Application of combination of nicotinic acid and metformin in blocking progress of esophageal squamous carcinoma
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-03-31
AI Technical Summary
Existing treatments for esophageal squamous cell carcinoma, such as radiotherapy and chemotherapy, have limited efficacy, and immunotherapy has a low response rate. Furthermore, patients with esophageal squamous cell carcinoma are often in the advanced stage and have a high metastasis rate, resulting in poor prognosis. There is a lack of effective multi-stage suppression strategies.
The combined use of niacin and metformin inhibits FRMD8 expression and blocks the JAK1-STAT3 axis through different mechanisms, synergistically inhibiting the proliferation, invasion and migration of esophageal squamous cell carcinoma cells, regulating the tumor immune microenvironment, and significantly inhibiting tumor growth and metastasis.
The combination of niacin and metformin significantly inhibits the proliferation, invasion and migration of esophageal squamous cell carcinoma cells in vitro and in vivo, increases the proportion of T cells, inhibits immune escape, covers multiple stages of the disease, is especially effective for lung metastases, has high safety, and is easy to promote in clinical practice.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of niacin combined with metformin in blocking the progression of esophageal squamous cell carcinoma. Background Technology
[0002] Esophageal cancer is one of the most common malignant tumors of the upper gastrointestinal tract in my country, with the vast majority of patients having esophageal squamous cell carcinoma (ESCC). In clinical treatment, radiotherapy and chemotherapy have limited efficacy, and emerging immunotherapies have generally low response rates and high resistance rates. Furthermore, most ESCC patients are diagnosed at an advanced stage, and the high metastasis and recurrence rates lead to poor prognosis. Therefore, exploring new treatment drugs and strategies is of great significance.
[0003] The development of esophageal squamous cell carcinoma is a multi-step, multi-stage continuous process: a morphological and molecular evolutionary process from normal epithelium → mild / moderate / severe dysplasia → carcinoma in situ → invasive carcinoma, including progressive morphological changes and cumulative genetic and epigenetic variations at the molecular level. Overactivation of the JAK / STAT pathway is often associated with various malignant phenotypes of tumors. After STAT3 is activated by JAK1 phosphorylation, it enters the nucleus, promotes target gene transcription, and contributes to tumor growth, metastasis, drug resistance, and immune escape. Targeted therapy research targeting the JAK1-STAT3 axis holds great promise.
[0004] Metformin is the first-line drug of choice for the clinical treatment of type 2 diabetes. In recent years, it has been found that metformin can inhibit the proliferation and metastasis of tumor cells and enhance the effect of immunotherapy. However, the mechanism of action of metformin in anti-tumor therapy and the sensitive population still need further research and screening. At the same time, better combination strategies of metformin with other drugs such as radiotherapy, chemotherapy and immunotherapy need to be explored.
[0005] Niacin is a vitamin Niacin is one of the main components of niacin and can be obtained from various dietary sources. It participates in the regulation of carbohydrate, lipid, and protein metabolism and plays a key role in cellular processes such as ATP synthesis, mitochondrial homeostasis, and DNA damage repair.
[0006] The structural formula of nicotinic acid is:
[0007]
[0008] Recent studies have found that niacin can inhibit tumor growth and sensitize chemotherapy drugs. However, it is currently unknown whether niacin can be used in combination with metformin to enhance its anti-cancer effect. Summary of the Invention
[0009] This invention reveals that niacin and metformin can inhibit FRMD8 expression through different mechanisms, thereby suppressing the activation of the JAK1-STAT3 axis. The combination of niacin and metformin significantly inhibited the proliferation and invasion / migration of esophageal squamous cell carcinoma cells in vitro compared to either drug alone; in a mouse subcutaneous tumor model, the combination of niacin and metformin significantly inhibited the infiltration of PMN-MDSCs cells and improved... The proportion of T cells has a better effect on inhibiting tumor growth than single drugs, thus the present invention provides the application of niacin combined with metformin in blocking the progression of esophageal squamous cell carcinoma.
[0010] The objective of this invention is achieved through the following technical solution:
[0011] The first aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of esophageal squamous cell carcinoma, the pharmaceutical composition comprising nicotinic acid and metformin.
[0012] Furthermore, the nicotinic acid is nicotinic acid or its pharmaceutically acceptable salt, prodrug, and metabolite.
[0013] Furthermore, in the pharmaceutical composition, the ratio of nicotinic acid to metformin, based on the mass of nicotinic acid and metformin, is 0.1 to 3:1.
[0014] Furthermore, the pharmaceutical composition also includes a carrier or excipient, and the nicotinic acid, metformin and the carrier or excipient are mixed to prepare a pharmaceutically acceptable dosage form.
[0015] Furthermore, the pharmaceutical composition is in the form of a solid dosage form or a liquid dosage form.
