Application of pinoresinol in treatment of rectal cancer
By using pinoresinol as an inhibitor of the CaLM1/CaMKII/CREB signaling pathway, the proliferation of rectal cancer cells was inhibited and their apoptosis was promoted, which solved the problems of side effects and drug resistance of existing treatments and provided a new direction for low-toxicity natural anticancer drugs.
Patent Information
- Application Number
- CN202511273244.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-08
- Publication Date
- 2025-11-21
AI Technical Summary
Existing treatments for rectal cancer, such as surgery, chemotherapy, and radiotherapy, have side effects such as decreased immune function and liver damage, and also face problems of tumor recurrence and drug resistance. Therefore, finding highly effective and low-toxic natural ingredients as anti-cancer drugs has become an important direction.
Pinoresinol was used as an inhibitor of the CaLM1/CaMKII/CREB signaling pathway. By inhibiting the expression of CaLM1, the signaling pathway was blocked, thereby inhibiting the proliferation of rectal cancer cells and promoting their apoptosis.
Pinoresinol significantly inhibits the CaLM1/CaMKII/CREB signaling pathway. In vitro experiments showed a dose-dependent reduction in the expression of CaMKII and p-CREB, inhibiting cancer cell proliferation and promoting apoptosis. In vivo experiments showed an inhibitory effect similar to that of the chemotherapy drug cisplatin, with better safety and tolerability.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of pharmaceutical technology, in particular to the application of pinoresinol in the treatment of rectal cancer. BACKGROUND
[0002] In recent years, rectal cancer (READ) has attracted extensive attention worldwide due to its high incidence and mortality. The main treatment methods for rectal cancer are still surgery, chemotherapy and radiotherapy. Although these treatments can prolong the survival period of patients to some extent, they have serious side effects such as immune function decline and liver damage. In addition, they also face problems such as tumor recurrence and drug resistance. Therefore, searching for natural ingredients with high efficiency and low toxicity has become an important direction of anti-cancer research. Studies have shown that the CaLM1 (calmodulin 1) / CaMKII (calmodulin-dependent protein kinase II) / CREB (CaMP response element binding protein) signaling pathway plays an important role in the occurrence and development of tumor cells. The over-activated CaLM1 / CaMKII / CREB pathway is closely related to the migration and invasion of tumor cells. CaLM1 can form a complex with Ca 2+ and activate CaMKII, and further regulate the cell cycle and promote tumor cell proliferation. In rectal cancer cells, activated CaMKII can promote the phosphorylation of CREB, which acts as a transcription factor to activate various genes related to growth and survival, helping cancer cells to escape apoptosis and enhance invasion ability. Therefore, targeting the CaLM1 / CaMKII / CREB signaling pathway is expected to become a new strategy for the treatment of rectal cancer. SUMMARY
[0003] Therefore, based on the above background, the present application provides the application of pinoresinol in the treatment of rectal cancer. It is found for the first time that pinoresinol can inhibit the expression of CaLM1, further inhibit the CaLM1 / CaMKII / CREB signaling pathway, and further use pinoresinol as a CaLM1 / CaMKII / CREB signaling pathway inhibitor to inhibit the proliferation of rectal cancer cells and promote the apoptosis of rectal cancer cells, so as to achieve effective treatment of cancer, especially rectal cancer, and provide a new direction for the treatment of rectal cancer.
[0004] The technical scheme provided by the present application is as follows:
[0005] The application of pinoresinol in the preparation of a CaM inhibitor or a CaLM1 / CaMKII / CREB signaling pathway inhibitor.
[0006] Further, the pinoresinol inhibits the CaLM1 / CaMKII / CREB signaling pathway by inhibiting the expression of CaLM1.
[0007] Based on the same inventive concept, the present application provides a CaM inhibitor or CaLM1 / CaMKII / CREB signaling pathway inhibitor, which comprises an effective amount of pinoresinol as the only active ingredient.
[0008] Based on the same inventive concept, the present application provides the use of a CaM inhibitor or CaLM1 / CaMKII / CREB signaling pathway inhibitor or pinoresinol in at least one of the following, which comprises:
[0009] a) preparing a product for promoting apoptosis of tumor cells by inhibiting CaLM1 / CaMKII / CREB signaling pathway;
[0010] b) preparing a product for inhibiting proliferation of tumor cells by inhibiting CaLM1 / CaMKII / CREB signaling pathway;
[0011] c) preparing a product for treating and / or assisting in treating cancer by inhibiting CaLM1 / CaMKII / CREB signaling pathway.
[0012] Further, the tumor cells comprise rectal cancer tumor cells, and the cancer comprises rectal cancer.
