Research ArticleExperimental Studies

Lenvatinib Suppresses Protein Kinase B Signaling and Induces Apoptosis in Osteosarcoma Cells

CHUN-YI LI, HSIN-CHUAN CHEN, CHIH-YING LIAO, FEI-TING HSU, KUANG-CHEN HUNG, KUO-CHING LIU, JAW-CHYUN CHEN and MING-CHOU KU
Anticancer Research January 2024, 44 (1) 85-92; DOI: https://doi.org/10.21873/anticanres.16790

Abstract

Background/Aim: Lenvatinib, an oral multikinase inhibitor, has demonstrated promising activity in patients with osteosarcoma (OS). Therefore, it is worth exploring the inhibitory efficacy and mechanism of action of lenvatinib in osteosarcoma. The primary goal of this study was to examine the inhibitory effectiveness and mechanism of lenvatinib on the growth and invasion of OS cells. Materials and Methods: The effects of lenvatinib on cell viability, apoptosis, protein kinase B (AKT) activation, its downstream effector proteins involved in tumor progression, and invasion capability were assessed using MTT assay, flow cytometry, western blotting, and invasion/migration assay on U-2 OS and MG63 cells. Results: Lenvatinib effectively induced cytotoxicity, apoptosis, as well as extrinsic and intrinsic apoptotic signaling in OS cells. Lenvatinib also significantly decreased the invasion/migration capability, AKT activation, and downstream effector proteins. Conclusion: The anti-OS effect of lenvatinib may be associated with the induction of apoptosis and the inactivation of AKT.

Key Words:

Osteosarcoma (OS) stands as the predominant bone malignancy, with a primary occurrence in children, adolescents, and individuals over 50 years of age (1, 2). Chemoresistance and distant recurrence impose constraints on the effectiveness of conventional OS treatments, which encompass surgery, chemotherapy, and radiotherapy (3, 4). The research area of improving outcomes for OS patients who have failed conventional treatments is crucial.

Owing to the significant success of tyrosine kinase inhibitors (TKIs) in cancer treatment, for OS, TKIs may be a highly promising treatment strategy (5). For instance, both sorafenib and regorafenib have been reported to provide therapeutic benefits for OS patients following chemotherapy failure (6, 7). Based on evidence from cells and animal models, the inactivation of oncogenic kinases, such as AKT and extracellular signal-regulated kinase (ERK), is linked to the inhibition of OS progression by these drugs (8, 9).

Constitutive activation of angiogenic and oncogenic kinases drives tumor progression through the upregulation of downstream effector molecules associated with cell proliferation, survival, angiogenesis, and metastasis (10-12). Lenvatinib, an oral tyrosine kinase inhibitor, exerts tumor suppression by targeting angiogenic and oncogenic kinases, including vascular endothelial growth factor (VEGF) receptors 1-3, platelet-derived growth factor receptor alpha, fibroblast growth factor (FGF) receptors 1-4, and rearranged during transfection (RET) protein (13, 14).

Single-agent lenvatinib has demonstrated both antitumor activity and safety in young adults with osteosarcoma (15). Lenvatinib demonstrates potential as a promising treatment option for osteosarcoma, and further comprehensive research is warranted to investigate its effects and mechanism of action in osteosarcoma. Therefore, the primary objective of the current study was to evaluate the inhibitory efficacy and mechanism of lenvatinib on the growth and invasion of osteosarcoma cells.

