Lovastatin inhibits the proliferation of HNSCC cells by downregulating the MCM complex after PPARγ activation

doi:10.21203/rs.3.rs-3795016/v1

Download PDF

Research Article

Lovastatin inhibits the proliferation of HNSCC cells by downregulating the MCM complex after PPARγ activation

https://doi.org/10.21203/rs.3.rs-3795016/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose

Statins have a protective effect overall cancer risk. However, the effect of lovastatin on head and neck squamous cell carcinoma (HNSCC) and the underlying mechanism remain unclear. Here, we focused on the potential role of PPARγ in the lovastatin-mediated inhibition of malignant development in HNSCC.

Methods

The postoperative survival of HNSCC patients treated with or without lovastatin was statistically analyzed, and the expression of Ki67 was detected via immunohistochemistry. EdU expression in the tumor tissue of nude mice was detected by immunofluorescence. The proliferation and migration of HNSCC cells were detected by CCK-8, scratch healing and Transwell assays. Cell apoptosis and cell cycle distribution were detected via flow cytometry. The expression levels of PPARγ and MCM in cells and human HNSCC tissues were detected by western blotting and immunohistochemistry, respectively. The contribution of PPARγ to the effect of lovastatin on HNSCC was verified by treatment with a PPARγ antagonist (GW9662) and PPARγ knockdown.

Results

A case‒control study revealed that patients who used lovastatin had a better prognosis and lower Ki67 expression in tumor tissues than patients who did not receive lovastatin. Lovastatin also inhibited HNSCC growth in nude mice (n = 5) and decreased EdU expression in tumors. Lovastatin inhibited proliferation by activating PPARγ and downregulating MCM complexes in HNSCC cells. In addition, after PPARγ activation, lovastatin inhibited migration and enhanced the cisplatin sensitivity of CaL-27 cells.

Conclusions

Our findings suggest that PPARγ may be a target of lovastatin in inhibiting the proliferation of HNSCC cells and improving patient prognosis.

Head and neck squamous cell carcinoma (HNSCC)

PPARγ

MCM complex

Cell proliferation

Prognosis

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world[1]. In the United States, 63,000 patients are diagnosed each year, and approximately 13,000 people die from HNSCC [2]. In recent years, surgery, chemotherapy, radiotherapy, and comprehensive treatment have improved as the most common treatments for HNSCC. However, the 5-year overall survival rate of HNSCC has not significantly improved and is still only 50% [3]. Risk factors for HNSCC have been identified, and these include smoking, alcohol consumption, and infection with high-risk human papilloma virus (HPV) [4]. Moreover, abnormal lipid metabolism is considered closely associated with the occurrence [5–6] and development [7] of HNSCC, as well as with the prediction of patient prognosis [8–11].

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors belonging to the nuclear receptor superfamily; PPARs include PPARα, PPARβ/PPAR δ, and PPARγ [12]. PPARs are critical for the regulation of cell differentiation, lipid metabolism, and tumorigenesis [13, 14]. PPARγ has been recommended as a chemopreventive target for HNSCC [15]. In laryngeal squamous cell carcinoma, PPARγ expression is correlated with the degree of tumor cell differentiation. PPARγ expression was enhanced in well-differentiated epithelium compared to basal undifferentiated epithelium [16]. PPARγ expression, which is associated with longer overall survival, is regarded as a prognostic marker in tongue squamous cell carcinoma [17]. As a tumor suppressor gene, PPARγ can cause cell cycle arrest to inhibit the proliferation of HNSCC cells [18, 19], and it can also enhance the chemosensitivity of HNSCC cells [20, 21].

Statins, small-molecule inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, are the most commonly used drugs for the treatment of geriatric hyperlipidemia, hypercholesterolemia, or atherosclerotic disease [22]. Furthermore, statins are closely related to cancer incidence and potentially exert a protective effect on overall cancer risk [23–26]. Notably, statins exert antiproliferative, proapoptotic and antimetastatic effects by activating PPARγ [27–29]. Recent studies have shown that statins have positive effects on HNSCC, improving overall survival and prognosis [30–31]. However, whether statins inhibit the progression of HNSCC by regulating PPARγ remains unclear.

Our case‒control study investigated the effect of lovastatin in human HNSCC. KI67 expression in tumor tissue was significantly reduced in participants taking lovastatin. Moreover, lovastatin inhibited the proliferation and migration of HNSCC cells through PPARγ activation and enhanced the cisplatin sensitivity of tumor cells.

2.1. Study population and samples

This case‒control study involved 60 patients with HNSCC at Inner Mongolia Medical University between January 2016 and June 2022. This study was approved by the Ethics Committee of Inner Mongolia Medical University (approval number: YKD202302013). Patients were individually matched (1:1) according to disease site (lips, tongue, gums, cheeks, neck, etc.), age (within 5 years), sex, and operation time (within 2 years). The specific information of the patients is shown in Table S1.

Paraffin-embedded clinical specimens were collected, and pathological information (age, sex, histological type, clinical stage, lymph node metastasis status, distant metastasis status, differentiation status, and family history) was retrieved from the archives of the Department of Pathology of the Affiliated Hospital of Inner Mongolia Medical University. None of the patients received antitumor therapy before diagnosis. Clinical staging and systemic therapy were performed in accordance with the American Joint Committee on Cancer (AJCC) Cancer Staging Manual and the National Comprehensive Cancer Network (NCCN) guidelines.

2.2 Animal care and treatment

Eight-week-old male BALB/c nude mice (Charles River, Beijing, China) were kept in a sterile, individually ventilated, constant temperature environment with 12 hours of light/dark per day. A total of 1×10⁶ cells were resuspended in 0.1 mL of serum-free medium and injected subcutaneously into the dorsal side of nude mice to establish CaL-27 xenografts. After 5 days, the nude mice were randomly divided into three groups and administered an intraperitoneal injection of DMSO (15 mg/kg or 30 mg/kg lovastatin) for 30 days. Tumor volume was measured with a caliper every 3 days and calculated according to the following formula: volume = π/6 × length × width². Body weight was measured every week. At the endpoint, the animals were sacrificed according to the standard protocol. Tumor specimens were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

2.3 Cell culture

The human tongue squamous cell line CaL-27 (Shanghai Institutes for Biological Sciences, Shanghai, China) and the human laryngeal squamous cell line FaDu (Shanghai Institutes for Biological Sciences, Shanghai, China) were cultured in a humidified environment at 37°C with 95% air and 5% CO2. DMEM (Gibco, New York City, USA) supplemented with 1% penicillin G sodium/streptomycin sulfate and 10% fetal bovine serum (Bioindustries, Kibbutz Beit-Haemek, Israel) was used. When the cells were 80% confluent, the culture medium was changed every 3 days.

