Application of lactobacillus rhamnosus H23A017 combined with trametinib in preparation of medicine for treating melanoma

Abstract

The invention provides application of lactobacillus rhamnosus H23A017 combined with trametinib in preparation of drugs for treating melanoma, lactobacillus rhamnosus H23A017 is combined with MEK inhibitor trametinib in an intratumoral injection mode, it is found that compared with a single medication group, combined treatment can improve the expression level of IFN-gamma and TNF-alpha in a tumor microenvironment, and the effect of treating melanoma is achieved. The application has the advantages that tumor cell apoptosis is promoted, tumor cell infiltration is increased, the tumor volume is further reduced, and a new strategy is provided for development of low-toxicity and high-efficiency melanoma immune drug combination.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of Lactobacillus rhamnosus H23A017 combined with trametinib in the preparation of a drug for treating melanoma. Background Art

[0002] Melanoma is a rare heterogeneous disease caused by the malignant transformation of melanocytes in the skin. Among patients who died from skin malignancies, melanoma accounts for more than 80% of the deaths, making it the skin cancer with the highest mortality rate. Melanoma not only develops rapidly, but is also prone to recurrence and metastasis. Once the tumor spreads, the 5-year relative survival rate of tumor patients is low.

[0003] At present, there are the following treatment methods for melanoma development stages: 1) Localized melanoma and locoregional melanoma, which are in the early stages of development, are mostly treated with resection and regional lymph node dissection; 2) In addition to resection treatment, locally advanced melanoma will also use adjuvant treatments such as interferon α-2b, BRAF inhibitors combined with MEK inhibitors, and PD-1 monoclonal antibodies after surgery; 3) For patients with advanced melanoma, tumors often metastasize during this period, and treatment methods include molecular targeted therapy, immunotherapy, chemotherapy, and radiotherapy. However, these treatments have the disadvantages of limited efficacy, high cost, and large side effects. Therefore, it is necessary to explore new treatments. Summary of the invention

[0004] The present invention aims to provide an application of Lactobacillus rhamnosus H23A017 combined with trametinib in the preparation of a drug for treating melanoma, so as to explore a new approach for the use of probiotics in tumor treatment.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The present invention provides a combined drug for treating melanoma, wherein the combined drug is Lactobacillus rhamnosus H23A017 combined with trametinib, and the Lactobacillus rhamnosus H23A017 has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of May 14, 2025, a deposit number of GDMCC No: 66326, and a classification name of Lactobacillus rhamnosus.

[0007] Preferably, the Lactobacillus rhamnosus H23A017 is in an injection form, and the trametinib is in an oral form.

[0008] The present invention also provides the use of Lactobacillus rhamnosus H23A017 in combination with trametinib in the preparation of a medicament for treating melanoma. Lactobacillus rhamnosus H23A017 has been deposited with the Guangdong Provincial Culture Collection Center of Microorganisms (GDMCC), located at the 5th Floor, Building 59, No. 100 Dashanyuan, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit date is May 14, 2025, and the deposit number is GDMCC No: 66326, with the taxonomic name Lactobacillus rhamnosus.

[0009] Preferably, Lactobacillus rhamnosus H23A017 is in an injection dosage form, and trametinib is in an oral dosage form.

[0010] More preferably, the administration method of Lactobacillus rhamnosus H23A017 is intratumoral injection.

[0011] Preferably, the combination of Lactobacillus rhamnosus H23A017 and trametinib can significantly delay the growth rate of melanoma.

[0012] Preferably, the combination of Lactobacillus rhamnosus H23A017 and trametinib can significantly increase the levels of IFN-γ and TNF-α in serum and tumor tissues, thereby inhibiting tumor growth.

[0013] Preferably, the combination of Lactobacillus rhamnosus H23A017 and trametinib can significantly change the tissue morphology of melanoma and inhibit tumor growth.

[0014] Preferably, the combination of Lactobacillus rhamnosus H23A017 and trametinib can inhibit tumor growth by promoting apoptosis of tumor cells.

[0015] Preferably, the combination of Lactobacillus rhamnosus H23A017 and trametinib can inhibit tumor growth by increasing the infiltration of immune cells in tumor tissues.