[0013] Further, the rectal cancer tumor cells comprise rectal cancer cells HCT116 and SW620.
[0014] Further, the product is a food, a health product, a medicine or an experimental reagent for basic research.
[0015] Further, when the product is a medicine, the pinoresinol is the only active ingredient.
[0016] Based on the same inventive concept, the present application provides a medicine for treating and / or assisting in treating cancer by inhibiting CaLM1 / CaMKII / CREB signaling pathway, which is characterized in that it comprises an effective amount of pinoresinol and a medically acceptable auxiliary agent.
[0017] Further, the cancer comprises rectal cancer.
[0018] Based on the same inventive concept, the present application provides a method for inhibiting expression of CaLM1 in tumor cells for non-diagnostic and therapeutic purposes, which comprises co-culturing tumor cells with an effective concentration of pinoresinol for 24 hours or more.
[0019] Preferably, the concentration of the pinoresinol is 2-8 μM.
[0020] The tumor cells comprise human rectal cancer cell lines HCT116 and SW620.
[0021] The present application achieves the following beneficial effects:
[0022] The present application first discovers that rosinol can inhibit the expression of CaLM1, and then inhibits the CaLM1 / CaMKII / CREB signaling pathway by reducing the expression levels of CaMKII and p-CREB, and based on this, first uses rosinol as
[0023] The experiment on the effect of the inhibitor of the CaLM1 / CaMKII / CREB signaling pathway on rectal cancer cells and their solid tumors in vitro and in vivo finds that rosinol reduces the expression of CaLM1, CaMKII and p-CREB in a dose-dependent manner in vitro, significantly inhibits the proliferation of cancer cells and promotes their apoptosis, and makes the tumor cell cycle arrest at the G0-G1 phase. The in vivo experiment finds that rosinol reduces the tumor volume and weight in a dose-dependent manner, and in the animal experiment, it is found that high-concentration rosinol (20.0 mg / kg) has a similar inhibitory effect on rectal cancer as cisplatin (8.0 mg / kg), which is a known chemotherapeutic drug; and the TUNEL experiment and immunohistochemical analysis further support that rosinol promotes apoptosis and anti-proliferation by inhibiting the CaLM1 / CaMKII / CREB signaling pathway.
[0024] Compared with traditional chemotherapeutic drugs, plant medicine extracts generally have the characteristics of multi-target and low toxicity, so rosinol may have better safety and tolerability in anti-tumor treatment, and thus, it can provide a new direction for rosinol to play an anti-tumor role by inhibiting the CaLM1-dependent signaling pathway, and for it to be used as a potential anti-cancer drug in the treatment of rectal cancer and the multi-target combination therapy of rectal cancer. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 The bioinformatics analysis of the embodiments of the present application screens out the potential gene CaLM1 which may be related to poor prognosis of rectal cancer:
[0027] APPENDIX Figure 1 A is a general outline of bioinformatics analysis;
[0028] APPENDIX Figure 1 B is PCA analysis;
[0029] APPENDIX Figure 1 C is a volcano plot of differential genes in READ;
[0030] APPENDIX Figure 1 D is WGCNA analysis;
[0031] APPENDIX Figure 1 E is correlation analysis between READ gene modules and clinical characteristics;
[0032] APPENDIX Figure 1 F is a module division tree diagram;
[0033] APPENDIXFigure 1 G is the gene number distribution of the READ gene co-expression module;
[0034] Appendix Figure 1 H is the correlation analysis of gene significance and module member relationship in the purple module;
[0035] Appendix Figure 1 I is the correlation analysis of gene significance and module member relationship in the light blue module.
[0036] Appendix Figure 2 The relationship between CALM1 related genes and tumors: Appendix Figure 2 A-Appendix Figure 2 C is the survival analysis of the central genes in READ; Appendix Figure 2 D is the result graph for verifying the role of CaLM1, MTHFD1L and DKC1 in the proliferation of HCT116 cells; Appendix Figure 2 E is the result graph for verifying the role of CaLM1, MTHFD1L and DKC1 in the proliferation of SW620 cells; Appendix Figure 2 F is the Ca 2+ and the results of unactivated CaM binding to promote cell proliferation; Appendix Figure 2 G-Appendix Figure 2 J is the influence of the expression level of CaLM1 gene on the survival rate of patients with bladder cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma and uveal melanoma.
