Ribonucleic acid interference knockdown of IL-6 enhances the efficacy of cisplatin in laryngeal cancer stem cells by down-regulating the IL-6/STAT3/HIF1 pathway
Fu et al. Cancer Cell Int
Ribonucleic acid interference knockdown of IL-6 enhances the efficacy of cisplatin in laryngeal cancer stem cells by down-regulating the IL-6/STAT3/HIF1 pathway
Qiang Fu 1 3
Pengruofeng Liu 1 2
Xiumei Sun 0
Shanshan Huang 0
Fengchan Han 3
Lili Zhang 0
Yannan Xu 0
Tingyan Liu 0
0 Department of Otolaryngology, Yantai Affiliated Hospital of Binzhou Medical University , Yantai 264003 , China
1 Qiang Fu and Pengruofeng Liu are Co-first authors
2 Department of Stomatology, The First Affiliated Hospital, School of Medicine, Zhejiang University , Hangzhou 310003 , China
3 College of Basic Medicine, Binzhou Medical University , Yantai 264003 , China
Background: Cisplatin has been used in the treatment of many cancers, including laryngeal cancer; however, its efficacy can be reduced due to the development of drug resistance. This study aimed to investigate whether interleukin-6 (IL-6) knockdown may enhance the efficacy of cisplatin in laryngeal cancer stem cells (CSC) and the potential involvement of the signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1 (HIF1) in this effect. Methods: The ALDH+ and CD44+ CSC in Hep2 human laryngeal squamous cancer cells were identified by the fluorescence-activated cell sorting technique. IL-6, STAT3 and HIF1 mRNA and protein expressions were examined with quantitative real-time polymerase chain reaction and Western blot, respectively. Cell proliferation was measured by MTT assay. Tumorigenicity was measured by a colony formation assay and invasion was determined by a cell invasion assay. Apoptotic cells were counted by flow cytometry. Immunohistochemistry was performed to detect immunoreactive IL-6, STAT3 and HIF1 cells in xenografts. Results: The mRNA and protein levels of IL-6, STAT3 and HIF1 were significantly increased in Hep2-CSC as compared with those from Hep2 cells. Application of siRNA-IL-6 to knockdown IL-6 resulted in significantly decreased IL-6, STAT3 and HIF1 mRNA and protein levels. IL-6 knockdown reduced cell proliferation, tumorigenicity and invasion and increased apoptosis within CSC. Enhanced degrees of suppression in these parameters were observed when IL-6 knockdown was combined with cisplatin in these CSC. Results from the xenograft study showed that the combination of IL-6 knockdown and cisplatin further inhibited the growth of xenografts as compared with that obtained in the cisplatin-injected group alone. Immunoreactive IL-6, STAT3 and HIF1 cell numbers were markedly reduced in IL-6 knockdown tumor tissues. IL-6, STAT3 and HIF1 immunoreactive cell counts were further reduced in tissue where IL-6 knockdown was combined with cisplatin treatment as compared with tissue receiving cisplatin alone. Conclusions: IL-6 knockdown can increase chemo-drug efficacy of cisplatin, inhibit tumor growth and reduce the potential for tumor recurrence and metastasis in laryngeal cancer. The IL-6/STAT3/HIF1 pathway may represent an important target for investigating therapeutic strategies for the treatment of laryngeal cancer.
Head and neck cancers represent the seventh most
common cancer worldwide [
]. In particular, head and neck
squamous cell carcinoma (HNSCC) is the eighth
leading cause of cancer mortality [
], with laryngeal
squamous carcinoma (LSCC) being the most common type
of HNSCC or head and neck cancer [
Chemoradiotherapy and surgery remain the major treatment
modalities for head and neck cancers. Despite improvements
in overall life quality achieved with the use of combined
therapies, survival rates of the cancer patients have not
advanced significantly over the past several decades [
The recurrence and metastasis of head and neck cancer
are often accompanied with chemo-drug resistance
generated during the cancer therapy, with the result that
therapeutic outcomes are unsatisfactory.
Cancer stem cells (CSC) have become a theoretical
foundation for chemo-resistance and cancer recurrence
studies. CSC represent a small population of tumor cells
that can uniquely self-renew, regenerate, sustain tumor
growth, and thus play an important role in the growth
and spread of the tumor [
]. During chemotherapy,
CSC can mutate or experience abnormal
differentiation, which may lead to tumor recurrence and metastasis
and serve as the basis for drug resistance [
Findings from recent studies have revealed that CSC can be
identified and isolated through distinct cell surface
markers, such as CD44 and CD133 [
], which are found
in laryngeal carcinoma cells [
]. In addition, certain
intracellular protein molecules have also been used for
isolating and detecting CSC. For example, aldehyde
dehydrogenase 1 (ALDH1), a soluble protein is used to
detect CSC in various cancers, including leukemia [
], colon [
], liver [
], lung [
] cancers. In fact, the ALDH assay has served as a
means to estimate stem cell features [
]. As CSC exhibit
tumor growth and drug resistance, they provide a
valuable model in which to investigate chemo-drug effects.