[0016] The beneficial effects of the present invention are as follows:

[0017] In the present invention, Lactobacillus rhamnosus H23A017 is combined with the MEK inhibitor trametinib by intratumoral injection. It is found that compared with the single-drug group, the combination therapy can increase the expression levels of IFN-γ and TNF-α in the tumor microenvironment, promote apoptosis of tumor cells, increase tumor cell infiltration, and further reduce the tumor volume, providing a new strategy for the development of low-toxicity and highly effective immunotherapy combination drugs for melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Effect of combined treatment with H23A017 and trametinib on the growth of murine melanoma. A: Comparison of the growth rates of murine melanoma. B: Comparison of tumor volumes at the end of the experiment, **P < 0.0025, ***P < 0.00025; C: Changes in tumors after treatment.

[0019] Figure 2 Effect of combined treatment with H23A017 and trametinib on IFN-γ in melanoma-bearing mice. A: Content of IFN-γ in serum. B: Content of IFN-γ in tumor tissues, *P < 0.025, *P < 0.0025.

[0020] Figure 3 Effect of combined treatment with H23A017 and trametinib on TNF-α in melanoma-bearing mice. A: Content of TNF-α in serum. B: Content of TNF-α in tumor tissues, *P < 0.025, **P < 0.0025.

[0021] Figure 4 Morphological changes in tumor tissues after treatment. A: Morphology of tumor tissues in the MOD group. B: Morphology of tumor tissues in the MED group. C: Morphology of tumor tissues in the Lr group.

[0022] Figure 5 Apoptosis changes in tumor tissues of mice in different groups after treatment. A: Morphology of tumor tissues in the MOD group. B: Morphology of tumor tissues in the MED group. C: Morphology of tumor tissues in the Lr group.

[0023] Figure 6 Changes in infiltration of immune cells in tumor tissues of mice in different groups after treatment. A: Infiltration of CD4+ T cells in tumor tissues of the MOD group. B: Infiltration of CD4+ T cells in tumor tissues of the MED group. C: Infiltration of CD4+ T cells in tumor tissues of the MOD group. D: Infiltration of CD8+ T cells in tumor tissues of the MOD group. E: Infiltration of CD8+ T cells in tumor tissues of the MED group. F: Tumor tissues of CD8+ T cells in the MOD group. Detailed implementation manners

[0024] The present invention will be described in detail below in conjunction with the specific implementation manners. The following specific examples are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0025] Examples

[0026] I. Materials and methods

[0027] 1.1 Materials and reagents

[0028] 1.1.1 Culture media and reagents

[0029] MRS broth medium; phosphate buffer solution (PBS); DMEM medium; trypsin (containing EDTA); fetal bovine serum (FBS); trametinib;

[0030] 1.1.2 Strains

[0031] Lactobacillus rhamnosus H23A017 was provided by the Innovation and Utilization Team of Tropical Probiotics, College of Food Science and Engineering, Hainan University. It has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC), located on the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou. The Institute of Microbiology, Guangdong Academy of Sciences. The deposit date was May 14, 2025, and the deposit number was GDMCC No: 66326. The taxonomic name is Lactobacillus rhamnosus.

[0032] The strain stored at -80°C was thawed and transferred into MRS medium in a laminar flow hood, and cultured at 37°C for 24 h. It was passaged 3 times before use.

[0033] 1.1.3 Cells

[0034] Melanoma cell line B16F10 (Cat No.FH0361) was purchased from Shanghai Fuheng Biotechnology Co., Ltd. 1.2 Cell culture and animal experiment model

[0035] 1.2.1 Cell culture

[0036] The cryopreserved B16F10 cells were rapidly thawed at 37°C, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The B16F10 cell pellet was resuspended with 1 mL of DMEM, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The B16F10 cell pellet was resuspended with 1 mL of DMEM, and then all were transferred into a cell culture flask and supplemented with DMEM containing 10% fetal bovine serum to 5 mL. When the B16F10 cells grew to 80%-90%, they were passaged into a new cell culture flask; after culturing enough B16F10 cells, the culture medium was carefully aspirated, washed twice with 5 mL of PBS, and then digested with 3 mL of trypsin for about 1 min until the B16F10 cells dispersed. 3 mL of DMEM medium containing 10% fetal bovine serum was added to terminate the digestion and pipetted evenly. The B16F10 cell suspension was aspirated into a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, resuspended with 5 mL of PBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, resuspended with 1 mL of PBS, and 100 μL of the B16F10 cell suspension was diluted evenly in 990 μL of PBS. The cell concentration was diluted to 5×10 6 cells / mL.