2.4 CCK-8 experiment

CaL-27 and FaDu cells were seeded in a 96-well plate (3,000 cells per well). After 24 h of incubation, the cells were treated with lovastatin (0, 20, 40, 60, 80 and 160 µmol/L) (Sigma‒Aldrich, Missouri, United States) for 24 h, 48 h and 72 h. Then, 10 µL of CCK-8 (Dojindo Laboratories, Japan) was added to each well at 37°C for 1–4 h. The absorbance at 450 nm was measured by enzyme labeling. CCK-8 assays revealed "relative cell proliferation" (relative cell proliferation = absorbance values of lovastatin-treated cells/absorbance values of lovastatin-untreated cells).

2.5 Wound healing experiments

CaL-27 and FaDu cells were grown in 6-well plates for 24 h (1×10⁶ cells per well) and then starved for 24 h. We created a wound in the Petri dish with a 200 µL pipette tip and rinsed the plate twice with PBS to remove the suspended cells. Medium containing different concentrations of lovastatin (0, 20, 40, and 80 µmol/L) was added. After 6, 12, and 24 hours, premarked points were observed under a 50X microscope, and the following formula was applied: Migration rate = (initial scratch distance-average distance between two edges after migration)/initial scratch distance.

2.6 Transwell experiment

CaL-27 and FaDu cells were treated with lovastatin (0, 20, 40, or 80 µmol/L) for 24 h, after which the cell density was adjusted to 2×10⁶ cells/mL. Cell suspensions (100 µL) in serum-free medium (Corning, USA) were added to the upper chamber. The lower chamber contained medium supplemented with 15% FBS. After 24 h, the cells were fixed with 4% PFA and stained with 0.1% crystal violet. Migrating cells were counted by phase contrast microscopy and subjected to statistical analysis.

2.7 Flow-cell cycle analysis

Cell cycle assessment was performed using a Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China). CaL-27 and FaDu cells treated with lovastatin (0, 20, 40, or 80 µmol/L) for 48 h were digested, centrifuged at 500 × g for 5 min, and washed twice with precooled PBS. Then, the cells (1×10⁶ cells/group) were fixed with 70% cold ethanol (− 20°C, overnight). Next, the sections were washed with cold PBS. Cells were treated with RNase A (20 µg/mL) and stained with propionic acid (50 µg/ml) for 30 min. Finally, the cell cycle distribution of these cells was assessed by flow cytometry.

2.8 Flow-type cell apoptosis analysis

We assessed apoptosis using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. CaL-27 and FaDu cells treated with lovastatin (0, 20, 40, or 80 µmol/L) were harvested and washed twice in cold PBS. The samples were centrifuged at 500 × g for 5 min and resuspended in 195 µL of binding buffer. The samples were incubated with FITC-Annexin V and PI for 15 min at room temperature and subsequently placed on ice. The cells were analyzed by flow cytometry.

2.9 Quantitative real-time PCR (qRT‒PCR)

Total RNA was isolated from CaL-27 and FaDu cells using TRIzol Reagent (Takara, Shiga, Japan). Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer's instructions. The forward and reverse primer sequences are listed in Table S2 (Biotend, Shanghai, China). qRT‒PCR was performed using TB GREEN Premix EX Taq (Takara, Shiga, Japan). The effect of the relative expression of PPARγ on GAPDH was examined.

2.10 Protein analysis

CaL-27 and FaDu cells treated with lovastatin (0, 20, 40, or 80 µmol/L) for 48 h were immersed in RIPA buffer containing protease inhibitors and phosphatase inhibitors for 30 min. The cell lysates were collected and centrifuged at 12,000 × g for 15 min. The supernatant was collected. The protein concentration was determined with a BCA kit (Thermo Fisher Scientific, Massachusetts, United States). Total proteins were separated by 10–15% SDS‒PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h at room temperature. Next, the sections were incubated with the primary antibody (Table S3) overnight at 4°C. After the PVDF membrane was washed, it was incubated with a horseradish peroxidase-conjugated secondary antibody (Table S4) at room temperature for 1 h. Finally, the bands were detected with a chemiluminescence enhancement kit (Thermo Fisher Scientific, Massachusetts, United States).

2.11 PPAR antagonist treatment

CaL-27 and FaDu cells were seeded in 96-well plates. After 24 h, the cells were treated with the PPARγ antagonist GW9662 (0, 20, 40 and 60 µmol/L) (Sigma‒Aldrich, Missouri, United States) for 24 h. Then, lovastatin was added to the cells.

2.12 Construction and transfection of small interfering RNA (siRNA)

PPARγ siRNA was purchased from Biotend (Shanghai, China). The siRNA sequence is shown in S1. CaL-27 and FaDu cells were seeded in 6-well plates. When the cell confluence reached 30–50%, the cells were transfected with PPARγ siRNA and Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Massachusetts, United States). After 48 h, the knockout efficiency of PPARγ was detected by western blotting.

2.13 Construction and transfection of the overexpression plasmid

The MCM2 overexpression plasmid (GeneChem, Shanghai, China) was transfected into CaL-27 and FaDu cells with Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Massachusetts, United States) according to the manufacturer's protocol. After 48 h, the transfection efficiency was detected by western blotting.

2.14 Combined use of the drugs

CaL-27 and FaDu cells were seeded in 96-well plates (3,000 cells per well). After 24 h, the cells were treated with cisplatin (0, 5, 10, 20, 40 or 80 µmol/L) and lovastatin (20 µmol/L) for 24 h. Cell viability was subsequently determined using CCK-8 assays.

