Novel therapeutic strategy for melanoma based on albendazole and the CDK4/6 inhibitor palbociclib

Although an increasing number of patients benefit from immunotherapy and targeted therapies, melanoma remains incurable with increasing incidence. Drug repositioning and repurposing is an alternative strategy to discover and develop novel anticancer drugs or combined therapeutic regimens. In this study, we demonstrated that albendazole (ABZ), an Food and Drug Administration (FDA)-approved broad-spectrum antiparasitic agent, significantly inhibits the proliferation of melanoma cells in vitro and in vivo. RNA sequencing and flow cytometry analysis revealed that ABZ arrests melanoma cells at the G2/M phase of the cell cycle and induces cell apoptosis. More importantly, the CDK4/6 inhibitor palbociclib, as a member of the first and only class of highly specific CDK inhibitors approved for cancer treatment to date, showed significant synergistic effects with ABZ treatment in melanoma cells and mouse models. Taken together, we revealed a previously unappreciated function of ABZ in antimelanoma proliferation by inducing cell cycle arrest and apoptosis and provided a novel combined therapeutic regimen of ABZ plus CDK4/6 inhibitor treatment in melanoma.


Scientific Reports
| (2022) 12:5706 | https://doi.org/10.1038/s41598-022-09592-0 www.nature.com/scientificreports/ whether ABZ can be used as a compound or an adjuvant for the treatment of melanoma alone or in combination and the underlying mechanism remains unknown. Selective cyclin kinase 4/6 (CDK4/6) inhibitors, including palbociclib (Ibrance, PD-0332991; Pfizer), are the first and only class of highly specific CDK inhibitors that have passed the clinical development stage and have been approved for cancer treatment to date. Palbociclib is an orally available, specific small-molecule inhibitor of CDK4/6 that is used in combination with aromatase inhibitors in the therapy of postmenopausal women with metastatic breast cancer positive for the estrogen receptor (ER+) but negative for human epidermal growth factor receptor 2 (HER2−) 10 . Moreover, phase 2 trials have shown palbociclib single-agent activity in mantlecell lymphoma, breast cancer, liposarcoma, and teratoma with reversible neutropenia as the main toxic effect 11 . CDK4/6 regulates the cellular transition from the G1 to the S phase of the cell cycle. Inhibition of this transition by CDK4/6 inhibitors arrests the progression of the cell cycle in the G1/S phase and blocks cell growth 12 .
In this study, we demonstrated that ABZ inhibited the proliferation of melanoma cells in vitro and in vivo. To provide a better understanding of the precise mechanisms of the antimelanoma effect of ABZ, we performed global transcriptomic profiling of the ABZ-treated and untreated human melanoma A375 cells via RNA sequencing. Our results suggest that ABZ inhibited cell growth by arresting the cell cycle at the G2/M phase. More importantly, we found that combining ABZ and palbociclib synergistically decreased cell viability and tumor growth in mouse models. Overall, we provided potential melanoma therapeutic regimens by treatment with ABZ alone or in combination with CDK4/6 inhibitors.

Materials and methods
Cell culture. Human melanoma cells (A375 and SK-MEL-28) and human epidermal cells HaCaT were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All cells were cultured in DMEM included with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, USA) and incubated at 37 °C with 5% CO 2 . ABZ and palbociclib (Selleck, USA) were added to the complete medium at the indicated concentrations and times.
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cells were seeded in triplicate in 96-well plates at a density of 3000 cells per well for 48 h before being subjected to ABZ treatment. Subsequently, MTT (Coolaber, Beijing) was added in each well to obtain a final concentration of 5 mg/mL and further incubated for 3 h. At last, the cells and MTT crystals were dissolved in DMSO (Sigma-Aldrich, Shanghai) and used to determine the absorbance at 570 nm by a microplate reader.