[0037] 1.2.2 Animal model construction

[0038] Specific pathogen-free (SPF) mice used in the experiment were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The experimental mice were divided into 3 groups: model group (MOD, n = 7), drug group (MED, n = 7), and probiotic intervention group (Lr, n = 7). All mice were adaptively fed for one week, during which maintenance feed for experimental mice was provided.

[0039] After the adaptive feeding period ended, all mice were subcutaneously injected with 100 μL of B16F10 cells at a concentration of 10 6 cells / mL on the right lower abdomen. After 7 days, it was observed whether the modeling was successful; after successful modeling, all mice were gavaged with 1 mg / kg trametinib daily according to their body weight. The MOD group and the MED group were injected with 50 μL of PBS every 2 days, and the Lr group was injected with 50 μL of Lactobacillus rhamnosus H23A017 at a concentration of 10 9 CFU / mL into the tumor every 2 days. The administration continued for 7 days.

[0040] 1.3 Index detection

[0041] 1.3.1 Tumor size measurement

[0042] On the 7th day after subcutaneous injection of B16F10 and every 2 days thereafter, a vernier caliper was used to measure the tumor size. The vernier caliper was perpendicular to the mouse skin, and the longest axis of the tumor was recorded as a, and the shortest axis was b. Then the tumor volume V was recorded as:

[0043] V = ab 2 / 2 (mm 3 )

[0044] 1.3.2 Detection of serum and tissue multi-factors using kits

[0045] Mouse blood was collected, allowed to stand at room temperature for 6 h, then centrifuged at 4000 rpm at 4 °C for 15 min, and the upper clear serum was carefully collected and stored at -40 °C for later use.

[0046] The tumor tissue was excised, 100 mg of the tissue was cut out, 1 mL of PBS was added, and it was ground using a grinder at 70 Hz for 30 s, then paused for 15 s, and this was repeated three times; then centrifuged at 3000 rpm at 4 °C for 15 min.

[0047] Referring to the Elisa kit instructions, the levels of IFN-γ and TNF-α in the serum and tissue samples were detected respectively. All kits were purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0048] 1.3.3 H&E staining of tumor tissue

[0049] The tumor tissue was excised. After being dehydrated with gradient ethanol and cleared with xylene, it was immersed in paraffin at 60 °C for 2 hours to complete embedding. Then the wax block was cut into 5-μm thin sections, which were spread on a polylysine-coated slide in ultrapure water at 42 °C and then attached to the slide. The slide was baked at 70 °C for 2 h. After baking, the slide was dewaxed with xylene for 20 min, then washed with gradient ethanol, stained with hematoxylin for 6 min, rinsed with running water, blued for 10 min, stained with eosin for 10 - 15 min, dehydrated with ethanol gradient, cleared with xylene, and sealed with neutral gum, and observed under a microscope.

[0050] 1.3.4 TUNEL sections of tumor tissue

[0051] The paraffin tumor tissue sections prepared in the previous step were dewaxed with xylene (5 min × 2 times), hydrated with gradient ethanol (from 100% to 50%), and then washed with 0.85% NaCl and PBS for 5 min each; fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, permeabilized with Proteinase K at room temperature for 15 min, fixed again for 5 min and washed. Subsequently, a wet box was used to balance with the equilibration solution for 10 min. Reaction solutions for the treatment group (containing rTdT + biotin-dUTP), negative control (without rTdT), and positive control (pretreatment with DNase 1) were prepared respectively. The TUNEL mixture was incubated in a wet box at 37 °C for 1 h in the dark, the reaction was terminated with 2×SSC for 15 min, and washed three times with PBS. After blocking with 0.3% H2O2 for 15 min, streptavidin-HRP (diluted 1:500 with PBS) was labeled for 30 min, and DAB was developed in the dark for 10 min until a light brown background appeared. It was rinsed 5 times with ultrapure water, counterstained with hematoxylin for 5 s, rinsed with running water, dehydrated with ethanol gradient, cleared with xylene, sealed with neutral gum, and observed under a microscope.

