Application of arsenic trioxide in preparation of medicine for reversing cetuximab resistance of head and neck squamous cell carcinoma

Abstract

The invention discloses an application of arsenic trioxide in preparation of a medicine for reversing cetuximab resistance of head and neck squamous cell carcinoma, arsenic trioxide ATO can be specifically combined with CLIP170, the ATO targets a zinc finger structure region of the CLIP170, arsenic trioxide can reverse and treat the cetuximab resistance of HNSCC by taking interference on the zinc finger structure of the CLIP170 as a target spot, the sensitivity of the cetuximab resistance of HNSCC is improved, and the application of arsenic trioxide in preparation of a medicine for reversing cetuximab resistance of HNSCC is realized. And a new thought is provided for attacking HNSCC cetuximab resistance chemosensitization.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for head and neck squamous cell carcinoma, and in particular to application of arsenic trioxide in the preparation of a drug for reversing cetuximab resistance in head and neck squamous cell carcinoma. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant tumor worldwide, accounting for 90% of head and neck malignancies and 16%-40% of systemic malignancies. There are 600,000 new cases worldwide each year, and its incidence and mortality rates are increasing annually. Only 30% of HNSCC patients are diagnosed early, and these patients can be cured with surgery and radiotherapy for a single, completely localized lesion. The remaining 70% of HNSCC patients will suffer varying degrees of local recurrence or distant metastasis of the cancer nest throughout the course of the disease. Therefore, finding effective treatments for mid- and late-stage HNSCC is an important issue that needs to be addressed urgently.

[0003] Since the beginning of the 21st century, monoclonal antibodies have been used clinically to treat many diseases, including head and neck cancer. For patients with locally advanced and metastatic HNSCC who have missed the optimal time for surgery, epidermal growth factor receptor (EGFR) inhibitors are often used as first-line treatment. Among them, cetuximab monoclonal antibody has been approved by the FDA (Food and Drug Administration) as a standard drug for the treatment of patients with advanced progressive and metastatic HNSCC. This type of drug can specifically bind to EGFR, inhibit tumor cell growth, invasion, and metastasis, and can prolong patients' overall survival and progression-free survival. However, after 12-18 months of treatment, drug insensitivity or tolerance will occur. Therefore, in-depth exploration of the molecular mechanisms of the occurrence and development of cetuximab-resistant HNSCC and identification of key factors that can be used for early diagnosis of molecular markers and drug targets are particularly important for the clinical treatment of HNSCC patients.

[0004] Currently, resistance mechanisms to cetuximab in advanced head and neck squamous cell carcinoma (HNSCC) include two main components: primary or secondary activation of the EGFR downstream signaling pathways RAS-RAF-MAPK and PI3K-AKT; and secondary resistance mechanisms caused by other signaling molecules closely related to EGFR outside the EGFR pathway. However, even taking these factors that can cause cetuximab resistance into account, the efficacy of the EGFR-targeted monoclonal antibody in the remaining HNSCC patients is only approximately 40%, indicating that other extremely important resistance mechanisms still exist. Predicting the efficacy of cetuximab in HNSCC patients remains a significant challenge. Summary of the Invention

[0005] The present invention provides an application of arsenic trioxide in preparing a drug for reversing cetuximab resistance in head and neck squamous cell carcinoma, so as to solve the above problems.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] On the one hand, the present invention provides the use of arsenic trioxide in preparing a drug for reversing cetuximab resistance in head and neck squamous cell carcinoma.

[0008] Furthermore, arsenic trioxide targets the zinc finger structure of the cytoplasmic connexin CLIP170 in head and neck squamous cell carcinoma by displacement.

[0009] Furthermore, the drug is an injection-type arsenic trioxide, wherein the concentration of the arsenic trioxide is 1-2 μM.

[0010] Another aspect of the present invention provides the use of arsenic trioxide in the preparation of a drug for enhancing the sensitivity of head and neck squamous cell carcinoma to cetuximab.

[0011] Furthermore, the drug is an injection-type arsenic trioxide, wherein the concentration of the arsenic trioxide is 1-2 μM.