2.15 Statistical analysis

All the statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, California, USA). Statistical significance was determined by using an unpaired t test. The data are presented as the mean ± standard deviation (SD). The threshold for statistical significance was set at p < 0.05. * P < 0.05, * * P < 0.01.

3.1. Lovastatin is associated with improved outcomes in HNSCC patients

To investigate the potential relationship between lovastatin and the development of HNSCC, 30 HNSCC patients and 30 individual-matched controls were selected for this study (1:1). Moreover, there was no significant difference in the TNM stage of the tumors of the matched patients. HNSCC patients with a history of lovastatin use exhibited better overall survival (OS) than did those without lovastatin use (Fig. 1a). Subsequently, we performed immunofluorescence staining of the patient's tumor tissue. The results showed decreased expression of Ki67 in tumor tissue from HNSCC patients treated with lovastatin (Fig. 1.b). These findings suggested that lovastatin administration may be associated with a better prognosis in HNSCC patients.

(a) Kaplan‒Meier curves of overall survival in HNSCC patients according to whether they were not taking lovastatin (n = 30) or were taking lovastatin (n = 30) (P < 0.05); (b) Immunohistochemistry analysis of Ki67 in human HNSCC tumor tissues from clinical cases. Scale bar = 100 µm.

3.2 Lovastatin inhibited the formation of HNSCC in nude tumor-bearing mice

We observed that lovastatin was associated with reduced Ki67 expression in human HNSCC. To investigate the possible protective role of lovastatin in the development of HNSCC, CaL-27 xenografts (Fig. 2a) were established in nude mice (n = 5). After 5 days, lovastatin was administered at different doses (15 mg/kg or 30 mg/kg) for 30 consecutive days. During this period, the body weights of the three groups of mice increased gradually, but there was no significant difference among the groups. On the 30th day, mice intravenously injected with 30 mg/kg lovastatin showed a trend toward weight loss, which may be related to dyspepsia (Fig. 2b). With lovastatin intervention, tumor growth and proliferation were inhibited (Fig. 2c, d). Lovastatin significantly inhibited tumor growth in mice at increasing doses (Fig. 2e) and decreased EDU expression in mouse tumor tissue (Fig. 2f). These results suggest that exposure to lovastatin inhibits the proliferation of HNSCC cells in nude mice.

(a) Establishment of the tumor-bearing model in nude mice; (b) changes in the body weight of nude mice; (c) volume of HNSCC tumors in nude mice during lovastatin treatment; (d) size of HNSCC tumors in each group at the endpoint; and (d) volume of HNSCC tumors in nude mice at the endpoint. (f) Immunohistochemistry of EDU in mouse tumor tissues. Scale bar = 100 µm. * P ≤ 0.05; **P ≤ 0.01.

3.3 Lovastatin inhibited the proliferation of HNSCC cells

To determine the effect of lovastatin on HNSCC proliferation, human tongue squamous cells (CaL-27) and laryngeal squamous cells (FaDu) were stimulated with lovastatin at concentrations ranging from 20 µmol/L to 160 µmol/L. Lovastatin significantly inhibited the growth of CaL-27 and FaDu cells in a concentration- and time-dependent manner (Fig. 3a, b, c). Apoptosis induction is the most common mechanism by which drugs inhibit cell proliferation; therefore, we assessed cell apoptosis. We found that lovastatin dose-dependently increased the apoptosis rate of CaL-27 and FaDu cells (Fig. 3d, e). Among them, the proapoptotic effect of lovastatin on CaL-27 cells was even more prominent. Lovastatin (20 µmol/L) significantly increased the apoptosis rate of CaL-27 cells (Fig. 3d), while 60 µmol/L was the dose at which lovastatin began to increase the apoptosis rate of FaDu cells (Fig. 3e). A close relationship exists between the apoptosis rate and the cell cycle [32]. Therefore, we investigated the effect of lovastatin on the cell cycle distribution. After 48 hours of treatment, compared with untreated cells, CaL-27 and FaDu cells were in G0/G1 phase and had a decreased percentage of cells in S phase (Fig. 1f, g). The above results indicated that lovastatin inhibited the proliferation of HNSCC cells, likely by causing cellular G0/G1 phase arrest.

Viability of CaL-27 (a) and FaDu (b) cells treated with lovastatin for 24, 48, or 72 hours; (c) IC50 of CaL-27 and FaDu cells treated with lovastatin. (d) Flow cytometry analysis of CaL-27 cell apoptosis after 48 h of lovastatin treatment. (e) Flow cytometry apoptosis analysis of FaDu cells treated with lovastatin for 48 h. Apoptosis was assessed by Annexin V-FITC and propidium iodide (PI) staining. Flow cytometry analysis of the cell cycle in CaL-27 (f) and FaDu (g) cells after 48 h of treatment with lovastatin. Each experiment was performed in triplicate. * P ≤ 0.05; **P ≤ 0.01.

3.4 Lovastatin inhibits the migration of HNSCC cells

We examined the effect of lovastatin on cell migration using wound healing and transwell assays. Wound healing assays showed that lovastatin significantly inhibited the lateral migration of CaL-27 and FaDu cells (Fig. 4a, b) and that it also significantly inhibited the longitudinal migration of cells (Fig. 4c, d). The migration of tumor cells is closely related to epithelial stromal transformation [33]; therefore, we further examined the expression of EMT-related proteins in CaL-27 (Fig. 4e) and FaDu (Fig. 4f) cells. Treatment with lovastatin increased the expression of the epithelial marker E-cadherin and reduced the expression of the mesenchymal markers N-cadherin and Snail. These data suggest that lovastatin may suppress the migration of HNSCC cells by inhibiting EMT.

(a) CaL-27 cell scratch healing assay; (b) FaDu cell scratch healing assay; (c) CaL-27 cell transwell assay (without Matrigel); (d) FaDu cell transwell assay (without Matrigel); (d) western blot of EMT-related proteins in CaL-27 cells; and (f) western blot of EMT-related proteins in FaDu cells. * P ≤ 0.05; **P ≤ 0.01. Scale bar = 500 µm.

