Application of decitabine combined with DZNep in the preparation of drugs for the treatment of oral squamous cell carcinoma

Abstract

This invention relates to the field of biomedical technology, disclosing the application of decitabine combined with DZNep in the preparation of a drug for treating oral squamous cell carcinoma (OSCC). At the cellular level, the drug concentration of decitabine is 0.5-2 μM, and the drug concentration of DZNep is 1-5 μM; at the animal level, the drug concentration of decitabine is 1 mg / kg, and the drug concentration of DZNep is 2 mg / kg. The decitabine combined with DZNep described in this invention inhibits the migration and invasion of OSCC by upregulating the expression of E-cadherin and downregulating the expression of N-cadherin and Vimentin, providing a new technical solution for the treatment of OSCC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of decitabine in combination with DZNep in the preparation of a drug for treating oral squamous cell carcinoma. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is a common type of head and neck malignancy, with consistently high incidence and mortality rates. Due to difficulties in early diagnosis and its tendency to invade and metastasize, the 5-year survival rate for OSCC patients has long been low. Metastasis is one of the most critical reasons for treatment failure in OSCC, with most patients with metastasis experiencing a poor prognosis within one year of diagnosis. Therefore, elucidating the molecular mechanisms of OSCC invasion and metastasis and identifying effective therapeutic targets are of significant clinical importance.

[0003] Epithelial-mesenchymal transition (EMT) is a core biological process by which tumor cells acquire the ability to migrate and invade, initiating a metastatic cascade, and is also a key mechanism for the malignant progression of epithelial tumors. EMT is typically characterized by decreased epithelial marker E-cadherin and increased mesenchymal markers N-cadherin and Vimentin, accompanied by activation of transcription factors such as Snail, Twist, and ZEB1.

[0004] Previous studies by our research group have found that DEPDC1 is significantly overexpressed in OSCC tissues and is closely related to poor patient prognosis. Cell function experiments have confirmed that DEPDC1 overexpression can significantly enhance the invasive ability of OSCC cells, suggesting that DEPDC1 is a key molecule regulating OSCC invasion and metastasis.

[0005] Building upon previous findings, this study further confirms that aberrant upregulation of DEPDC1 in OSCC can induce epithelial-mesenchymal transition (EMT) by activating the EMT program: on the one hand, it downregulates E-cadherin, disrupting cell adhesion; on the other hand, it upregulates key molecules such as N-cadherin, Vimentin, and Snail, driving EMT activation and enhancing the migration and invasion potential of OSCC. These results indicate that DEPDC1-mediated EMT activation is an important mechanism promoting OSCC invasion and metastasis, but targeted intervention strategies have not yet been reported.

[0006] Decitabine is a DNA methyltransferase inhibitor, and DZNep (3-deazaneplanocin A) is a histone methylation inhibitor. Both are involved in epigenetic regulation and can affect the expression of EMT-related genes. Currently, there are no reports of decitabine and DZNep being used in combination for OSCC, especially targeting the DEPDC1-EMT axis to exert an anti-metastatic effect. Summary of the Invention

[0007] The purpose of this invention is to provide the application of decitabine combined with DZNep in the preparation of drugs for treating oral squamous cell carcinoma. Decitabine combined with DZNep downregulates the expression of DEPDC1, thereby upregulating the expression of E-cadherin and downregulating the expression of N-cadherin and Vimentin. That is, by targeting the DEPDC1-EMT axis, it inhibits the migration and invasion of OSCC, providing a new technical solution for the treatment of OSCC.

[0008] To achieve the above objectives, on the one hand, the present invention provides the application of decitabine in combination with DZNep in the preparation of a drug for treating oral squamous cell carcinoma.

[0009] Furthermore, ① at the cellular level, the drug concentration of decitabine was 0.5-2 μM, and the drug concentration of DZNep was 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

[0010] On the other hand, the present invention provides the use of formulations containing decitabine and DZNep in the preparation of medicaments for treating oral squamous cell carcinoma.

[0011] Furthermore, ① at the cellular level, the drug concentration of decitabine was 0.5-2 μM, and the drug concentration of DZNep was 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

[0012] Furthermore, decitabine combined with DZNep upregulated the expression of E-cadherin and downregulated the expression of N-cadherin and Vimentin, thereby inhibiting the migration and invasion of OSCC.

[0013] On the other hand, the present invention provides a drug for treating oral squamous cell carcinoma, wherein the active ingredients of the drug are decitabine and DZNep.

[0014] Furthermore, ① at the cellular level, the drug concentration of decitabine was 0.5-2 μM, and the drug concentration of DZNep was 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] The advantages and positive effects of the application of decitabine combined with DZNep in the preparation of a drug for treating oral squamous cell carcinoma as described in this invention are: 1. In this invention, decitabine combined with DZNep downregulates the expression of DEPDC1, thereby upregulating the expression of E-cadherin and downregulating the expression of N-cadherin and Vimentin. In other words, by targeting the DEPDC1-EMT axis, it inhibits the migration and invasion of OSCC, providing a new technical solution for the treatment of OSCC.