Of particular relevance to the present report is the use
of cisplatin in this CSC model. Cisplatin is a well-known
anticancer drug used against a variety of malignancies,
including laryngeal cancer [
Serum interleukin-6 (IL-6) levels are increased in
laryngeal cancer patients as compared with healthy volunteers,
and these serum levels show further increases as a
function of malignancy progression [
]. Elevated levels
of IL-6 are also observed in tissue specimens of
laryngeal cancer . Secretion of IL-6 has been suggested to
act as a potential biomarker for assessing the aggressive
tumor phenotype in laryngeal carcinoma. Findings from
recent studies have indicated that the expressions of CSC
markers are significantly upregulated in IL-6
expressing lung cancer cells and cell-derived tumor xenograft
tissues after cisplatin treatment. However, these CSC
markers were not upregulated in IL-6 knockdown cells
and in IL-6 knockdown cell-derived tumor tissue [
Negative effects of IL-6 signaling in triggering increased
tumor growth and drug resistance in lung cancer
during cisplatin treatment have been reported [
signal transducer and activator of transcription 3 (STAT3)
and hypoxia-inducible factor 1 (HIF1) are the
downstream molecules of the IL-6 signaling pathway, STAT3
activation has been observed in cancers and its
activation in tumor cells plays a crucial role in mediating and
promoting tumorigenesis [
hypoxiainducible factor 1α (HIF-1α), as the core of the
hypoxiarelated response network [
], can bind to downstream
molecules to induce the formation of angiogenesis and
multidrug resistance genes [
]. While IL-6/STAT3/HIF1
signaling has been reported to play an important role
in the treatment of ovarian cell cancer [
], the issue of
whether the IL-6/STAT3/HIF1 pathway may play a role
in laryngeal cancer remains uncertain.
In the present study, we aimed to explore whether
IL-6 knockdown enhances the effectiveness of
cisplatin in laryngeal CSCs and the potential involvement
of IL-6/STAT3/HIF1 signaling. To accomplish this
goal, we used hep2, the laryngeal squamous cancer
cell line, and isolated ALDH+ and CD44+ CSC from
hep2 cells along with siRNA technology to silence IL-6
gene expression. We observed that in response to
IL6-knockdown, laryngeal CSC characteristics show
marked changes and enhanced effects of IL-6
knockdown on anti-tumor effects of cisplatin were
demonstrated upon a number of parameters including cell
proliferation, invasion, tumorigenesis, apoptosis and
tumors in xenograft studies.
Materials and methods
A human laryngeal squamous cancer cell line, Hep2,
was purchased from ATCC (Manassas, VA, USA), and
cultured in Dulbecco’s modified Eagle’s medium /F12
supplemented with 10% fetal bovine serum. Cells were
maintained at 37 °C in a humidified incubator with a
mixture of 95% air (20% O2) and 5% CO2 environment.
When applicable, cisplatin was used at an optimal dose of
5 μg/mL in cultured cells as suggested from our previous
Fluorescence‑activated cell sorting
Flow cytometry assays for the CD44+ and, subsequently
ALDH+ cells were performed in this study. Briefly,
Hep-2 cells were collected and rinsed with
phosphatebuffered solution (PBS). The number of dissociated cells
was counted, then treated with fluorochrome-conjugated
CD44+ antibody for 30 min at 4 °C and protected from
light. Once completed, cells were then washed and
analyzed using a flow cytometer. The CD44+ and CD44−
cells were sorted by the fluorescence-activated cell
sorting (FACS) technique and the proportion of CD44+
cells were recorded. The CD44+ cells were further
treated with PBS containing fluorochrome-conjugated
ALDH+ antibody for 30 min at 4 °C. Once completed,
cells were washed and sorted by FACS and analyzed for
the proportion of ALDH+ and CD44+ cancer stem cells.
IL-6 siRNA expression plasmids were purchased from
Sigma-Aldrich (St Louis, MO). ALDH+/CD44+
Hep2CSC were transfected with siRNA-IL-6 to knock down
IL-6. The siRNA was transfected into the CSC using
Lipofectamine 2000 (invitrogen life technologies)
according to the manufacturer’s instructions. Total RNA was
prepared 24 h post-transfection and the results of gene
knockdown were determined by reverse
transcriptionquantitative polymerase chain reaction (RT-qPCR).
Hep2-CSC cell proliferation with or without
siRNAIL-6 was measured with use of the MTT assay. In brief,
MTT (20 μL) was added to each well of the plate and
cells were incubated for 4 h at 37 °C. After incubation,
DMSO (150 μL) was added to the well in the dark for 2 h
to develop coloration. The absorbance values (490 nm) of
each well were measured using an automatic multi-well
spectrophotometer. Data were obtained from triplicate
wells per condition and representatives of at least three
Colony formation assay
Cell suspensions were diluted to a density of 200 cells
per culture plate and then placed in the incubator for
2 weeks. Incubation was terminated when the colonies
were visually perceptible. The colonies were then fixed
in 1:3 acetic acid/methanol for 15 min and stained with
Giemsa staining solution for 10–30 min. The number of
colonies was counted when viewed microscopically.
Cell invasion assay
The effect of cisplatin with/without siRNA-IL-6 on the
invasion of Hep2-CSC was analyzed using Boyden
chambers with coated Matrigel as instructed by the
manufacturer (BD Biosciences, San Jose, CA). The invasive
cancer cells were stained with crystal violet and
visualized microscopically. All experiments were performed at
least twice in triplicates.
