7 siRNA (Figure
S3 in the Supporting Information) or inhibited CDK7 activity by
THZ1 in GBC-SD and NOZ cells. Both CDK7 inhibition
strategies significantly reduced the growth of GBC-SD and NOZ
cells in a time-dependent manner (Figure 1C and 1D). In
addition, CDK7 inhibition significantly increased cell apoptosis
in GBC-SD and NOZ cells (Figure 1E, 1F, and Figures S4 and
S5 in the Supporting Information). CDK7 inhibition also caused
cell cycle block in the G2/M phase in GBC cells (Figure 1G and
1H). Previous reports showed that THZ1 inhibited the activity
of CDK12/13 kinase.7,15 We found that knockdown of CDK12
or CDK13 had mild effects on cell proliferation and apoptosis,
and had little effect on cell cycle in GBC cells (Figure S6 in the
Supporting Information), suggesting that THZ1 affects the
phenotype of GBC cells mainly by inhibiting the CDK7 kinase
activity.
Suppression of Transcriptional Addiction by CDK7
Inhibition in GBC. We observed that the RNAPII CTD
Scheme 1. Schematic Diagram of Preparation of THZ1@8P4 NPs and THZ1@8P4 NPs Suppressing GBC through Inhibiting
Transcriptional Addiction
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phosphorylation in GBC-SD and NOZ cells dramatically
decreased upon THZ1 treatment in a time-dependent manner
(Figure 2A). To elucidate the underlying molecular mechanisms
by which THZ1 suppressed GBC progression, we conducted
RNAPII ChIP-seq combined with RNA-seq analyses to identify
the change of gene expression upon THZ1 treatment in GBCSD cells. As shown in Figure 2B, THZ1 treatment led to a robust
reduction of RNAPII binding at the transcription start site
(TSS) in a genome-wide fashion. Integrative analysis of both
RNA-seq and ChIP-seq analyses indicated that 788 genes
Figure 1. CDK7-targeted treatment inhibited cell proliferation, promoted apoptosis, and blocked the cell cycle in GBC. (A) CDK7 expression in
different TNM stages (stage I/II vs. III/IV) of GBC. (B) Kaplan−Meier survival curves of disease-free survival (DFS) and overall survival (OS)
in 120 GBC patients, stratified by CDK7 IHC score (low expression of CDK7, n = 53 vs high expression of CKD7, n = 67). The log-rank test was
used to calculate the P-value. HR = hazard ratio. (C) Cell growth curve of GBC-SD and NOZ cells transfected with CDK7 siRNA or control. (D)
GBC-SD and NOZ cells were treated with THZ1 at indicated concentration for 24, 48, and 72 h. The relative cell viability related to DMSO
group is shown. (E) Apoptosis analysis of GBC-SD and NOZ cells transfected with CDK7 siRNA or control for 48 h. (F) Effects of different
concentrations of THZ1 on apoptosis of GBC-SD and NOZ cells. (G) Cell cycle analysis of GBC-SD and NOZ cells transfected with CDK7
siRNA or control for 48 h. (H) Cell cycle analysis of GBC-SD and NOZ cells treated with different concentrations of THZ1. The results are
presented as mean ± standard deviation (SD) of three independent experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001,
according to a Student’s t-test.]
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C
showed mRNA expression downregulation and the binding of
RNAPII reduction after THZ1 treatment (Figures 2C and 2D).
Using gene ontology (GO) analysis, we identified these 788
genes as being significantly enriched in GO terms of transcription regulation (Figure 2E). The gene set included many
well-established master transcriptional factors related with
oncogenic transcription, such as FOSL1 and JUN (Figures 2F
and 2G). Knockdown of FOSL1 or JUN (Figure S7 in the
Supporting Information) significantly reduced the cell growth in
both GBC-SD and NOZ cells (Figures 2H and 2I). These data
indicated that THZ1 suppresses proliferation of GBC cells
through blocking oncogenic transcriptional programs transcription.
Abnormal super enhancer (SE) recruits transcription
machines to drive the high expression of oncogenes, which has
been found to be an important mechanism of carcinogenesis.16
Some studies have shown that THZ1-sensitive transcripts are
often associated with the SEs.17−19 We found that THZ1
treatment led to selective inhibition of some carcinogenic
transcripts, including FOSL1 and JUN. Both FOSL1 and JUN are
members of AP-1, and AP-1 has been found to be an important
module of the core transcriptional regulatory circuit (CRC) of
SE in other cancers.20 The inhibitory effect of THZ1 on the
proliferation of GBC cells may be achieved by repressing the
transcription of oncogenic transcriptional factors. Whether
THZ1 affects SE-driven genes in GBC must be further
investigated.