[0017] 2. In this invention, decitabine and DZNep have a significant synergistic antitumor effect in OSCC, with a CDI≈0.7, which is stronger than that of single drugs.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This invention relates to the in vitro antitumor effects of decitabine and DZNep (3-deazaneplanocin A) monotherapy in the embodiments of the present invention, wherein A represents the in vitro antitumor effect of decitabine monotherapy on the SCC-9 cell line, B represents the in vitro antitumor effect of decitabine monotherapy on the SCC-25 cell line, C represents the in vitro antitumor effect of DZNep monotherapy on the SCC-9 cell line, and D represents the in vitro antitumor effect of DZNep monotherapy on the SCC-25 cell line. Figure 2 This is a CDI heatmap for screening the optimal concentration of decitabine synergistic with DZNep in an embodiment of the present invention, where A is the SCC-9 cell model and B is the SCC-25 cell model. Figure 3 The results of in vitro inhibition of OSCC by decitabine combined with DZNep in this embodiment of the invention are shown, where A is the SCC-9 cell model and B is the SCC-25 cell model. Figure 4 This is the result of in vivo inhibition of OSCC by decitabine combined with DZNep in an embodiment of the present invention; Figure 5 The relative body weight of mice in each group during the drug administration period in this embodiment of the invention; Figure 6The results of the effects of decitabine combined with DZNep on OSCC migration and invasion in this embodiment of the invention are shown, where A is the migration distance and B is the percentage of invasive cells. Figure 7 This embodiment of the invention uses Western blotting to detect changes in the expression of epithelial-mesenchymal transition (EMT) related biomarkers, where A is E-cadherin, B is N-cadherin, C is Vimentin, and D is Snail. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this invention. The SCC-9 and SCC-25 cell lines were purchased from ATCC.

[0024] Example 1: In vitro antitumor effects of decitabine and DZNep (3-deazaneplanocin A) monotherapy To evaluate the in vitro antitumor effects of decitabine and DZNep, decitabine concentrations of 0, 0.5, 1, 2, 5, and 10 μM and DZNep concentrations of 0, 1.25, 2.5, 5, and 10 μM were set. SCC-9 and SCC-25 cell lines were treated with these drugs for 72 hours, and the effects of the two drugs on cell proliferation were analyzed using the IncuCyte® S3 live-cell imaging system.

[0025] The results are as follows Figure 1 As shown, during the dosing cycle, both decitabine and DZNep significantly inhibited the growth of OSCC in a dose- and time-dependent manner. Compared with the control group, the cell proliferation curves of each treatment group showed a clear concentration gradient inhibition effect, indicating that both drugs have significant in vitro antitumor activity.

[0026] Example 2: Screening for the optimal concentration of decitabine and DZNep for synergistic antitumor effects To screen for the optimal synergistic antitumor concentrations of decitabine and DZNep, SCC-9 and SCC-25 cells were used as models, and concentration gradients of decitabine (0, 0.5, 1, 2, 5, 10 μM) and DZNep (0, 1.25, 2.5, 5, 10 μM) were established for combined administration. Cells were seeded in 96-well plates and incubated with decitabine and / or DZNep for 48 hours. Cell viability was then assessed using the cellTiter-Glo® luminescent cell viability assay kit, and the interaction properties were quantitatively evaluated using the combination effect index (CDI) model.

[0027] The CDI value for decitabine combined with DZNep treatment is calculated using the following formula: CDI = AB / (A × B); The results are calculated based on the number of viable cells (absorbance value). AB is the ratio of the combined drug treatment group to the control group, A is the ratio of decitabine to the control group, and B is the ratio of DZNep to the control group. If CDI < 1, it indicates that the two drugs have a synergistic effect; if CDI < 0.7, the synergistic effect is very significant. If CDI = 1, the effect of the two drugs is additive; if CDI > 1, the effect of the two drugs is antagonistic.

[0028] The results show that ( Figure 2 At all tested concentration combinations, the CDI value of the two drugs was less than 1, indicating that decitabine and DZNep have a broad synergistic effect in inhibiting OSCC proliferation. Among them, the combination of 2 μM decitabine and 5 μM DZNep showed the most significant synergistic antitumor effect, with the lowest CDI value among all combinations, suggesting that this concentration ratio is the optimal synergistic dosing regimen for subsequent in vitro experiments.

[0029] Example 3: Combined use of decitabine and DZNep to inhibit OSCC proliferation To dynamically verify the antiproliferative effect of the optimized concentration combination, OSCCs were seeded in 96-well plates and cultured for 12 hours, followed by treatment with 2 μM decitabine and 5 μM DZNep, alone or in combination, for 72 hours. During the drug treatment, the process was continuously monitored using the IncuCyte® S3 live-cell imaging system, with images acquired every 2 hours. Data analysis of cell confluence formation curves was performed using IncuCyte® S3 software.

[0030] Compared with the control group, both 2 μM decitabine and 5 μM DZNep significantly inhibited the proliferation of OSCC. Furthermore, the combined treatment with these two drugs exhibited the strongest inhibitory effect on cell growth, with a significantly lower cell confluence curve compared to the individual drug groups. Figure 3 ).

