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Application of homoharringtonine in preparation of preparations for inhibiting tumor cells PD-L1

A technology of homoharringtonine and PD-L1, applied in the fields of biomedicine and molecular biology

Pending Publication Date: 2021-11-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And many studies have confirmed that this alkaloid has anti-tumor activity, but the search found that the effect of HHT on PD-L1 has not been reported yet

Method used

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  • Application of homoharringtonine in preparation of preparations for inhibiting tumor cells PD-L1
  • Application of homoharringtonine in preparation of preparations for inhibiting tumor cells PD-L1
  • Application of homoharringtonine in preparation of preparations for inhibiting tumor cells PD-L1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1: Homoharringtonine (HHT) down-regulates PD-L1 protein level in lung cancer cells

[0138] Inoculate H460, H1792, and A549 cells in 12-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, treat with homoharringtonine (HHT) (0, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM) for 20 hours, and then place the culture plate (dish) with cells on ice on the plate, aspirate the medium; wash the cells once with pre-cooled 1×PBS, and then aspirate the liquid in the plate; add an appropriate volume of cell lysate (PIC) containing 1% mixed protease inhibitors, lyse on ice for 30min, and collect The cell lysate was centrifuged at 13200r / min at 4°C for 15min, and the supernatant was taken for sample preparation. Then make a standard curve and measure the protein concentration. First, take five 1.5mL EP tubes, add 10 μL deionized water to each, and then add (0, 1, 2, 4, 8) μL BSA (concentration: 2 mg / mL). For the preparation of the standard...

Embodiment 2

[0141] Example 2: Homoharringtonine (HHT) down-regulates PD-L1 protein levels through the ubiquitin-proteasome pathway

[0142]A549 and H1792 cells were seeded in 6-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, use 1 μM and 2 μM HHT to treat for 20 hours, then extract total RNA. Before extracting RNA, use a vacuum pump to suck out the medium solution in the culture plate, and then add 1×PBS phosphate buffer solution to wash it once. Remove residual liquid; add 500 μL of TRIZOL solution to each 6-well plate for lysis, use a pipette to repeatedly blow the cells during lysis to completely rupture the cells, and then place the 6-well plate at room temperature for 5 minutes; After standing still, put the lysate obtained above into an autoclaved RNase-free 1.5 mL centrifuge tube. Then add 100 μL of chloroform solution and shake fully for about 15 seconds, and finally let it stand at room temperature for 2 minutes; after standing for ...

Embodiment 3

[0149] Example 3: Homoharringtonine (HHT) upregulates TRIM13 protein levels.

[0150] A549 cells were seeded in 12-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, they were treated with different concentrations (0, 0.125 μM, 0.25 μM, 0.5 μM) of homoharringtonine (HHT) for 20 hours, and then the cells were collected and lysed (see Example 1 for specific steps). Then, the protein levels of PD-L1, TRIM13, CHIP, SYVN1 and ACTB were detected by Western blot (see Example 1 for the steps). Inoculate H460 and H1792 cells in 12-well plates (cell culture dishes) at 37°C, CO 2 After culturing in the incubator for 24 hours, they were treated with different concentrations (0, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM) of homoharringtonine (HHT) for 20 hours, and then the cells were collected for lysis (see Example 1 for specific steps) ). The protein levels of PD-L1, TRIM13 and ACTB were detected by Western blot (see Example 1 for steps).

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Abstract

The invention discloses an application of homoharringtonine in preparation of preparations for inhibiting tumor cells PD-L1. The concentration at which the level of tumor cells PD-L1 is effectively down-regulated by homoharringtonine is 0.5-2 [mu] mol / L. In addition, the invention proves that TRIM13 mediates ubiquitination of PD-L1, and HHT increases the level of lung cancer cells TRIM13 and enhances the combination of TRIM13 and PD-L1, indicating that HHT promotes degradation of PD-L1 through an ubiquitin-proteasome pathway by up-regulating the protein level of TRIM13 and enhancing the combination of TRIM13 and PD-L1. Meanwhile, mice experiments prove that HHT can inhibit expression of PD-L1 in LLC tumor-bearing mice, increase the number of killer T cells and inhibit tumor growth. The invention achieves inhibition on development of tumors by down-regulating the PD-L1 level in the tumors, indicating that HHT can participate in tumor immunotherapy as a traditional chemotherapy drug, and has good prospects and practical values. The invention provides reference and inspiration for secondary development of traditional drugs in the field of tumor treatment, and offers a new idea for combined medication of immunotherapy.

Description

technical field [0001] The present invention relates to the application of the non-specific anti-tumor plant alkaloid Homoharringtonine (Homoharringtonine, HHT) in the cell cycle, in particular to the application of Homoharringtonine in the preparation of down-regulated tumor cell PD-L1 (Programmed Cell Death 1 Ligand 1, programmed cell death The application of the receptor ligand 1 (also known as CD274 or B7-H1) level preparation belongs to the technical field of biomedicine and molecular biology. Background technique [0002] PD-L1 is a glycoprotein present in the plasma membrane of tumor cells. After PD-L1 binds to PD1 (Programmed Death Receptor1, programmed death receptor-1) on the surface of T cells, it can induce T cell apoptosis, incapacity, and exhaustion. Inhibition of tumor antigen-specific CD8 + Activation, proliferation and anti-tumor functions of T cells to achieve tumor immune escape. So far, some monoclonal antibodies have achieved good results in cancer tre...

Claims

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Application Information

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IPC IPC(8): A61K31/55A61K36/13A61P35/00
CPCA61K31/55A61K36/13A61P35/00
Inventor 苏玲刘相国刘超李晓鹏王莹莹
Owner SHANDONG UNIV
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