Preparation and Characterization of THZ1@8P4 NPs.
To improve tumor targeting efficiency of THZ1 and achieve
effective THZ1 delivery, a phenylalanine-based poly(ester
amide) (8-Phe-4, 8P4) was formulated into NPs (8P4 NPs)
via nanoprecipitation method and encapsulated THZ1 to form
THZ1-loaded 8P4 NPs (THZ1@8P4 NPs) (Figure 3A). The
chemical structure and molecular weight of 8P4 were
determined by 1
H nuclear magnetic resonance (NMR) (Figure
S8A in the Supporting Information) and gel permeation
chromatography (GPC) (Figure S8B in the Supporting
Information), respectively. THZ1@8P4 NPs showed an average
size of 100.00 ± 1.54 nm (Figure 3B), a narrow polydispersity
index (PDI) and an acceptable drug loading capacity (see Table
2). In addition, the particle size of THZ1@8P4 NPs fluctuated
slightly in PBS and PBS containing 10% fetal bovine serum
(FBS) within 7 days, with sizes ranging from 100 nm to 120 nm
(Figure 3C), reflecting that THZ1@8P4 NPs were able to
remain stable in physiological environment. Next, we evaluated
the drug release behavior of THZ1@8P4 NPs. As shown in
Figure 3D, under the condition of pH 7.4, the cumulative release
of THZ1 from THZ1@8P4 NPs was only ∼20%, while under
the condition of pH 5.0, the release of THZ1 increased
significantly, indicating the effective release of THZ1 in tumor
tissues.
To investigate the pharmacokinetics of THZ1 and THZ1@
8P4 NPs, free THZ1 or THZ1@8P4 NPs were intravenously
injected into SD rats, respectively. As shown in Figure 3E,
concentration of free THZ1 reduced rapidly in circulation,
whereas the systematic clearance rate of THZ1@8P4 NPs
slowed significantly.
The fluorescent images of GBC-SD cells incubated with
Coumarin 6-loaded 8P4 NPs (C6@8P4 NPs) for different times
were detected by confocal laser scanning microscopy (CLSM).
After 1 h of incubation, the green fluorescence of C6@8P4 NPs
and the red fluorescence of lysosomes were dramatically
overlapped, indicating that the NPs were successfully internalized by GBC cells. Intriguingly, C6@8P4 NPs could escape from
the lysosomes in GBC-SD cells with time increasing to 8 h
(Figure 3F).
Biodistribution and tumor accumulation of 8P4 NPs.
The C6@8P4 NPs showed improved distribution of C6 in the
cytoplasm than free C6 at 2 h in GBC cells (see Figures S9A and
S9B in the Supporting Information), suggesting 8P4 NPs may
improve the intracellular accumulation of THZ1 in GBC cells.
We then studied the uptake of C6@8P4 NPs and free C6 in
GBC-SD and NOZ tumor spheroids, which are more likely to
simulate 8P4 NPs uptake by GBC tumors in vivo. The
fluorescence intensity of C6@8P4 NPs in the same sections of
GBC-SD and NOZ tumor spheroids was markedly stronger than
that in free C6 groups (see Figure S9C in the Supporting
Information).
Then, we used GBC PDX model (PDX0018) to evaluate the
biodistribution and tumor accumulation of 8P4 NPs in vivo. A
fluorescent dye, DiR was used to label the NPs. DiR-loaded 8P4
NPs (DiR@8P4 NPs) or free DiR were injected into the tumorbearing mice, respectively. As shown in Figure 4A, the
fluorescence intensity of DiR@8P4 NPs group was mainly in
the tumors. In contrast, the fluorescence intensity was mainly
accumulated in the liver but hardly detected in the tumors in the
free DiR group, indicating the higher accumulation of 8P4 NPs
in the GBC tumors. The tumors and main organs of the two
groups were dissected for quantitative analysis (Figure 4B). The
fluorescence intensity of DiR@8P4 NPs group was significantly
higher in tumors compared with free DiR group (Figure 4C).
DiR@8P4 NPs were also found in the liver, spleen, and other
tissues rich in sinuses. To determine the distribution of THZ1 in
major organs, we treated the PDX mice with free THZ1 or
THZ1@8P4 NPs and collected the major organ tissues to
measure THZ1 by HPLC-MS. The concentration of THZ1 in
tumors in THZ1@8P4 NPs treatment group was significantly
Table 1. Correlation between CDK7 Expression and Clinical
Pathological Characteristics in 120 GBC Patients
Number of Patients
I/II 14 24 0.006 III/IV 53 29
χ2 tests.