[0016] Furthermore, the drug composition can be administered via any one of the following routes: gastrointestinal, rectal, injection, mucosal, and respiratory tract.
[0017] The second aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for blocking the progression of esophageal squamous cell carcinoma.
[0018] Furthermore, the blocking of esophageal squamous cell carcinoma progression includes inhibiting in situ growth of esophageal squamous cell carcinoma and metastasis to any organ.
[0019] Furthermore, the metastases include at least one of the following: lymph node metastases, lung metastases, liver metastases, bone metastases, and brain metastases.
[0020] The advantages of this invention compared to the prior art are as follows:
[0021] 1. This application is the first to discover that nicotinic acid and metformin inhibit FRMD8 expression through a differential mechanism. Specifically, nicotinic acid downregulates its protein level, while metformin inhibits its mRNA transcription. The two work together to block the JAK1-STAT3 signaling pathway, with complementary and precise targeting effects.
[0022] 2. The combination of niacin and metformin effectively inhibits the proliferation, invasion, and migration of esophageal squamous cell carcinoma cells in vitro. In mouse models, it significantly inhibits tumor growth and regulates the tumor immune microenvironment (reducing PMN-MDSCs infiltration and enhancing tumor function). The T-cell ratio inhibits immune escape, and its effect is far superior to single-drug therapy.
[0023] 3. The combination of niacin and metformin can inhibit the growth of esophageal squamous cell carcinoma and has a clear inhibitory effect on the high incidence of lung metastasis, covering multiple stages of the disease, including the advanced stage and the metastatic stage.
[0024] 4. Niacin (vitamin) Both the active ingredient (the active ingredient) and metformin (a commonly used clinical drug) have well-established safety profiles and good biocompatibility. They do not require the development of entirely new compounds, have low development costs and low translational risks, and are better tolerated than radiotherapy, chemotherapy and immunotherapy, making them easy to promote in clinical practice.
[0025] 5. This application breaks through the existing treatment bottlenecks and provides a new multi-dimensional strategy to inhibit tumor progression in response to the limited efficacy of radiotherapy and chemotherapy, low response rate of immunotherapy, and high rate of metastasis and recurrence in esophageal squamous cell carcinoma, which helps to improve patient prognosis. Attached Figure Description
[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments:
[0027] Figure 1 Schematic diagram of how nicotinic acid and metformin inhibit the FRMD8-JAK1-STAT3 axis through different pathways;
[0028] Figure 2 A schematic diagram illustrating how the combination of niacin and metformin significantly inhibited the proliferation of esophageal squamous cell carcinoma cells in vitro compared to single-agent therapy.
[0029] Figure 3 A schematic diagram illustrating how the combination of niacin and metformin significantly inhibited the invasion and migration of esophageal squamous cell carcinoma cells in vitro compared to single-agent therapy.
[0030] Figure 4 This diagram illustrates how the combination of niacin and metformin significantly inhibited the growth of subcutaneous esophageal squamous cell carcinoma in immunocompetent mice compared to monotherapy. Detailed Implementation
[0031] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.
[0032] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0033] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.
[0034] Example 1: Detection of the inhibition of FRMD8 expression and downstream JAK1-STAT3 signaling pathway by nicotinic acid and metformin.
[0035] This embodiment uses Western blot to detect the effects of nicotinic acid and metformin on FRMD8 protein levels, and RT-PCR to analyze their effects on FRMD8 mRNA levels, specifically including:
[0036] Lentiviral cells stably overexpressing FRMD8 were packaged into 293T cells. Esophageal squamous cell carcinoma cells in logarithmic growth phase were seeded into six-well plates, achieving a confluence of 30%-40% at infection. Approximately 24 hours after seeding, lentiviral infection was performed: 1 ml of complete culture medium, polybrene reagent (working concentration 5 μg / ml), and 1 ml of virus solution were added, mixed thoroughly, and incubated at 37°C for 24 hours. After 24 hours, selection was performed by adding fresh complete culture medium containing Puromycin or G418. The working concentration of Puromycin was 1 μg / ml.
[0037] We seeded KYSE30 cells stably overexpressing FRMD8 into six-well plates. After adhesion, we treated them with 20 mM nicotinic acid or 10 mM metformin for 48 hours. We then extracted total cell protein for Western blot experiments. The results showed that both nicotinic acid and metformin could inhibit the JAK1-STAT3 signaling pathway by downregulating FRMD8 protein levels. Figure 1 ).
[0038] Total protein extraction from adherent cells: Discard the culture medium, wash 3 times with pre-cooled PBS, add an appropriate amount of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, scrape off all adherent cells and transfer them to a 1.5 ml centrifuge tube, lyse on ice for 15 min, centrifuge at 12,000 rpm for 15 min at 4°C, and collect the supernatant as total cell protein.