3.5 Lovastatin inhibits HNSCC progression by regulating the MCM complex

To decipher the molecular mechanism by which lovastatin inhibits tumor proliferation and migration, we performed a gene transcriptome analysis of treated CaL-27 cells by mRNA sequencing and KEGG pathway enrichment analysis. The results showed consistent genetic trends across samples (n = 3) of untreated or lovastatin-treated cells (Fig. 5a, b, c). Among the downregulated pathways, DNA replication and the cell cycle were the most common (Fig. 5d). This finding is consistent with the finding that lovastatin inhibits cell proliferation. Gene Ontology (GO) analysis (Fig. 5e) revealed that the expression of genes encoding members of the tiny chromosome maintenance protein (MCM) complex, which is closely related to the cell cycle, was strongly altered.

Therefore, we confirmed that the expression of MCM2, MCM3, MCM4, MCM5, MCM6, MCM7 and MCM8 was inhibited in both the CaL-27 and FaDu cell lines (Fig. 5f). The expression of cell cycle-related proteins associated with the MCM complex was also altered (Fig. 5g). Treatment with lovastatin increased the expression of the cyclin-dependent kinase inhibitors cyclin-dependent kinase inhibitor 2B (CDKN2B, p15) and cyclin-dependent kinase inhibitor 1A (CDKN1A, p21); decreased the expression of cyclin D1 (CCND1) and cyclin E2 (CCNE2); and subsequently reduced the level of phosphorylated retinoblastoma protein (p-RB) without affecting the total RB level, resulting in reduced expression of chromatin licensing and DNA replication factor 1 (CDT1) and cell division cycle 6 (CDC6), which are necessary for MCM2-7-mediated formation of a prereplication complex (preRC). These findings are similar to previous results showing that lovastatin upregulated p21, downregulated CCND 1 and MCM2, and induced cell cycle arrest [34].

MCM2 and MCM3 are closely associated with HNSCC proliferation and prognosis [35, 36]. Immunofluorescence analysis revealed that lovastatin decreased MCM expression in mouse tumor tissue (Figure S1). To further determine whether the MCM2 and MCM3 genes are altered in human HNSCC tissues, we performed gene expression profiling interactive analysis (GEPIA) (http://gepia.cancer-pku.cn/). We found that, similar to MCM2 and MCM3 (Fig. 5h), MCM4-8 was highly upregulated in HNSCC tissues compared with adjacent normal tissues (Figure S2). Furthermore, in the case‒control study, MCM2 and MCM3 were overexpressed in HNSCC tumor tissues compared with control tissues (Fig. 5i). Taken together, these results suggest that the MCM complex mediates the inhibitory effect of lovastatin on HNSCC.

(a) Heatmap of differentially expressed genes in CaL-27 cells before and after lovastatin treatment. Red indicates upregulated genes, and green indicates downregulated genes. (b) Volcano map and (c) PCA map. (d) Bubble diagram of the downregulated pathways. (e) GO analysis results. (f) Changes in MCM protein expression levels in CaL-27 and FaDu cells. (g) Changes in the expression levels of cell cycle-related proteins in the CaL-27 and FaDu cell lines. (h) Expression of MCM2 and MCM3 in human HNSCC tissues and normal tissues in GEPIA; tumor tissues are shown in red, and normal tissues are shown in gray. (i) Immunohistochemistry analysis of MCM2 and MCM3 in human HNSCC tumor tissues from clinical cases.

3.6 Lovastatin increases the sensitivity of HNSCC cells to cisplatin

Decreased expression of MCM2 has been reported to be associated with increased sensitivity of cancer cells to cisplatin in many cancers [37–38]. Therefore, we overexpressed MCM2 in CaL-27 and FaDu cells. We found that the changes in the expression of EMT-associated proteins, which included decreased E-cadherin expression and increased CDC6, N-cadherin and Snail expression, in HNSCC cells were similar to those observed in resistant cell lines (Fig. 6a, b). We subsequently assessed the effect of lovastatin on cellular sensitivity to cisplatin. After 24 h of cisplatin treatment, the MCM2-overexpressing cells had a greater percentage of viable cells than did the negative control cells (empty plasmid). However, after combined treatment with lovastatin and cisplatin, the viability of HNSCC cells overexpressing MCM2 was significantly reduced (Fig. 6c, d). These results suggested that lovastatin can cooperate with cisplatin to kill HNSCC cells and enhance the cisplatin sensitivity of tumor cells by regulating the MCM2 protein.

(a) Western blot after overexpression of MCM2 in the CaL-27 and (b) FaDu cell lines; (c) viability of Cal-27 and (d) FaDu cells measured 24 hours after combined treatment with different concentrations of cisplatin and lovastatin. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

3.7 PPARγ inhibitors attenuate the antitumor effects of lovastatin mediated by MCM

Through mRNA sequencing and KEGG pathway enrichment analysis, we found significant alterations in PPAR expression in the upregulated pathways (Fig. 7a). Therefore, we confirmed that the expression of PPARγ was significantly promoted in CaL-27 (Fig. 7b) and FaDu (Se) cells and that PPARδ expression was slightly increased after lovastatin treatment. To determine whether PPARγ expression varies in human HNSCC, we searched the GEPIA database and found that PPARγ expression was lower in HNSCC tumor tissues than in normal adjacent tissues (Fig. 7c). If‒control studies, PPARγ was highly expressed in HNSCC tissues from lovastatin-treated patients (Fig. 7d). It has been reported that lovastatin can induce cell cycle arrest by regulating cell cycle-related proteins through activation of PPARγ [39]. These findings suggested that lovastatin may regulate the cell cycle signaling pathway and MCM by regulating PPARγ.

To test this hypothesis, we evaluated the influence of a PPARγ inhibitor (GW9662) on the antitumor effects of lovastatin. We found that GW9662 restored the migration of CaL-27 (Fig. 7e) and FaDu (Sa) cells, reduced the number of cells in G0/G1 phase (Fig. 7f and Sb), and inhibited the changes in PPAR, cell cycle-related proteins and MCM expression caused by lovastatin treatment in CaL-27 (Fig. 7g) and FaDu (Sf) cell lines. Compared with those in lovastatin-treated cells, the expression levels of PPARγ, PPARδ and P15 were lower, while the expression levels of CCND1, CCNE2, MCM2, MCM3 and E-cadherin were increased in cells treated with GW9662.

