Research ArticleExperimental Studies
Open Access

Anlotinib Inhibits Ovarian Cancer and Enhances Cisplatinum Sensitivity via Suppressing NOTCH2 Expression and Stemness

XIAOSHENG XU, QUN WANG, LIFEI SHEN, YUHONG SHEN, HUA LIU, YAN LIU, ZHIJIAN YANG, ROBERT M. HOFFMAN and WEIWEI FENG
Anticancer Research April 2024, 44 (4) 1399-1407; DOI: https://doi.org/10.21873/anticanres.16936

Abstract

Background/Aim: The prognosis of ovarian cancer (OC) patients is especially poor for patients with chemotherapy resistance. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor, has shown encouraging clinical efficacy in several tumor types. The aim of the present study was to examine the inhibitory efficacy and mechanism of anlotinib on the proliferation and chemosensitivity of OC cells. Materials and Methods: The inhibitory effects of Anlotinib on SKOV3 and OVCAR3 OC cells were examined using CCK-8 cell-viability, colony-formation, flow-cytometry, transwell-migration and sphere-formation assays. A xenograft mouse model was used for in vivo studies. RT-qPCR and western blotting were used to detect gene expression. Results: Molecular targets of anlotinib were elevated in OC patient tumors. Anlotinib significantly inhibited ovarian cancer cell proliferation and migration in vitro. Anlotinib enhanced the sensitivity of ovarian cancer cells to cisplatinum both in vitro and in vivo. Anlotinib suppressed sphere formation and the stemness phenotype of OC cells by inhibiting NOTCH2 expression. Conclusion: Anlotinib inhibits ovarian cancer and enhances cisplatinum sensitivity, suggesting its future clinical promise.

Key Words:

Ovarian cancer (OC) is the 7th most prevalent malignant cancer type in women globally and is the leading cause of death from gynecological cancer, accounting for 4% of cancer-related deaths (1). Cytoreductive surgery and platinum-based cytotoxic chemotherapy have not improved the prognosis of advanced OC patients (2, 3). Approximately 80% of OC patients will relapse and most will die due to chemotherapy resistance (4, 5). Therefore, the discovery and application of improved therapy for OC is necessary.

Anlotinib is a multi-targeted tyrosine-kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and has also shown activity against platelet-derived growth factor receptors (PDGFR) and the stem-cell factor receptor (c-kit) (6, 7). Results of phase I-III clinical trials have shown promising clinical efficacy of anlotinib in the treatment of advanced non-small-cell lung cancer (NSCLC) (8), metastatic small-cell lung cancer (9), soft-tissue sarcoma (STS) (10), metastatic renal-cell carcinoma (11), glioblastoma (12, 13) and thyroid cancer (14). A partial response was observed in an elderly woman with advanced OC after six cycles of anlotinib monotherapy (15). Two independent retrospective observational studies showed that anlotinib improved overall survival of patients with platinum-resistant OC (16, 17). These previous studies indicate that further research on the efficacy of anlotinib is critical.

In the present study, we tested the efficacy of anlotinib at the cellular level and in mouse tumor models, and its ability to enhance cisplatinum efficacy. Additionally, we tested the effect of anlotinib on NOTCH2 signaling and suppressing the OC cell-stemness phenotype.

Materials and Methods

Cell lines and culture. The human ovarian-cancer cell lines SKOV3 and OVCAR8 were purchased from ATCC (Rockville, MD, USA). SKOV3 cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. OVCAR8 cells were cultured in McCoy’s 5a Medium Modified (GIBCO) supplemented with 10% FBS and penicillin/streptomycin. All cells were grown and maintained at 37°C in a humidified incubator with 5% CO2. Anlotinib was a kind of gift from Chia Tai Tianqing Co., Ltd (Nanjing, PR China).