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higher than that in free THZ1 treatment group (Figure 4D).
Moreover, we modified THZ1 with biotin (bio-THZ1) without
harming its covalent binding ability (Figures S10 and S11 in the
Supporting Information). Bio-THZ1 could also inhibit cell
Figure 2. THZ1 inhibited oncogenic transcription in GBC. (A) Immunoblotting analyses of total and phosphorylation of C-terminal repeat
domain (CTD) of RNAPII in GBC-SD and NOZ cells treated with THZ1 (500 nM and 50 nM, respectively) at different time points. (B)
Average binding intensity of RNAPII at promoters in GBC-SD cells treated with or without THZ1 (500 nM for 24 h). (C) Venn diagram
depicting shared down-regulated genes in ChIP-seq and RNA-seq assays in GBC-SD cells upon THZ1 (500 nM for 24 h) treatment. (D)
Heatmap of 788 down-regulated genes in ChIP-seq and RNA-seq assays in GBC-SD cells upon THZ1 (500 nM for 24 h) treatment. (E) GO
enrichment analysis of 788 down-regulated genes in ChIP-seq and RNA-seq assays. (F) Scatter plot of RNAPII binding strength and
transcriptional regulatory gene expression in GBC-SD cells treated with THZ1 (500 nM for 24 h). (G) Integrative Genomics Viewer (IGV)
showing RNAPII ChIP-seq and RNA-seq profiles of JUN and FOSL1 in GBC-SD cells upon THZ1 treatment. (H) Cell growth curve of GBC-SD
and NOZ cells after silencing JUN by siRNA transfection. (I) Cell growth curve of GBC-SD and NOZ cells after silencing FOSL1 by siRNA
transfection. The results are presented as mean ± SD of three independent experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001,
according to a Student’s t-test.]
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viability (see Figures S12A and S12B in the Supporting
Information) and RNAPII CTD phosphorylation (Figure
S12C in the Supporting Information) in GBC-SD and NOZ
cells. Then, we treated the GBC PDX mice with bio-THZ1 or
bio-THZ1@8P4 NPs for 4 h. The pulldown assay was
conducted to assess the target binding of THZ1 to CDK7 in
vivo (Figure 4E). Indeed, we found that both bio-THZ1 and bioTHZ1@8P4 NPs were specifically bound to CDK7 in tumor
and major organs (kidney, liver, spleen) (Figure 4F).
Inhibitory Effects of THZ1@8P4 NPs GBC Cells in Vitro.
As expected, THZ1@8P4 NPs robustly decreased the viability
of GBC cells (GBC-SD and NOZ) in a dose-dependent manner
(Figures 5A and 5B). Note that the half-inhibitory concentration
(IC50) of THZ1@8P4 NPs was significantly lower than that of
free THZ1 (Figure 5A), which reflected the enhanced
therapeutic effects of THZ1@8P4 NPs on GBC cells. In
addition, THZ1@8P4 NPs significantly increased cell apoptosis
in GBC-SD and NOZ cells (see Figure 5C, as well as Figure
S13A in the Supporting Information) and caused cell cycle block
Figure 3. Characterization of THZ1@8P4 NPs. (A) Representative transmission electron microscopy (TEM) images of THZ1@8P4 NPs and
8P4 NPs. Scale bar = 100 nm. (B) Size distribution of THZ1@8P4 NPs. (C) Stability of THZ1@8P4 NPs in PBS and PBS containing 10% FBS.
(D) Cumulative release characteristics of THZ1 from THZ1@8P4 NPs under different pH conditions. (E) Serum concentration of THZ1 in SD
rats after 10 mg/kg intravenous (iv) injection of free THZ1 or THZ1@8P4 NPs. (F) Representative intracellular distribution images of C6 in
GBC-SD cells treated with C6@8P4 NPs for indicated time points. The lysosomes were labeled by Lysotracker-red. Scale bar = 10 μm. The
results are presented as mean ± SD of three independent experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, according to a
Student’s t-test.]
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in the G2/M phase (see Figure 5D, as well as Figure S13B in the
Supporting Information). Then, we investigated the effect of
THZ1@8P4 NPs on the tumor formation capacity of GBC-SD
and NOZ cells. We found that the number of colonies formed by
the cells treated with THZ1@8P4 NPs were significantly less
than those formed by the cells treated with free THZ1 (Figure
5E). Consistently, THZ1@8P4 NPs reduced the mRNA
expression of FOSL1 and JUN in GBC cells (Figure 5F). In
addition, THZ1@8P4 NPs also reduced the enrichment of
RNAP II in the promoter region of JUN or FOSL1 (Figure 5G).