Apoptotic cells were measured with use of Annexin V/
PI double staining. Briefly, cells were harvested in 0.25%
trypsin, washed with PBS, resuspended in 250 µL of
binding buffer and adjusted to 1 × 106/mL. Staining solution
containing annexin V/FITC and propidium iodide was
added to the cell suspension. After incubation for 30 min
at room temperature in the dark, cells were analyzed by
flow cytometry (FACSAria, Becton–Dickinson, USA).
Quantitative real‑time PCR
Total RNA was extracted from cells or tumor tissue using
the RNeasy Mini kit (Qiagen) according to the
manufacturer’s instructions. Quantitative real-time PCR
experiments were performed using appropriate primers and
SYBR green power master mix (applied biosystems)
to determine the mRNA expression levels of genes of
interest. GAPDH served as a reference gene to
normalize other genes. The GAPDH, IL-6, STAT3 and HIF1
fragments were amplified using the following primer
GAPDH, forward 5′-GGTGGTCTCCTCTGACTTCAA
CA-3′ and reverse 5′-GTTGCTGTAGCCAAATTCG
IL-6, forward 5′-ACCTTCCAAAGATGGCTGAA-3′ and
STAT3, forward 5′-CTGGTGTCTCCACTGGTCTA
TCT-3′ and reverse 5′-AAACTTGGTCTTCAGGTA
HIF1, forward 5′-CATCTCCATCTCCTACCCACA-3′;
reverse 5′-CTGCTCTGTTTGGT GAGGC-3′.
Protein levels of interested targets were measured using
Western blot. Briefly, cells or tumor tissues were
homogenized and diluted with RIPA lysis buffer (50 mM Tris–Cl
at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL
aprotinin, 0.2 mM PMSF). Lysates containing equal amounts of
proteins (20–40 μg) were separated on 8–10% SDS/PAGE
gel and then transferred onto PVDF membranes
(Millipore, Billerica, MA, USA). After blocking, membranes
were incubated with primary antibodies for IL-6, STAT3,
HIF1 or β-Actin and horseradish peroxidase-conjugated
secondary antibodies (1:5000). Immunoreactive proteins
were visualized in Imager (Bio-Rad) using the ECL system
(Thermo Fisher Scientific, Rochester, NY, USA).
In vivo xenograft studies
Tumor growth of xenografts was examined as developed
from the ALDH+/CD44+ CSC with/without siRNA-IL-6.
The cells (5 × 105 cells in 100 µL of PBS) were
subcutaneously injected into the right dorsal area of nude mice.
When tumor size achieved approximately 150 mm3,
animals were randomly assigned into one of the following four
groups (N = 5/group): (1) CSC, (2) CSC + IL-6-siRNA,
(3) CSC + cisplatin and (4) CSC + IL-6-siRNA +
cisplatin. Cisplatin was administered to the mice via an
intraperitoneal injection at 10 mg/kg daily for 10 days. Tumor
development was monitored daily. Drug toxicity effects,
such as weight loss, behavioral change and feeding pattern
were continuously monitored during the treatment period.
At the end of the experiment, mice were euthanized and
tumor tissues were removed for determination of gene or
protein expression levels in the tissue. All animal studies
were performed in accordance with the recommendations
in the guide for the care and use of laboratory animals of
the national institute of health. The protocol was approved
by the Institutional Animal Care and Use Committee of
Binzhou Medical University.
After dissection, tumor tissues were fixed in 2%
paraformaldehyde overnight at 4 °C and then soaked in 30%
sucrose solution for an additional 4 h at 4 °C. The frozen
tumors were cut at 8 μm thickness by MICROM
cryostat (MICROM International, Walldorf, Germany) and
examined for the levels of targeted proteins. Briefly,
sections were washed in PBS and incubated in the blocking
buffer followed by primary antibodies for IL-6, STAT3 or
HIF1. Sections were then incubated with the secondary
antibody. The 3,3’-Diaminobenzidine was used as a
substrate for staining. Staining was observed, photographed
and density measured.
The data were presented as the mean ± SEM. Differences
in mean values between two groups were analyzed by
two-tailed Student’s t-tests. Differences in three or more
than three groups were analyzed by one-way ANOVA,
followed by the post hoc Fisher’s least significant
difference test. A p < 0.05 was required for results to be
considered statistically significant.
IL‑6, STAT3 and HIF1 mRNA and protein expressions in Hep2‑CSC
Similar to our previous study [
], a high yield of
ALDH+/CD44+ was obtained in this current study.
IL-6, STAT3 and HIF1 mRNA levels in Hep2-CSC were
significantly increased as compared to the IL-6, STAT3
and HIF1 mRNA expression levels in Hep2 cells and
Hep2-derived tumor tissue, which were obtained from
the tumor tissue after Hep2-cells were injected into
dorsal area of nude mice (p < 0.001, Fig. 1a–c). In addition,
IL-6, STAT3 and HIF1 protein levels in Hep2-CSC were
consistently and significantly increased as compared
with that from Hep2 cells or Hep2-derived tumor tissue
(p < 0.001, Fig. 1d–g). The protein levels were obtained
from three independent experiments and were compared
among the groups after being normalized to β-actin
IL‑6 siRNA effects upon mRNA and protein levels of IL‑6,
STAT3 and HIF1
As compared to mRNA levels of IL-6, STAT3 and
HIF1 in unaltered Hep2-CSC, siRNA-IL-6 significantly
reduced mRNA expression levels of IL-6, STAT3 and
HIF1 (Fig. 2a–c, p < 0.001). Although the siRNA-IL-6
vector also affected gene expression levels as compared
to Hep2-CSC, the levels of reduction were less than
that of siRNA-IL-6 (p < 0.05, Fig. 2a–c). Consistently,
IL-6, STAT3 and HIF1 protein levels were significantly
decreased after siRNA-IL-6 administration in Hep2-CSC
(p < 0.001). As compared with that of the siRNA-IL-6
empty vector control, IL-6, STAT3 and HIF1 protein
levels were significantly lower than that of the siRNA-IL-6
group (p < 0.01, Fig. 2d–g).