[0012] The beneficial effects of the present invention are:

[0013] The present invention determines that arsenic trioxide (ATO) can specifically bind to CLIP170. ATO targets the zinc finger structure region of CLIP170. Arsenic trioxide can reverse the cetuximab resistance in HNSCC by interfering with the CLIP170 zinc finger structure, thus realizing the transformation from basic research to clinical application and providing new ideas for overcoming the chemotherapy sensitization of HNSCC cetuximab resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a graph showing the relationship between CLIP170 expression in late-stage metastatic HNSCC and overall survival and progression-free period of patients in Example 1 of the present invention, wherein: Figure 2A. Overall survival (OS) of HNSCC patients based on the expression of CLIP170 by Kaplan-Meier analysis; 2B. Metastasis-free survival (MS) of HNSCC patients based on the expression of CLIP170 by Kaplan-Meier analysis;

[0016] Figure 2 This is a result diagram of the expression of CLIP170 in HNSCC cetuximab-resistant strains in Example 2 of the present invention, wherein: Figure 2 A: Western blot method was used to detect the protein expression level of CLIP170 in different cells, and statistical analysis was performed; Figure 2 B is the result of scratch test to detect the horizontal migration ability of HNSCC cetuximab-resistant strain (Cal27-Cet) and knockdown or overexpression of CLIP170 in HNSCC cetuximab-resistant strain (Cal27-Cet). Figure 2 C is Figure 2 Statistical analysis results of tumor cell migration and invasion ability in B; Figure 2 D is a Transwell assay to detect the migration and invasion abilities of tumor cells in HNSCC cetuximab-resistant strain (Cal27-Cet) with CLIP170 knocked down or overexpressed in Cal27-Cet. Figure 2 E is Figure 2 Statistical analysis results of tumor cell migration and invasion abilities in D (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0017] Figure 3 This is a graph showing the cell viability of HNSCC cells after treatment with arsenic trioxide (ATO) at different concentrations in Example 3 of the present invention. Figure 3 A is the result of CCK8 assay to detect cell viability after Cal27 cells were treated with different concentrations of ATO (0 μM, 1 μM, 2 μM, 4 μM from left to right) for 6 hours, 12 hours, and 24 hours; Figure 3 B is the result of CCK8 assay of cell viability after Cal27-cet was treated with different concentrations of ATO (0-4 μM) for 6 hours, 12 hours and 24 hours (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0018] Figure 4 This is a graph showing the interaction between arsenic trioxide (ATO) and CLIP170 in Example 4 of the present invention, wherein: Figure 4A: Cal27 cells were treated with ATO for 0-24 hours, and the protein CLIP170 was extracted by IP method. The contents of Zn and As in CLIP170 at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h were detected by ICP-MS. Figure 4 B is the result of zinc ion fluorescence assay to detect the zinc ion content in CLIP170 after Cal27 cells were treated with TPEN, ATO, and ATO+Zn for 12 hours (*, P<0.05; **, P<0.01; ***, P<0.001);

[0019] Figure 5 This is a graph showing the effect of ATO on cetuximab-resistant HNSCC strains in Example 5 of the present invention, wherein: Figure 5 A is the effect of 1 μM ATO on the morphology of cetuximab-resistant tumor cells observed under a microscope. Figure 5 B is the Transwell experiment to detect the effects of 1μM and 2μM ATO on the invasion and metastasis ability of HNSCC cetuximab-resistant strain (Cal27-Cet). Figure 5 C is the changes of EMT-related indicators in HNSCC cetuximab-resistant strain (Cal27-Cet) after treatment with 2μM ATO detected by RT-qPCR (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0020] Figure 6 The effect of ATO on the therapeutic effect of cetuximab was detected in the human primary HNSCC mouse tumor model in Example 7 of the present invention. Figure 6 A is the tumor picture of each group after the mice were euthanized; Figure 6 B is the tumor growth curves of the Cal27 control group, Cetuximab group, Cetuximab group + 1 μM ATO, and Cetuximab group + 2 μM ATO group in the tumor-bearing model; Figure 6 C shows the results of Ki-67 immunohistochemical staining to detect tumor proliferation activity. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0022] Example 1: Relationship between CLIP170 expression and overall survival and progression-free survival in patients with advanced metastatic HNSCC