(a) Bubble map of downregulated pathways identified by mRNA sequencing and KEGG pathway enrichment analysis; (b) PPAR expression in CaL-27 cells after lovastatin treatment. (c) Statistical plot of differences in PPARγ expression between human HNSCC tissues and normal tissues in the GEPIA database, with tumor tissues in red and normal tissues in gray. (d) Immunohistochemistry analysis of PPARγ in human HNSCC tumor tissue from clinical cases. (e) The CaL-27 wound healing assay showed that PPARγ inhibitors attenuated the effect of lovastatin. The left histogram shows the extent of wound healing in each group. (f) Flow cytometric analysis of CaL-27 cells showing significant recovery of cell cycle arrest after treatment with the PPARγ inhibitor; the histogram shows the relative proportion of cells in each cycle. (g) The expression of key PPARs and cell cycle signaling proteins in CaL-27 cells was determined via western blot analysis after the addition of a PPARγ inhibitor. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale bar = 100 µm.

3.8 Lovastatin downregulates MCM2 and MCM3 via PPARγ

We knocked down PPARγ in CaL-27 and FaDu cells using small interfering RNA and examined the knockdown efficiency (Fig. 8a, b). PPARγ knockdown abolished the inhibition of CaL-27 (Fig. 8c) and FaDu (Sc) cell line migration by lovastatin and restored the cell cycle arrest caused by lovastatin (Fig. 8d and Sd). Furthermore, western blot analysis showed that lovastatin activated PPARγ in CaL-27 cells, enhanced P21 expression and attenuated the expression of CCND1, CCNE2, MCM2 and MCM3. In CaL-27 cells with PPARγ knockout, the changes in the levels of these proteins were attenuated (Fig. 8e). These data suggest that lovastatin inhibits HNSCC cell proliferation by regulating cycle-related proteins (P15, P21, CCND1, CCNE2, P-RB, and E2F1) and MCM2-7 after activation of PPARγ while blocking EMT to inhibit tumor cell migration (Fig. 8f).

The efficiency of PPARγ knockdown in the cells was determined by (a) qRT‒PCR and (b) western blotting. (c) Wound healing assay in CaL-27 cells; the left histogram shows the extent of wound healing in each group. These results suggested that the inhibitory effect of lovastatin on migration was significantly reduced following the knockdown of PPARγ. (d) Flow cycle analysis of CaL-27 cells; the histogram shows the percentage of cells in each phase of the cell cycle. Lovastatin-induced cell cycle arrest was clearly attenuated after knockout of PPARγ. (e) Western blotting after PPARγ knockout was performed in CaL-27 cells to detect the expression of PPARs and key proteins in the cell cycle signaling pathway. (f) (Mechanism by which lovastatin inhibits HNSCC. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Scale = 100 µm.

Statins have potential protective effects in terms of overall cancer risk, particularly for lung, prostate, kidney, and colorectal cancers [40–43]. Recent studies have demonstrated that statins can improve the overall survival and prognosis of HNSCC patients [44–45]. In a case‒control study, Ki67 expression was low in the tumor tissues of patients exposed to lovastatin. Consistent with these findings, lovastatin inhibited the growth of HNSCC in nude mice. In vitro studies revealed that lovastatin inhibited the proliferation and migration of CaL-27 and FaDu cells, promoted tumor cell apoptosis, and caused cell cycle arrest in HNSCC cells, and these results support the therapeutic effect of statins on HNSCC. However, the molecular mechanisms underlying the antitumor effects of statins are still poorly understood, so we further investigated the mechanism by which lovastatin affects HNSCC.

PPARγ, a peroxisome proliferator-activated receptor, is a major regulator of adipocyte differentiation and can exert inhibitory effects on cell proliferation and growth. This property makes the activation of PPARγ by natural and synthetic ligands an attractive tool for cancer treatment and prevention [46]. Statins activate PPARγ by regulating cholesterol or lipid metabolism pathways [40, 47]. Statins also activate PPARγ through crosstalk with other pathways [41–42]. PPARγ activation can upregulate P21 and downregulate CCND1, thus inducing G0/G1 phase cell cycle arrest to play an antitumor role [43]. These findings are similar to our findings that lovastatin treatment activated PPARγ and PPARγ in CaL-27 and FaDu cells, upregulated P21 and P15, and downregulated CCND1 and CCNE2. In addition, we further found that lovastatin caused changes in the expression of downstream proteins (P-RB, CDT1, CDC6, MCM2-7), especially MCM family proteins.

MCM proteins, downstream targets of P-RB and E2F1, are the basis of DNA replication [44]. MCMs have been reported to be essential for regulating DNA replication to maintain normal cellular processes, especially during the DNA initiation and elongation stages [45, 48]. MCM2-7, as a functional DNA helicase, binds to several key factors in the cell cycle (such as CDC6 and CDT1) to form a prereplication complex that triggers the initial phase of DNA replication [49–50]. However, abnormal expression of MCM may be a driving force for tumorigenesis. MCMs are frequently activated in various premalignant lesions and malignancies and lead to uncontrolled cell cycle progression. Our study revealed that lovastatin reduced CDT1 and CDC6 expression in the transcription initiation complex and decreased MCM2-7 expression while inhibiting DNA replication. A reduction in MCM expression is highly important, as the application of MCM as a biomarker for cancer diagnosis and surveillance could improve cancer treatment [51–52].