siRNA‑IL‑6 knockdown enhances inhibitory effects
of cisplatin on colony‑formation and cell invasion
The in vitro tumorigenicity of hep2-CSC with/without
IL-6-siRNA plus cisplatin was determined using a
softagar assay. Fewer colonies were formed in the hep2-CSC
cells treated with siRNA-IL-6 or cisplatin as compared
to their corresponding vector controls. Maximal colony
reduction was obtained when IL-6 siRNA was combined
with cisplatin as compared to that observed with
cisplatin or siRNA-IL-6 alone (p < 0.001, Fig. 3a–e). In
addition, while siRNA-IL-6 knockdown or cisplatin both
inhibited cell invasion when used alone, significantly
greater reductions in cell invasion were obtained when
cisplatin was combined with siRNA-IL-6 as compared
with effects resulting from their individual application
(p < 0.001, Fig. 3f–j).
siRNA‑IL‑6 knockdown enhances inhibitory effects
of cisplatin on cell proliferation
To test the effect of siRNA-IL-6 on cell proliferation,
Hep2-CSC cells exhibiting stable expressing control
vectors, were exposed to either siRNA-IL-6, 10 µM cisplatin
or siRNA-IL-6 combined with 10 µM cisplatin and
examined with use of a MTT assay. Cisplatin or siRNA-IL-6
significantly inhibited Hep2-CSC cell proliferation in a
temporally-dependent manner. However, their combined
treatment substantially enhanced this inhibitory effect
and produced the lowest cell proliferation rates as
compared with that of the controls (Fig. 4).
IL‑6 knockdown enhances cisplatin mediated apoptotic effects
To evaluate siRNA-mediated apoptotic effects, Hep2-CSC
cells exhibiting stable expressing control vectors, were
exposed to either siRNA-IL-6, cisplatin or siRNA-IL-6
combined with cisplatin and were subjected to FACS
analysis. The siRNA-IL-6 or cisplatin alone resulted in similar
rates of apoptosis as that seen in controls, while
siRNAIL-6 combined with cisplatin significantly enhanced cell
apoptosis rates compared with controls (p < 0.01, Fig. 5).
Cisplatin combined with IL‑6 knockdown enhances antitumor effects in xenografts
We next examined tumor growth of xenografts
developed from CSC cells with/without siRNA-IL-6
knockdown. Our data show that siRNA-IL-6 or cisplatin
1 2 3 4 5 6 7
Incubation Time (Day)
Fig. 4 Proliferation results after siRNA-IL-6 knock down. Cisplatin
or siRNA-IL-6 significantly inhibited Hep2-CSC cell proliferation in a
temporally-dependent manner. The combined treatment of cisplatin
and siRNA-IL-6 significantly enhanced the inhibitory effect of
siRNAIL-6. *p < 0.05, **p < 0.01 vs hep2-CSC control
(See figure on previous page.)
Fig. 5 Cisplatin increases the pro-apoptotic effect of siRNA-IL-6. Representative images showing apoptosis in control (a), siRNA-IL-6-treated (b),
cisplatin-treated (c), and siRNA-IL-6 and cisplatin-treated (d) hep2-CSC. Relative apoptosis rates (e) were slightly promoted by siRNA-IL-6 or
cisplatintreated hep2-CSC. The combination of cisplatin and siRNA-IL-6 significantly increased the pro-apoptotic effect of siRNAIL-6 or cisplatin. **p < 0.01 vs
injection slowed tumor development, with tumor sizes
in these groups being much smaller than that of the CSC
group. Maximal reductions in tumor size were observed
in the siRNA-IL-6 knockdown + cisplatin treated group
(p < 0.001, Fig. 6).
Decreased IL‑6, STAT3 and HIF1 protein levels after siRNA‑IL‑6 knockdown in xenografts
Immunohistochemistry staining demonstrated lower
numbers of positive-stained IL-6, STAT3 and HIF1 cells
in tumor tissues developed from siRNA-IL-6
knockdown cells, as compared with that of the CSC-derived
xenografts. The IL-6+, STAT3+ and HIF1+ cell numbers
were also decreased in the cisplatin-treated xenografts.
