Application of compound ZL-n-91 in preparation of medicine for treating oral cancer

Abstract

The invention discloses an application of a novel phosphodiesterase 4 (PDE4) inhibitor ZL-n-91 in preparation of medicines for treating growth and metastasis of oral cancer. In-vitro cytology experiments and in-vivo zoology experiments show that the phosphodiesterase 4 inhibitor ZL-n-91 can significantly inhibit proliferation and migration of the oral cancer, reveals that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to treat the oral cancer, and has good development and application prospects.

Description

Technical Field

[0001] This invention relates to the application of a phosphodiesterase 4 inhibitor, and more particularly to the application of a phosphodiesterase 4 inhibitor ZL-n-91, which belongs to the field of tumor biology. Background Technology

[0002] Oral cancer is one of the most common malignant tumors of the head and neck. Although surgical treatment combined with radiotherapy, chemotherapy, and targeted therapy has significantly improved the survival rate of oral cancer patients, the prognosis has not changed much, with a five-year survival rate of only 40%-50%. Currently, treatment methods for oral cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Due to the limitations of head and neck surgery, surgery may produce severe functional side effects. Patients undergoing surgery experience significant and severe impacts on swallowing, speech function, and facial appearance, resulting in obvious and severe disfigurement, eating and speech difficulties, and aesthetic and functional impairments, leading to a decline in the patient's quality of life. Therefore, radiotherapy plays an important role in the treatment of oral cancer. In addition, chemotherapy drugs such as cisplatin, 5-fluorouracil (5-FU), and docetaxel have been approved for the treatment of oral squamous cell carcinoma. These chemotherapy drugs can also act as radiosensitizers to improve the efficacy of radiotherapy. However, both chemotherapy and radiotherapy have serious side effects, and tumor cells are prone to developing drug resistance. The development of new and innovative drugs or treatment strategies remains crucial in addressing oral cancer.

[0003] Phosphodiesterases (PDEs) hydrolyze intracellular second messengers cAMP or cGMP, thereby affecting the signaling pathways mediated by these second messengers and regulating cellular function. PDEs are classified into 11 subtypes, among which phosphodiesterase 4 (PDE4) specifically hydrolyzes cAMP. PDE4 is mainly distributed in various inflammatory cells, including mast cells, macrophages, lymphocytes, and epithelial cells, and participates in promoting monocyte and macrophage activation, neutrophil infiltration, vascular smooth muscle proliferation, vasodilation, and myocardial contraction, affecting central nervous system function, cardiovascular function, the inflammatory / immune system, and cell adhesion. Studies have shown that PDE4 inhibitors (PDE4i) have anti-inflammatory, anti-allergic, and anti-platelet activation effects. Its mechanisms of action mainly involve: 1) inhibiting the release of various inflammatory mediators / cytokines, and suppressing the expression of IL-4 and IL-5 genes; 2) inhibiting leukocyte activation (such as respiratory burst) and leukocyte migration; 3) inhibiting or upregulating the expression of cell adhesion factors; 4) inducing the production of cytokines with inhibitory activity, such as IL-6; 5) inducing apoptosis; and 6) stimulating the release of endogenous hormones and catecholamines. PDE4 inhibitors that have been developed or are under development primarily target chronic obstructive pulmonary disease (COPD), asthma, inflammatory bowel disease, and arthritis.

[0004] Existing PDE4 inhibitors mainly include Rolipram, Cilomilast, and Roflumilast. However, Rolipram and Cilomilast cause gastrointestinal adverse reactions such as dizziness, headache, nausea, and vomiting, hindering their widespread clinical application. One possible reason for these gastrointestinal adverse reactions is the poor specificity of PDE4 inhibitors, resulting in moderately selective inhibition of the entire PDE family. For example, Cilomilast's Ki for PDE4 is 92 nM, only 500 to 1000 times that of PDE1, 2, 3, and 5. Therefore, higher doses of Cilomilast can interact with other PDE family members, leading to side effects. In fact, vomiting is a common side effect of most PDE4 inhibitors at high doses. While Roflumilast has been approved by the FDA for the treatment of COPD, reducing lung inflammation, combating oxidative stress, effectively alleviating pulmonary fibrosis, enhancing mucosal clearance, and promoting airway remodeling, its effects are limited. However, adverse reactions also exist, mainly manifested as diarrhea, weight loss, nausea, atrial fibrillation, and exacerbation of mental illnesses (such as insomnia, anxiety, and depression). The novel phosphodiesterase inhibitor ZL-n-91 is designed based on second-generation PDE4 inhibitors, with IC50 values ​​of 12 nM and 20 nM for PDE4D2 and PDE4B2, respectively, more than 5000 times higher than other members of the PDE family, demonstrating strong specificity. Compared to other PDE4 inhibitors, it exhibits neuroprotective activity and fewer side effects, effectively reducing or even avoiding adverse reactions such as vomiting. Like other PDE4 inhibitors, initial research on ZL-n-91 focused primarily on immunity, neurology, and inflammation. Studies have found that ZL-n-91 inhibits IL-17 production in human memory Th17 cells without significantly affecting cell viability. Ya-juan Wang et al. found that ZL-n-91 can inhibit inflammatory responses in a COPD-like rat model.