[0039] Western blotting of proteins: Prepare SDS-PAGE separating gels of the required concentration according to the molecular weight of the target protein. Perform electrophoresis and membrane transfer according to standard methods, and block with 5% skim milk powder on a level shaker at room temperature for 1 h. Incubate the primary antibody overnight at 4°C, and then incubate with a diluted secondary antibody corresponding to the species of the primary antibody at room temperature for 60 min. Immerse the PVDF membrane in freshly prepared chemiluminescence solution for 10 s, remove it, place it on an exposure plate, aspirate excess chemiluminescence solution, and collect the results using an exposure instrument.
[0040] We seeded KYSE30 and KYSE150 cells in six-well plates. After adhesion, we treated them with 20 mM nicotinic acid or 10 mM metformin for 48 hours, respectively. RNA was then extracted for RT-PCR experiments. The results showed that nicotinic acid treatment did not affect the mRNA level of FRMD8, while metformin significantly downregulated the mRNA level of FRMD8. Therefore, nicotinic acid and metformin inhibited FRMD8 expression through different mechanisms. Figure 1 ).
[0041] Total RNA extraction from cells using the TRIzol method: Cells were washed twice with pre-chilled PBS. After aspirating the liquid, 1 ml of TRIzol was added, and the mixture was repeatedly pipetted into an RNase-free Eppendorf tube. The tube was incubated at room temperature for 30 min, followed by the addition of 200 µl of chloroform and vigorous shaking for 3 min. The tube was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was carefully aspirated and transferred to a new Eppendorf tube. 500 µl of isopropanol was added, and the mixture was inverted and incubated at room temperature for 15 min. The tube was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was carefully discarded, leaving the feathery precipitate. The precipitate was washed with 75% ethanol, centrifuged to remove the supernatant, and air-dried at room temperature. 30 μl of RNase-free ultrapure water was added, and the concentration and purity of the sample were determined using Nanodrop.
[0042] RNA reverse transcription was performed using the Yessen Hifair® AdvanceFast 1st Strand cDNA Synthesis Kit. The real-time PCR system consisted of: 5 μl of 2×SYBR PCR master Mix, 1 μl of cDNA template diluted 5-10 times, 0.3 μl of forward primer (10 μmol / L), 0.3 μl of reverse primer (10 μmol / L), and 3.4 μl of ultrapure water. Each sample was prepared in triplicate. The Ct method is used to calculate the relative expression level of the target gene.
[0043] Example 2: Detection of the inhibitory effect of niacin combined with metformin on the in vitro proliferation of esophageal squamous cell carcinoma cells.
[0044] This embodiment uses the CCK-8 assay with KYSE30 and KYSE150 esophageal squamous cell carcinoma cells as the research subjects to compare the inhibitory effects of niacin monotherapy, metformin monotherapy, and the combination of two drugs on cell proliferation, and to verify whether the combination of two drugs has a more significant anti-proliferative activity in vitro than that of monotherapy. Specifically, it includes:
[0045] KYSE30 and KYSE150 cells were seeded in 96-well plates. After adhesion, 20 mM nicotinic acid and 10 mM metformin were added. Four replicates were set up for each group. 10 μl of CCK-8 was added every 24 hours. After incubation at 37°C for 2 hours, the cells were removed and the wavelength at 450 nm was measured to plot the cell growth curve.
[0046] like Figure 2 The results showed that both niacin and metformin monotherapy could inhibit the proliferation of esophageal squamous cell carcinoma cells, while the combination of the two drugs had a more significant inhibitory effect than the monotherapy.
[0047] Example 3: Detection of the effect of niacin combined with metformin on the in vitro invasion and migration ability of esophageal squamous cell carcinoma cells.
[0048] This embodiment uses transwell assays to detect migration and invasion abilities, comparing the blocking effects of different drug groups (single drug and combination therapy) on the invasion and migration abilities of KYSE30 and KYSE150 cells. Specifically, it includes:
[0049] KYSE30 and KYSE150 cells were seeded in six-well plates. After adhesion, 20 mM nicotinic acid and 10 mM metformin were added, and after 48 hours of treatment, transwell assays were performed. 100 μl of free culture medium was added to the migration chamber, and 100 μl of free culture medium containing Matrigel was added to the invasion chamber (3 μl Matrigel was added to every 100 μl of medium and mixed well). The chambers were then incubated for activation. Cells were digested and resuspended in free culture medium. 10 μl of the cell suspension was slowly added to the counting chamber of a cell counting chamber, and the cells were counted under a microscope. The cell concentration was adjusted to [specific value missing] using free culture medium. Cells / ml, mix well. Remove the transwell chamber from the incubator and discard the liquid. Add 100 μl of cell suspension to the upper chamber, so that each well contains 20,000 cells. Add 600 μl of culture medium containing 20% FBS to the lower chamber and incubate for 24–48 h in a constant temperature incubator. Afterward, remove the transwell chamber, fix with 4% paraformaldehyde at room temperature for 20 min, stain with crystal violet solution for 5 min, observe and image under an inverted microscope.