The siRNA-IL-6 + cisplatin group showed the fewest
number of IL-6+, STAT3+ and HIF1+ cells (p < 0.001,
Using in vitro cell lines and an in vivo xenograft model,
we investigated whether knockdown of IL-6, as achieved
using a siRNA technique, can increase the
chemodrug efficacy of cisplatin in laryngeal cancer. Our
results show that siRNA-IL-6 combined with cisplatin
reduced cell proliferation, colony formation and
invasion and increased apoptosis to a greater degree than
that obtained when either siRNA-IL-6 or cisplatin were
administered alone. Similarly, results from our xenograft
study showed greater efficacy upon suppressing the rate
of tumor growth when siRNA-IL-6 was combined with
cisplatin as compared with siRNA-IL-6 or cisplatin
treatment alone. Taken together, our results suggest that IL-6
knockdown can increase chemo-drug efficacy, reduce
drug resistance, inhibit tumor growth and reduce the
potential for tumor recurrence and metastasis in
laryngeal cancer. These siRNA-IL-6 effects were accompanied
with decreased STAT3 and HIF1 mRNA and protein
IL-6 levels in serum and cancer tissue are increased in
laryngeal cancer patients as compared with healthy
], suggesting that IL-6 can act as a potential
biomarker for assessing tumor growth and malignancy
progression. However, IL-6 has also been found to alter the
susceptibility of tumor cells to apoptosis by
chemotherapeutic drugs . Results from a recent study have revealed
that IL-6 treatment was found to be associated with
increased cisplatin resistance in lung CSC and increased
CSC stemness [
]. When lung CSC were treated with
neutralizing IL-6 antibody, cisplatin resistance decreased [
Using CSC from a different cancer source, we found that
silencing IL-6 gene expression with siRNA significantly
enhanced the cisplatin effect in laryngeal tumor cells as
-2epSCCH Ii--s6LSNARCC i-sSCCC Ii---6sLSARNCC isC -2epSCCH Ii--6sLSANRCC i-sSCCC Ii---6sLSARNCC isC -2epSCCH Ii--6sLSNARCC i-sSCCC Ii---6sLSARNCC isC
Fig. 7 IL-6 (a–d), STAT3 (e–h) and HIF1 (i–l) protein levels as detected with immunohistochemistry in xenograft cancer tissues derived from
hep2CSC, hep2-CSC-siRNA-IL-6, hep2-CSC + cisplatin and hep2-CSC-siRNA-IL-6 + cisplatin. Image analysis results showed that the number of IL-6+,
STAT3+ and HIF1+ cells (m–o) was decreased in response to siRNA-IL-6 or cisplatin-treated xenografts. Maximal reductions in IL-6+, STAT3+ and
HIF1+ cell numbers were obtained in the group receiving the combined treatment of siRNA-IL-6 and cisplatin. *p < 0.05, ***p < 0.001 vs hep2-CSC
indicated by reductions in cell proliferation, colony
formation, invasion, and an increase in the number of apoptotic
cells. Accordingly, with the use of CSC, which is considered
a very effective model for investigating drug-resistance, we
demonstrate the importance of IL-6 signaling in triggering
increased cisplatin efficacy in laryngeal cancer. In specific,
we show that reducing IL-6 is beneficial for cisplatin
efficacy particularly in a drug-resistant condition.
In this study, we identified and isolated ALDH+ and
CD44+ CSC from laryngeal cancer cells as an approach
to better predict the role of IL-6 in chemo-drug
resistance. Cancer stem cell theory is one of the most likely
explanations for chemoresistance and recurrence in
]. Resistance of CSC to conventional therapies
has been shown to result from multiple mechanisms ,
including increased expression of detoxifying enzymes
such as ALDH. With the administration of chemotherapy
and irradiation, ALDH alters aldehydes (oxygen, carbon,
and hydrogen) within a cell to prevent DNA damage.
Increased ALDH enzyme activity has been found in CSC
derived from colon, ovarian, prostate, and breast cancers
]. ALDH+ CSC has also been found to mediate
metastasis and result in poor clinical outcomes in
inflammatory breast cancer , as well as predict engraftment
of primary breast tumors [
This effect of IL-6 on cisplatin efficacy was
accompanied by decreased STAT3 in these laryngeal cancer cells.
The IL-6 signaling factor induces STATs
tyrosine-phosphorylation and initiation by activating members of the
janus kinase (JAK) family [
]. STAT3 is required and
essential for tumorigenesis as shown in a variety of
cancers. STAT3 has been reported to play a pivotal role in
maintenance of stem cell-like breast cancer cells, which
have been shown to be related to tumor recurrence,
metastasis and chemo-resistance [
]. In addition,
STAT3 has been shown to be constitutively activated or
over expressed in head and neck squamous cell carcinoma
 and lung [
] cancers. Our results show that IL-6 and
STAT3 expressions are increased in laryngeal CSC and
decreased after IL-6 knockdown. These findings are
consistent with what has been observed in prostate cancer,
and blocking of STAT3 suppresses clonogenicity in stem
cell-like cells from high grade prostate cancer patients
]. STAT3 has also been reported to be involved in
IL6-induced proliferation of renal cancer cells [
The findings that HIF1 is increased in laryngeal CSC
may indicate another factor that contributes to
drugresistance in CSC. HIF1 is comprised of α subunit which
is oxygen-dependent and β subunit which is
continually expressed. Under normal oxygen
pressure/conditions, α subunit is rapidly degraded by the proteasome
pathway, while under hypoxic conditions this subunit
remains stable. Activated HIF-1α, when transferred
into the nucleus, binds to downstream molecules, such
as the anti-apoptotic factors Bcl-2, Survivin and Xiap.