[0023] Kaplan-Meier survival analysis was used to obtain the survival rates of HNSCC patients at different time points, and to understand the relationship between CLIP170 expression and overall survival and progression-free survival in patients with advanced metastatic HNSCC. Figure 1 As shown in the figure, it can be seen that the expression of cytoplasmic junction protein CLIP170 in HNSCC patients is positively correlated with the overall survival of patients;

[0024] The overall survival (OS) and metastasis-free survival (MFS) of HNSCC patients with high CLIP170 expression were significantly shorter than those with low CLIP170 expression (P = 0.041 and P = 0.036, respectively). Figure 1 A, Figure 1 B), the above results indicate that CLIP170 can serve as a potential therapeutic target for advanced HNSCC.

[0025] Example 2: CLIP170 expression in cetuximab-resistant HNSCC strains affects tumor cell invasion and metastasis

[0026] (1) Construction of the HNSCC cetuximab-resistant strain Cal27-Cet:

[0027] Cal27 cells (a human tongue squamous cell carcinoma cell line, a cell model for head and neck squamous cell carcinoma (HNSCC)) were cultured in medium containing varying concentrations of cetuximab. The IC50 of the cetuximab was measured using WST-8. Subsequently, cetuximab was added to the Cal27 cells, starting at a concentration slightly below the IC50. During passaging, the most viable cells were selected, and the cetuximab concentration was increased using a two-generation frequency gradient. When the effective concentration of cetuximab reached 10 μM, this concentration was maintained for five stable passages, resulting in the successful construction of a drug-resistant strain (Cal27-Cet).

[0028] (2) The invasion and metastasis abilities of the cetuximab-resistant HNSCC cell line Cal27 were evaluated by performing a metastasis and invasion assay in a 24-well Transwell chamber (pore size 8 μm, Corning, USA). The experimental steps were as follows:

[0029] The HNSCC cetuximab-resistant strain Cal27-Cet cells obtained above (5×10 3Cells were suspended in serum-free culture medium and directly seeded into the upper chamber, while normal culture medium was used in the lower chamber. After 24 hours of incubation, the cells in the upper chamber were swabbed with a cotton swab. The Transwell chamber was fixed with 4% paraformaldehyde (Solarbio) for 15 minutes and stained with 1% crystal violet for 10 minutes. Five randomly selected areas of each sample were counted under an inverted fluorescence microscope.

[0030] Western blot analysis was used to measure protein concentration in the samples. Tumor cells were lysed on ice using RIPA, and protein concentration was measured using a BCA assay. Proteins were denatured by adding SDS loading buffer and heating each sample to 96°C for 5 minutes. Proteins were then electrophoresed on SDS-PAGE, separated on a PAGE gel, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1.5 hours and incubated with the primary antibody (Anti-CLIP170) overnight at 4°C. The secondary antibody was incubated for 1 hour before color development.

[0031] Meanwhile, CLIP170 was knocked down or overexpressed in Cal27 non-resistant strains and cetuximab-resistant strains (Cal27-Cet) as comparisons;

[0032] Western blot results are as follows Figure 2 As shown, Figure 2 Results A showed that CLIP170 expression was significantly upregulated in Cal27-Cet cells compared with Cal27 non-resistant strains; Figure 2 The B-2E results showed that knocking down CLIP170 in Cal27-Cet reduced the invasion and migration abilities of drug-resistant tumor cells, while overexpressing CLIP170 in Cal27-Cet significantly improved the invasion and migration abilities of drug-resistant tumor cells. This indicates that CLIP170 plays a role in regulating the invasion and migration of tumor cells in cetuximab-resistant HNSCC strains.