[0050] like Figure 3 The results showed that when niacin and metformin were used in combination to treat KYSE30 and KYSE150 cells, their invasion and migration abilities in vitro were more effectively inhibited.
[0051] Example 4: Verification of the effects of niacin combined with metformin on the growth of subcutaneous esophageal squamous cell carcinoma and the tumor immune microenvironment in mice.
[0052] This embodiment establishes a C57BL / 6 mouse subcutaneous esophageal squamous cell carcinoma model and compares the inhibitory effects of single-drug and combination therapies on in situ tumor growth through in vivo drug administration experiments; simultaneously, flow cytometry is used to detect PMN-MDSCs cell infiltration in tumor tissue and The study aimed to investigate the T-cell ratio and its regulatory effect on the tumor immune microenvironment through dual-drug combination therapy, thereby verifying its synergistic tumor-suppressive effect in vivo. Specifically, this included:
[0053] mEC25 cells were seeded subcutaneously in C57BL / 6 mice, and each mouse was injected with... Cells were selected and treated with control, niacin monotherapy, metformin monotherapy, and niacin / metformin combination therapy. Niacin was administered by gavage at a dose of 600 mg / kg every two days, and metformin was administered by intraperitoneal injection at a dose of 250 mg / kg every two days. The combination of niacin and metformin significantly inhibited the growth of subcutaneous tumors compared to niacin monotherapy or metformin monotherapy. At the experimental endpoint, tissue samples were collected for flow cytometry analysis: subcutaneous tumor tissue was collected from the back of mice, pus and ulcerated scabs were removed, and the tissue was placed in a 5 ml tube. 3 ml of digestion solution (prepared by mixing 1 ml DNase + 1 ml collagenase IV + 8 ml DMEM) was added, and the tissue was minced and digested at 37°C for 2000 rpm for 30 min on a shaker. Cells were sieved through a 200-mesh sieve, centrifuged at 4°C for 2500 rpm for 5 min, and the supernatant was discarded. Cells were washed once with flow cytometry washing buffer. Add 1 ml of erythrocyte lysis buffer and incubate on ice for 10 min to lyse red blood cells. Centrifuge and wash once with flow cytometry wash buffer. Resuspend the cell pellet with flow cytometry wash buffer and divide into fractions. Add 0.25 μg CD16 / 32 antibody to each tube containing 100 μl of cells and incubate on ice for 10 min. Add 1.5 μl of surface marker antibody to each tube and incubate at room temperature in the dark for 30 min. Centrifuge and wash twice with flow cytometry buffer. Analyze using Cytoflex.
[0054] Comparison Figure 4 Compared with the single-drug group, the combination therapy group significantly inhibited the infiltration of PMN-MDSCs cells and significantly upregulated [their activity]. The proportion of T cells. The combination of niacin and metformin is more effective than single-agent therapy in inhibiting the growth of esophageal squamous cell carcinoma.
[0055] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. 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 be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.
Claims
1. A pharmaceutical composition for the prevention or treatment of esophageal squamous cell carcinoma, characterized in that, The pharmaceutical composition comprises nicotinic acid and metformin.
2. The pharmaceutical composition of claim 1, wherein, The nicotinic acid is nicotinic acid or pharmaceutically acceptable salts, prodrugs and metabolites thereof.
3. The pharmaceutical composition of claim 1, wherein, In the pharmaceutical composition, the ratio of nicotinic acid to metformin is 0.1-3:1 by mass of nicotinic acid and metformin.
4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The pharmaceutical composition further comprises carriers or excipients, and the nicotinic acid, metformin and carriers or excipients are mixed to prepare a pharmaceutically acceptable dosage form.
5. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition is in a solid dosage form or a liquid dosage form.
6. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition is administered by any one of the following routes: gastrointestinal administration, rectal administration, injection administration, mucosal administration and respiratory administration.
7. Use of the pharmaceutical composition according to any one of claims 1-6 in the preparation of a drug for blocking the progression of esophageal squamous cell carcinoma.
8. Use according to claim 7, characterized in that, The blocking of the progression of esophageal squamous cell carcinoma includes inhibition of the in situ growth of esophageal squamous cell carcinoma and metastasis to any organ.
9. Use according to claim 7, characterized in that, The metastasis includes at least one of the following: lymph node metastasis, lung metastasis, liver metastasis, bone metastasis and brain metastasis.