These anti-apoptotic factors can then induce the
formation of angiogenesis and multi-drug resistance genes
]. We found that the enhanced cisplatin efficacy after
knockdown of IL-6 with siRNA was accompanied with
decreased HIF1 levels. These results suggest that this
reduction in HIF1 may be important component for
increased cisplatin efficacy in laryngeal cancer.
To the best of our knowledge, our study represents the
first to examine the effect of IL-6 knockdown in
combination with cisplatin in drug-resistance laryngeal
cancer using ALDH+ and CD44+ CSC. Due to the
limitations of cancer chemotherapy resulting from drug
resistance, siRNA-based therapeutics has emerged as
a promising new anticancer tactic. A small number of
Phase I clinical trials that have been completed [
and discussions regarding the benefits and limitations
of siRNA for cancer therapy have been ongoing [
]. The results of our present study demonstrate
distinct beneficial effects of IL-6 knockdown in
combination with cisplatin treatment, and provide a theoretical
base for applying siRNA techniques in the treatment of
CSC: cancer stem cells; LSCC: laryngeal squamous carcinoma; ALDH: aldehyde
dehydrogenase; HIF1: hypoxia-inducible factor 1; HNSCC: head and neck
squamous cell carcinoma; IL-6: interleukin-6; STAT3: signal transducer and activator
of transcription 3; HIF1: hypoxia-inducible factor 1; JAK: janus kinase; FACS:
fluorescence-activated cell sorting; PBS: phosphate buffer solution.
QF, PL performed the experiments and wrote the manuscript. XS, SH, FH and
LZ contributed to the technical support. YX participated in the statistical
analysis. QF, TL participated in the coordination of the study. TL designed the
study. All authors read and approved the final manuscript.
The authors would like to thank ED-IT Editorial Service Ltd for their professional
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study available from the
corresponding author on reasonable request.
Consent for publication
All animal studies were performed in accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of the National
Institute of Health. The protocol was approved by the Institutional Animal Care
and Use Committee of Binzhou Medical University.
This work was supported by grants from National Science Foundation of
China (NSFC81571512, NSFC81570927), Shandong Provincial Natural Science
Foundation of China (ZR2015JL027), Zhejiang Provincial Natural Science
Foundation of China (LY12H13004), Medicine and Health Care in Zhejiang Province
Science and Technology Plan (2013ZDA018) and Roadmap for Science and
Technology Development of Hangzhou (20120533Q31).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Jemal A , Bray F , Center MM , Ferlay J , Ward E , Forman D . Global cancer statistics. CA Cancer J Clin . 2011 ; 61 ( 2 ): 69 - 90 .
2. Ragin CC , Modugno F , Gollin SM . The epidemiology and risk factors of head and neck cancer: a focus on human papillomavirus . J Dent Res . 2007 ; 86 ( 2 ): 104 - 14 .
3. Jemal A , Siegel R , Ward E , Murray T , Xu J , Thun MJ . Cancer statistics, 2007 . CA Cancer J Clin. 2007 ; 57 ( 1 ): 43 - 66 .
4. Marioni G , Marchese-Ragona R , Cartei G , Marchese F , Staffieri A . Current opinion in diagnosis and treatment of laryngeal carcinoma . Cancer Treat Rev . 2006 ; 32 ( 7 ): 504 - 15 .
5. Al-Hajj M , Clarke MF . Self-renewal and solid tumor stem cells . Oncogene . 2004 ; 23 ( 43 ): 7274 - 82 .
6. Cho RW , Clarke MF . Recent advances in cancer stem cells . Curr Opin Genet Dev . 2008 ; 18 ( 1 ): 48 - 53 .
7. Lobo NA , Shimono Y , Qian D , Clarke MF . The biology of cancer stem cells . Annu Rev Cell Dev Biol . 2007 ; 23 : 675 - 99 .
8. Prince ME , Sivanandan R , Kaczorowski A , Wolf GT , Kaplan MJ , Dalerba P , Weissman IL , Clarke MF , Ailles LE . Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma . Proc Natl Acad Sci USA . 2007 ; 104 ( 3 ): 973 - 8 .
9. Reya T , Morrison SJ , Clarke MF , Weissman IL . Stem cells, cancer, and cancer stem cells . Nature . 2001 ; 414 ( 6859 ): 105 - 11 .
10. Wicha MS , Liu S , Dontu G . Cancer stem cells: an old idea-a paradigm shift . Cancer Res . 2006 ; 66 ( 4 ): 1883 - 90 .
11. Tomao F , Papa A , Rossi L , Strudel M , Vici P , Lo Russo G , Tomao S . Emerging role of cancer stem cells in the biology and treatment of ovarian cancer: basic knowledge and therapeutic possibilities for an innovative approach . J Exp Clin Cancer Res . 2013 ; 32 : 48 .
12. Dean M , Fojo T , Bates S. Tumour stem cells and drug resistance . Nat Rev Cancer . 2005 ; 5 ( 4 ): 275 - 84 .
13. Donnenberg VS , Donnenberg AD . Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis . J Clin Pharmacol . 2005 ; 45 ( 8 ): 872 - 7 .
14. Thapa R , Wilson GD. The importance of CD44 as a stem cell biomarker and therapeutic target in cancer . Stem Cells Int . 2016 ; 2016 : 2087204 .