[0005] We have for the first time used the novel phosphodiesterase 4 inhibitor ZL-n-91 to treat oral cancer cells, demonstrating that ZL-n-91 can dose-dependently inhibit the proliferation and migration of oral cancer cells, arrest the cell cycle, and induce apoptosis. This is also the first time that ZL-n-91 has been used in vivo to treat oral cancer, providing a theoretical basis for ZL-n-91 treatment of oral cancer and offering a new strategy for the clinical treatment of oral cancer. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the application of the phosphodiesterase 4 inhibitor ZL-n-91 in the preparation of oral cancer anti-proliferation and metastasis.

[0007] To solve the above technical problems, the following technical solution is adopted:

[0008] The use of the phosphodiesterase 4 inhibitor ZL-n-91 in the preparation of drugs against oral cancer proliferation and metastasis is within the scope of protection of this invention.

[0009] This invention utilizes in vitro tumor cell culture and in vivo subcutaneous tumor models to investigate the pathophysiological effects of ZL-n-91 through cell proliferation, cell cycle, cell scratch assays, and animal subcutaneous tumor experiments. Experiments confirmed that this inhibitor can significantly inhibit the proliferation of human oral cancer cells CAL-27 and HSC-6; significantly arrest the cell cycle of oral cancer cells CAL-27 and HSC-6; significantly induce the migration of oral cancer cells CAL-27 and HSC-6; and significantly inhibit the growth of oral cancer CAL-27 subcutaneous tumors, with minimal side effects in mice. This lays the foundation for research into anti-oral cancer proliferation drugs.

[0010] The preferred administration methods for the above applications are oral, injection, or inhalation.

[0011] The phosphodiesterase 4 inhibitor ZL-n-91 described in this invention can be purchased directly or synthesized by oneself. For example, it can be synthesized by oneself with reference to the literature [Ruihong Ma, Bin-yan Yang, Chang-you Wu. A selective phosphodiesterase 4 (PDE4) inhibitor Zl-n-91 suppresses IL-17 production by human memory Th17 cells. International Immunopharmacology, 2008, 8(10): 1408-1417.].

[0012] Beneficial Effects: The selective PDE4 inhibitor ZL-n-91 described in this invention can significantly inhibit the proliferation and migration of oral cancer cells, producing unexpected effects and providing a new approach for the treatment of oral cancer with the PDE4 inhibitor ZL-n-91. The inhibitory potency of ZL-n-91 against PDE4B and PDE4D is more than 5000 times that of other members of the PDE family. Compared with other PDE4 inhibitors, this compound exhibits higher selectivity and specificity for PDE4B and PDE4D, produces fewer side effects, and can effectively reduce or even avoid adverse reactions such as vomiting, further enhancing the application prospects of PDE4 inhibitors in the treatment of oral cancer. Attached Figure Description

[0013] Figure 1The effect of ZL-n-91 on the viability of oral cancer cells; (A) and (C) the effect of different concentrations of ZL-n-91 on the activity of oral cancer cells CAL-27 and HSC-6; (B) and (D) the half-maximal inhibitory concentration (IC50) of ZL-n-91 on oral cancer cells CAL-27 and HSC-6; mean ± standard error, n = 3, comparison of significant differences: compared with the control group, ***P < 0.001.

[0014] Figure 2 The effect of ZL-n-91 on the proliferation of oral cancer cells; (A) and (C) Effect of different concentrations of ZL-n-91 on the proliferation of oral cancer cells CAL-27 and HSC-6; (B) and (D) Statistical analysis of the number of EdU positive cells; mean ± standard error, n = 3, comparison of significant differences: compared with the control group, ***P < 0.001.