RNA extraction and real-time quantitative PCR (RT-qPCR). RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. PrimeScript™ RT Master Mix (Takara, Shiga, Japan) was used to synthesize cDNA using 1 μg RNA as template. RT-PCR was performed in a 2X SYBR Green Gene Expression PCR Master Mix (Takara) in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative quantification (RQ) was derived from the difference in the cycle threshold (Ct) between the target gene and GAPDH (ΔCt) using the formula RQ=2−ΔΔCt. GAPDH was used as internal control.

Western blotting. Harvested OC cells were extracted with RIPA lysis solution (Beyotime, Shanghai, China). Protein concentration was measured using the BCA Protein Detection Kit (Beyotime). Proteins (30 μg) were separated using 10% SDS–PAGE gel electrophoresis and electrotransferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 5% non-fat milk to prevent non-specific binding for 1 h followed by blotting with primary antibodies overnight at 4°C and horse-radish peroxidase (HRP)-labeled secondary antibodies for 2 h. The antibodies included: anti-gamma H2A.X (1:500), (ABCAM, Cambridge, UK); anti-NOTCH1 (1:500), (Cell Signaling Technology, Boston, MA, USA); anti-NOTCH2 (1:500), (Cell Signaling Technology); anti-beta-catenin (1:500), (Cell Signaling Technology); anti-phos-RelA (1:500), (Cell Signaling Technology); and anti-GAPDH (1:2,000), (Cell Signaling Technology).

CCK-8 assay. Cell viability was evaluated using a CCK-8 Kit (Beyotime). Cells were seeded in 96-well plate at a density of 5,000 cells/well, and different concentrations of anlotinib or cisplatinum were added to the culture medium. Optical density (OD) at 450 nm was measured using a spectrophotometer every 24 h. Before detection, CCK-8 at 1:10 dilution was added to each well and incubated for an additional 3 h, protected from light.

Colony formation assay. OC cells were seeded in 6-well plates at a density of 1000 cells/well, and different drug concentrations were added to the culture medium. After 7 days, cells were fixed and stained with 0.1% crystal violet for 10 min at room temperature, washed with distilled water, and scanned to count the colonies.

Flow cytometry with 5-ethynyl-2′ deoxyuridine (EdU) and Annexin V-7AAD staining for apoptosis and cell-cycle analysis. OC cells were seeded in 6-well plates at a density of 1×105 cells per well and treated with anlotinib or cisplatinum for 48 h. The cells were harvested and stained with Annexin-V-FITC (1:200; BD Biosciences; Franklin Lakes, NJ, USA; 556420) and 7-AAD (1:100; BD Biosciences; 559925) for apoptosis analysis. For cell-cycle analysis, OC cells were seeded in 6-well plates at a density of 5×104 cells per well. After treatment of cells with or without anlotinib for 24 h, EdU (10 mM) (RiboBio, Guangzhou, China) was added to the culture. Two hours later the cells were collected and fixed with 4% formaldehyde, treated with 0.5% Triton X-100 for permeabilization and processed for EdU staining and AnnexinV-7AAD staining. Flow cytometry was performed, and data were analyzed with FlowJo 7.6 (Treestar Inc, Fenton, MI, USA).

Sphere formation assay. OC cells were harvested with 0.025% trypsin-EDTA, washed and counted. Then cells were plated in ultra-low-attachment 6-well plates (Corning Inc., Corning, NY, USA) at a density of 2,000 viable cells per well. The cultured medium contained serum-free DMEM/F12 (GIBCO) supplemented with 1:50 B27 (GIBCO), 20 ng/ml bFGF (PeproTech; Cranbury, NJ, USA, 100-18B), 10 ng/ml EGF (PeproTech; AF-100-15), 100 U/ml penicillin and 100 μg/ml streptomycin. Spheres were photographed and quantitated under a microscope after 7 days culture.

Transwell migration assay. Cell migration assays were conducted using Transwell inserts (Costar, Cambridge, MA, USA) containing 8-μm-pore polycarbonate membrane filters in 24-well culture plates. SKOV3 or OVCAR3 cells (5×104) were suspended and seeded in the upper chamber of transwell plates (Corning) in serum-free medium with or without anlotinib. DMEM, containing 10% FBS (500 μl), was added to the lower chamber. After incubation for 24 h, cells were fixed with 4% polyoxymethylene and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The cells which had migrated were counted and images of 3 random fields were captured under a light microscope.