15. Yan Y , Zuo X , Wei D . Concise review: emerging role of CD44 in cancer stem cells: a promising biomarker and therapeutic target . Stem Cells Transl Med . 2015 ; 4 ( 9 ): 1033 - 43 .
16. Zhou JY , Chen M , Ma L , Wang X , Chen YG , Liu SL . Role of CD44high/ CD133high HCT-116 cells in the tumorigenesis of colon cancer . Oncotarget . 2016 ; 7 ( 7 ): 7657 - 66 .
17. Sim MW , Grogan PT , Subramanian C , Bradford CR , Carey TE , Forrest ML , Prince ME , Cohen MS . Effects of peritumoral nanoconjugated cisplatin on laryngeal cancer stem cells . Laryngoscope . 2016 ; 126 ( 5 ): E184 - 90 .
18. Cheung AM , Wan TS , Leung JC , Chan LY , Huang H , Kwong YL , Liang R , Leung AY . Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential . Leukemia . 2007 ; 21 ( 7 ): 1423 - 30 .
19. Ginestier C , Hur MH , Charafe-Jauffret E , Monville F , Dutcher J , Brown M , Jacquemier J , Viens P , Kleer CG , Liu S , et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome . Cell Stem Cell . 2007 ; 1 ( 5 ): 555 - 67 .
20. Carpentino JE , Hynes MJ , Appelman HD , Zheng T , Steindler DA , Scott EW , Huang EH . Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer . Cancer Res . 2009 ; 69 ( 20 ): 8208 - 15 .
21. Ma S , Chan KW , Lee TK , Tang KH , Wo JY , Zheng BJ , Guan XY . Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations . Mol Cancer Res . 2008 ; 6 ( 7 ): 1146 - 53 .
22. Jiang F , Qiu Q , Khanna A , Todd NW , Deepak J , Xing L , Wang H , Liu Z , Su Y , Stass SA , et al. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer . Mol Cancer Res . 2009 ; 7 ( 3 ): 330 - 8 .
23. Rasheed ZA , Yang J , Wang Q , Kowalski J , Freed I , Murter C , Hong SM , Koorstra JB , Rajeshkumar NV , He X , et al. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma . J Natl Cancer Inst . 2010 ; 102 ( 5 ): 340 - 51 .
24. Karatas OF , Suer I , Yuceturk B , Yilmaz M , Hajiyev Y , Creighton CJ , Ittmann M , Ozen M. The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells . Tumour Biol . 2016 ; 37 ( 3 ): 4183 - 92 .
25. Chen Z , Malhotra PS , Thomas GR , Ondrey FG , Duffey DC , Smith CW , Enamorado I , Yeh NT , Kroog GS , Rudy S , et al. Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer . Clin Cancer Res . 1999 ; 5 ( 6 ): 1369 - 79 .
26. Hao W , Zhu Y , Zhou H . Prognostic value of interleukin-6 and interleukin-8 in laryngeal squamous cell cancer . Med Oncol . 2013 ; 30 ( 1 ): 333 .
27. Zhang F , Duan S , Tsai Y , Keng PC , Chen Y , Lee SO , Chen Y. Cisplatin treatment increases stemness through upregulation of hypoxia-inducible factors by interleukin-6 in non-small cell lung cancer . Cancer Sci . 2016 ; 107 ( 6 ): 746 - 54 .
28. Marotta LL , Almendro V , Marusyk A , Shipitsin M , Schemme J , Walker SR , Bloushtain-Qimron N , Kim JJ , Choudhury SA , Maruyama R , et al. The JAK2/STAT3 signaling pathway is required for growth of CD44(+) CD24(−) stem cell-like breast cancer cells in human tumors . J Clin Invest . 2011 ; 121 ( 7 ): 2723 - 35 .
29. Grivennikov S , Karin E , Terzic J , Mucida D , Yu GY , Vallabhapurapu S , Scheller J , Rose-John S , Cheroutre H , Eckmann L , et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer . Cancer Cell . 2009 ; 15 ( 2 ): 103 - 13 .
30. Gao SP , Mark KG , Leslie K , Pao W , Motoi N , Gerald WL , Travis WD , Bornmann W , Veach D , Clarkson B , et al. Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas . J Clin Invest . 2007 ; 117 ( 12 ): 3846 - 56 .
31. Yu H , Kortylewski M , Pardoll D . Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment . Nat Rev Immunol . 2007 ; 7 ( 1 ): 41 - 51 .
32. Huang C , Yang G , Jiang T , Huang K , Cao J , Qiu Z. Effects of IL-6 and AG490 on regulation of Stat3 signaling pathway and invasion of human pancreatic cancer cells in vitro . J Exp Clin Cancer Res . 2010 ; 29 : 51 .
33. Liu Y , Song X , Wang X , Wei L , Liu X , Yuan S , Lv L . Effect of chronic intermittent hypoxia on biological behavior and hypoxia-associated gene expression in lung cancer cells . J Cell Biochem . 2010 ; 111 ( 3 ): 554 - 63 .
34. Middleton K , Jones J , Lwin Z , Coward JI . Interleukin-6: an angiogenic target in solid tumours . Crit Rev Oncol Hematol . 2014 ; 89 ( 1 ): 129 - 39 .