Materials and Methods

Chemicals, reagents, and antibodies. Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lenvatinib were obtained from Sigma (St. Louis, MO, USA). CaspGLOW™ Fluorescence active caspase-3, -8, and -9 staining kits were obtained from BioVision (Milpitas, CA, USA). Annexin-V/propidium iodide (PI) staining kit and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) were obtained from Vazyme Biotech Co. Ltd (Nanjing, PR China) and Enzo Life Sciences (Farmingdale, NY, USA). Primary antibodies, including protein kinase B (PKB/AKT), AKT (ser473), x-linked inhibitor of apoptosis protein (XIAP), Cyclin D1, and β-actin, were obtained from Elabscience (Houston, TX, USA) and Matrix metallopeptidase-2 (MMP-2) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Cell culture. In this study, the human osteosarcoma cell lines MG-63 and U-2 OS were used. MG-63 cells were cultured in a RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The cells were then incubated in a humidified incubator at 37°C with 5% CO2. U-2 OS cells were maintained in McCoy’s 5A medium with 10% FBS and 1% PS under the same incubation conditions.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell viability following lenvatinib treatment was assessed using the MTT assay. Cells (5×103/well) were plated in 96-well plates and allowed to adhere overnight. Subsequently, the cells were exposed to varying concentrations (0-60 μM) of lenvatinib for 24 or 48 h. Following treatment, 0.5 mg/ml of MTT solution was added, and the cells underwent an additional 2-hour incubation. Subsequently, 100 μl DMSO was used to replace the medium before further assessment. Absorbance at 570 nm was determined by using a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) (16, 17).

Annexin V and PI double staining for assessing apoptosis using flow cytometry. Cells were placed in 6-well plates with 2×105 cells per well and allowed to adhere overnight. An Annexin-V/PI staining kit was used to assess apoptosis in cells following exposure to lenvatinib (0-20 μM) for 48 h. The Annexin-V/PI dual staining protocol and the detection of Annexin-V/PI signaling using flow cytometry were performed as previously described (16).

Caspase activation analysis using flow cytometry. Cells (2×105) were cultured in a 6-well dish and incubated overnight. The cells were then exposed to lenvatinib (0, 10, and 20 μM) for 48 h. Afterward, the cells were harvested and subjected to staining using a caspase-3 activation probe (FITC-DEVE-FMK), a caspase-8 activation probe (Red-IETD-FMK), or a caspase-9 activation probe (FITC-LEHD-FMK) for 30 min at 37°C. The staining procedures were performed according to the manufacturer’s protocols (18, 19).

Detection of mitochondrial membrane potential (ΔΨm) using flow cytometry. The cells were seeded in 6-well plates at a density of 2×105 cells per well, incubated overnight, and then exposed to lenvatinib at concentrations 0, 10, and 20 μM for 48 h. Following treatment, cells were harvested and subjected to staining with 1 μM DiOC6 in 500 μl of PBS for 30 min at 37°C. The detection of DiOC6 signal was performed using the FL-1 channel (20).

Invasion and migration assay. The cells (2×106) were seeded in 10 cm dishes and incubated overnight. Subsequently, cells were exposed to 0, 10, and 20 μM lenvatinib for 48 h. After treatment, the cells were harvested, and 2×105 cells were resuspended in 200 μl of serum-free medium before being added to the upper transwell chambers (Corning, Corning, NY, USA) that were pre-coated with matrigel for the assessment of cell invasion. After 48 h the matrigel was removed and the transwell inserts were immersed in 4% paraformaldehyde for 30 min at 4°C to fix the cells. Subsequently, the cells were stained with a crystal violet solution for 2 h, photographed using a microscope (Nikon ECLIPSE Ti-U, Minato, Tokyo, Japan), and invaded cells were counted using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA). For evaluating cell migration, the upper transwell chambers were not pre-coated with Matrigel. The subsequent steps were identical to those of the invasion assay (16, 21).

Western blotting assay. Two million cells were seeded in 10 cm dishes and incubated overnight. Afterward, the cells were treated with 0, 10, and 20 μM lenvatinib for 48 h. The protein expression of AKT (Ser473), AKT, XIAP, Cyclin D1, and MMP2 was evaluated using western blotting assay as described previously (16).

Statistical analysis. We used analysis of variance (ANOVA) to assess the significance of differences between each experimental group and the control group. Data is presented as mean±standard error, and a p-value less than 0.05 was deemed to be statistically significant.

