†These authors contributed equally.
Academic Editors: Nicholas Pavlidis and Stergios Boussios
Background: Anlotinib, a multi-target tyrosine kinase inhibitor, has
significant anti-cancer effects on breast cancer (BC), lung cancer, colon cancer
and ovarian cancer, but its mechanism has not been investigated in BC.
Methods: The cell viability and growth of human non-triple negative BC
cell line MCF-7 and triple negative BC cell line MDA-MB-231 with the treatment of
anlotinib were tested by Cell Counting Kit-8 (CCK-8) assay and Ki67 staining. The
alteration of genes related to apoptosis and autophagy were investigated by
quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR),
western blots and immunocytochemistry (ICC). Cell apoptosis was valued by TUNEL
staining and flow cytometry. Further, mouse breast tumour cell lines AT-3 cells
were subcutaneously injected into C57BL/6 mice, and the effect of anlotinib
intragastrically on tumour growth in vivo was examined.
Results: We found that anlotinib suppressed the cell viability and
depressed Ki67 staining in MCF-7 and MDA-MB-231 cell lines. Besides, the drug
also enhanced cell autophagy and apoptosis of MCF-7 and MDA-MB-231 cell lines,
which could be rescued by autophagy inhibitors wortmannin (wort) and
3-methyladenine (3-MA), and BECN1 knockdown. Furthermore, Akt/GSK-3
Breast cancer (BC) is one of the most common and high-risk female malignant tumors worldwide. Its incidence rises each year, and the age of onset tends to be younger. At present, BC had surpassed lung cancer and became the most common cancer in the world, accounting for 11.7% of all new cancer cases in 2020 [1]. BC was also the most frequent cancer in female in 2020 in China and was estimated to be the fourth most common cancer diagnosed, replacing liver cancer [2]. Although the treatment of BC has improved in recent years, postoperative tumour cell recurrence, metastasis and drug resistance have resulted in short tumour-free survival and low 5-year survival rates in high-risk populations, which seriously threaten the lives and health of patients [2, 3, 4]. Targeted therapy, as a new therapeutic strategy, has the advantages of strong specificity, remarkable curative effects and few side effects. Hence, targeted therapy has been recognized as an effective and selective method to kill tumour cells, and it is gradually becoming a hot spot and trend in the field of cancer therapy.
Anlotinib hydrochloride, a novel oral multi-target tyrosine kinase receptor inhibitor that targets vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR), shows broad-spectrum inhibitory effects on tumour angiogenesis and growth [5, 6]. In vitro studies have shown that anlotinib selectively inhibits VEGFR2/kinase insert domain receptor (KDR) and VEGFR3, with an inhibition rate 20 times that of sunitinib and 500 times that of sorafenib, respectively. In addition, the dysregulated FGF/FGFR axis promotes cancer progression and enhances the angiogenic potential of the tumour microenvironment, leading to an invasive phenotype of cancer cells [7, 8, 9], and FGF/FGFR signalling changes are associated with chemoresistance and adverse clinical prognosis of cancers. Part of reports shows that anlotinib inactivates FGFRs obviously, especially FGFR2, compared with the effects of sorafenib in preclinical trials. Recently, it is found that anlotinib also inhibits c-Kit receptor, glial cell-derived neurotrophic factor receptor tyrosine kinase, Aurora-B kinase, c-Fms kinase and discoid domain receptor 1, which are involved in the cell proliferation of colon cancer, lymphoma, and acute T cell leukaemia or in the progression of lung, breast, and ovarian cancers [10, 11, 12, 13]. In vivo experiments also demonstrates that anlotinib has broad inhibitory effects against xenograft tumors from transplanted human colon, ovarian, kidney and lung cancer cells and glioma cells [14].
It has been shown that anlotinib has anti-cancer effects on many malignant tumors remarkably, such as non-small cell lung cancer, renal cell carcinoma and hepatocellular carcinoma in clinical trials. Recently, the clinical trials of triple-negative BC and metastatic HER2-negative BC have been carried out, however, there is still a lack of in-depth research on the role of anlotinib in BC and its underlying mechanism. Especially, BC can be divided into three main sub-types at least, including triple negative BC, HER2 Enriched BC and Luminal (A and B) BC. Hence, in this study, we used triple negative BC cell line MDA-MB-231 and non-triple negative, luminal, BC cell line MCF-7 to explore the effects of anlotinib on the growth of different types of BC and its underlying mechanism, which provided a new choice for targeted therapy of BC.