Furthermore, our results revealed the inhibitory effect of lovastatin on CaL-27 and FaDu cell migration and EMT. These effects can regulate important cellular functions, dramatically alter cell morphology, and cause the loss of intercellular tight junctions. Increasing evidence indicates that EMT participates in tumor invasion and metastasis [53–54]. Our study revealed that lovastatin-treated CaL-27 and FaDu cells had increased E-cadherin (epithelial marker) levels and decreased N-cadherin (mesenchymal marker) levels. Given that EMT is closely related to cell drug resistance [55], we explored the effect of lovastatin on the cisplatin sensitivity of resistant cells. The MCM2 protein is a potential marker for HNSCC. When MCM2is overexpressed, the expression levels of N-cadherin and E-cadherin increase synchronously. Moreover, compared with control cells, MCM2-overexpressing cells showed enhanced resistance to cisplatin. Therefore, we overexpressed MCM2 in CaL-27 and FaDu cells to mimic the features of drug-resistant cell lines. The results showed that lovastatin and cisplatin synergistically kill tumor cells and increase their cisplatin sensitivity.

In conclusion, our study demonstrated that lovastatin can decrease the levels of MCM complexes by regulating the cell cycle signaling pathway through the activation of PPARγ. This triggers cell cycle arrest at the G0/G1 phase and inhibits HNSCC cell proliferation. Lovastatin also inhibited tumor cell migration by inhibiting EMT and increasing the cisplatin sensitivity of tumor cells. This finding supports the rationale that lovastatin could be used to improve survival and treatment outcomes in patients with HNSCC.

Statements and Declarations

Conflict of interest: The authors report no conflicts of interest.

Ethics approval and consent to participate: All experiments were approved by the Ethics Review Committee of Inner Mongolia Medical University (approval number: YKD202302013). All animal experiments and experimental protocols were in accordance with the provisions of the Inner Mongolia Medical University Committee. All patients involved in this study provided written informed consent.

Data availability: The microRNA-seq data are accessible from the Gene Express Omnibus (accession number: GSE225931). The datasets used and analyzed in the current study are available from the author upon reasonable request.

Author Contribution

Qi Li and Xuebing Zhang conceived and designed the study. Xuebing Zhang designed and conducted the majority of the experiments, and interpreted and analyzed the data. Sujuan Niu and Ying Gao performed some of the experiments and wrote the manuscript. Sujuan Niu, La Na and YiJie Bai performed some of the experiments and analyzed the data. XiaoHui Xu, Zhehui Bi and JianMing Hou assisted in writing the manuscript. Qi Li and Xuebing Zhang reviewed and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgments:

We thank Geng Yajun (Renji hospital of shanghai jiaotong university Shanghai, China) for excellent technical assistance. This study was supported by the Department of Stomatology, Affiliated Hospital of Inner Mongolia Medical University and the Laboratory of Oral Microecology and Systemic Diseases, Clinical Research Center, Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine.