In vivo study. Five-week-old male BALB/c nude (nu/nu) mice were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and bred under specific pathogen-free conditions. For the xenograft mouse tumor model, SKOV3 cells (1×106) in 100 μl PBS were subcutaneously injected into the right armpits of the nude mice. On day-7 after injection, the mice with SKOV3 tumors were randomly divided into four groups (n=5 per group). Cisplatinum at 5 mg/kg or anlotinib at 5mg/kg mouse body weight, or their combination, were injected intraperitoneally. Tumor size was measured every 7 days according to the following formula: V=1/2(length × width2). The animal study was approved by the Ethics Committee of Ruijin Hospital (RHEC-2021-0313).

Statistical analysis. Data in the figures are presented as mean±standard error of the mean (SEM). Statistical analysis was performed using R (http://www.r-project.org/), and statistical significance was determined by the 2-tailed Student’s t-test, one-way ANOVA and post-hoc test as indicated in the figure legends. For all statistical tests, a p-value of <0.05 was considered to be statistically significant (*p<0.05).

Results

VEGFR, FGFR2 and PDGFRB are overexpressed and associated with poor outcome in OC. Anlotinib is a multi-targeted inhibitor that exerts its efficacy via targeting VEGFR, FGFR and PDGFR tyrosine kinase receptors in OC. VEGFR (Figure 1A), FGFR2 (Figure 1B) and PDGFRB (Figure 1C) expression was significantly elevated in human OC biopsies compared to associated non-tumor tissues. Furthermore, high expression of VEGFR (Figure 1D), FGFR2 (Figure 1E) and PDGFRB (Figure 1F) was associated with a poor outcome in OC cases. These findings suggested that inhibition of VEGFR, FGFR2 and PDGFRB by anlotinib might have efficacy against OC.

Figure 1.
Figure 1.

Vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor 2 (FGFR2) and platelet-derived growth factor receptors (PDGFRB) are overexpressed and associated with poor outcomes in ovarian cancer (OC). (A to C) VEGFR, FGFR2 and PDGFRB mRNA expression was examined by qRT-PCR in OC tissue (n=38) and associated normal tissue (n=24). *p<0.05 using the two-tailed Student’s t-test. (D to F) Kaplan-Meier plots of overall survival of OC patients based on VEGFR, FGFR2 and PDGFRB expression levels using the Kaplan-Meier plotter database. p-Values are derived from the log-rank test.

Anlotinib inhibits cell proliferation and migration in OC. Anlotinib inhibited cell proliferation in a dose-dependent manner for both OVCAR8 and SKOV3 (Figure 2A). Anlotinib significantly reduced the colony number compared with the control group for both OVCAR8 and SKOV3 (Figure 2B and C). and resulted in a G2/M cell cycle arrest in both OC cell lines (Figure 2D and E). Anlotinib also significantly inhibited the migration of both OC cell lines (Figure 2F and G).

Figure 2.
Figure 2.

Anlotinib inhibits cell proliferation and migration in ovarian cancer (OC) OVCAR8 and SKOV3 cells. (A) Cell viability of OC cells treated with anlotinib. (B and C) Colony formation of OC cells treated with anlotinib. (D and E) Cell cycle arrest of OC cells treated with anlotinib. (F and G) Migration of OC cells treated with anlotinib. *p<0.05 using one-way ANOVA with the post-hoc Scheffe test.