[0037] Appendix Figure 3 CaLM1 regulates the proliferation and apoptosis of cancer cells through the CaMKII and p-CREB signaling pathways:
[0038] Appendix Figure 3 A is the cell state observed under a microscope in the normal expression group, overexpression group and knockdown group of CaLM1 gene;
[0039] Appendix Figure 3 B is Hoechst staining;
[0040] Appendix Figure 3 C is the Western blot detection of CaLM1, CaMKII, CREB, p-CREB gene protein expression;
[0041] Appendix Figure 3 D is the Western blot detection of Cyclin D1, Bcl-2, Caspase-3, Caspase-9, Cleaved Caspase-3, Cleaved Caspase-9 protein expression.
[0042] Appendix Figure 4To confirm the relevant characterization results of pinocembrol as a CaM inhibitor:
[0043] Figure 1 Figure 4 A is the working principle of phosphodiesterase kit;
[0044] Figure 2 Figure 4 B is the screening of CaM inhibitors from 14 extracts of nightshade and comparison with two known CaM inhibitors;
[0045] Figure 3 Figure 4 C is the comparison of PIN and CGS-9342B inhibition rates;
[0046] Figure 4 Figure 4 D is the determination of PIN IC 50 value.
[0047] Figure 5 Figure 5 Pinocembrol blocks the cycle of tumor cells and induces apoptosis:
[0048] Figure 6 Figure 5 A is the effect of adding different concentrations of PIN on cell state;
[0049] Figure 7 Figure 5 B and Figure 5 C are cell colony formation experiments.
[0050] Figure 8 Figure 6 Flow cytometry determination of cell cycle and apoptosis results:
[0051] Figure 9 Figure 6 A and Figure 6 B are SW620 flow cytometry apoptosis;
[0052] Figure 10 Figure 6 C and Figure 6 D are SW620 flow cytometry cycle;
[0053] Figure 11 Figure 6 E and Figure 6 F are HCT116 flow cytometry apoptosis;
[0054] Figure 12 Figure 6 G and Figure 6 H are HCT116 flow cytometry cycle.
[0055] Figure 13 Figure 7 Pinocembrol inhibits tumor cell proliferation and induces apoptosis through CaLM1 / CaMKII / CREB-dependent signaling pathway:
[0056] Figure 14 Figure 7 A to Figure 7 D are the effects of PIN on the expression levels of CaLM1, CaMKII, CREB, and p-CREB;
[0057] Figure 2 Figure 7 Figure 3
[0058] Figure 4 Figure 8 Figure 5
[0059] Figure 6 Figure 8 Figure 7
[0060] Figure 8 Figure 8 Figure 9
[0061] Figure 10 Figure 8 Figure 11
[0062] Figure 12 Figure 8 Figure 13 Figure 8 Figure 14 Figure 8 Figure 15 Figure 8 Figure 16 Figure 8 Figure 17 Figure 8 Figure 18 DETAILED DESCRIPTION
[0063] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative labor fall within the protection scope of the present application.
[0064] Embodiment 1
[0065] Ethical statement on the following animal experiments
[0066] Experimental animals were from Vital River Laboratory Animal Technology (Beijing, China). Experimental animals were bred in Shandong Xindai Pharmaceutical Co., Ltd. (Linyi, China). The methods of all animal experiments followed the National Research Council [U.S.] Committee for the Update of the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the CNDSE Animal Ethics Committee of Shandong Xindai Pharmaceutical Co., Ltd. (Approval date: March 19, 2022, AN-IACUC-2022-024). Human tissue samples were collected from the Department of Pathology of Linyi Tumor Hospital and approved by the Biomedical Research Ethics Committee of Linyi Tumor Hospital (Batch No. KY-2204).
[0067] Some chemicals and reagents involved in the following experiments: Pinoresinol (PIN, #HY-N6253, purity 98.35%) was purchased from MedChemExpress (MCE, Shanghai, China), diluted to 200 mM with DMSO, and stored at -40 °C. Cisplatin (CDDP) was purchased from MedChemExpress (MCE, Shanghai, China), diluted to 3.3 mM with sterile water, and stored at -40 °C. The primary and secondary antibodies of CaLM1, CaMKII, CREB, p-CREB, Bcl-2, caspase-3, and caspase-9 were purchased from Abeam (Cambridge, UK). Anti-CyclinD1 antibody (#ab16663) was purchased from Cell Signaling Technology (CST, Shanghai, China).
[0068] Hoechst 33342 staining solution (1.0 mg / ml, #C0031), CCK-8 cell proliferation and cytotoxicity assay kit (#CA1201), Annexin V-FITC apoptosis detection kit (#CA1020), and DNA content quantification assay (CellCycle, #CA1510) were purchased from Life Sci Biences (Beijing, China).
[0069] (1) Data sources and bioinformatics
[0070] All clinical data were obtained from TCGA database (The Cancer Genome Atlas, http. / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga). RNA sequencing data of READ patients were downloaded from TCGA database, including 168 tumor samples and 16 normal samples. The above data were used to identify the expression of different genes and to perform survival analysis.