[0052] 1.3.5 Immunohistochemical sections of tumor tissue

[0053] Paraffin tumor sections were incubated with 3% hydrogen peroxide at room temperature for 15 min to inactivate endogenous enzymes. After washing on a shaker with PBS (8 min, 3 times), they were heat-repaired with citrate buffer in a microwave (maintained for 10 min after boiling). Washed three times again with PBS, blocked with goat serum for 15 min, and after discarding the blocking solution, the diluted primary antibody was added and incubated in a wet box at 4 °C for 12 h. After rewarming, washed three times with PBS, incubated with biotin-labeled secondary antibody at room temperature for 15 min, washed three times with PBS, and blocked with HRP-labeled streptavidin for 15 min. DAB was developed for 10 s and immediately rinsed with running water for 30 min to blue. After counterstaining with hematoxylin, dehydrated with ethanol gradient, cleared with xylene, sealed with neutral gum, and observed under a microscope. During microscopy, a significant yellowish-brown staining in the nucleus / cytoplasm / interstitium compared to the background was determined as positive.

[0054] II. Results

[0055] 2.1 The combination of Lactobacillus rhamnosus H23A017 and trametinib significantly delays the growth rate of melanoma in mice. After successful modeling, the major axis and minor axis of the tumors in the mice were recorded, and the tumor volume was calculated.

[0056] The results showed that compared with the MOD group, the tumor volume in the MED group was significantly reduced (***P < 0.00025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 1 ), indicating that trametinib can significantly slow down the growth rate of melanoma and inhibit the growth of melanoma in mice. Compared with the MED group, the tumor volume in the Lr group was further reduced, and the tumor volume was significantly smaller than that in the MED group (**P < 0.0025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 1 ), indicating that compared with the intervention with trametinib alone, intratumoral injection of Lactobacillus rhamnosus H23A017 combined with treatment can further inhibit tumor growth.

[0057] 2.2 The combination of Lactobacillus rhamnosus H23A017 and trametinib treatment significantly increases the content of serum and tumor cytokines in melanoma mice

[0058] Interferon-γ (IFN-γ) is mainly secreted by natural killer cells, natural killer T cells, and CD8+ T cells. Its functions mainly include inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and reducing angiogenesis. The results showed that the serum IFN-γ levels in the MED group (*P < 0.025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 2 A) and the Lr group (**P < 0.0025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 2 A) were significantly higher than those in the MOD group. In addition, in the tumor tissue, the IFN-γ levels in the MED group (*P < 0.025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 2 B) and the Lr group (**P < 0.0025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 2 B) were significantly higher than those in the MOD group. The above results indicate that compared with single drug use, Lactobacillus rhamnosus H23A017 intervention can significantly increase the IFN-γ levels in the serum and tumors of mice, thereby inhibiting tumor growth.

[0059] Tumor necrosis factor α (TNF-α) is a type of small molecule protein mainly secreted by monocytes and macrophages. In the body, TNF-α can not only resist tumors through direct killing or inhibitory effects, but also promote the proliferation and differentiation of T cells, block tumor angiogenesis, thereby playing an inhibitory role on tumors.

[0060] After trametinib intervention, the serum TNF-α level in the MED group of mice was lower than that in the MOD group, while the serum TNF-α level in the Lr group showed a significant increase compared with the MED group (**P<0.0025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 3 A). In tumor tissues, the TNF-α level in the MED group was higher than that in the MOD group. After the intervention of Lactobacillus rhamnosus H23A017, the TNF-α level in the tumor tissues of mice further increased (**P<0.0025, Mann-Whitney U test, Bonferroni correction, n = 2 comparisons, Figure 3 B). The above results indicate that compared with single drug use, after the intervention of Lactobacillus rhamnosus H23A017, the serum and tumor TNF-α levels in mice can be significantly increased, thereby inhibiting tumor growth.