Anlotinib enhances cisplatinum-induced OC-cell apoptosis. Resistance to platinum-based cytotoxic chemotherapy is a major challenges in OC and is closely correlated with tumor recurrence and patient survival. Anlotinib significantly enhanced the efficacy of cisplatinum on OC cell viability even when anlotinib was administered at a low concentration (2 μM) to both OVCAR8 and SKOV3 (Figure 3A). The proportion of apoptotic cells induced by cisplatinum was further increased when cisplatinum treatment was in combination with anlotinib, confirming that anlotinib and cisplatinum had synergy (Figure 3B and C). γH2A.X focus formation is a hallmark of apoptosis induced by platinum-drug treatment (including cisplatinum) (18). Western blot analysis showed that anlotinib increased cisplatinum-induced γH2A.X expression (Figure 3D). Together, these results demonstrate that anlotinib could enhance the chemosensitivity of OC cells.

Figure 3.
Figure 3.

Anlotinib enhances cisplatinum-induced apoptosis of both OVCAR8 and SKOV3 OC cell lines. (A) Cell viability of OC cells treated with different concentrations of cisplatinum and anlotinib. (B and C) Apoptosis of OC cells treated with cisplatinum alone and in combination with anlotinib. *p<0.05 using one-way ANOVA with the post-hoc Scheffe test. (D) γH2A.X protein expression of OC cells treated with cisplatinum alone and in combination with anlotinib.

Anlotinib inhibited OC progression and enhanced cisplatinum sensitivity in a mouse tumor model. Administration of anlotinib led to a significant reduction in SKOV3 tumor growth (Figure 4A) compared with the control mice, as assessed by tumor volume and tumor weight (Figure 4B and C). The combination of cisplatinum and anlotinib led to a significant reduction in tumor growth compared with the mice treated with cisplatinum alone (Figure 4B and C). Histopathological analysis showed a significant increase of apoptotic cells (TUNEL-positive) and significant decrease of a cell proliferation indicator (Ki67) in the tumors treated with anlotinib alone or in combination with cisplatinum compared with the respective control groups (Figure 4D, E and F).

Figure 4.
Figure 4.

Anlotinib inhibits ovarian cancer (OC) progression and enhances cisplatinum sensitivity in a mouse tumor model of SKOV3. (A) Representative SKOV3 tumor images of different treatment groups. (B) Tumor growth curve and (C) tumor weight of different treatment groups. *p<0.05 using one-way ANOVA with post-hoc Scheffe test. (D-F) Hematoxylin and eosin, TUNEL and Ki67 staining of the tumors from treated groups. *p<0.05 using two-tailed Student’s t-test.

Anlotinib suppresses OC cell stemness. Sphere formation ability, when cells are plated at low density in nonadherent culture, is a cancer-cell stemness phenotype (19). Sphere-forming ability decreased significantly with anlotinib treatment for both OVCAR8 and SKOV3 (Figure 5A and B). Accordingly, we found that mRNA expression of several cancer-cell stemness-related genes (or cancer stem-cell markers) including OCT4, SOX2, NANOG, CD44 and ALDH1A1 were downregulated by anlotinib for both OVCAR8 and SKOV3 (Figure 5C). Anlotinib significantly downregulated protein expression of SOX2, NANOG and ALDH1A1 for both OVCAR8 and SKOV3 (Figure 5D). These results demonstrate that anlotinib suppressed the OC cell stemness phenotype and inhibited expression of cancer-cell stemness-related genes.

Figure 5.
Figure 5.

Anlotinib suppresses ovarian cancer (OC) cell stemness of both OVCAR8 and SKOV3. (A and B) Sphere formation of OC cells treated with different concentrations of anlotinib. (C) mRNA expression of cancer stem cell-related genes in OC cells treated with different concentrations of anlotinib. (D) Protein expression of SOX2, NANOG and ALDH1A in OC cells treated with different concentrations of anlotinib. *p<0.05 using one-way ANOVA with post-hoc Scheffe test.

Anlotinib-induced inhibition of OC stemness is associated with inhibition of NOTCH2 expression. NOTCH2 expression was significantly downregulated by anlotinib for both OVCAR8 and SKOV3 (Figure 6A). Higher NOTCH2 expression is associated with poor clinical outcome in OC (Figure 6B). Furthermore, we found that overexpression of NOTCH2 could significantly reduce sensitivity to cisplatinum in anlotinib-treated OC cells (Figure 6C).