[0015] Figure 3 The effect of ZL-n-91 on the cell cycle of oral cancer cells; (A) and (C) flow cytometry distribution of cell cycle; (B) and (D) statistical analysis of cell cycle distribution of oral cancer cells; mean ± standard error, n = 3, comparison of significant differences: compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0016] Figure 4 The effect of ZL-n-91 on the expression of cell cycle-related proteins in oral cancer cells; (A) and (C) Western blot analysis of the expression of cell cycle-related proteins P21, CDK2, and CDK4 in oral cancer cells; (B) and (D) Statistical graphs of cell cycle-related protein expression in oral cancer cells; mean ± standard error, n = 3, comparison of significant differences: compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0017] Figure 5 ZL-n-91 induces apoptosis in oral cancer cells in vitro; (A) and (C) flow cytometry distribution of apoptosis; (B) and (D) statistical analysis of total apoptosis in oral cancer cells; mean ± standard error, n = 3, comparison of significant differences: compared with the control group, *P < 0.05, **P < 0.01.

[0018] Figure 6 The effect of ZL-n-91 on the migration of oral cancer cells; (A), (C) The effect of ZL-n-91 on the metastatic ability of oral cancer cells; (B), (D) Statistical graph of the size of the area of ​​healing after migration of oral cancer cells; mean ± standard error, n=3, comparison of significant differences: compared with the control group, **P<0.01, ***P<0.001.

[0019] Figure 7The effects of ZL-n-91 on the CAL-27 oral cancer subcutaneous xenograft model were as follows: (A) Body weight change curve of nude mice with CAL-27 oral cancer subcutaneous xenograft after administration; (B) Tumor volume change curve of nude mice with CAL-27 oral cancer after treatment; (C) Photographing and recording of CAL-27 oral cancer subcutaneous xenograft tissue after treatment; (D) Weighing and statistical analysis of the excised CAL-27 oral cancer subcutaneous xenograft tissue; Mean ± standard error, n = 3, Significant differences were compared: Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0020] Figure 8 This is HE staining of CAL-27 oral cancer subcutaneous xenograft tissue. Detailed Implementation

[0021] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0022] Example 1: Cell counting method to detect the effect of ZL-n-91 on the proliferation of CAL-27 and HSC-6 cells.

[0023] 1) Cell plating

[0024] To ensure the cells are in good condition before plating, a new complete DMEM solution was used the day before plating. The next day, the culture medium was discarded, and the cells were digested, centrifuged, washed, and then the healthy oral cancer cells were collected. After cell counting, 1 × 10⁵ cells per well were seeded into 2 mL of complete DMEM in 6-well plates, with 3 replicates per group.

[0025] 2) Drug treatment

[0026] After the cells adhered, the culture medium was discarded, and the cells were washed with 0.5 mL of PBS. 2 mL of fresh complete DMEM was added to each well, followed by the addition of phosphodiesterase 4 inhibitor ZL-n-91, to achieve final concentrations of 0, 25, 50, 100, 150, 200, 300, and 400 μM. The six-well plates containing ZL-n-91 were then incubated in a 5% CO2, 37°C incubator for 48 h.

[0027] 3) Counting

[0028] After 48 hours, the culture medium was discarded, and the cells were transferred to 1.5 mL centrifuge tubes after digestion. After centrifugation and washing, the cells were collected, and 1 mL of culture medium was added to each tube. The cell suspension was gently pipetted to mix thoroughly, and the cells were counted using a hemocytometer. Each well was repeated three times, and the average value was taken. The number of cells counted was the cell proliferation rate. The cell inhibition rate was calculated as 1 - cell proliferation rate × 100%. The half-maximal inhibitory concentration (IC50) of ZL-n-91 against oral cancer cells was further fitted using Graphpad software.

[0029] The results are as follows Figure 1 As shown, ZL-n-91 significantly inhibited the cell viability of both oral cancer cell lines. With increasing ZL-n-91 concentration, cell viability also decreased significantly. The IC50 values ​​of ZL-n-91 against CAL-27 and HSC-6 oral cancer cell lines were 143.3 μM and 204.2 μM, respectively. These results indicate that the phosphodiesterase 4 inhibitor ZL-n-91 can significantly inhibit the proliferation activity of both oral cancer cell lines.