R. Kleszcz, A. Szymańska, V. Krajka-Kuźniak, W. Baer-Dubowska, J. Paluszczak, Inhibition of CBP/β-catenin and porcupine attenuates Wnt signaling and induces apoptosis in head and neck carcinoma cells. Cell. Oncol. (Dordr). 42(4), 505–520 (2019)
R.L. Siegel, K.D. Miller, A. Jemal, Cancer Stat. 2017 CA Cancer J Clin. 67(1), 7–3 (2017)
E. Ebnoether, L. Muller, Diagnostic and Therapeutic Applications of Exosomes in Cancer with a Special Focus on Head and Neck Squamous Cell Carcinoma (HNSCC). Int. J. Mol. Sci. 21(12), 4344 (2020)
S. Miyauchi, S.S. Kim, J. Pang, K.A. Gold, J.S. Gutkind, J.A. Califano, L.K. Mell, E. Cohen, A.B. Sharabi, Immune Modulation of Head and Neck Squamous Cell Carcinoma and the Tumor Microenvironment by Conventional Therapeutics. Clin. Cancer Res. 25(14), 4211–4223 (2019)
X. Li, X. Xie, Y. Gu, J. Zhang, J. Song, X. Cheng, Y. Gao, Y. Ai, Fat mass and obesity-associated protein regulates tumorigenesis of arecoline-promoted human oral carcinoma. Cancer Med. 10(18), 6402–6415 (2021)
X. Li, W. Chen, Y. Gao, J. Song, Y. Gu, J. Zhang, X. Cheng, Y. Ai, Fat mass and obesity-associated protein regulates arecoline-exposed oral cancer immune response through programmed cell death-ligand 1. Cancer Sci. 113(9), 2962–2973 (2022)
L.Y. Li, Q. Yang, Y.Y. Jiang, W. Yang, Y. Jiang, X. Li, M. Hazawa, B. Zhou, G.W. Huang, X.E. Xu, S. Gery, Y. Zhang, L.W. Ding, A.S. Ho, Z.S. Zumsteg, M.R. Wang, M.J. Fullwood, S.J. Freedland, S.J. Meltzer, L.Y. Xu, E.M. Li, H.P. Koeffler, D.C. Lin, Interplay and cooperation between SREBF1 and master transcription factors regulate lipid metabolism and tumor-promoting pathways in squamous cancer. Nat. Commun. 12(1), 4362 (2021)
Y.W. Su, P.S. Wu, S.H. Lin, W.Y. Huang, Y.S. Kuo, H.P. Lin, Prognostic Value of the Overexpression of Fatty Acid Metabolism-Related Enzymes in Squamous Cell Carcinoma of the Head and Neck. Int. J. Mol. Sci. 21(18), 6851 (2020)
X. Gao, N. Zhao, L. Dong, X. Zheng, Y. Zhang, C. Ding, S. Zhao, Z. Ma, Y. Wang, A Novel Lipid Prognostic Signature of ADCY2, LIPE, and OLR1 in Head and Neck Squamous Cell Carcinoma. Front. Oncol. 11,735993(2021)
Q. Hu, J. Peng, X. Chen, H. Li, M. Song, B. Cheng, T. Wu, Obesity and genes related to lipid metabolism predict poor survival in oral squamous cell carcinoma. Oral Oncol. 89, 14–22 (2019)
P. Du, Y. Chai, S. Zong, J. Yue, H. Xiao, Identification of a Prognostic Model Based on Fatty Acid Metabolism-Related Genes of Head and Neck Squamous Cell Carcinoma. Front. Genet. 13, 888764 (2022)
A.K. Reka, M.T. Goswami, R. Krishnapuram, T.J. Standiford, V.G. Keshamouni, Molecular cross-regulation between PPAR-γ and other signaling pathways: implications for lung cancer therapy. Lung Cancer. 72(2), 154–159 (2011)
A. Belfiore, M. Genua, R. Malaguarnera, PPAR-γ agonists and their effects on IGF-I receptor signaling: Implications for cancer. PPAR Res. 2009,830501(2009)
J. Berger, D.E. Moller, The mechanisms of action of PPARs. Annu. Rev. Med. 53, 409–435 (2002)
M. Burotto, E. Szabo, PPARγ in head and neck cancer prevention. Oral Oncol. 50(10), 924–929 (2014)
K. Kourelis, G. Sotiropoulou-Bonikou, G. Vandoros, I. Varakis, Peroxisome proliferatoractivated receptor gamma expression correlates with the differentiation level of normal, premalignant, and malignant laryngeal squamous cells. J. Otolaryngol. 36(5), 282–290 (2007)
S. Theocharis, J. Klijanienko, C. Giaginis, J. Rodriguez, T. Jouffroy, A. Girod, D. Point, G. Tsourouflis, X. Satre-Garau, Peroxisome proliferator-activated receptor-γ in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients survival. J. Cancer Res. Clin. Oncol. 137(2), 251–259 (2011)
J.A. Hall, M. Rusten, R.D. Abughazaleh, B. Wuertz, V. Souksavong, P. Escher, F. Ondrey, Effects of PPAR-γ agonists on oral cancer cell lines: Potential horizons for chemopreventives and adjunctive therapies. Head Neck. 42(9), 2542–2554 (2020)
G. Harris, R.A. Ghazallah, D. Nascene, B. Wuertz, F.G. Ondrey, PPAR activation and decreased proliferation in oral carcinoma cells with 4-HPR. Otolaryngol. Head Neck Surg. 133(5), 695–701 (2005)
M. Lian, J. Chen, X. Shen, L. Hou, J. Fang, Pparg may Promote Chemosensitivity of Hypopharyngeal Squamous Cell Carcinoma. PPAR Res. 2020,6452182(2020)
M. Lian, Y. Tao, J. Chen, X. Shen, L. Hou, S. Cao, J. Fang, Variation of PPARG Expression in Chemotherapy-Sensitive Patients of Hypopharyngeal Squamous Cell Carcinoma. PPAR Res. 2021,552509 (2021)
K.K. Chan, A.M. Oza, L.L. Siu, The statins as anticancer agents. Clin. Cancer Res. 9(1), 10–19 (2003)
L. Blais, A. Desgagné, J. LeLorier, 3-Hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors and the risk of cancer: a nested case‐control study. Arch. Intern. Med. 160(15), 2363–2368 (2000)
M.R. Graaf, A.B. Beiderbeck, A.C. Egberts, D.J. Richel, H.J. Guchelaar, The risk of cancer in users of statins. J. Clin. Oncol. 22(12), 2388–2394 (2004)
S. Friis, A.H. Poulsen, S.P. Johnsen, J.K. McLaughlin, J.P. Fryzek, S.O. Dalton, H.T. Sorensen, J.H. Olsen, Cancer risk among statin users: a population-based cohort study. Int. J. Cancer. 114(4), 643–647 (2005)
H. Xiao, C.S. Yang, Combination regimen with statins and NSAIDs: a promising strategy for cancer chemoprevention. Int. J. Cancer. 123(5), 983–990 (2008)
G. Wang, R. Cao, Y. Wang, G. Qian, H.C. Dan, W. Jiang, L. Ju, M. Wu, Y. Xiao, X. Wang, Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway. Sci. Rep. 6, 35783 (2016)
R.E. Teresi, C.W. Shaiu, C.S. Chen, V.K. Chatterjee, K.A. Waite, C. Eng, Increased PTEN expression due to transcriptional activation of PPARgamma by Lovastatin and Rosiglitazone. Int. J. Cancer. 118(10), 2390–2398 (2006)
U. Walther, K. Emmrich, R. Ramer, N. Mittag, B. Hinz, Lovastatin lactone elicits human lung cancer cell apoptosis via a COX-2/PPARγ-dependent pathway. Oncotarget. 7(9), 10345–10362 (2016)
N.L. Lebo, R. Griffiths, S. Hall, J. Dimitroulakos, S. Johnson-Obaseki, Effect of statin use on oncologic outcomes in head and neck squamous cell carcinoma. Head Neck. 40(8), 1697–1706 (2018)
A. Gupta, W. Stokes, M. Eguchi, M. Hararah, A. Amini, A. Mueller, R. Morgan, C. Bradley, D. Raben, J. McDermott, S.D. Karam, Statin use associated with improved overall and cancer specific survival in patients with head and neck cancer. Oral Oncol. 90, 54–66 (2019)
G.I. Evan, K.H. Vousden, Proliferation, cell cycle and apoptosis in cancer. Nature. 411(6835), 342–348 (2001)
G. Pan, Y. Liu, L. Shang, F. Zhou, S. Yang, EMT-associated microRNAs and their roles in cancer stemness and drug resistance. Cancer Commun. (Lond). 41(3), 199–217 (2021)
X. Zhang, Y. Teng, F. Yang, M. Wang, X. Hong, L.G. Ye, Y.N. Gao, G.Y. Chen, MCM2 is a therapeutic target of lovastatin in human non-small cell lung carcinomas. Oncol. Rep. 3(5), 2599–2605 (2015)
I. Kodani, M. Osaki, K. Shomori, K. Araki, E. Goto, K. Ryoke, H. Ito, Minichromosome maintenance 2 expression is correlated with mode of invasion and prognosis in oral squamous cell carcinomas. J. Oral Pathol. Med. 32(8), 468–474 (2003)
K. Nowinska, M. Chmielewska, A. Piotrowska, B. Pula, W. Pastuszewski, T. Krecicki, M. Podhorska-Okołow, M. Zabel, P. Dziegiel, Correlation between levels of expression of minichromosome maintenance proteins, Ki-67 proliferation antigen and metallothionein I/II in laryngeal squamous cell cancer. Int. J. Oncol. 48(2), 635–645 (2016)
G. Pan, Y. Liu, L. Shang, F. Zhou, S. Yang, EMT-associated microRNAs and their roles in cancer stemness and drug resistance. Cancer Commun. (Lond). 41(3), 199–217 (2021)
K. Macleod, P. Mullen, J. Sewell, G. Rabiasz, S. Lawrie, E. Miller, J.F. Smyth, S.P. Langdon, Altered ErbB receptor signaling and gene expression in cisplatin-resistant ovarian cancer. Cancer Res. 65(15), 6789–6800 (2005)
G. Wang, R. Cao, Y. Wang, G. Qian, H.C. Dan, W. Jiang, L. Ju, M. Wu, Y. Xiao, X. Wang, Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway. Sci. Rep. 6, 35783 (2016)
Y.W. Qin, P. Ye, J.Q. He, L. Sheng, L.Y. Wang, J. Du, Simvastatin inhibited cardiac hypertrophy and fibrosis in apolipoprotein E-deficient mice fed a Western-style diet by increasing PPAR α and γ expression and reducing TC, MMP-9, and Cat S levels. Acta Pharmacol. Sin. 31(10), 1350–1358 (2010)
A.S. Paintlia, M.K. Paintlia, A.K. Singh, I. Singh, Modulation of Rho-Rock signaling pathway protects oligodendrocytes against cytokine toxicity via PPAR-α-dependent mechanism. Glia. 61(9), 1500–1517 (2013)
Z. Zhang, H.Y. Zhang, Y. Zhang, H. Li, Inactivation of the Ras/MAPK/PPARγ signaling axis alleviates diabetic mellitus-induced erectile dysfunction through suppression of corpus cavernosal endothelial cell apoptosis by inhibiting HMGCS2 expression. Endocrine. 63(3), 615–631 (2019)
C. Sun, G. Zhang, S. Luan, C. Luan, H. Shao, F. Dong, X. Liu, Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARγ) pathway. J. Recept Signal. Transduct. Res. 36(4), 422–428 (2016)
J. Klawitter, T. Shokati, V. Moll, U. Christians, J. Klawitter, Effects of lovastatin on breast cancer cells: a proteo-metabonomic study. Breast Cancer Res. 12(2), R16 (2010)
B.K. Tye, MCM proteins in DNA replication. Annu. Rev. Biochem. 68, 649–686 (1999)
A.K. Reka, M.T. Goswami, R. Krishnapuram, T.J. Standiford, V.G. Keshamouni, Molecular cross-regulation between PPAR-γ and other signaling pathways: implications for lung cancer therapy. Lung Cancer. 72(2), 154–159 (2011)
A.S. Paintlia, M.K. Paintlia, A.K. Singh, J.K. Orak, I. Singh, Activation of PPAR-γ and PTEN cascade participates in lovastatin-mediated accelerated differentiation of oligodendrocyte progenitor cells. Glia. 58(14), 1669–1685 (2010)
S.L. Forsburg, M.C.M. Eukaryotic, proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68(1), 109–131 (2004)
Y. Wang, H. Chen, J. Zhang, A.S.L. Cheng, J. Yu, K.F. To, W. Kang, MCM family in gastrointestinal cancer and other malignancies: From functional characterization to clinical implication. Biochim. Biophys. Acta Rev. Cancer. 1874(2), 188415 (2020)
L. Fei, H. Xu, Role of MCM2-7 protein phosphorylation in human cancer cells. Cell. Biosci. 8, 43 (2018)
K. Gou, J. Liu, X. Feng, H. Li, Y. Yuan, C. Xing, Expression of minichromosome maintenance proteins (MCM) and cancer prognosis: a meta-analysis. J. Cancer. 9(8), 1518–1526 (2018)
M.R. Alison, T. Hunt, S.J. Forbes, Minichromosome maintenance (MCM) proteins may be pre-cancer markers. Gut. 50(3), 290–291 (2002)
F. Song, B. Hu, J.W. Cheng, Y.F. Sun, K.Q. Zhou, P.X. Wang, W. Guo, J. Zhou, J. Fan, Z. Chen, X.R. Yang, Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma. Cell. Death Dis. 11(7), 573 (2020)
G. Wang, R. Cao, Y. Wang, G. Qian, H.C. Dan, W. Jiang, L. Ju, M. Wu, Y. Xiao, X. Wang, Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway. Sci. Rep. 6, 35783 (2016)
B. Du, J.S. Shim, Targeting Epithelial-Mesenchymal Transition (EMT) to Overcome Drug Resistance in Cancer. Molecules. 21(7), 965 (2016)

