Use of ldn-212320 in the preparation of an anti-tumor medicament

Abstract

The present application relates to the technical field of medicine, in particular to the application of LDN-212320 in the preparation of anti-tumor drugs. 50 The present application finds that LDN-212320 has good in-vitro anti-tumor effect, can significantly inhibit the proliferation of tumor cells (IC 50 is 15.89-21.00 μM), induces tumor cell cycle arrest, and promotes tumor cell apoptosis. It is shown that LDN-212320 provides a new treatment for tumors and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of LDN-212320 in the preparation of antitumor drugs. Background Technology

[0002] Currently, traditional treatments for cancer include surgical resection, radiotherapy, and chemotherapy, but chemotherapy drugs have significant toxic side effects. Therefore, developing new therapeutic drugs remains a hot topic in cancer treatment. Excessive proliferation of tumor cells is one of the important reasons for high mortality and poor prognosis. Screening for new anti-tumor drugs based on the phenotype of inhibiting tumor cell proliferation will provide new options for cancer treatment. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides the application of LDN-212320 in the preparation of antitumor drugs. This invention reveals that LDN-212320 can significantly inhibit the in vitro proliferation of tumor cells, induce tumor cell cycle arrest, and promote tumor cell apoptosis, thus serving as a novel antitumor drug.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of LDN-212320 in the preparation of antitumor drugs, wherein the chemical structural formula of LDN-212320 is shown in Formula I:

[0006]

[0007] Preferably, the anti-tumor effect is to inhibit the proliferation of tumor cells, induce tumor cell cycle arrest, and promote tumor cell apoptosis.

[0008] Preferably, the tumor includes lung cancer and liver cancer.

[0009] The present invention provides an antitumor drug, the active ingredient of which includes LDN-212320, the chemical structural formula of which is shown in Formula I;

[0010]

[0011] Preferably, the drug has an IC50 effect on tumor cells. 50 The range is 15.89–21.00 μM.

[0012] Preferably, the tumor includes lung cancer and liver cancer.

[0013] Preferably, the active ingredient of the drug is LDN-212320.

[0014] Preferably, the drug includes pharmaceutically acceptable excipients.

[0015] Preferably, the dosage form of the drug includes oral or injectable formulations.

[0016] Beneficial effects:

[0017] This invention has revealed that LDN-212320 exhibits good in vitro antitumor activity, significantly inhibiting the proliferation of tumor cells (IC50). 50 At concentrations ranging from 15.89 to 21.00 μM, LDN-212320 induces cell cycle arrest and promotes apoptosis in tumor cells. This indicates that LDN-212320 offers a novel treatment option for tumors and has promising application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 Results of the effect of LDN-212320 on tumor cell viability;

[0020] Figure 2 Results of the effect of LDN-212320 on tumor cell cycle distribution;

[0021] Figure 3 The results show the effect of LDN-212320 on tumor cell apoptosis. Detailed Implementation

[0022] This invention provides the application of LDN-212320 in the preparation of antitumor drugs, wherein the chemical structural formula of LDN-212320 is shown in Formula I:

[0023]

[0024] In this invention, the anti-tumor effect is preferably the inhibition of tumor cell proliferation, the induction of tumor cell cycle arrest, and the promotion of tumor cell apoptosis; the tumor preferably includes lung cancer and liver cancer; the lung cancer preferably includes non-small cell lung cancer.

[0025] This invention has revealed that LDN-212320 exhibits good in vitro antitumor activity, significantly inhibiting the proliferation of tumor cells (IC50). 50 At concentrations ranging from 15.89 to 21.00 μM, LDN-212320 induced tumor cell cycle arrest in a concentration-dependent manner and promoted tumor cell apoptosis in a concentration-dependent manner. This indicates that LDN-212320 provides a new therapeutic option for tumors and has good application prospects.

[0026] This invention provides an antitumor drug, the active ingredient of which includes LDN-212320, the chemical structural formula of which is shown in Formula I. In this invention, the active ingredient of the drug is preferably LDN-212320; the drug has an IC50 inhibitory effect on tumor cells. 50 The preferred concentration is 15.89–21.00 μM; the tumor cells preferably include lung cancer cells and liver cancer cells; the lung cancer cells are preferably lung cancer cells H460 or H1299; the liver cancer cells are preferably liver cancer cells HepG2; the drug preferably includes pharmaceutically acceptable excipients; the dosage form of the drug preferably includes oral or injectable formulations.

[0027] To further illustrate the present invention, the application of LDN-212320 provided by the present invention in the preparation of antitumor drugs is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] 1. Materials

[0030] Human lung cancer cells H460 and H1299, and human hepatocellular carcinoma cells HepG2 were all purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; LDN-212320 (#T15728) was purchased from Shanghai Taoshu Biotechnology Co., Ltd.; RPMI 1640 medium (#SH3080901, HyClone), MEM medium (#SH30024.01, HyClone), PBS buffer (#SH30256.01, HyClone), and penicillin / streptomycin antibiotic solution (SV30010, HyCl) were also used. All of the following were purchased from Beijing Northern Tongzheng Biotechnology Development Co., Ltd.; 0.25% trypsin (#FG301-01) was purchased from Beijing TransGen Biotechnology Co., Ltd.; fetal bovine serum (#1027-106) was purchased from Gibco, Inc. (USA); MTT (#M2003) and PI (#P4170) were purchased from Sigma-Aldrich, Inc. (USA); apoptosis kit (#FXP022) was purchased from Sizhengbai Co., Ltd.; dimethyl sulfoxide and ethanol were purchased from Beijing Tongguang Fine Chemical Co., Ltd.; Scientific 3131 CO2 cell culture incubator was purchased from Thermo Fisher Scientific, Inc. (USA); Synergy H1 microplate reader was purchased from BioTek, Inc. (USA); and BD FACS Calibur flow cytometer was purchased from BD Biosciences, Inc. (USA).