[0033] Example 3: Effects of different concentrations of arsenic trioxide (ATO) on the viability of HNSCC cells and HNSCC cetuximab-resistant strains

[0034] Cal27 cells and Cal27-Cet cells were treated with different concentrations of ATO (0-4 μM) for 6 hours, 12 hours and 24 hours, and cell viability was detected by CCK8 assay. Figure 3 As shown, combined Figure 3 As shown in Table 1, at all time points of 6 hours, 12 hours, and 24 hours, low concentrations of ATO (0.5 μM) had no cytotoxic effect on the cell viability of Cal27 cells, and 1 μM and 2 μM had little cytotoxic effect on Cal27 cells. However, when the ATO concentration reached 4 μM, at 24 hours, the cell viability of Cal27 cells was significantly inhibited. Cell viability Therefore, ATO at a concentration of 2 μM or lower had no or little cytotoxicity to HNSCC cells within 24 hours;

[0035] Table 1 Effects of different concentrations of arsenic trioxide on Cal27 cell activity

[0036]

[0037] like Figure 3 As shown in B, at all time points of 6 hours, 12 hours, and 24 hours, low concentrations of ATO (0.5 μM) had no cytotoxic effect on the cell viability of Cal27-Cet cells, and 1 μM and 2 μM ATO had little effect on cell activity. However, when the ATO concentration reached 4 μM, a significant decrease in cell viability was observed at 24 hours. These results indicate that ATO at a concentration of 2 μM or less has no cytotoxicity or low cytotoxicity to HNSCC cells within 24 hours. At the same time, compared Figure 3 A and Figure 3 B, It can be seen that the effect of ATO on Cal27-Cet cell viability is significantly greater than that on Cal27, indicating that HNSCC cetuximab-resistant strains are more sensitive to ATO and ATO can serve as a selective inhibitor of resistant cells.

[0038] Example 4: Interaction between arsenic trioxide (ATO) and CLIP170

[0039] (1) ATO (1 μM) was used to treat the HNSCC cetuximab-resistant strain Cal27 cells in Example 2 for 0-24 hours. CLIP170 protein was extracted from the cells by immunoprecipitation at different time points. The changes in the content of zinc ions and arsenic ions in CLIP170 were then detected by inductively coupled plasma mass spectrometry (ICPMS) to verify whether the arsenic ions in arsenic trioxide ATO bind to the zinc finger structure in CLIP170. The results are shown in Figure 2. Figure 4 As shown in A, the arsenic ion content reached its peak at 4 hours and lasted until 24 hours, while the zinc ion content showed a significant downward trend and did not rise significantly after 24 hours;

[0040] (2) After treating Cal27 cells with different concentrations of ATO and ATO+Zn for 12 hours, CLIP170 was extracted and the zinc ion content in CLIP170 of different groups at the 12-hour time point was detected using zinc ion fluorescence assay. Specifically, a zinc ion solution with a concentration of 1 mM was prepared, and the CLIP170 ligand and zinc ion solution of each group were mixed in DMF buffer, and the fluorescence intensity was measured by fluorescence spectrometer. By comparing the fluorescence changes under different conditions, the presence and concentration of zinc ions were determined. The results are shown in Figure 2. Figure 4 As shown in Figure 2, zinc ion chelators TPEN (5 μM) and ATO can both reduce the zinc ion content in CLIP170. When zinc ions were added to the ATO group, the zinc ion content in CLIP170 was significantly increased (i.e., arsenic ions can competitively occupy the zinc ion binding site, and exogenous zinc ion supplementation can replace arsenic ions and restore the zinc ion binding state of CLIP170). This indicates that in HNSCC resistant cells, ATO can target CLIP170, and trivalent arsenic ions have a replacement effect on zinc ions in CLIP170.

[0041] Example 5: Effect of ATO on HNSCC Cetuximab-resistant Strain Cal27-Cet

[0042] The HNSCC cetuximab-resistant strain Cal27-Cet cells (Example 3) treated with ATO at concentrations of 0 μM, 1 μM, and 2 μM were observed and tested. Figure 5 As shown, from Figure 5 The microscopic results of A showed that 1 μM ATO had an effect on the morphology of Cal27-Cet cells. After ATO administration, the pseudopodia of Cal27-Cet cells were reduced. Figure 5 BTranswell results showed that 1μM ATO could significantly inhibit the invasion and metastasis of Cal27-Cet cells, and 2μM ATO was even more significant. Figure 5 In C, RT-qPCR detection results showed that the expression levels of EMT-related indicators (N-cad, Vimentin, Snail) in Cal27-Cet were significantly inhibited after treatment with 1-2 μM ATO.