[0061] 2.3 The combined treatment of Lactobacillus rhamnosus H23A017 and trametinib significantly changes the tissue morphology of melanoma mice

[0062] Observation of H&E sections of tumor tissues in melanoma mice found that in the MOD group, tumor cells were dense, with complete morphology and clear nuclear morphology; in the MED group, the cell nuclei were sparser and the morphology was not as complete as that in the MOD group; in the Lr group, the cell nuclei were dispersed and the cell morphology was incomplete ( Figure 4 ). The above results indicate that trametinib can inhibit the growth of melanoma in mice. Under the combined treatment of Lactobacillus rhamnosus H23A017, the activity of tumor growth and division is further reduced, and tumor growth is inhibited.

[0063] 2.4 The combined treatment of Lactobacillus rhamnosus H23A017 and trametinib accelerates apoptosis in tumor tissues of melanoma mice

[0064] To explore the effects of trametinib and Lactobacillus rhamnosus H23A017 on tumor tissue apoptosis, the present invention performed TUNEL apoptosis section observation on melanoma in mice. The results showed that compared with the MOD group, the apoptosis level of cells in the tumor tissues of mice in the MED group and the Lr group increased. Among them, the Lr group had the most significant effect on the increase in the apoptosis level of cells in tumor tissues ( Figure 5 ), indicating that the combined treatment of Lactobacillus rhamnosus H23A017 and trametinib can promote the apoptosis of tumor cells to a certain extent to inhibit tumor growth.

[0065] 2.5 Effects of Lactobacillus rhamnosus H23A017 combined with trametinib on immune cell infiltration in murine melanoma

[0066] To investigate the effects of trametinib and Lactobacillus rhamnosus H23A017 on tumor tissue cell infiltration, immunohistochemical sections of murine melanoma were observed in this invention. The experimental results showed that compared with the MOD group, the MED group and the treatment with Lactobacillus rhamnosus H23A017 combined with trametinib increased the infiltration of CD4+ and CD8+ T cells in the tumor tissue. The infiltration of immune cells in the tumor tissue of the Lr group mice was the most obvious ( Figure 6 ). It indicates that the combination therapy can inhibit tumor growth by increasing the infiltration of immune cells in the tumor tissue.

[0067] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly explaining the present invention, rather than limiting the implementation modes of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A combined drug for treating melanoma, characterized in that, The combined drug is Lactobacillus rhamnosus H23A017 combined with trametinib. The Lactobacillus rhamnosus H23A017 has been deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of May 14, 2025, a deposit number of GDMCC No: 66326, and a classification name of Lactobacillus rhamnosus.

2. The combination drug according to claim 1, wherein, The Lactobacillus rhamnosus H23A017 is in an injection form, and the trametinib is in an oral form.

3. Use of Lactobacillus rhamnosus H23A017 combined with trametinib in the preparation of a medicament for treating melanoma, characterized in that, The Lactobacillus rhamnosus H23A017 has been deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with a deposit date of May 14, 2025, a deposit number of GDMCC No: 66326, and a classification name of Lactobacillus rhamnosus.

4. The application according to claim 3, characterized in that The Lactobacillus rhamnosus H23A017 is in an injection form, and the trametinib is in an oral form.

5. The application according to claim 4, wherein The administration method of the Lactobacillus rhamnosus H23A017 is intratumoral injection.

6. The application according to claim 3, wherein The Lactobacillus rhamnosus H23A017 combined with trametinib can significantly slow down the growth rate of melanoma.

7. The application according to claim 3, characterized in that, The combination of Lactobacillus rhamnosus H23A017 and trametinib can significantly increase the levels of IFN-γ and TNF-α in serum and tumors, thereby inhibiting tumor growth.

8. The application according to claim 3, wherein The combination of Lactobacillus rhamnosus H23A017 and trametinib can significantly change the tissue morphology of melanoma and inhibit tumor growth.

9. The application according to claim 3, characterized in that The Lactobacillus rhamnosus H23A017 combined with trametinib can promote the apoptosis of tumor cells to inhibit tumor growth.

10. The application according to claim 3, characterized in that, The Lactobacillus rhamnosus H23A017 combined with trametinib can inhibit tumor growth by increasing the infiltration of immune cells in tumor tissues.

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