[0071] The following are the specific operations of the related experiments:
[0072] (1) Cell culture
[0073] Human colorectal cancer cell lines HCT116 and SW620 were provided by the Chinese Academy of Sciences and cultured in DMEM (HCT116) and L-15 (SW620) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U / mL). They were placed in a humidified cell incubator with a CO2 concentration of 5% and a temperature setting of 37°C.
[0074] (2) Cell viability assay
[0075] Cells were seeded in 96-well plates at a density of 5.0 x 10 3 cells per well, and HCT116 and SW620 cells were treated with different concentrations (concentrations of 2, 4, 8 micromoles) of pinoresinol for 24 hours. The control group was treated with serum-free medium without pinoresinol. After different treatment times, 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for 1 hour, followed by determination of the absorbance value at 450 nm using an enzyme marker (Infinite M200 Pro, Tecan, Switzerland).
[0076] (3) Transfection
[0077] Small interfering RNA (siRNA) targeting CaLM1 was purchased from IBSBIO (Integrated Biotech Solutions Co., Ltd., Shanghai, China). CaLM1 expression plasmid (D / N, A / N, D / A, A / A) was purchased from MiaoLing Bio Company in Wuhan, China. According to the instructions, siRNA transfection was performed under optimal conditions. Western blot was used to detect the related proteins to verify whether the transfection was successful.
[0078] (4) Phosphodiesterase kit screening CaM inhibitors:
[0079] SW620 and HCT116 cells were seeded in 96-well plates at a density of 5.0 x 10 3HCT116 and SW620 cells were seeded in 96-well plates at a density of 200 cells / well and treated with 2 mM of Solanum-derived substances (Solasonine, Solamargine, β-Solamargine, Solasodine, N-Methylsolasodine, Pinoresinol) for 24 hours. After treatment, 10 μl of CCK-8 reagent was added and incubated for 1 hour. Cell proliferation was determined by measuring the absorbance (OD value) at 450 nm wavelength using a microplate reader (Infinite M200 Pro, Tecan, Switzerland).
[0080] 12β-Hydroxysolasodine, α-Solanigrine, Solanigridine, Solanine, Uttroside A, Uttroside B, Dumoside, Quercetin-3-O-rhamnoside, Pinoresinol) were used to treat cells, respectively. Blank control group (without Solanum-derived substances) was set. After 24 hours of treatment, 10 μl of CCK-8 reagent was added and incubated for 1 hour. Cell proliferation was determined by measuring the absorbance (OD value) at 450 nm wavelength using a microplate reader (Infinite M200 Pro, Tecan, Switzerland).
[0081] (5) Colony formation assay:
[0082] To detect the proliferation ability of cells, colony formation assay was performed. HCT116 and SW620 cells (treated with no drug (0 mM), low concentration of drug (2 mM Pinoresinol), medium concentration of drug (4 mM Pinoresinol), and high concentration of drug (4 mM Pinoresinol), respectively) were seeded in 12-well plates (200 cells per well). When the colonies were visible to the naked eye and the number of cells in each group was greater than 50, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet. After washing and air-drying, the number of colonies was photographed and counted.
[0083] (6) Flow cytometry cycle assay:
[0084] The cell cycle detection was performed according to the operation steps of the DNA content quantitative detection kit. The specific steps were as follows: the cells were fixed with pre-cooled 70% ethanol at 4°C for 2 hours. After centrifugation (250g, 5 minutes), the cells were resuspended with PBS, 100 μl of RNase A solution was added and incubated at 37°C for 30 minutes, and then 400 μl of PI staining solution was added and incubated at 4°C for 30 minutes.
[0085] (7) Flow cytometry apoptosis assay:
[0086] Annexin V-FITC apoptosis detection kit. The specific steps are as follows: resuspend the cells with 1x Annexin V binding buffer, then add staining solution (10 μl Annexin V and 10 μl PI), incubate at room temperature for 10 minutes in the dark. And within 10 minutes, the machine is detected. The above results are detected by BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA).