Results

Lenvatinib inhibits growth and triggers apoptosis in OS cells. Figure 1A illustrates the inhibitory potency of lenvatinib on the survival of OS cells. Treatment with lenvatinib significantly suppressed cell viability in both cell lines. After a 48-hour exposure to 20 μM of lenvatinib, MG63 and U-2 OS cells exhibited an approximately 50% reduction in cell viability compared to the control group. This substantiates that 20 μM lenvatinib represents its half-maximal inhibitory concentration (IC50). For additional experiments, 5 μM (IC75) and 20 μM of lenvatinib were chosen in MG63 cells, and 10 μM (IC75) and 20 μM of lenvatinib were selected for further investigation in U-2 OS cells. Induction of apoptosis leads to tumor growth inhibition. Consequently, we evaluated whether lenvatinib induces apoptosis in OS cells using a caspase-3 activation probe and annexin V/PI double Staining. Figure 1B and C indicates that lenvatinib effectively induced apoptosis while upregulated caspase-3 activation. Based on these results, we conclude that the induction of apoptosis may correlate with lenvatinib-mediated growth inhibition of OS cells.

Figure 1.
Figure 1.

Inhibition of cell growth and induction of apoptosis by lenvatinib in OS cells. MG-63 and U-2 OS cells were treated with 0-60 μM lenvatinib for 24 or 48 h, and then cell viability was evaluated using the MTT assay (A). Apoptotic events (B) and activation of cleaved caspase-3 (C) are analyzed using flow cytometry after treating MG-63 cells with 0, 5, and 20 μM lenvatinib, and U-2 OS cells with 0, 10, and 20 μM lenvatinib, following a 48-hour incubation period (** p<0.01, *** p<0.0001 vs. 0 μM Lenvatinib).

Lenvatinib initiates extrinsic and intrinsic apoptotic pathways in OS cells. The extrinsic and intrinsic pathways are the main two pathways that initiate apoptosis (22). We used caspase-8 and caspase-9 activation probes, along with ΔΨm measurement, to elucidate the association between lenvatinib-induced apoptosis and these pathways. The results indicated that treatment with lenvatinib for 48 h significantly upregulated the activation of caspase-8 and caspase-9, while the ΔΨm was significantly reduced compared to the control (Figure 2A-C). Based on these findings, we propose that lenvatinib-induced apoptosis is associated with the initiation of both the extrinsic and intrinsic apoptosis pathways in OS cells.

Figure 2.
Figure 2.

Lenvatinib enhances the activation of cleaved caspase-8 and -9, while it induces loss of ΔΨm in OS cells. After treating MG-63 cells with 0, 5, or 20 μM lenvatinib, and U-2 OS cells with 0, 10, or 20 μM lenvatinib, the activation of cleaved caspase-8 (A), cleaved caspase-9 (B), and the alteration in ΔΨm (C) were assessed using flow cytometry, with measurements taken following a 48-hour incubation period.

Lenvatinib suppresses the activity of AKT and its downstream effector proteins, as well as the invasion/migration capability of osteosarcoma (OS) cells. Cyclin D1, XIAP, and MMP2 are downstream effector proteins of AKT that promote tumor growth, survival, and invasion (8, 23). We employed western blotting and invasion assays to confirm the impact of lenvatinib on the expression of these proteins and invasion capability. The results showed that the protein levels of phospho-AKT (p-AKT), MMP2, Cyclin D1, and XIAP were significantly decreased by treatment with lenvatinib compared to the control group (Figure 3A and B). The results of the invasion/migration assay indicated that the invasion/migration capability of both U-2 OS and MG63 were significantly reduced following treatment with lenvatinib (Figure 3C and D).

Figure 3.
Figure 3.

Lenvatinib facilitates AKT inactivation, suppresses downstream effector proteins of AKT, and inhibits invasion potential in OS cells. Protein levels of p-AKT, MMP2, Cyclin-D1, and XIAP were assessed using western blotting in MG-63 (A) and U-2OS (B) cells. Invasion and migration capacities of MG-63 (C) and U-2OS (D) cells were investigated using invasion and migration assays after treatment with 0, 5, or 20 μM lenvatinib for MG-63 cells and 0, 10, or 20 μM lenvatinib for U-2 OS cells, with a subsequent 48-hour incubation.