Plate clone formation assay. Cells were seeded in 6-well plates (1,000 cells per well) in a growth medium and, where applicable, subject to albendazole or/and palbociclib treatment. When colonies reached a considerable size, cells were fixed in 4% paraformaldehyde for 10 min, followed by staining with crystal violet solution (Sigma-Aldrich, Shanghai) for another 15 min. Colonies were counted manually using ImageJ software.
Flow cytometry assay. For cell cycle analysis, cells were harvested and fixed in 70% ethanol at 4 °C for 24 h. The fixed cells were washed with phosphate-buffered saline (PBS) and then incubated PBS containing 20 μg/mL propidium iodide (PI) and 0.2 mg/mL RNase A at room temperature for 15 min. Flow cytometer FACSDxp AthenaTM and AuroraTM (Cytek, USA) was used to detect the stained cells. Flow Jo 10.0 software was used to further analyze the stained cells.
For apoptosis analysis, cells were collected in an EP tube and washed with PBS. Then, 5 µL annexin V fluorescein isothiocyanate (FITC) was added and followed by a 10 min incubation. The mixture was centrifuged at 100g for 5 min and the supernatant was discarded. Annexin V FITC and PI staining solution were added to the precipitated cells and the mixture was incubated on ice for 20 min. Flow cytometer FACSDxp AthenaTM and AuroraTM (Cytek, USA) was used to detect the apoptosis cells. Flow Jo 10.0 software was used to further analyze the apoptosis cells.
RNA-sequencing assay. mRNA, from total RNA extracted using Trizol reagent, was enriched using magnetic beads with Oligo (dT). Then, the obtained mRNA was fragmented to short length via adding Fragmentation Buffer. mRNA fragments with random primers were needed in the first chain cDNA synthesis. Subsequently, Buffer, dNTPs, RNase H and DNA polymerase I were added for the second chain cDNA synthesis. Purification of PCR products was performed using a kit. EB Buffer was used to eluting the products at the end of the repair and processing of A bases. After adding sequencing connectors, agarose gel electrophoresis was used to recover the target size fragments. The resulting fragments were used as a template for PCR amplification to complete the library preparation. The quality control steps included bioanalyzer RNA chip analysis and qRT-PCR for library quantification. Data were provided by GEO. An Illumina sequencer was used to sequence the constructed library. www.nature.com/scientificreports/ Mice tumorigenicity assay. All experiments involving animals were following the guidelines of the Animal Ethics Committee from the Third Xiangya Hospital of Central South University and procedures approved by the committee (SYXK 2020-2019, license no. 2019sydw011). All experimental procedures were conducted in strict accordance with the ARRIVE guidelines. A735 cells (1 × 10 6 ) were inoculated subcutaneously into 6-weekold female nude athymic BALB/c nude mice. When the tumor grew to 100 mm 3 , the mice were injected with ABZ (50 mg/kg, dissolved in 100 μL sesame oil, 2 Day i.p., Selleck Chemicals, Cat# S1640), a combination of ABZ and palbociclib (Selleck Chemicals, Cat# S1579) or negative control DMSO respectively. The tumor volume and mice weight were measured every 2 days. The tumor volume was estimated according to the following formula: V = width 2 × length/2. After 3 weeks, the mice were sacrificed by cervical dislocation, and the tumors were dissected from mice, and tissue samples were fixed with 10% formaldehyde for immunohistochemical staining Ki67 (abcam, ab15580), CDK1 and p-RB1 according to standard protocols. Images were obtained using an Olympus photomicroscope (Olympus, Japan).
Statistical analysis. SPSS was used to analyze the data, which are expressed as the mean ± standard deviation. Univariate analysis of variance was used to determine that the difference in experimental data between groups was statistically significant, and p < 0.05 was considered statistically significant.