[0087] (8) Western Blot analysis:
[0088] Western blot analysis was used to detect the expression of different proteins. Total protein extraction was performed using total protein extraction kit (Solarbio, BC3710) according to the instructions, and the proteins in cytoplasm and nucleus were extracted by NE-PER nuclear and cytoplasmic extraction reagent (ThermoFisher, 78835). Cell protein samples (20 μg / well) and tissue protein samples (30 μg / well) were loaded into polyacrylamide gel for electrophoresis. Protein samples were subjected to the following steps: after SDS-PAGE separation, transfer to PVDF membrane, block with 5% bovine serum albumin at room temperature for 3 hours, then add the corresponding primary antibody and incubate at 4°C overnight, wash with Tris-buffered saline with Tween (TBST) (10 minutes each, a total of 3 times), then add secondary antibody and incubate at room temperature for 45 minutes, wash with TBST for 3 times. Finally, incubate with SuperSignal West Pico PLUS chemiluminescent substrate (#34580, ThermoFisher Scientific, Shanghai, China) for 2 minutes, and take pictures to record the results by UVP imaging system (BioSpectrum Imaging System, Upland, CA, USA). TM West Pico PLUS chemiluminescent substrate (#34580, ThermoFisher Scientific, Shanghai, China) for 2 minutes, and take pictures to record the results by UVP imaging system (BioSpectrum Imaging System, Upland, CA, USA).
[0089] (9) Hoechst staining (33342 staining solution)
[0090] SW620 and HCT116 were seeded in 12-well plates to about 60% density, the culture medium was removed, washed with PBS for 2 times, and Hoechst staining solution was added to ensure uniform coverage of the cells. The samples were incubated in a 37°C incubator for 8 minutes in the dark, then washed with PBS for 2 times, and the staining effect was observed under a fluorescence microscope (Olympus FV100, Olympus, Tokyo, Japan).
[0091] (10) Animal model:
[0092] Forty-eight 5-week-old male BALB nude mice were selected and injected subaxillarily with 2×10 6 HCT116 cells were placed in PBS solution. After 10 days, tumor size was measured twice a week using digital calipers, and tumor volume was calculated using the following formula: (D 2 ×d) / 2, where D is the large diameter of the tumor and d is the small diameter of the tumor.
[0093] Randomly divided into 4 groups:
[0094] Control group (intraperitoneal injection of normal saline 8.0 mg / kg, once daily);
[0095] One CDDP treatment group (CDDP dissolved in normal saline, administered intraperitoneally at a concentration of 8.0 mg / kg, once daily);
[0096] Two PIN treatment groups were established (pinoresinol was dissolved in physiological saline; one group received pinoresinol at 10.0 mg / kg via intraperitoneal injection, and the other group received pinoresinol at 20.0 mg / kg via intraperitoneal injection, once daily). The administration was performed intraperitoneally for 21 consecutive days. On day 22, the mice were euthanized, the tumors were removed, and the mice were immediately weighed and their volume calculated.
[0097] (11) TUNEL cell apoptosis assay
[0098] Apoptosis in xenografted rectal cancer tissues was assessed using the Beyotime (Shanghai, China) TUNEL kit. Rectal cancer tissue from mice was dissected, fixed in 4% paraformaldehyde for 24 hours, routinely embedded in paraffin, and sectioned to a thickness of 5 μm. Sections were dewaxed with xylene, dehydrated with graded ethanol, washed with distilled water, and incubated with 20 μg / mL proteinase K at 37°C for 20 minutes. Staining followed by incubation at 37°C in the dark for 1 hour, DAB staining for 3-5 minutes, termination of the reaction with distilled water, and counterstaining with hematoxylin. Positive controls were treated with DNase I, while the enzyme solution was omitted for negative controls. The number of brown TUNEL-positive cells was counted under an optical microscope, and statistical analysis was performed using GraphPad Prism software. p < 0.05 was considered statistically significant.
[0099] (12) Immunohistochemical staining
[0100] Tumor tissues from each group were dissected, and the protein expression of CaLM1, CaMKII, CREB, and p-CREB in mouse tumor-bearing FFPE (formalin-fixed paraffin-embedded) samples was detected by immunohistochemical (IHC) staining. Fluorescence was observed using a fluorescence microscope (Olympus FV100, Olympus, Tokyo, Japan). Histological evaluation was performed using a BX53 optical microscope (Olympus, Tokyo, Japan).
[0101] Data statistical analysis:
[0102] All experiments in this example were performed at least three times. Flow data were analyzed using Flowjo 10 software, statistical analysis was performed using SPSS 21.0 software, and data were expressed as mean ± standard deviation (S.D.). Significant differences were tested by one-way analysis of variance (ANOVA), and p < 0.05 indicated that the difference was statistically significant.