THZ1@8P4 NPs Enhanced Therapeutic Effects against
GBC Xenograft in Vivo with Negligible Systemic Toxicity.
To test whether THZ1@8P4 NPs exhibit improved therapeutic
effects in vivo, we used the PDX model of GBC (PDX0018) to
mimic the tumor environment of human GBC. The PDX tumorbearing mice were treated with THZ1@8P4 NPs, THZ1, 8P4
NPs, or vehicle for 17 days. The growth rate of tumor treated
with THZ1@8P4 NP was significantly slower than that of other
groups (Figures 6A and 6B). THZ1@8P4 NPs treatment
significantly reduced the tumor weight (Figure 6C), but had no
significant toxic effect on weight loss (Figure 6D), liver function
(see Figure 6E , as well as Figure S14A in the Supporting
Information) and renal function (Figures S14B and S14C in the
Supporting Information)). After THZ1@8P4 NPs treatment of
PDX, the expression of JUN and FOSL1 decreased more
significantly than that of free drugs (Figure 6F). Taken together,
THZ1@8P4 NPs will be a potent therapeutic strategy that
targets CDK7-mediated transcriptional addiction in GBC.
THZ1 has been proved to have a good clinical application
prospect in a variety of cancers. However, its clinical application
is limited by its strong hydrophobicity, low bioavailability, and
fast metabolism in vivo. In this study, 8P4 NPs were formulated
and assembled with THZ1 to form THZ1-loaded 8P4 NPs. The
THZ1@8P4 NPs have improved pharmacokinetic behavior and
superior antitumor properties with negligible systemic toxicity.
THZ1@8P4 NPs can effectively carry a large amount of THZ1,
to promote drug uptake and control drug release in GBC.
Compared with THZ1 alone, THZ1@8P4 NPs have longer
half-life and improved antitumor effect with no detectable side
effects. Therefore, THZ1@8P4NPs provides an effective
treatment strategy for CDK7-mediated GBC transcription
addiction.
CONCLUSIONS
In this study, we found that the high expression of CDK7 was
closely associated with the clinical outcomes and poor prognosis
of patients with GBC. Knockdown of CDK7 or treatment with
CDK7 covalent inhibitor, THZ1, exerted tumor-suppressive
effects on GBC. Mechanistically, THZ1 is capable of repressing
the transcription of key oncogenes, such as FOSL1 or JUN,
through inhibiting the RNAPII CTD phosphorylation, thereby
contributing to the suppression on GBC progression. On this
basis, we developed 8P4 NPs to deliver THZ1 with the aim to
enhance tumor accumulation and improve the therapeutic
effects of THZ1 against GBC. As expected, compared with free
THZ1, THZ1@8P4 NPs exhibited prolonged half-life and
exerted stronger antitumor effect in PDX model without causing
obvious side effects.
METHODS
GBC Cell Lines and Patient Tissues. The GBC cell lines GBC-SD
and NOZ, purchased from Guangzhou Cellcook Biotech Co., Ltd.
(Guangzhou, China), were maintained in DMEM high glucose medium
(Gibco, USA) supplemented with 10% FBS. Cells were cultured at 37
°C in a humidified atmosphere of 5% CO2.
A total of 120 paraffin-embedded tissues of GBC patients were
obtained from the Department of Pathology of the First Affiliated
Hospital of Sun Yat-sen University. IHC staining were conducted as
previously described.21 Two experienced pathologists performed
histological scores according to tissue positive staining and staining
intensity. This study was approved by the Human Ethics Committee of
the First Affiliated Hospital of Sun Yat-sen University.
Antibodies and Chemicals. THZ1 (Catalog No. S7549) was
purchased from Selleckchem (Houston, TX, USA). Anti-CDK7
antibody (Catalog Nos. ab243863 and 27027-1-AP) were purchased
from Abcam (Cambridge, U.K.) and Proteintech (Wuhan, China),
respectively. Anti-RNA polymerase II CTD antibody (Catalog No.
ab26721), Anti-RNA polymerase II CTD pSer2 (Catalog No. ab5095)
and pSer5 antibody (Catalog No. ab5131) were purchased from Abcam
(Cambridge, U.K.). Anti-CDK12 antibody (Catalog No. 44460) was
purchased from Signalway Antibody (College Park, MD, USA). AntiCDK13 antibody (Catalog No. A10258) was purchased from ABclonal
(Wuhan, China). Anti-JUN antibody (Catalog No. ET1608−3) was
purchased from Hua’an Biotech (Hangzhou, China). Anti-FOSL1
antibody (Catalog No. ab124722) was purchased from Abcam
(Cambridge, U.K.). The small-interfering RNA (siRNA) that
specifically target human CDK7, FOSL1 and JUN were purchased
from GenePharma (Suzhou, China). The siRNA sequences used in this
study are listed in Table S1 in the Supporting Information. The
compound 1,2-distearoylsn-glycero-3-phosphoethanolamine poly-
(ethylene glycol) 2000 (DSPE-PEG 2k) was purchased from Avanti
Polar Lipids. Dimethyl sulfoxide (DMSO) and triethylamine (ET3N)
were purchased from Aladdin. Coumarin 6 (C6) was purchased from
Sigma-Aldrich.