No competing interests reported.

Supplementary.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Lovastatin inhibits the proliferation of HNSCC cells by downregulating the MCM complex after PPARγ activation

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusions

Figures

1. Introduction

2. Materials and Methods

2.1. Study population and samples

2.2 Animal care and treatment

2.3 Cell culture

2.4 CCK-8 experiment

2.5 Wound healing experiments

2.6 Transwell experiment

2.7 Flow-cell cycle analysis

2.8 Flow-type cell apoptosis analysis

2.9 Quantitative real-time PCR (qRT‒PCR)

2.10 Protein analysis

2.11 PPAR antagonist treatment

2.12 Construction and transfection of small interfering RNA (siRNA)

2.13 Construction and transfection of the overexpression plasmid

2.14 Combined use of the drugs

2.15 Statistical analysis

3. Results

3.1. Lovastatin is associated with improved outcomes in HNSCC patients

3.2 Lovastatin inhibited the formation of HNSCC in nude tumor-bearing mice

3.3 Lovastatin inhibited the proliferation of HNSCC cells

3.4 Lovastatin inhibits the migration of HNSCC cells

3.5 Lovastatin inhibits HNSCC progression by regulating the MCM complex

3.6 Lovastatin increases the sensitivity of HNSCC cells to cisplatin

3.7 PPARγ inhibitors attenuate the antitumor effects of lovastatin mediated by MCM

3.8 Lovastatin downregulates MCM2 and MCM3 via PPARγ

4. Discussion and conclusion

Declarations

Author Contribution

Acknowledgments:

References

Additional Declarations

Supplementary Files

Status:

Version 1