[0103] Experimental results
[0104] (1) Based on bioinformatics analysis, potential regulatory gene CaLM1 that may lead to poor prognosis of rectal cancer was screened out
[0105] The clinical characteristics and gene expression data of 168 tumor samples and 16 normal samples were extracted from the TCGA database. Principal component analysis (PCA) showed that there was a significant separation between principal component 1 (PC1, 13.99%) and principal component 2 (PC2, 8.26%), indicating that there were obvious differences in gene expression between tumor and normal samples Figure 1 B). Differential expression gene analysis identified 1337 significantly up-regulated genes and 2911 significantly down-regulated genes Figure 1 C). Subsequently, based on gene expression data, clustering analysis was performed on the samples, and tumor samples and normal samples were divided into different groups according to gene expression patterns Figure 1 D), and a heat map showed the significant differences between the two groups. Through WGCNA weighted gene co-expression network analysis, it was found that the gene modules related to rectal cancer were significantly related to clinical characteristics such as tumor type, pathological stage, etc. Figure 1 E). Cluster tree analysis was performed on the results of WGCNA analysis, and multiple key gene modules that may play an important role in the development of rectal cancer were identified Figure 1 F). The analysis results showed that the purple and light blue modules contained more genes, which may play a key role in the development of rectal cancer Figure 1 G, H, I).
[0106] Analysis of the genes in the purple and light blue modules Figure 1 H and Figure 1 I, three genes CaLM1, MTHFD1L and DKC1 related to survival were screened out (see Figure 2 A, Figure 2 B, Figure 2C). To evaluate the effect of up- or down-regulation of each gene expression on cell proliferation, each experiment of SW620 and HCT116 cell lines was divided into 3 experimental groups. The results showed that overexpression (OE) of CaLM1 gene significantly increased the proliferation index of HCT116 and SW620 cells in the regulation of CaLM1 gene expression. In contrast, the expression of si-CaLM1 interference group significantly reduced the cell proliferation index, indicating that CaLM1 played a promoting role in cell proliferation. For DKC1 and MTHFD1L genes, no significant change in proliferation index was observed after overexpression or down-regulation treatment, indicating that the roles of these two genes in the proliferation process of these two cells were small or less relevant to proliferation Figure 2 D, Figure 2 E). These experimental results showed that CaLM1 played a key regulatory role in the proliferation of HCT116 and SW620 colorectal cancer cells, while MTHFD1L and DKC1 had limited effect on proliferation. This is consistent with previous studies that the expression of CaLM1 gene can promote cell proliferation, and its mechanism is that Ca 2+ binds with unactivated calmodulin to form an activated complex, which further acts on downstream signaling molecules to promote cell proliferation Figure 2 F). Survival analysis showed that high expression of CaLM1 in various cancers was associated with shorter survival, suggesting that it can be used as a prognostic marker and target for personalized treatment Figure 2 G、 Figure 2 H、 Figure 2 I、 Figure 2 J).
[0107] (2) CaLM1 regulates colorectal cancer cell proliferation and apoptosis through the CaMKII / p-CREB pathway
[0108] Ca 2+ and can bind with unactivated CaM to form an activated complex
[16] , which is the starting point of the CaLM1 / CaMKII / CREB signaling pathway. To observe the effect of CaLM1 gene expression level on cell growth state, CaLM1 gene low expression and high expression in SW620 and HCT116 cells were cultured respectively. Under the microscope, cells in the control group showed normal morphology and density; the cell density increased in the CaLM1-OE group; while the number of cells in the siCaLM1 group decreased significantly, the morphology changed, and some cells showed signs of apoptosis or death Figure 3 A). Hoechst staining results showed that the number of cell nuclei increased in the CaLM1 overexpression group, and the density of cell nuclei decreased in the CaLM1 low expression group, indicating that the CaLM1 overexpression group could promote cell proliferation Figure 3B) western blot results showed that CaLM1 overexpression slightly enhanced the expression of CaMKII, while the expression was reduced in the siRNA group, suggesting that the expression of CaMKII might be regulated by CaLM1. In addition, p-CREB was significantly enhanced in the overexpression group, while it was significantly reduced in the siRNA knockdown group, indicating that the expression level of CaLM1 had a significant impact on the phosphorylation of CREB. The total protein expression of CREB did not change significantly, indicating that CaLM1 mainly regulates the activity of CREB by regulating its phosphorylation Figure 3 C).
[0109] Cyclin D1, as a cell cycle regulatory protein, helps cells enter the S phase from the G1 phase, promoting cell proliferation
[17] ; Bcl-2 is a classic anti-apoptotic protein, and its overexpression can inhibit apoptosis; the reduction of Caspase-3 and Caspase-9 activity also inhibits apoptosis. Western blot results showed that the expression of Cyclin D1 and Bcl-2 was enhanced in the CaLM1 high expression group. Conversely, in the CaLM1 low expression group, the expression of Cyclin D1 and Bcl-2 was reduced, while the expression of Cleaved Caspase-3 and Cleaved Caspase-9 was enhanced Figure 3 D), indicating that CaLM1 may promote the apoptosis and inhibit the proliferation of tumor cells by reducing the expression of Cyclin D1 and Bcl-2 and promoting the cleavage of Caspase-3 and Caspase-9.