ABZ inhibited the growth of melanoma cells in vitro and in vivo.. To investigate the antitumor role
of ABZ in melanoma, MTT assays were first performed in melanoma cell lines and human normal keratinocyte HaCaT cells. As shown in Fig. 1A,B, ABZ suppressed the viability of A375 and SK-MEL-28 cells by 50% at very low concentrations, at 0.8751 and 1.599 μM respectively. However, ABZ showed no obvious survival rate change in HaCaT cells, indicating that ABZ exhibits low cytotoxicity in normal cells (Fig. 1C). Colony formation assays also showed that ABZ significantly inhibited melanoma cell proliferation (Fig. 1D,E). To further investigate the antitumor role of ABZ in vivo, we generated subcutaneous melanoma tumor xenografts in nude mice using A375 cells and treated them with ABZ and DMSO (Fig. 1F). As shown in Fig. 1G-I, ABZ treatment significantly reduced the growth and weight of xenografted melanoma tumors compared with DMSO treatment. However, ABZ administration did not result in significant changes in mouse body weight ( Fig. 1J), suggesting limited toxicity of ABZ treatment. Taken together, these results suggested that ABZ could significantly inhibit the growth of melanoma cells in vitro and in vivo with low toxicity.

ABZ arrested the melanoma cells at the G2/M phase and induced cell apoptosis..
To explore the mechanism of action of ABZ in melanoma growth inhibition, we performed RNA-seq analysis to identify the signaling pathways altered by ABZ in A375 cells. We found 1478 significantly upregulated genes and 887 downregulated genes in response to treatment with ABZ ( Fig. 2A). In particular, both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the upregulated genes were significantly enriched in cell cycle-related genes (Fig. 2B,C). To test whether ABZ inhibits melanoma cell growth in a cell cycle-dependent manner, we first performed flow cytometry analysis and showed significant enrichment of cells in the sub-G1 and G2/M phase and a significant reduction in cells in the G0/G1 phase when A375 and SK-MEL-28 cells were treated with ABZ, indicating that ABZ arrested the melanoma cells at the G2/M phase of the cell cycle and induced cell apoptosis (Fig. 2D,E, Supplementary Figure 1A). Furthermore, western blot was performed to analyze cell cycle-associated molecules. As shown in Fig. 2F and Supplementary Figure 1B, the expression levels of CDK1 and Cyclin B1, which are regulatory proteins related to the G2/M phase of the cell cycle, and the level of G1/S phase regulatory protein phospho-RB1 (p-RB1), were significantly reduced in cells treated with ABZ at 0.625 and 1.25 μM compared with DMSO. The expression level of p53, another mediator of the G2/M checkpoint, was increased in cells treated with ABZ. However, the levels of phospho-H3 (p-H3) and γ-H2AX, which are regulatory proteins related to the chromosome condensation during mitosis and DNA damage respectively, were not obviously changed unless at the very high concentration of ABZ (Supplementary Figure 2). In particular, to investigate the apoptosis-inducing effect of ABZ, we analyzed the percentage of apoptotic A375 and SK-MEL-28 cells using flow cytometry. As shown in Fig. 2G,H, the percentage of apoptotic melanoma cells was significantly increased after treatment with the indicated doses of ABZ. Moreover, ABZ treatment significantly increased the Cleaved Caspase-3 in A375 and SK-MEL-28 cells ( Supplementary Figure 1B). Taken together, we found that ABZ inhibited the growth of melanoma cells by inducing G2/M phase arrest and apoptosis.