[0031] 2. Cell Culture

[0032] Human lung cancer cells H460 and H1299 were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 U / mL streptomycin. Human liver cancer cells HepG2 were cultured in MEM medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 U / mL streptomycin. All cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0033] 3. Effects of LDN-212320 on tumor cell viability

[0034] Human lung cancer cells H460 and H1299, and human liver cancer cells HepG2, were seeded in 96-well plates (5000 cells / 100 μL / well) and cultured at 37°C with 5% CO2 for 24 h. Different concentrations (0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μM) of LDN-212320 were added, and the cells were incubated for 48 h. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 3–4 h. The supernatant was discarded, and 150 μL of dimethyl sulfoxide was added to each well. After shaking at room temperature for 10 min, the absorbance at 490 nm was measured using a microplate reader to calculate cell viability. With the cell viability of the solvent control group (without drug) as 100%, cell viability was calculated as follows: (absorbance value of the drug-treated group - absorbance value of the blank control group) / (absorbance value of the control group - absorbance value of the blank control group) × 100%. The IC50 of the drug on the cells was calculated using GraphpadPrism 8 software. 50 The results are shown below. Figure 1 .

[0035] Depend on Figure 1 It is known that LDN-212320 has an IC50 effect on human lung cancer cells H460 and H1299 and human liver cancer cells HepG2. 50 At concentrations of 15.89 μM, 17.65 μM, and 21.00 μM, respectively, all showed good inhibitory effects. This indicates that LDN-212320 can inhibit tumor cell proliferation in vitro.

[0036] 4. Effects of LDN-212320 on tumor cell cycle distribution

[0037] Human lung cancer cells H460 and H1299 were seeded in 6-well plates (4 × 10⁻⁶ m²). 5Cells were cultured (cells / well) for 24 h, and then treated with LDN-212320 at final concentrations of 10 μM and 20 μM, respectively. After 48 h of treatment, cells were collected by trypsin digestion, centrifuged at 1000 rpm for 5 min, resuspended in 1 mL of PBS buffer, centrifuged again and the supernatant was discarded. Cells were then resuspended in 1 mL of pre-chilled 70% ethanol and fixed overnight at 4°C. The next day, cells were centrifuged at 1000 rpm for 5 min, washed once with PBS buffer, centrifuged again and the supernatant was discarded. The cell pellet was resuspended in 400 μL of PBS buffer containing RNase A (final RNase A concentration of 100 μg / mL) and incubated in a 37°C water bath for 30 min to allow for complete RNA lysis. Then, 4 μL of LPI staining solution (final concentration of 50 μg / mL) was added and incubated in a 37°C water bath in the dark for 30 min. After filtering the cells through a 300-mesh cell sieve, the fluorescence intensity of PI was detected by flow cytometry, and cell cycle analysis was performed using Flowjo software. The results are shown in [Figure 1]. Figure 2 .

[0038] Depend on Figure 2 It was found that after treatment with LDN-212320, the proportion of H460 and H1299 cells in the S phase significantly increased. This indicates that LDN-212320 can induce S phase arrest in tumor cells in a concentration-dependent manner.

[0039] 5. Effects of LDN-212320 on tumor cell apoptosis

[0040] Human lung cancer cells H460 and H1299 were seeded in 6-well plates (4 × 10⁻⁶ m²). 5 Cells were cultured (cells / well) for 24 h, and then treated with LDN-212320 at final concentrations of 10 μM and 20 μM, respectively. After 48 h of treatment, cells and culture medium were collected by trypsin digestion. Cells were centrifuged at 1000 rpm for 5 min, and resuspended in 1 mL of PBS buffer. The supernatant was discarded after centrifugation. Following the apoptosis kit instructions, binding buffer was diluted with deionized water at a ratio of 1:3. Cells were resuspended in 100 μL of the diluted binding buffer, stained with 5 μL of Annexin V in the dark for 30 min, and then 10 μL of PI staining solution at a concentration of 20 μg / mL was added. After mixing, 400 μL of PBS buffer was added, and the cells were filtered through a 300-mesh sieve and analyzed by flow cytometry. The apoptosis rate was analyzed using Flowjo software. Results are shown in [Figure number missing]. Figure 3 .

[0041] Depend on Figure 3 It was found that LDN-212320 treatment significantly increased the apoptosis rate of H460 and H1299 cells. This indicates that LDN-212320 promotes tumor cell apoptosis in a concentration-dependent manner.

[0042] In conclusion, LDN-212320 can significantly inhibit the proliferation of tumor cells, induce tumor cell cycle arrest, and promote tumor cell apoptosis, and can be used as a new anti-tumor drug.

[0043] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of LDN-212320 in the preparation of antitumor drugs, wherein the chemical structural formula of LDN-212320 is shown in Formula I: Formula I: ; The tumors are non-small cell lung cancer and liver cancer.

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