[0110] These experimental results suggest that CaLM1 may regulate the proliferation and apoptosis of cancer cells through the CaMKII and p-CREB signaling pathways. By targeting the inhibition of CaLM1, it may interfere with its related calcium signaling, thereby reducing the activation of CaMKII and p-CREB, and then inhibiting the proliferation and promoting the apoptosis of cancer cells.
[0111] (3) Pinoresinol blocks the cell cycle and promotes cell apoptosis by inhibiting the CaLM1 signaling pathway
[0112] Combined with the results of bioinformatics analysis, 14 kinds of single compounds were screened from Solanum nigrum, and the CaM inhibitor was screened by phosphodiesterase kit. It was found that pinoresinol could play a similar role to known CaM inhibitors Figure 4 A, Figure 4 B).
[0113] Next, the pinoresinol found to have the effect of CaM inhibitor was further tested to verify the effect of pinoresinol on rectal cancer cells and whether the effect was dependent on CaLM1.
[0114] HCT116 cell lines were treated with low expression of the CaLM1 gene using small interfering RNA (siRNA) transfection technology. Results showed that in the control group (Ctrl), the cell proliferation inhibition rate significantly increased with increasing concentrations of PIN and CGS-9342B; while in the siCaLM1 group, the inhibitory effect of the drugs was significantly weakened. Figure 4 C). This indicates that when CaLM1 gene expression is downregulated, the proliferation-inhibiting effects of PIN and CGS-9342B are significantly reduced, suggesting that the proliferation-inhibiting effect of PIN depends on CaLM1 expression.
[0115] To further verify the antitumor activity of PIN, the effect of PIN on cell viability was detected by the CCK-8 assay to determine its half-maximal inhibitory concentration (IC50) against rectal cancer. 50 The results showed that PIN's inhibitory effect on rectal cancer cells was dose-dependent. Although its inhibitory effect was not as strong as the positive control CGS-9342B, PIN still significantly inhibited cell activity, and both showed a similar dose-dependent trend. Figure 4 D).
[0116] Microscopic observation showed that as PIN concentration increased, the number of cells decreased, morphology changed, and some cells showed signs of death. Figure 5 A). Cell colony formation assays showed that higher PIN concentrations resulted in fewer cell colonies, confirming that PIN has an inhibitory effect on tumor cell proliferation in a dose-dependent manner. Figure 5 B and Figure 5 C). The effect of PIN on the cell cycle of SW620 and HCT116 cells was detected by flow cytometry. After treatment with different concentrations of PIN, SW620 and HCT116 cells showed obvious cell cycle arrest at the G0 / G1 phase. Figure 6 C Figure 6 D、 Figure 6 G, Figure 6 H). Furthermore, the effect of PIN on apoptosis in SW620 and HCT116 cells was examined, and the results showed that the apoptosis rate increased with increasing PIN concentration. Figure 6 A, Figure 6 B. Figure 6 E, Figure 6 F).
[0117] (4) PIN inhibits tumor cell proliferation and induces apoptosis through the CaLM1-dependent signaling pathway.
[0118] To verify the results of flow cytometry and further explore the related signaling pathways, Western blot technique was used to detect the protein expression levels of Cyclin D1, Bcl-2, Caspase-3, Caspase-9, CaLM1, CaMKII, p-CREB and CREB in the following experiments Figure 7 E). The results showed that the expression levels of Cyclin D1 and Bcl-2 were significantly reduced, while the cleavage levels of Caspase-3 and Caspase-9 were significantly increased after PIN treatment. This indicates that PIN inhibits cell proliferation and promotes tumor cell apoptosis by down-regulating the expression of Cyclin D1 and Bcl-2, and activating Caspase-3 and Caspase-9.
[0119] In addition, the expression levels of CaLM1, CaMKII and p-CREB were significantly decreased after PIN treatment, while the expression level of CREB remained basically unchanged Figure 7 A、 Figure 7 B、 Figure 7 C、 Figure 7 D). This result indicates that PIN may inhibit the proliferation of tumor cells and induce their apoptosis in a dose-dependent manner through CaLM1 / CaMKII / CREB-dependent signaling pathway.
[0120] CaLM1 / CaMKII / CREB-dependent signaling pathway.