Cell Proliferation, Cell Cycle, and Apoptosis Analyses. Cell
proliferation was assessed by the CellTiter-Glo Luminescent Cell
Viability Assay Kit (G7570, Promega, USA) according to the
manufacturer’s instruction. Cell cycle assays and apoptosis assays
were conducted using the Cell Cycle Staining Kit (CCS012, Multi
Sciences, China) or Annexin V-FITC/PI Apoptosis Detection Kit
(AP101−100-kit, Multi Sciences, China), respectively. The stained
cells were analyzed by CytoFLEX flowcytometry (Beckman Coulter).
3D Tumor Spheroids. The GBC-SD or NOZ cells were seeded at a
density of 2 × 103 cells per 100 μL per well in the 96-well Ultra Low
Attachment Microplate. After several days, the 3D tumor spheroids
were treated with different drugs. After 48 h, the tumor spheroids were
imaged and counted.
Immunoblot Analysis. The immunoblot analysis were conducted
as previously described.21 Briefly, proteins were separated by SDSPAGE, then transferred to PVDF blotting membrane (Millipore), and
analyzed by immunoblot. All blots were detected using the enhanced
chemiluminescence (ECL) with ChemiDoc XRS+ imaging system
(Bio-Rad, Hercules, CA, USA). Images were analyzed with Image Lab
Software (Bio-Rad, Hercules, CA, USA).
Quantitative Real-Time PCR (qRT-PCR). The qRT-PCR analysis
was performed according to previously described procedure.21 Briefly,
TRIzol reagent (Invitrogen, USA) was used to extract the total RNA.
Maxima First Strand cDNA synthesis kit (Thermo Scientific, USA) was
used for qRT-PCR. The mRNA level was measured using the Takana
Table 2. Physiochemical Properties and Drug Loading
Capacity of THZ1@8P4 NPs
parameter value/remark
tested sample THZ1@8P4 NPs
diametera 100.00 ± 1.54 nm
polydispersity indexa 0.264 ± 0.006
zeta potentiala 10.10 ± 0.31 mV
loading capacityb 6.56% ± 0.52%
encapsulation efficiencyb 26.24% ± 2.07%
Determined via DLS; b
Analyzed via HPLC.
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Figure 4. Biodistribution of the THZ1@8P4 NPs in GBC PDX model. (A) Fluorescence imaging of the biodistribution of free DiR or DiR@8P4
NPs in mice (n = 3) bearing GBC PDX tumors (PDX0018). (B) Fluorescence imaging of major organs and tumors from mice (n = 3) bearing
GBC PDX tumors (PDX0018) treated with free DiR or DiR@8P4 NPs. (C) Quantitative analysis of radiant efficiency in major organs and
tumors. (D) PDX mice were treated with free THZ1 or THZ1@8P4 for 4h. Biodistribution of the free THZ1 or THZ1@8P4 NPs in major
organs of GBC PDX mice was detected by HPLC-MS. (E) Flowchart of the pulldown assay, which was conducted to assess the target binding of
THZ1 to CDK7 in vivo. (F) THZ1 was labeled with biotin (bio-THZ1). The GBC PDX mice were treated with bio-THZ1 or bio-THZ1@8P4
NPs for 4 h. Tumor, liver, kidney, and spleen tissue samples were collected. Streptavidin agarose was used for bio-THZ1 pulldown assay. IB =
immunoblot. The results are presented as mean ± SD of three independent experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001,
according to a Student’s t-test.]