[0043] Combined with the experimental results of Examples 1-5, ATO targets CLIP170 protein, affects its zinc ion content, and thereby inhibits the function of CLIP170, ultimately achieving the inhibition of invasion, metastasis and related indicators of HNSCC cetuximab-resistant strains, indicating that ATO targeting CLIP170 protein can significantly inhibit / reverse HNSCC cetuximab resistance and increase the sensitivity of HNSCC cetuximab resistance, and ATO at a concentration of 1-2 μM has a good inhibitory effect on Cal27-Cet and low cytotoxicity.

[0044] Example 6: Preparation of arsenic trioxide medicine:

[0045] Arsenic trioxide was purchased from Sigma-Aldrich and a 10 μM stock solution was prepared according to the instructions. Based on the optimal concentration of ATO determined in Example 5, ATO was diluted to different concentrations of 1 μM and 2 μM using the stock solution to complete the preparation of ATO drug.

[0046] Example 7: Animal Experiment

[0047] The effect of ATO on the therapeutic efficacy of cetuximab was tested using a human primary HNSCC mouse tumor model. The mice were divided into the following four groups: Cal27 group (control group); Cal27 + cetuximab group (Cetuximab group); Cal27 + cetuximab group + 1μM ATO drug (Cetuximab + ATO 1μM); Cal27 + cetuximab group + 2μM ATO drug (Cetuximab + ATO 2μM);

[0048] A. Cal27 cells were inoculated subcutaneously into nude mice to establish an HNSCC tumor-bearing mouse model. The tumor size was observed and measured daily.

[0049] B. After the tumors grew to an appropriate size, the HNSCC tumor models of mice in each group were treated with cetuximab or cetuximab + ATO, respectively. The cetuximab group: cetuximab dose 10 mg / kg, twice a week; the cetuximab + ATO group: cetuximab dose 10 mg / kg, twice a week, and ATO drug (prepared in Example 6) dose 15 mg / kg, three times a week; the control group was intraperitoneally injected with PBS twice a week. The tumor volume and body weight of the model were continuously measured until 5 weeks (tumor volume was less than 1000 mm 3 ) The mice were euthanized to evaluate the drug performance, and the proliferation activity of the tumor was detected by HE staining and Ki-67 immunohistochemical staining.

[0050] Figure 6 A is the tumor picture of each group after mice were euthanized; Figure 6 B. Statistics of tumor volume showed that compared with the single-drug group and the blank control group, the ATO-administered group could significantly inhibit tumor growth; Figure 6 C is the result of Ki-67 immunohistochemistry staining. It can be seen that the tumor volume of the cetuximab monotherapy group decreased compared with the control group, while the tumor volume of the combined drug group decreased most significantly, among which the 2μM ATO drug group was more significant, indicating that ATO drug can effectively reverse cetuximab resistance in head and neck squamous cell carcinoma.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of arsenic trioxide in the preparation of drugs to reverse cetuximab resistance in head and neck squamous cell carcinoma.

2. The use of arsenic trioxide according to claim 1 in the preparation of a drug for reversing cetuximab resistance in head and neck squamous cell carcinoma, characterized in that: Arsenic trioxide targets the zinc finger structure of the cytoplasmic connexin CLIP170 in head and neck squamous cell carcinoma by displacement.

3. The use of arsenic trioxide in reversing cetuximab resistance in head and neck squamous cell carcinoma according to claim 1, characterized in that: The drug is arsenic trioxide in the form of an injection, wherein the concentration of the arsenic trioxide is 1-2 μM.

4. Application of arsenic trioxide in the preparation of drugs to enhance the sensitivity of head and neck squamous cell carcinoma to cetuximab.

5. The use of arsenic trioxide in a drug for enhancing the sensitivity of head and neck squamous cell carcinoma to cetuximab according to claim 4, characterized in that: The drug is arsenic trioxide in the form of an injection, wherein the concentration of the arsenic trioxide is 1-2 μM.

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