[0030] Example 2: EdU assay to detect the effect of ZL-n-91 on the proliferation of CAL-27 and HSC-6 cells

[0031] 1) CAL-27 and HSC-6 cells in good growth condition were introduced into the culture medium at a density of 2 × 10⁶ cells per well. 5 Cells were seeded into six-well plates and cultured in a cell culture incubator. After overnight incubation, the cells were treated with three drugs: a control group, a ZL-n-91 (50 μM) treatment group, and a ZL-n-91 (100 μM) treatment group.

[0032] 2) After 48 hours, remove the culture medium from the six-well plate and add 2 mL of preheated EdU working solution (37°C) to each well. Place the six-well plate in an incubator and continue incubation for 2 hours.

[0033] 3) After EdU labeling of cells, remove the culture medium and add 1 mL of fixative to each well to fix at room temperature for 30 minutes.

[0034] 4) Remove the fixative, add 1 mL of immunostaining blocking solution to each well, and wash the cells 3 times for 5 minutes each time.

[0035] 5) Remove the washing solution, add 1 mL of immunostaining permeabilization buffer to each well, and incubate at room temperature for 15 minutes.

[0036] 6) Remove the immunostaining permeabilization buffer, add 1 mL of immunostaining blocking buffer to each well, and wash the cells twice, 5 minutes each time.

[0037] 7) Remove the washing buffer. Add 500 μL of Click reaction solution to each well, gently shake the six-well plate to ensure the reaction solution evenly covers the sample, and incubate at room temperature in the dark for 30 minutes. Remove the Click reaction solution. Add 1 mL of immunostaining blocking solution to each well and wash the cells three times for 5 minutes each time. Remove the washing buffer. Add 1 mL of 1X Hoechst solution to each well for nuclear staining and incubate at room temperature in the dark for 10 minutes. Remove the 1X Hoechst solution. Add 1 mL of immunostaining blocking solution to each well and wash the cells three times for 5 minutes each time. Remove the washing buffer. Add an appropriate amount of anti-fluorescence quenching mounting solution to each well to cover the bottom of the six-well plate. Place the six-well plate in a light-protected box for detection or short-term storage at 4°C.

[0038] 8) Fluorescence detection was performed using an inverted fluorescence microscope. EdU showed red fluorescence, and Hoechst showed blue fluorescence; photographs were taken and recorded.

[0039] 9) Use ImageJ software to analyze the EdU positivity rate of the images, and use GraphPad Prism software to perform statistical analysis on the data.

[0040] The results are as follows Figure 2 As shown: Red staining represents EdU-labeled proliferating cells, and blue staining represents the nuclei of all cells labeled with Hoechst. Compared with the control group, the proportion of red EdU-positive cells in the ZL-n-91 treatment group was significantly reduced in a dose-dependent manner. This further demonstrates that the phosphodiesterase 4 inhibitor ZL-n-91 can significantly inhibit the proliferation of both oral cancer cell lines.

[0041] Example 3: Detection of the effect of ZL-n-91 on the cell cycle of oral cancer cells by PI staining flow cytometry and Western blotting (1) Detection of the effect of ZL-n-91 on the cell cycle of oral cancer cells by PI staining flow cytometry

[0042] 1) Cell plating

[0043] To ensure the cells are in good condition before plating, a new complete DMEM solution was used the day before plating. The next day, the culture medium was discarded, and the cells were digested, centrifuged, washed, and then the healthy oral cancer cells were collected. After cell counting, 2 × 10⁵ cells per well were seeded into 2 mL of complete DMEM in 6-well plates, with 3 replicates per group.

[0044] 2) Starvation treatment

[0045] After the cells adhered, the culture medium was discarded, and the cells were washed twice with 0.5 mL PBS. Then, 2 mL of serum-free basal DMEM was added to each well for starvation treatment for 24 h.

[0046] 3) Drug treatment

[0047] After 24 hours, the culture medium was discarded, and the plates were washed with PBS. 2 mL of fresh complete DMEM was added to each well, followed by the addition of phosphodiesterase 4 inhibitor ZL-n-91 to make the final concentrations of ZL-n-91 0, 100, and 200 μM. The six-well plates containing ZL-n-91 were then placed in a 5% CO2 incubator at 37°C and incubated for 48 hours.

[0048] 4) Cell fixation

[0049] After 48 hours, discard the culture medium, digest and centrifuge, add 1 mL of pre-chilled PBS, gently pipette to mix the cells, transfer to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 1 min, carefully discard the PBS, avoiding aspirating the cell pellet. Slowly add 1 mL of pre-chilled 70% ethanol to the cell pellet, gently agitate the cell deposit to achieve uniform distribution and prevent cell aggregation, then fix at 4°C for at least 2 hours or overnight.