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Figure 5. Inhibitory effects of THZ1@8P4 NPs on GBC cells in vitro. (A) Cell viability of GBC-SD and NOZ cells treated with different
concentrations of THZ1 or THZ1@8P4 NPs for 48 h. (B) Cell growth curve of GBC-SD and NOZ cells upon different treatments (DMSO, 8P4
NPs, THZ1, or THZ1@8P4 NPs). (C) Apoptosis analysis of GBC-SD and NOZ cells upon different treatments (DMSO, 8P4 NPs, THZ1, or
THZ1@8P4 NPs) for 48 h. (D) Cell cycle analysis of GBC-SD and NOZ cells upon different treatments (DMSO, 8P4 NPs, THZ1, or THZ1@
8P4 NPs) for 48 h. (E) Tumor sphere formation assay of GBC-SD and NOZ cells upon different treatments (DMSO, 8P4 NPs, THZ1, or
THZ1@8P4 NPs) on day 5. The sphere number was counted on day 7. (F) mRNA expression of FOSL1 and JUNin GBC-SD cells upon different
treatments (8P4 NPs or THZ1@8P4 NPs) for 24 h. (G) ChIP-qPCR analysis of RNAP II enrichment in the JUN or FOSL1 promoter region in
GBC-SD cells upon different treatments (8P4 NPs or THZ1@8P4 NPs) for 24 h. The concentration of DMSO is 1%. The concentration of
THZ1 and THZ1@8P4 used in GBC-SD and NOZ were 500 nM and 50 nM, respectively. The results are presented as mean ± SD of three
independent experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, according to a Student’s t-test.]
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SYBR Primix Ex Taq Kit (Takana, China). The primes used in this
study are also listed in Table S1.
ChIP-seq and RNA-seq. The cells cultured with or without THZ1
(500 nM) for 24 h were cross-linked with 1% (v/v) formaldehyde at
room temperature for 10 min and processed with the SimpleChIP
Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA),
according to manufacturers’ instructions. Anti-RNA polymerase II
CTD antibody (Catalog No. ab26721) were purchased from Abcam.
The RNA sequencing (RNA-seq) and the Chromatin immunoprecipitation sequencing (ChIP-seq) samples were performed by Novogene
(Beijing, China). DESeq package was used in RNA-seq assay for
differential analyses. Bowtie2, MACS2, and ChIPseeker were used in
ChIP-seq assay for mapping, peak calling, and annotation, respectively.
The clusterProfiler package was used for gene ontology (GO) analysis.
Synthesis of 8P4 Polymer and Preparation of THZ1@8P4
NPs. The synthesis of 8-Phe-4 (8P4) polymer was performed as
described previously (Figure S1 in the Supporting Information).22 The
chemical structure data of 8P4 polymer was collected by 1
H NMR
(Bruker Avance III HD 400 MHz NMR spectrometer). Gel permeation
chromatography (GPC) was used to characterize the molecular weight
of 8p4 polymer. 8P4 NPs were prepared as follows: DSPE-PEG 2k and
8P4 were dissolved in 10 mg/mL dimethyl sulfoxide, then dropwise
added to stirring water to form 8P4 NPs. The particle size and
distribution were measured by dynamic light scattering (DLS)
(Malvern, Model Zetasizer Nano-ZS90). The morphology of NPs
was characterized by transmission electron microscopy (TEM) (Model
JEM-1400 Plus, 120 kV, JEOL). For stability test, the particle size of
THZ1@8P4 NPs in PBS and PBS containing 10% FBS was measured at
predetermined time points.
In Vitro Drug Release. The drug release of THZ1@8P4 NPs was
performed under two pH conditions (pH 7.4 and pH 5.0) at 37 °C
using the dialysis bag method. The amount of drug released from
THZ1@8P4 NPs was determined using HPLC (Agilent, Model 1260
Infinity II).
Pharmacokinetic Study of THZ1@8P4 NPs. Six SD rats were
randomly divided into two groups (n = 3). The rats in each group were
intravenously injected with THZ1@8P4 NPs or free THZ1 at an
equivalent THZ1 dose of 10 mg/kg. Blood samples were collected at
predesigned time points, then immediately centrifugated at 3500 rpm
for 15 min at 4 °C to get the plasma. The concentrations of THZ1 in
serum were determined by high-performance liquid chromatography−
mass spectrometry (HPLC-MS).
Cellular Uptake Assay. For drug uptake assay of GBC cells, GBCSD or NOZ cells at a density of 1.5 × 105 cells per well were cultured in a
six-well plate or a confocal dish. After incubated with C6@8P4 NPs for
Figure 6. Therapeutic effects of THZ1@8P4 NPs in vivo. (A) Images of tumors derived from the PDX-tumor (PDX0018) bearing mice (n = 6)
treated with Vehicle, 8P4 NPs, THZ1 or THZ1@8P4 NPs for 17 days, respectively. (B) Tumor growth curves of each group (n = 6, Vehicle, 8P4
NPs, THZ1, or THZ1@8P4 NPs). (C) Tumor weight (n = 6) in different groups (Vehicle, 8P4 NPs, THZ1, or THZ1@8P4 NPs). (D) Body
weight of the tumor bearing mice (n = 6) during the treatment period (Vehicle, 8P4 NPs, THZ1, or THZ1@8P4 NPs). (E) Serum AST level of
the mice (n = 6) in different groups (Vehicle, 8P4 NPs, THZ1, or THZ1@8P4 NPs). (F) qPCR analysis of JUN or FOSL1 expression in tumor
tissues from different groups (Vehicle, 8P4 NPs, THZ1, or THZ1@8P4 NPs). The results are presented as mean ± SD of three independent
experiments. [Legend: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, according to a Student’s t-test.]