ABZ plus palbociclib synergistically suppresses cell growth in vitro. As mentioned above, ABZ inhibited the growth of melanoma cells by inducing G2/M phase arrest. Therefore, we tried to explore whether ABZ exhibits synergistic effects with the cell cycle inhibitor palbociclib, an FDA-approved specific small-molecule inhibitor of CDK4/6, or Vemurafenib (PLX4032), an FDA-approved BRAF kinase inhibitor (BRAFi) for melanoma patients harboring the constitutively active BRAF-V600E mutation. First, we investigated the inhibitory effects of palbociclib on A375 and SK-MEL-28 cell survival based on MTT assays. As shown in Fig. 3A,B, the cell survival ratio was significantly decreased as the concentration of palbociclib increased, and 50% inhibition after 48 h of treatment was observed at palbociclib concentrations of 1.588 and 3.183 μM in A375 (IC50 = 1.588 μM) and SK-MEL-28 (IC50 = 3.183 μM) cells, respectively. Western blot showed that treatment with palbociclib dramatically reduced the level of p-RB1, indicating that palbociclib destructed the G1/S phase of the cell cycle (Fig. 3C, Supplementary Figure 3A). Flow cytometry analysis showed that co-treated the A375 and SK-MEL-28 cells with ABZ and palbociclib significant enrichment of cells in the sub-G1 and G2/M phase and a significant reduction in cells in the G0/G1 phase (Fig. 3D). Next, we combined palbociclib and ABZ at   www.nature.com/scientificreports/ the indicated concentrations to treat A375 and SK-MEL-28 cells and used CompuSyn software to calculate the combination index. As expected, palbociclib and ABZ produced synergistic effects at very low concentrations (both < 1 μM, Fig. 3E, Supplementary Figure 3B). Meanwhile, combining palbociclib and ABZ decreased the colony number more significantly than either treatment alone in A375 and SK-MEL-28 cells (Fig. 3F). Co-treated the A375 and SK-MEL-28 cells with ABZ and palbociclib synergistically reduced the protein level of CDK1, Cyclin B1 and p-RB1, while increasing the accumulation of p53, CDK4 and Cyclin D (Fig. 3G, Supplementary  Figure 3C). However, combining ABZ and Vemurafenib (PLX4032) have no synergistic effect on the inhibition of cell growth (Supplementary Figure 4).
Synergistic effect of ABZ and palbociclib in the treatment of a melanoma xenograft mouse model. Based on the aforementioned results that ABZ plus palbociclib synergistically suppresses cell growth in vitro, we hypothesized that ABZ plus palbociclib may present a similar synergistic effect in the mouse model. To test this hypothesis, subcutaneous melanoma tumor xenografts in nude mice with A375 cells were generated and treated with ABZ, palbociclib, ABZ plus palbociclib, or the control (Fig. 4A). As shown in Fig. 4B, both ABZ or palbociclib alone significantly decreased tumor growth beginning on Day 13 after treatment compared to the control group, whereas ABZ and palbociclib combination treatment achieved better efficacy. Similarly, combining ABZ and palbociclib reduced tumor volume and tumor weight more significantly than either treatment alone (Fig. 4C,D). More importantly, regardless of whether the mice were treated with ABZ or palbociclib alone or together, no significant changes in mouse weight were noted (Fig. 4E). In addition, ABZ and palbociclib combination treatment markedly reduced the number of proliferative cells in tumors, as indicated by decreased Ki67-positive cells compared to the tumors treated with ABZ or palbociclib alone or control tumors. Furthermore, combining ABZ and palbociclib also dramatically decreased the expression levels of CDK1 and p-RB1 in tumors compared to those treated with ABZ or palbociclib alone or control (Fig. 4F,G). Overall, we suggest that ABZ plus palbociclib synergistically suppresses tumor growth in a melanoma xenograft mouse model with no side effects.

Discussion
Melanoma is highly aggressive with an increasing incidence, and the development of novel treatment strategies remains an urgent need 13 . In this study, we demonstrated that ABZ, an FDA-approved broad-spectrum anthelmintic drug, significantly inhibits the proliferation of melanoma cells in vitro and in vivo. Mechanistically, using RNA sequencing and flow cytometry analysis, we revealed that ABZ arrested the melanoma cell cycle at the G2/M phase and induced cell apoptosis. Preclinically, we showed that ABZ had a synergistic tumor-eliminating effect with the CDK4/6 inhibitor palbociclib in the treatment of nude mouse models (Fig. 5). Taken together, we revealed a previously unappreciated function of ABZ in antimelanoma proliferation by inducing cell cycle arrest and apoptosis and provided a novel combined therapeutic regimen of ABZ plus CDK4/6 inhibitor treatment in melanoma.