Figure 6.
Figure 6.

Anlotinib-induced inhibition of ovarian cancer (OC) stemness is associated with inhibition of NOTCH2 expression. (A) Expression of beta-catenin, phos-RelA, NOTCH1, NOTCH2, SOX2 and ALDH1A in OC cell lines OVCAR8 and SKOV3 24 h after anlotinib treatment. (B) Kaplan-Meier plot of overall survival of OC patients based on NOTCH2 expression levels using the Kaplan-Meier Plotter database. (C) Sensitivity of anlotinib-treated OC cells to cisplatinum in NOTCH2 overexpressed OC cell lines OVCAR8 and SKOV3. *p<0.05 using one-way ANOVA with post-hoc Scheffe test.

Discussion

Tumor metastasis and recurrence due to chemotherapy resistance are two major problems in the treatment of ovarian cancer (20-22). It was previously reported that anlotinib increased chemosensitivity and inhibited tumor growth in osteosarcoma through blockade of MET phosphorylation (23). Anlotinib suppressed metastasis and multidrug resistance via dual blockade of met/abcb1 in colorectal carcinoma cells (24). In a phase I clinical study, anlotinib in combination with platinum/pemetrexed-based chemotherapy, showed promising antitumor activity in advanced non-small-cell lung cancer (NSCLC) (25). In the present study, we found that anlotinib inhibited OC cell proliferation and enhanced sensitivity to cisplatinum in vitro and in a mouse tumor model, indicating the potential inhibitory efficacy of anlotinib on cisplatinum-resistant ovarian cancer.

In the present study, we also demonstrated that anlotinib inhibited the OC stemness phenotype which was associated with inhibition of NOTCH2 expression. NOTCH signaling is a conserved cell-fate-determination pathway that plays a crucial role in the maintenance and tumorigenicity of cancer stem cells (26). Glioblastoma stem-cell (GSC) proliferation and drug resistance are dependent upon NOTCH expression (27). NOTCH identifies cells with cancer stem cell-like properties and correlates with shorter survival in lung adenocarcinoma (28), while NOTCH3 expression promotes a stem-like phenotype in lung adenocarcinoma (29). Similarly, NOTCH-induced aldehyde dehydrogenase 1A1 (ALDH1A1) deacetylation is associated with promotion of breast-cancer stem cells (30).

Conclusion

In summary, the present results showed for the first time that anlotinib has efficacy in OC. Anlotinib significantly inhibited OC cell proliferation, migration, and enhanced cisplatinum chemosensitivity. Anlotinib inhibited the cancer stem-cell phenotype including sphere formation and stem-cell-associated gene expression by downregulating NOTCH2 expression. Thus, anlotinib has potential as a drug against ovarian cancer, especially for chemoresistant OC patients who lack an effective therapeutic option.

Acknowledgements

The research was supported by the grant from the National Natural Science Foundation of China (No. 82172934).

Footnotes

  • Authors’ Contributions

    Weiwei Feng, Yan Liu and Hua Liu designed the study and wrote the draft manuscript; Xiaosheng Xu, Qun Wang, Lifei Shen and Yuhong Shen performed experiments and analyzed the data; Zhijian Yang and Robert M. Hoffman revised the manuscript.

  • Conflicts of Interest

    None of the Authors have any conflict of interest with regard to this study.

  • Received November 15, 2023.
  • Revision received February 4, 2024.
  • Accepted February 15, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Anlotinib Inhibits Ovarian Cancer and Enhances Cisplatinum Sensitivity via Suppressing NOTCH2 Expression and Stemness
XIAOSHENG XU, QUN WANG, LIFEI SHEN, YUHONG SHEN, HUA LIU, YAN LIU, ZHIJIAN YANG, ROBERT M. HOFFMAN, WEIWEI FENG
Anticancer Research Apr 2024, 44 (4) 1399-1407; DOI: 10.21873/anticanres.16936
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