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2 h, cellular uptake efficiency was measured using CLSM or flow
cytometry.
For drug uptake assay of GBC spheroids, GBC-SD or NOZ cells at a
density of 2 × 103 cells per well were cultured in a 24-well Ultra Low
Attachment Microplate for several days. Then, the spheroids were
incubated with C6@8P4 NPs for 2 h. Cellular uptake efficiency in
spheroids was also determined with CLSM.
Intracellular Distribution of NPs in GBC-SD Cells. The GBCSD cells at a density of 1.5 × 105 cells per well were seeded in a confocal
dish. After 24 h, the cells were incubated in medium with C6@8P4 NPs
(2 μg/mL) for predesigned time (1, 4, and 8 h), then in medium with
Lysotracker-red (50 nM) and Hoechst 33342 (5 μg/mL) for 10 min.
Finally, after treatment with 4% formaldehyde, the cells were prepared
for CLSM observation.
Patient-Derived Xenograft (PDX) Model and THZ1@8P4 NPs
Treatment. PDX model from a patient (PDX0018, which relapsed in 6
months after R0 resection) were inoculated subcutaneously into the
right flank of 5-week-old female BALB/c nu/nu mice. Tumor volume
(V) was calculated using the following formula: V = 0.5 × length ×
width × width. When the tumor volume reached 100−150 mm3
, mice
were randomly divided into four groups and treated with vehicle (5%
DMSO + 45% PEG 300 + 50% PBS), THZ1 (4 mg/mL in vehicle
solution, 10 mg/kg, daily), 8P4 NPs, or THZ1@8P4 NPs (at an
equivalent THZ1 dose of 10 mg/kg, daily), respectively. Tumor volume
was determined every 4 days. Upon harvesting tumors, tumor
xenografts were excised, weighed, and photographed. The concentrations of serum aspartate aminotransferase (AST), creatinine (Cr),
alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were
detected. The animal experiments were approved by the Institutional
Review Board of the First Affiliated Hospital of Sun Yat-Sen University
([2019] No. 114).
Biodistribution of NPs by in Vivo Imaging System (IVIS). PDX
model from a patient (PDX0018, which relapsed in 6 months after R0
resection) were inoculated subcutaneously into the right flank of 5-
week-old female BALB/c nu/nu mice. After the tumors grow up to 200
mm3
, the mice were randomized into two groups and respectively
treated with DiR or DiR@8P4 NPs at an equivalent DiR dose of 0.5
mg/kg. The fluorescence distribution in mice was detected at 1, 4, 8, 12,
24 h post-injection using IVIS (Perkin−Elmer, USA). Ultimately, the
mice were sacrificed and the major organs and tumors were harvested
for ex vivo imaging.
Target Binding of THZ1 to CDK7 in Vivo. PDX model
(PDX0018) mice with a tumor size of ∼200 mm3 were randomly
divided into three groups and intravenously injected with vehicle (5%
DMSO + 45% PEG 300 + 50% PBS), bio-THZ1 (4 mg/mL in vehicle
solution, 10 mg/kg), or bio-THZ1@8P4 NPs (at an equivalent bioTHZ1 dose of 10 mg/kg), respectively. The mice were sacrificed at 4 h
post injection, and the major organs and tumors were weighed. After
digestion with tissue homogenizer, tissue lysates were incubated with
streptavidin beads overnight at 4 °C. Beads were washed three times
with lysis buffer and then boiled in SDS solution for 10 min at 100 °C.
Finally, the binding of THZ1 to CDK7 was determined by
immunoblotting.
Distribution of THZ1 in Major Organs and Tumors. To
determine the distribution of THZ1 in major organs and tumor tissues,
PDX model (PDX0018) mice with tumor size of ∼200 mm3 were
randomly divided into two groups and intravenously injected with
THZ1 or THZ1@8P4 NPs at an equivalent THZ1 dose of 10 mg/kg.
Then, the mice were sacrificed and the major organs and tumors were
collected at 4 h post-injection. The digested tissue samples were mixed
with acetonitrile to extract THZ1. Finally, the content of THZ1 in
different organs and tumors was determined by HPLC-MS.