[0031] Example 4: Decitabine combined with DZNep can enhance the antitumor activity of the OSCC PDX model. OSCC xenografted tumor tissue was cut into 2-3 mm pieces. 3 A small piece was implanted into the right side of the mouse. The tumor was allowed to grow to an average size of approximately 100-150 mm. 3 Mice bearing tumors were randomly divided into four groups (n=4-5): control group, decitabine monotherapy group, DZNep monotherapy group, and combination group (decitabine + DZNep). Decitabine was administered at 1 mg / kg intraperitoneally once daily for 5 consecutive days; DZNep was administered at 2 mg / kg intraperitoneally every other day for 2 consecutive weeks. Tumor size and body weight were measured twice weekly using calipers, and tumor volume was calculated using the formula: Tumor volume (mm²) 3 = length × width 2 ×0.5. The tumor growth inhibition (TGI) rate is calculated as follows: (1 - tumor volume in the treatment group / tumor volume in the control group) ×100%.

[0032] Mice were euthanized after treatment. All OSCC PDX xenograft tumors and major organs were collected for subsequent molecular and pathological analysis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were measured to assess liver and kidney function.

[0033] To evaluate the in vivo efficacy of decitabine and DZNep, a pharmacodynamic study was conducted in an OSCC PDX model. Results showed that the combination therapy was superior to either single agent in terms of therapeutic effect. Figure 4 The tumor growth inhibition rate at 30 days is shown in Table 1. Table 1 Tumor growth inhibition (TGI) rate

[0034] As shown in Table 1, the TGI of the combination group was 43.8%, which was significantly higher than that of any single-drug group.

[0035] In terms of safety, the combination therapy regimen demonstrated good tolerability. Throughout the administration period, no significant decrease in body weight was observed in any group of mice. Figure 5After treatment, serum biochemical parameters (ALT, AST, BUN, Cr) in mice were all within the normal range, and pathological examination of major organs (heart, liver, spleen, lung, and kidney) revealed no significant damage, suggesting that the combined treatment did not produce observable toxicity to liver and kidney function or major tissues. In vivo experimental results indicate that decitabine combined with DZNep synergistically and effectively inhibits tumor growth in the OSCC PDX model, with good safety, providing strong preclinical evidence for the clinical translation of this combined strategy.

[0036] Example 5: Decitabine combined with DZNep inhibits OSCC migration and invasion To verify the inhibitory effect of the combined treatment on the OSCC metastasis phenotype, Transwell assays were used to assess cell migration and invasion. OSCCs were seeded in the upper chamber of a Transwell assay (without Matrigel for migration assays, coated with Matrigel for invasion assays), and the lower chamber was supplemented with medium containing 10% FBS as a chemokine. Control, decitabine monotherapy (2 μM), DZNep monotherapy (5 μM), and combined treatment (2 μM + 5 μM) groups were set up. After 48 hours of treatment, cells that migrated or invaded to the lower chamber were fixed and stained, and counted under a microscope.

[0037] The results show that ( Figure 6 Compared with the single-drug group, the combination therapy group significantly reduced the number of migrating and invading cells. Simultaneously, Western blot analysis was used to detect changes in the expression of epithelial-mesenchymal transition (EMT) related markers. Figure 7 The study found that the combination therapy significantly upregulated E-cadherin expression and downregulated the expression of N-cadherin, Vimentin, and Snail. These results indicate that the combination therapy of decitabine and DZNep can effectively reverse the EMT phenotype, thereby inhibiting the migration and invasion of OSCC, providing direct evidence for its anti-metastatic effect.

[0038] Therefore, in this invention, decitabine combined with DZNep downregulates the expression of DEPDC1, thereby upregulating the expression of E-cadherin and downregulating the expression of N-cadherin and Vimentin. In other words, by targeting the DEPDC1-EMT axis, it inhibits the migration and invasion of OSCC, providing a new technical solution for the treatment of OSCC.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of decitabine combined with DZNep in the preparation of drugs for the treatment of oral squamous cell carcinoma.

2. The application according to claim 1, characterized in that: ① At the cellular level, the drug concentration of decitabine is 0.5-2 μM, and the drug concentration of DZNep is 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

3. Application of formulations containing decitabine and DZNep in the preparation of drugs for the treatment of oral squamous cell carcinoma.

4. The application according to claim 3, characterized in that: ① At the cellular level, the drug concentration of decitabine is 0.5-2 μM, and the drug concentration of DZNep is 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

5. The application according to claim 1 or 3, characterized in that: Decitabine combined with DZNep upregulates the expression of E-cadherin and downregulates the expression of N-cadherin and Vimentin, thereby inhibiting the migration and invasion of OSCC.

6. A drug for treating oral squamous cell carcinoma, characterized in that: The active ingredients of the drug are decitabine and DZNep.

7. The drug according to claim 6, characterized in that: ① At the cellular level, the drug concentration of decitabine is 0.5-2 μM, and the drug concentration of DZNep is 1-5 μM; ②At the animal level, the drug concentration of decitabine was 1 mg / kg; the drug concentration of DZNep was 2 mg / kg.

8. The drug according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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