Anlotinib was kindly given as a gift by Chia Tai Tianqing Co., Ltd. (Nanjing, JS, China). Wortmannin (Wort) and 3-methyladenine (3-MA) were purchased from APExBIO (Houston, TX, USA). The antibodies for western blotting, immunocytochemistry and immunohistochemistry were listed in the Supplementary Table 1.
The human BC cell lines MCF-7 and MDA-MB-231 were purchased from the cell bank
of the Chinese Academy of Science. MCF-7 was cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% foetal bovine serum (FBS) and antibiotics
(penicillin 100 U/mL and streptomycin 100 mg/mL) in a 37
For silencing of BECN1, siRNAs were transiently transfected into cells using jetPRIME® transfection reagent according to the manufacturer’s instructions (Polyplus transfection®SA, France).
A Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Briefly, 10000 cells for MCF-7 cell and 5000 cells for MDA-MB-231 cell per well were seeded in 96-well plates and incubated with anlotinib at various concentrations and for different time points. The CCK-8 reagent was then added at a 1:10 dilution and incubated for 1.5 h, and the absorbance at 450 nm was measured on a microplate reader to calculate the cell viability and IC50.
RNA was extracted from the cells or tissues using the
TRIzol-trichloromethane-isopropanol method and reverse transcribed into cDNA
using ReverTra Ace qPCR RT Kit (TOYOBO CO., LTD., Osaka, Japan). The mixtures
contained samples of 10 ng (1
Protein samples from cancer cells and tissues were resolved by SDS-PAGE (10% or 12%), electrotransferred onto Immobilon-P membranes, blocked, and incubated with primary and secondary antibodies. Densitometric quantification of the protein bands was analysed using ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA).
For apoptosis analysis, MCF-7 and MDA-MB-231 cells were collected and stained using Annexin V-FITC/PI apoptosis detection kit (Vazyme, Nanjing, JS, China). The samples were tested on a flow cytometer (FACSCalibur) and analysed by ModFit LT v.3.0 software (Verity Software House, Topsham, ME, USA).
Cells were fixed in 4% paraformaldehyde (PFA) for 15 min and then permeabilized
with 0.1% Triton X-100 for 20 min. After blocking with 5% goat serum for 2 h,
the cells were incubated with primary antibodies against Ki67, BECN1 and LC3B
overnight at 4
Dissected tumour tissues were preserved in 4% PFA at 4
Sixteen male wild-type C57BL/6 mice were purchased from the Laboratory Animal
Resources of Chinese Academy of Sciences (Shanghai, China). Mice were maintained
in specific pathogen free (SPF) conditions at the Fudan University Animal
Experimental Centre, compliant with the guidelines of the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. The experiment was
approved by the Institutional Animal Care and Utilization Committee of Fudan
University (Approval number: 20190703). AT-3 cells were harvested and resuspended
at 1
Statistical analysis was performed by Student’s t-test for comparisons
between the DMSO and anlotinib groups and two-factor Analysis of Variance (ANOVA)
for comparisons among the four groups, followed by a subsequent post-hoc test.
Growth curves descripted in the CCK-8 assay were analysed with two-factor ANOVA
(Treatment
The CCK-8 assay was used to assess cell viability and the half maximal
inhibitory concentration (IC50) in MCF-7 and MDA-MB-231 cells. The results showed
that anlotinib inhibited cell viability in a dose- and time-dependent manner. The
IC50 values of MCF-7 cells treated with anlotinib for 4 h, 12 h, 24 h and 48 h
were 34.02
Anlotinib suppressed the proliferation of human BC cells. (A
and B) Dose-response curves of anlotinib treatment. Cells were cultured with
anlotinib at various concentrations for 4, 12, 24 and 48 h, and cell viability
was detected by CCK-8 assay. (C and D) Representative images of Ki67 staining.