Statistical Analyses. Values were presented as mean ± standard
deviation (SD). The unpaired Student’s t-test was used to determine
the statistical significance between two groups. P < 0.05 was considered
statistically significant and marked with an asterisk (*), P < 0.01 marked
with two asterisks (**), and P < 0.001 marked with three asterisks
(***). All results are from at least three independent experiments.
ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsnano.1c04570.
The synthetic route of 8-Phe-4 (8P4) polymer (Figure
S1); correlation of CDK7 expression and T stage in
human GBC (Figure S2); knockdown efficiency of CDK7
siRNA (Figure S3); representative flow cytometry data of
apoptosis analysis of GBC cells after CDK7 inhibition
(Figure S4); apoptosis analysis of GBC cells treated with
siCONTROL+DMSO, siCDK7+DMSO, or siCONTROL+THZ1 for 48 h (Figure S5); the effects of
CDK12/13 siRNAs on cell proliferation, apoptosis, and
the cell cycle in GBC (Figure S6); the knockdown
efficiency of JUN or FOSL1 siRNA (Figure S7);
characterization of 8P4 polymer (Figure S8); cellular
uptake behavior of C6@8P4 NPs in GBC cells (Figure
S9); the synthetic route of bio-THZ1 (Figure S10);
characterization of bio-THZ1 (Figure S11); the cellbased assay of Bio-THZ1 (Figure S12); tumor targeting
efficiency of THZ1@8P4 NPs in vitro (Figure S13); the
concentrations of serum alanine aminotransferase (ALT),
creatinine (Cr), and blood urea nitrogen (BUN) in
different treatment groups (Figure S14); sequences of
siRNA and primers used for experiments in this study
(Table S1) (PDF)
AUTHOR INFORMATION
Corresponding Authors
Xiao-Yu Yin − Department of Pancreato-Biliary Surgery, The
First Affiliated Hospital of Sun Yat-sen University, Guangzhou
510080, China; orcid.org/0000-0002-5518-5984;
Email: [email protected]
Wei Zhao − Guangdong Provincial People’s Hospital,
Guangdong Academy of Medical Sciences, Guangzhou
510080, China; Email: [email protected]
Jun Wu − Key Laboratory of Sensing Technology and
Biomedical Instrument of Guangdong Province, School of
Biomedical Engineering, Sun Yat-sen University, Guangzhou
510006, China; orcid.org/0000-0002-9074-856X;
Email: [email protected]
Authors
Chen-Song Huang − Department of Pancreato-Biliary Surgery,
The First Affiliated Hospital of Sun Yat-sen University,
Guangzhou 510080, China
Qiong-Cong Xu − Department of Pancreato-Biliary Surgery,
The First Affiliated Hospital of Sun Yat-sen University,
Guangzhou 510080, China
Chunlei Dai − Key Laboratory of Sensing Technology and
Biomedical Instrument of Guangdong Province, School of
Biomedical Engineering, Sun Yat-sen University, Guangzhou
510006, China
Liying Wang − Key Laboratory of Sensing Technology and
Biomedical Instrument of Guangdong Province, School of
Biomedical Engineering, Sun Yat-sen University, Guangzhou
510006, China
Yi-Chih Tien − Department of Pancreato-Biliary Surgery, The
First Affiliated Hospital of Sun Yat-sen University, Guangzhou
510080, China
Fuxi Li − Guangdong Provincial People’s Hospital, Guangdong
Academy of Medical Sciences, Guangzhou 510080, China
ACS Nano www.acsnano.org Article
https://doi.org/10.1021/acsnano.1c04570
ACS Nano XXXX, XXX, XXX−XXX
K
Qiao Su − Department of Animal Experiment Center, First
Affiliated Hospital of Sun Yat-sen University, Guangzhou
510080, China
Xi-Tai Huang − Department of Pancreato-Biliary Surgery, The
First Affiliated Hospital of Sun Yat-sen University, Guangzhou
510080, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsnano.1c04570
Author Contributions
These authors contributed equally to this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors sincerely acknowledge the financial support from
National Natural Science Foundation of China (Nos. 81772522,
82002501, 51973243, 81972651, and 31771630), the Guangdong Basic and Applied Basic Research Foundation (Nos.
2021A1515010123, 2019A1515010096, and
2019A1515010686), National Science and Technology Major
Project of the Ministry of Science and Technology of China
(No. 2018ZX10301402), International Cooperation and
Exchange of the National Natural Science Foundation of
China (No. 51820105004), Science and Technology Planning
Project of Shenzhen (No. JCYJ20190807155801657), Guangdong Innovative and Entrepreneurial Research Team Program
(No. 2016ZT06S029).
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