[0050] 5) PI staining

[0051] The cell suspension fixed with 70% ethanol was centrifuged at 1000 rpm for 1 min, the ethanol was discarded, and the cells were washed twice with 1 mL of pre-cooled PBS and the cell pellet was collected. 0.5 mL of propidium iodide (PI) staining reagent was slowly added to the cell pellet, the cells were gently mixed by pipetting, and the cells were incubated at 37°C in the dark for 30 min before further analysis.

[0052] 6) Computer Lab

[0053] After staining, the cells are filtered (to avoid large clumps of cells clogging the flow cytometer) and transferred to flow cytometry tubes. After being thoroughly mixed, the cells can be detected using a flow cytometer in the dark (to ensure the accuracy of the experimental results, the detection should be completed within 5 hours).

[0054] 7) Data Processing

[0055] After the experiment was completed, the collected data were analyzed using ModFid LT5.0 software, and the cell population was divided into G0 / G1 phase, S phase, and G2 / M phase.

[0056] The results are as follows Figure 3 As shown, compared with the control group, ZL-n-91 treatment resulted in significant G0 / G1 phase arrest in both CAL-27 and HSC-6 cells, with a reduced proportion of cells in the S and G2 / M phases, in a dose-dependent manner. These results indicate that ZL-n-91 can arrest oral cancer cells in the G0 / G1 phase of the cell cycle and inhibit their proliferation.

[0057] (2) Western blot analysis of the effect of ZL-n-91 on oral cancer cell cycle

[0058] We used Western blotting to detect the expression of cell cycle-related genes and proteins. CDK2 and CDK4 are cyclin-dependent kinases (CDKs). The CDK2 / CyclinE1 and CDK4 / CyclinD complexes regulate the cell cycle and promote cell transition from G1 phase to S phase. CAL-27 and HSC-6 oral cancer cells were treated with different concentrations of ZL-n-91 (0, 100, and 200 μM) for 24 h, and the expression of cell cycle-related proteins CDK2, CDK4, and P21 was detected by Western blotting.

[0059] The results are as follows Figure 4 As shown, compared with the control group, with the increase of ZL-n-91 concentration, the expression of P21 was significantly upregulated, while the expression of CDK2 and CDK4 was significantly downregulated. The results indicate that ZL-n-91 may inhibit cell proliferation by upregulating P21 and downregulating the expression of CDK2 and CDK4, thereby arresting the cell cycle of oral cancer cells in the G0 / G1 phase.

[0060] Example 4: Flow cytometry detection of the effect of ZL-n-91 on apoptosis in CAL-27 and HSC-6 cells

[0061] 1) Cell plating

[0062] To ensure the cells are in good condition before plating, a new complete DMEM solution was used the day before plating. The next day, the culture medium was discarded, and the cells were digested, centrifuged, washed, and then the healthy oral cancer cells were collected. After cell counting, 2 × 10⁵ cells per well were seeded into 2 mL of complete DMEM in 6-well plates, with 3 replicates per group.

[0063] 2) Drug treatment

[0064] After the cells adhered, the culture medium was discarded, and the cells were washed with 0.5 mL of PBS. Then, 2 mL of fresh complete DMEM was added to each well, followed by the addition of phosphodiesterase 4 inhibitor ZL-n-91 to achieve final concentrations of 0, 100, and 200 μM. The six-well plates containing ZL-n-91 were then incubated in a 5% CO2, 37°C incubator for 48 h.

[0065] 3) Cell collection

[0066] After 48 hours, the culture medium was discarded, the cells were digested and centrifuged, 1 mL of pre-cooled PBS was added, the cells were gently pipetted to mix them, and then transferred to a 1.5 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 1 min. This process was repeated once more before discarding the PBS.

[0067] 4) Annexin V-FITC / 7-ADD staining

[0068] Resuspend the cells in the cell pellet with 0.5 mL of 1×Binding Buffer, then add 5 μL of Annexin V-FITC and 5 μL of 7-AAD staining solution. Gently pipette to mix thoroughly and incubate at room temperature in the dark for 15-20 min.

[0069] 5) Computer Lab

[0070] After the reaction is complete, the cells are filtered and transferred to flow cytometry tubes. After being thoroughly mixed, they can be detected using a flow cytometer in the dark (to ensure the accuracy of the experimental results, the detection should be completed within 1 hour).