The MCF-7 and MDA-MB-231 cells were stained after anlotinib treatment with 10
We next explored the apoptosis of MCF-7 and MDA-MB-231 cells with anlotinib
treatment. The results revealed that both early and late apoptosis in MCF-7 and
MDA-MB-231 cells were increased significantly after anlotinib treatment (Fig. 2A,
Anlotinib promoted apoptosis in human BC cells. The MCF-7 and
MDA-MB-231 cells were induced by anlotinib with 10
Anlotinib induced autophagy with 10
Cell autophagy is associated with cell proliferation and apoptosis. Then, we
detected the expression of autophagy-related markers in MCF-7 and MDA-MB-231
cells with anlotinib induction. We found that anlotinib upregulated LC3B,
BECN1and ATG4B mRNA levels in MCF-7 cells (Fig. 3A,
Autophagy is a double-edged sword in tumour progression and therapy and is
closely related with apoptosis in cancer cell growth. To confirm the role of
autophagy in anlotinib-induced apoptosis, MCF-7 and MDA-MB-231 cells were
pre-treated with the autophagy inhibitors wortmannin (wort, 1
Anlotinib induced apoptosis by promoting autophagy in MCF-7 and
MDA-MB-231 cells with the mount of 10
Given that anlotinib showed significant therapeutic efficacy in BC cells, we
further determined the underlying mechanism. Recent report revealed that the
level of Akt (S473) and GSK-3
Anlotinib regulated the Akt/GSK-3
To examine the therapeutic significance in vivo, mice were
subcutaneously injected with AT-3 cells to generate syngeneic tumors followed by
continuous 3-week intragastric treatment with anlotinib. The anlotinib group mice
showed significantly decreased body weight, tumour weight and tumour volume
compared to those of the DMSO group. The body weight of mice fed with anlotinib
was not significantly different from that of mice without anlotinib in the first
two weeks. However, the weight of mice treated with anlotinib was increased
significantly during the last two weeks. The result comprehensively demonstrated
that anlotinib inhibited tumour growth (Fig. 6A–D,
Anlotinib suppressed BC growth in vivo. (A) Images of
dissected tumors from C57BL/6 mice injected with AT-3 cells in the DMSO-treated
group (n = 6) and the anlotinib-treated group (n = 10). (B) The body weight of
the mice after injection over time. (C) Tumour volumes were decreased in the
anlotinib group compared to those of the DMSO group. (D) Tumour weights with
anlotinib treatment were heavier than those without anlotinib treatment. (E) The
mRNA level of autophagy-associated molecules was increased markedly in the
anlotinib-treated tumour tissues. (F and G) The ratio of LC3B II/LC3B I and the
expression level of Bcl-2 and Cl-Caspase 3 were detected by western blotting and
analysis. (H) Ki67 immunohistochemistry staining of tumour sections from DMSO and
anlotinib mice. Scale bars: 50
Anlotinib, an inhibitor of multiple tyrosine kinase receptors, inhibits tumour progression by inhibiting angiogenesis [17], but there is no published literature on the inhibitory mechanism of anlotinib on BC.
Many studies have reported that anlotinib exerts antitumour effects to inhibit cell viability and proliferation in hepatocellular carcinoma (HCC), lung cancer, thyroid cancer, and osteosarcoma [16, 18, 19, 20]. Our study found that anlotinib also inhibited the cell viability and proliferation of BC cells, which was consistent with the effects in other tumors.
Autophagy is an important cellular mechanism that plays a “housekeeping” role in normal physiological processes, including the removal of longevity, aggregation and misfolded proteins, removal of damaged organelles, and the regulation of growth and ageing. In tumour cells, autophagy is usually activated during anticancer treatments such as radiation therapy, chemotherapy, and targeted therapy. This may be a cytoprotective mechanism that also causes excessive autophagy in the cell, namely, excessive self-digestion, and induces phagocytic cell death, which is also known as type II programmed cell death [21]. A study found that anlotinib induced autophagy in human lung cancer cells in a time- and concentration-dependent manner and increased the ratio of LC3BII/I protein and the protein expression level of BECN1. The autophagy inhibitors 3-MA and small interfering RNA of BECN1 reversed the autophagy effect induced by anlotinib, unexpectedly, they both enhanced the inhibitory effect of anlotinib on cell proliferation, making the anticancer effect of anlotinib more sensitive and strengthening its inhibition of angiogenesis [19]. These findings suggested that the induction of autophagy in human lung cancer cells by anlotinib is a cytoprotective effect. In our study, anlotinib also induced autophagy in MCF-7 and MDA-MB-231 human BC cells. These results showed significantly increased mRNA and protein expression levels of LC3B and BECN1 and the ratio of protein LC3BII/I and BECN1 levels and decreased P62 levels, suggesting that anlotinib promotes autophagic cell death in BC cells.