In recent years, the clinical practice of new drug compounds, especially targeted therapies (i.e., BRAFis) and immune checkpoint inhibitors (ICIs), has largely improved the response rates and overall survival (OS) of advanced melanoma patients 14 . BRAF is an oncogene encoding a serine-threonine protein kinase that is mutated in approximately 50% of melanomas 15 . The most promising targeted therapies include the BRAF inhibitors vemurafenib and dabrafenib, which were approved for the treatment of metastatic and unresectable BRAF-mutated melanomas in 2011 and 2013, respectively 16,17 . However, a majority of patients develop secondary resistance to BRAFis within a relatively short amount of time 18 . The combination of BRAFis and MEK inhibitors (MEKi) has recently demonstrated prolonged survival in patients with advanced melanoma harboring activating BRAF mutations. This therapy strategy has demonstrated high anti-tumor activity with fast responses in most patients. However, tumor relapse commonly occurs 12-18 months after initiation of treatment due to the emergence of acquired resistance 19 . We investigated the combined inhibitory effect of the BRAFi vemurafenib and ABZ on wild-type A375 cells and vemurafenib-resistant A375 cells (A375R). Although ABZ could effectively inhibit the growth of vemurafenib-wildtype and -resistant melanoma cells, ABZ plus vemurafenib have no synergistic effect on the inhibition of cell growth, and the underlying mechanism needs to be further explored in our future work. In the presence of BRAF mutations, both BRAFi-targeted therapies and ICIs can be used; however, in the presence of wild-type BRAF, only ICIs can be prescribed 20 . Regarding ICIs, first-line immunotherapy is represented by anti-PD-1 blockade (nivolumab and pembrolizumab), while anti-CTLA-4 ipilimumab can be used as second-line therapy 21 . Although ICIs represent a breakthrough for melanoma therapeutics, a significant subset of patients does not respond to these drugs, and many patients who do respond develop secondary resistance 22,23 .
Considering the toxicity (largely due to off-target effects) and rapid development of adaptive resistance to targeted therapies in addition to the low response ratio and the enormous cost of immunotherapies, it is important to identify economical and effective existing drugs. Herein, we believe that ABZ is such a compound that exhibits great potential for future clinical use in melanoma therapy. We showed that ABZ inhibits melanoma cell proliferation in vitro with very low IC50 values of approximately 1000 nM. Moreover, ABZ significantly suppressed the growth of xenografted melanoma tumors with low toxicity. These results indicated that ABZ is an existing drug that may serve as a potentially effective melanoma therapy. However, whether ABZ can be used as an adjuvant for the treatment of melanoma in combination with other drugs still needs to be further explored.
Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that regulate cell cycle progression through the G1, S, G2 and M phases, and aberrant expression or mutations in CDKs consistently result in cell cycle dysregulation and cancer 24 . Therefore, CDKs have been recognized as promising therapeutic targets, fueling efforts in the development of CDK inhibitors as anticancer drugs 25 . Palbociclib, a selective CDK4/6 inhibitor, was approved for the treatment of postmenopausal women with metastatic breast cancer 26   www.nature.com/scientificreports/ the proliferation of melanoma cells by arresting the cell cycle at the G2/M phase, we further assessed whether ABZ combined with palbociclib, which arrested the cell cycle in the G1/S phase, could synergistically suppress cell growth. Our data showed that ABZ plus palbociclib synergistically suppresses cell growth in vitro at very low concentrations (500-1000 nM). More importantly, combination treatment with ABZ significantly enhanced the tumor elimination effect of palbociclib in mouse models. However, the molecular mechanism underlying the combination treatment needs more in-depth work to elucidate in the future. Taken together, our results suggest that ABZ inhibits the proliferation of melanoma cells by inducing cell cycle arrest and apoptosis, and combination treatment with ABZ and a CDK4/6 inhibitor exhibits potential as a novel therapeutic strategy for the treatment of melanoma.