[0071] 6) Data processing

[0072] After the experiment was completed, the collected data were analyzed using the software Flowjo 10.0 to classify the cells into normal cells, early apoptotic cells, late apoptotic cells, and dead cells.

[0073] The results are as follows Figure 5 As shown in the figure (Q1 represents dead cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q1 represents normal cells), compared with the control group, after ZL-n-91 treatment, the proportion of early apoptosis and late apoptosis (Q3+Q2, apoptotic cells) in CAL-27 and HSC-6 cells was significantly increased, indicating that ZL-n-91 promotes apoptosis of oral cancer cells.

[0074] Experimental Example 5: Detection of the effect of ZL-n-91 on the migration of CAL-27 and HSC cells using the scratch assay (1) Detection of the effect of ZL-n-91 on the migration of CAL-27 and HSC cells using the scratch assay

[0075] 1) CAL-27 and HSC cells in good growth condition were placed at a density of 9 × 10⁶ cells per well. 5 Cells were seeded into six-well plates and cultured in a cell culture incubator. CAL-27 and HSC oral cancer cells were treated with different concentrations of ZL-n-91 (0, 100, 200 μM) for 24 h.

[0076] 2) After 24 hours, scratch the well with a 200 μL pipette tip, add 1 mL of PBS to each well, wash twice, and add 2 mL of serum-free culture medium containing control solvent or drug to each well.

[0077] 3) Take photos of the same location on the scratch using an inverted microscope at 0h and 24h respectively.

[0078] 4) Use ImageJ software to analyze the changes in the scratch area of ​​the image and calculate the migration rate, and use GraphPadPrism software to perform statistical analysis on the data.

[0079] The results are as follows Figure 6 As shown, compared with the control group, the scratch width of oral cancer cells treated with ZL-n-91 was significantly larger in a dose-dependent manner. These results indicate that ZL-n-91 can significantly inhibit the migration of oral cancer cells.

[0080] Experiment 6: The effect of ZL-n-91 on the growth of oral subcutaneous carcinoma in nude mice

[0081] We established a subcutaneous xenograft model of oral cancer CAL-27. After modeling, the tumor-bearing mice were divided into a solvent control group and a control group and a control group receiving ZL-n-91 (2.5 mg / kg). The mice were administered the drug by gavage daily, and the tumor volume and weight of the mice were measured and recorded every two days. After 22 days of treatment, the tumors were removed, photographed, and weighed.

[0082] The results are as follows Figure 7 The results showed that during ZL-n-91 treatment, there was no significant difference in mouse body weight and no abnormal activity, indicating that a dose of 2.5 mg / kg of ZL-n-91 had no obvious toxic side effects on mice. Compared with the solvent control group, around day 11 of treatment, the tumor volume in the ZL-n-91 group was significantly smaller than that in the control group. After treatment, the tumors were removed, and the tumor volume in the ZL-n-91 treatment group was significantly smaller than that in the control group. Statistical analysis of tumor weight showed that the tumor weight in the ZL-n-91 treatment group was significantly smaller than that in the solvent control group. These experimental results indicate that ZL-n-91 significantly inhibits the growth of subcutaneous xenografts in nude mice in vivo and has no obvious toxic side effects on mice.

[0083] To clarify the reason why ZL-n-91 inhibits CAL-27 oral cancer subcutaneous xenografts in animals, we performed pathological analysis of CAL-27 subcutaneous xenograft tumor tissue using hematoxylin and eosin staining. Hematoxylin staining stained the cell nuclei blue-purple, while eosin staining marked the cytoplasm pink.

[0084] The results are as follows Figure 8 The results showed that tumor cells in the untreated control group remained morphologically intact and densely packed. In contrast, tumor samples treated with ZL-n-91 exhibited a significant decrease in cell density, reduced intercellular connections, and observed nuclear pyknosis. These results indicate that ZL-n-91 induces necrosis in CAL-27 oral cancer subcutaneous xenograft tissue.

[0085] The above research results indicate that the phosphodiesterase 4 inhibitor ZL-n-91 used in this invention can inhibit the proliferation of oral cancer and has a good anti-tumor effect.

Claims

1. Application of phosphodiesterase 4 inhibitor ZL-n-91 in inhibiting the proliferation and metastasis of oral cancer cells.

2. The application according to claim 1, characterized in that, It can be administered orally, by injection, or via nebulization.

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