Apoptosis is a common programmed cell death and plays a key role in the development of diseases, including cancer. Cancer cells evade apoptosis, thereby achieving excessive proliferation and surviving under hypoxic conditions and with drug resistance [22]; thus, promoting tumour cell apoptosis has become an important strategy for the treatment of cancer. Studies have shown that anlotinib exerts its antitumour effects on HCC, thyroid cancer, osteosarcoma, and lung cancer by promoting apoptosis [16, 18, 19, 20, 23]. Anlotinib significantly inhibited colony formation and promoted apoptosis in HCC and thyroid cancer in vitro [16, 18]. It upregulated the pro-apoptotic molecule Bax and inhibited the anti-apoptotic proteins Bcl-2 and Survivin to kill tumour cells. In addition, animal experiments demonstrated that anlotinib reduced the volumes and weights of transplanted tumors [18]. In thyroid cancer, anlotinib caused abnormal spindle assembly and G2/M arrest, promoted the activation of cleaved-Caspase 3 and cleaved PARP, and activated TP53 [16]. Like the above experimental results, we found that anlotinib increased the mRNA and protein levels of proapoptotic proteins and inhibited the mRNA and protein levels of the anti-apoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 BC cells, thereby exerting an antitumour effect.
Autophagy and apoptosis often occur in the same cells with the same upstream
cellular signals activated by the endoplasmic reticulum, such as extracellular
regulated protein kinases (ERK)/activating transcription factor 4 (ATF4),
Inositol-requiring enzyme-1
Our study indicates that anlotinib has both effects on triple negative breast cancer cells and non-triple negative breast cancer cells. The IC50 value of anlotinib in MCF-7 is smaller than that in MDA-MB-231 in a time-dependent manner, demonstrating that anlotinib has a stronger effect to kill neoplasm cells in non-triple negative breast cancer. The apoptosis induction analysis revealed that a larger proportion of early and late apoptosis was counted in the MCF-7 cells compared with MDA-MB-231 cells. These results may reveal that anlotinib is more effective in the treatment of non-triple negative breast cancer.
Evidence indicates that Akt is a key molecule in both autophagy and apoptosis
because it is the upstream signal of mammalian target of rapamycin complex and
JNK [25, 26, 27]. Previous studies reported that anlotinib inhibits Erk and Akt
signal transduction pathways to regulate cell growth in HCC cells [18]. Akt/GSK-3
signals can regulate cell autophagy, apoptosis, and cell cycle. Furthermore,
level of Akt (S473) and GSK-3
The mechanism by which anlotinib exerted its anticancer effect in BC.
In summary, our study demonstrated that anlotinib inhibited the growth of BC
in vitro and in vivo, especially in non-triple negative,
luminal, BC cell line MCF-7 cells. Anlotinib promoted cell apoptosis and
inactivated Akt/GSK-3
3-MA, 3-methyladenine; ATF4, activating transcription factor 4; ATG, autophagy-related protein; BC, Breast cancer; ERK, extracellular regulated protein kinases; FGFR, fibroblast growth factor receptor; PFA, paraformaldehyde; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor; wort, wortmannin.
PZ (Ping Zhou) and HY conceived the idea and designed the research study. SC designed and performed most experiments, analyzed the data, and wrote the manuscript. YG repeated some experiments. PZ (Ping Zhu) and YJ provided help and advice on animal experiment. XW and LZ analyzed the data. XZ provided advice on the study and manuscript.
All animal care and experimental procedures complied with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiment was approved by the Institutional Animal Care and Utilization Committee of Fudan University (Approval number: 20190703).
Thanks to all the peer reviewers for their opinions and suggestions.
This study was supported by Medical Science Research Foundation from Beijing Medical and Health Foundation (F2190E), and Medical Science Research Foundation from Bethune Charitable Foundation (B19358ET).
The authors declare no conflict of interest.