[0121] (5) In vivo anti-tumor effect of pinoresinol and verification of its molecular mechanism
[0122] To further verify the results of in vitro experiments, the following experiments constructed a HCT116 cell tumor-bearing mouse model. The results showed that PIN significantly inhibited tumor volume and weight at different doses. The inhibitory effect of high-dose PIN group (20.0 mg / kg) was close to that of cisplatin (8.0 mg / kg) Figure 8 A- Figure 8 C). And the TUNEL staining results showed that PIN could dose-dependently promote tumor cell apoptosis, and the number of TUNEL positive cells significantly increased with the increase of dose, and the pro-apoptotic effect of high-dose group was close to that of
[0123] CDDP group Figure 8 D). Finally, to verify the in vivo signaling pathway, immunohistochemical staining experiment was carried out Figure 8 , Figure 8 F- Figure 8The results showed that CaLM1, CaMKII, and p-CREB expression levels were high in the control group, manifested as a large number of brown positive cells in the tumor tissue. With increasing PIN dosage, the number of CaLM1, CaMKII, and p-CREB positive cells in the PIN-treated group significantly decreased. Furthermore, the inhibitory effect of the high-concentration PIN group (20.0 mg / kg) was similar to that of the cisplatin group (8.0 mg / kg). Statistical analysis indicated that the number of CaLM1, CaMKII, and p-CREB positive cells was significantly lower in both the PIN-treated and cisplatin groups than in the control group, with the high-dose PIN group showing a more significant inhibitory effect.
[0124] The above experiments show that pinoresinol can effectively target rectal cancer cells and rectal cancer by inhibiting the CaLM1 / CaMKII / CREB signaling pathway.
[0125] This invention is the first to discover that pinoresinol can inhibit CaM (calm1) and, for the first time, utilizes it as an agent in the CaLM1 / CaMKII / CREB signaling pathway to act on rectal cancer cells and rectal cancer itself. It was found that pinoresinol dose-dependently reduces the expression of CaLM1, CaMKII, and p-CREB, thereby inhibiting the expression of downstream proliferation-related proteins (such as Cyclin D1) and anti-apoptotic proteins (such as Bcl-2), and promoting the cleavage of apoptosis-related proteins Caspase-3 and Caspase-9. These results reveal the role of pinoresinol in inhibiting rectal cancer cell proliferation and promoting apoptosis. This provides a new approach to anti-tumor strategies targeting the CaLM1-dependent pathway. As a low-toxicity natural plant compound, pinoresinol exhibits good anti-tumor potential. Through in vitro and in vivo experiments, this study validated its efficacy in inhibiting rectal cancer, providing experimental evidence for the application of natural products in tumor therapy.
[0126] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. Application of pinoresinol in the preparation of CaM inhibitors or CaLM1 / CaMKII / CREB signaling pathway inhibitors.
2. The application according to claim 1, characterized in that, The pinoresinol can inhibit the expression of CaLM1, and can inhibit the CaLM1 / CaMKII / CREB signaling pathway by inhibiting the expression of CaLM1.
3. A CaM inhibitor or an inhibitor of the CaLM1 / CaMKII / CREB signaling pathway, characterized in that, It includes an effective amount of pinoresinol, which is the only active ingredient.
4. The use of the CaM inhibitor of claim 3, or the CaLM1 / CaMKII / CREB signaling pathway inhibitor, or pinoresinol in at least one of the following, characterized in that, The applications include: a) Prepare products that promote apoptosis of rectal cancer tumor cells by inhibiting the CaLM1 / CaMKII / CREB signaling pathway; b) Prepare products that inhibit the proliferation of rectal cancer tumor cells by inhibiting the CaLM1 / CaMKII / CREB signaling pathway; c) Prepare products for the treatment and / or adjuvant therapy of rectal cancer by inhibiting the CaLM1 / CaMKII / CREB signaling pathway.
5. The application according to claim 4, characterized in that, The product is food, health product, drug, or experimental reagent for basic research.
6. The application according to claim 5, characterized in that, When the product is a drug, pinoresinol is the only active ingredient.
7. A drug for treating and / or adjuvant treatment of cancer by inhibiting the CaLM1 / CaMKII / CREB signaling pathway, characterized in that, It includes an effective amount of pinoresinol and medically acceptable adjuvants.
8. A medicament for treating and / or adjuvant treatment of cancer by inhibiting the CaLM1 / CaMKII / CREB signaling pathway according to claim 7, characterized in that, The cancers mentioned include rectal cancer.
9. A method for inhibiting the expression of CaLM1 in tumor cells for non-diagnostic and non-therapeutic purposes, characterized in that, Tumor cells were co-cultured with an effective concentration of pinoresinol.
10. A method for inhibiting the expression of CaLM1 in tumor cells for non-diagnostic and non-therapeutic purposes according to claim 9, characterized in that, The concentration of pinoresinol is 2-8 μM.