35. Anglesio MS , George J , Kulbe H , Friedlander M , Rischin D , Lemech C , Power J , Coward J , Cowin PA , House CM , et al. IL-6 -STAT3-HIF signaling and therapeutic response to the angiogenesis inhibitor sunitinib in ovarian clear cell cancer . Clin Cancer Res . 2011 ; 17 ( 8 ): 2538 - 48 .
36. Liu T , Liu P , Li Y , Cui C , Dai L , Zhou X , Jin C , Fu Q. Inhibition of STAT3 with shRNA enhances the chemosensitization of cisplatin in laryngeal carcinoma stem cells . Int J Exp Pathol . 2017 ; 10 ( 6 ): 6512 - 9 .
37. Yusuf RZ , Duan Z , Lamendola DE , Penson RT , Seiden MV . Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation . Curr Cancer Drug Targets . 2003 ; 3 ( 1 ): 1 - 19 .
38. Visvader JE , Lindeman GJ . Cancer stem cells: current status and evolving complexities . Cell Stem Cell . 2012 ; 10 ( 6 ): 717 - 28 .
39. Islam F , Gopalan V , Smith RA , Lam AK . Translational potential of cancer stem cells: a review of the detection of cancer stem cells and their roles in cancer recurrence and cancer treatment . Exp Cell Res . 2015 ; 335 ( 1 ): 135 - 47 .
40. Morrison R , Schleicher SM , Sun Y , Niermann KJ , Kim S , Spratt DE , Chung CH , Lu B. Targeting the mechanisms of resistance to chemotherapy and radiotherapy with the cancer stem cell hypothesis . J Oncol . 2011 ; 2011 : 941876 .
41. Charafe-Jauffret E , Ginestier C , Iovino F , Tarpin C , Diebel M , Esterni B , Houvenaeghel G , Extra JM , Bertucci F , Jacquemier J , et al. Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer . Clin Cancer Res . 2010 ; 16 ( 1 ): 45 - 55 .
42. Charafe-Jauffret E , Ginestier C , Bertucci F , Cabaud O , Wicinski J , Finetti P , Josselin E , Adelaide J , Nguyen TT , Monville F , et al. ALDH1 -positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program . Cancer Res . 2013 ; 73 ( 24 ): 7290 - 300 .
43. Suzuki R , Sakamoto H , Yasukawa H , Masuhara M , Wakioka T , Sasaki A , Yuge K , Komiya S , Inoue A , Yoshimura A . CIS3 and JAB have different regulatory roles in interleukin-6 mediated differentiation and STAT3 activation in M1 leukemia cells . Oncogene . 1998 ; 17 ( 17 ): 2271 - 8 .
44. Ihle JN , Witthuhn BA , Quelle FW , Yamamoto K , Thierfelder WE , Kreider B , Silvennoinen O . Signaling by the cytokine receptor superfamily: JAKs and STATs . Trends Biochem Sci . 1994 ; 19 ( 5 ): 222 - 7 .
45. Ihle JN , Kerr IM . Jaks and Stats in signaling by the cytokine receptor superfamily . Trends Genet . 1995 ; 11 ( 2 ): 69 - 74 .
46. Wang X , Wang G , Zhao Y , Liu X , Ding Q , Shi J , Ding Y , Wang S. STAT3 mediates resistance of CD44(+)CD24(−/low) breast cancer stem cells to tamoxifen in vitro . J Biomed Res . 2012 ; 26 ( 5 ): 325 - 35 .
47. Liu S , Ye D , Wang T , et al. Repression of GPRC5A is associated with activated STAT3, which contributes to tumor progression of head and neck squamous cell carcinoma . Cancer Cell Int . 2017 ; 17 : 34 . doi: 10 .1186/ s12935-017-0406-x.
48. Alexandrow MG , Song LJ , Altiok S , Gray J , Haura EB , Kumar NB . Curcumin: a novel Stat3 pathway inhibitor for chemoprevention of lung cancer . Eur J Cancer Prev . 2012 ; 21 ( 5 ): 407 - 12 .
49. Kroon P , Berry PA , Stower MJ , Rodrigues G , Mann VM , Simms M , Bhasin D , Chettiar S , Li C , Li PK , et al. JAK-STAT blockade inhibits tumor initiation and clonogenic recovery of prostate cancer stem-like cells . Cancer Res . 2013 ; 73 ( 16 ): 5288 - 98 .
50. Horiguchi A , Oya M , Marumo K , Murai M. STAT3, but not ERKs, mediates the IL-6-induced proliferation of renal cancer cells , ACHN and 769P. Kidney Int . 2002 ; 61 ( 3 ): 926 - 38 .
51. Zuckerman JE , Davis ME. Clinical experiences with systemically administered siRNA-based therapeutics in cancer . Nat Rev Drug Discov . 2015 ; 14 ( 12 ): 843 - 56 .
52. Wu D , Han H , Xing Z , Zhang J , Li L , Shi W , Li Q. Ideal and reality: barricade in the delivery of small interfering RNA for cancer therapy . Curr Pharm Biotechnol . 2016 ; 17 ( 3 ): 237 - 47 .
53. Young SW , Stenzel M , Yang JL . Nanoparticle-siRNA: a potential cancer therapy ? Crit Rev Oncol Hematol . 2016 ; 98 : 159 - 69 .