Discussion

X-linked inhibitor of apoptosis protein (XIAP) is an anti-apoptotic protein that suppresses the activation of caspase-3, -7, and -9, thereby preventing apoptosis (24). Elevated XIAP expression mediates the development of resistance to chemotherapy and radiotherapy in tumor cells (25-27). Matrix metalloproteinase 2 is an invasion-related protein that enzymatically digests gelatin, as well as types I and IV collagens, thus fostering the invasion and metastasis of tumor cells (27). Increased levels of MMP2 have been associated with metastasis and a poor prognosis in OS patients (28). Our findings demonstrated that lenvatinib significantly triggered apoptosis and inhibited invasion in OS cells (Figure 1B, and Figure 3C and D). Furthermore, treatment with lenvatinib effectively decreased the expression of XIAP and MMP2 (Figure 3A and B).

Cyclin D1 has been observed to be overexpressed in advanced OS. It plays a vital role in promoting tumor growth by facilitating the cell cycle transition from G1 to S phase (29, 30). The silencing of Cyclin D1 has been shown to reverse chemoresistance and restore the growth inhibition induced by doxorubicin in OS cells (31). Based on the results of the MTT assay and western blot analysis, we concluded that lenvatinib treatment effectively suppressed cell proliferation and Cyclin D1 expression (Figure 3A and B).

Protein kinase B (PKB/AKT) serves as a downstream kinase in receptor tyrosine kinase (RTK) pathways, and its activation is essential for tumor progression driven by RTK signaling (32-35). Phospho-AKT (p-AKT) has been demonstrated as an unfavorable prognostic factor correlated with poorer survival in OS patients (35). Suppression of p-AKT has been demonstrated to lead to the decrease of downstream effector proteins, including MMP2, MMP9, Cyclin D1, and XIAP, along with the inhibition of invasion ability in OS cells (8, 23). Our data showed that lenvatinib effectively induced a decrease in p-AKT levels in OS cells (Figure 3A and B).

Apoptosis is a programmed cell death that contributes to the inhibition of tumor cell growth triggered by anticancer agents. This process can be initiated through both extrinsic and intrinsic pathways. The interaction between the death receptor and its ligand, as well as the loss of ΔΨm, results in the activation of caspase-8 and -9. Active caspase-8 and -9, functioning as effector caspases, elicit the cleavage of downstream executioner caspases, ultimately culminating in cell death. The results demonstrated that treatment with lenvatinib markedly enhanced the activation of caspase-3, -8, and -9, and induced the loss of ΔΨm in OS cells (Figure 1C and Figure 2A-C).

In conclusion, this study showed that lenvatinib induces apoptosis while concomitantly inhibiting the anti-apoptotic and invasive capabilities in OS cells. Our findings suggest that apoptosis induction and AKT inactivation are linked to the growth inhibition and reduced invasion of OS cells following treatment with lenvatinib.

Acknowledgements

Experiments and data analysis were partially conducted at the Medical Research Core Facilities Center, Office of Research & Development, China Medical University, Taichung, Taiwan, R.O.C.

Footnotes

  • Authors’ Contributions

    CHL, HCC, CYL, and FTH conducted all experiments, performed statistical analyses, and summarized the data. KCH, KCL, JCC, and MCK drafted the initial version of the article. JCC, and MCK conceptualized the presented idea, supervised the findings of this work, conducted the literature review, and prepared the final version of the paper.

  • Conflicts of Interest

    The Authors affirm that they have no financial interests that might be construed as conflicting with the findings or conclusions presented in this study.

  • Funding

    The study received support from the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-111044).

  • Received November 15, 2023.
  • Revision received December 2, 2023.
  • Accepted December 4, 2023.

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Lenvatinib Suppresses Protein Kinase B Signaling and Induces Apoptosis in Osteosarcoma Cells
CHUN-YI LI, HSIN-CHUAN CHEN, CHIH-YING LIAO, FEI-TING HSU, KUANG-CHEN HUNG, KUO-CHING LIU, JAW-CHYUN CHEN, MING-CHOU KU
Anticancer Research Jan 2024, 44 (1) 85-92; DOI: 10.21873/anticanres.16790
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