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Application of small-molecule inhibitor minoxidil of PLOD2 in tumor treatment

A dose of minoxidil, a technology for tumor treatment, and the application field in the preparation of tumor treatment drugs

Inactive Publication Date: 2020-08-21
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are currently no reports of PLOD2 inhibition as a therapeutic target in colorectal cancer, or that minoxidil can be used in the treatment of colorectal cancer

Method used

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  • Application of small-molecule inhibitor minoxidil of PLOD2 in tumor treatment
  • Application of small-molecule inhibitor minoxidil of PLOD2 in tumor treatment
  • Application of small-molecule inhibitor minoxidil of PLOD2 in tumor treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Proteomic differences between normal colorectal tissue and colorectal cancer tissue

[0022] 1. Sample collection

[0023] Paraffin-embedded tissue specimens (FFPE) from 20 normal colorectal tissues and 30 colorectal cancer tissues were collected and approved by the Institutional Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (Zhejiang Province, China) (SYXK2018-0012) .

[0024] 2. Sample preparation and detection

[0025] Two biological replicates were taken from 50 cases of FFPE specimens. FFPE tissues were dewaxed and decrosslinked, followed by tissue lysis using a cyclic pressure-based technique (PCT). After reduction and alkylation, the sample protein was digested sequentially with lysC enzyme and trypsin (Hualishi Scientific, China) in a Barocycler (Pressure BioSciences. Inc, USA). Use Sep-Pak Vac 1cc (50mg) C18 column (Waters, MA, USA) to desalt and dry the acidified peptide to obtain purified peptide, r...

Embodiment 2

[0029] Example 2 Verification of PLOD2 expression in colorectal cancer and normal tissues

[0030] 1. Verification of PLOD2 expression by Western blotting (WB)

[0031] Four pairs of fresh tissue samples (colorectal cancer and its negative margin tissue) were used to obtain protein. 40ug protein samples were separated by electrophoresis and separated by gel electrophoresis using a 10% SDS-polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane (Bio-Rad), followed by a mouse anti-PLOD2 antibody (1 :2000, Invitrogen, MA5-26585) and mouse anti-GAPDH antibody (1:2000, Invitrogen, MA5-15738) for detection.

[0032] 2. Validation of PLOD2 expression using tissue chips

[0033] The tissue microarray (TMA) consisted of 79 normal colon and 119 colorectal cancer tissue samples. Tissue samples were used in accordance with national and institutional codes of ethics. TMA was stained using an antibody against PLOD2 (mouse monoclonal antibody, 1:150, MA5-26585, Invitrog...

Embodiment 3

[0035] Example 3 Construction of PLOD2 Knockout Colorectal Cancer Cell Lines

[0036] 1. Selection of cell lines

[0037] Human CRC cell lines SW620 (CCL-227, LOT: 61886714) and SW480 (CCL-228, LOT: 63424079) were purchased from American Type Culture Collection (ATCC). HCT116, HT-29, LoVo and RKO were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). And all cell lines were identified by short tandem repeat markers. The above cell lines were cultured in ATCC's recommended appropriate medium containing 10% fetal bovine serum (Gibco), supplemented with 100 IU / ml sodium penicillin and 100 mg / ml streptomycin in the recommended ATCC. Extract cellular protein, detect PLOD2 expression with the above-mentioned method with Western Blot (such as image 3 Middle a).

[0038] 2. Construction of PLOD2 knockout colorectal cancer cell lines HCT116 and HT-29 CRISPR-Cas9 system (Invitrogen, Cat: A36496), a stable ribonucleoprotein (RNP) transduction complex, i...

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Abstract

The invention provides an application of a small molecule inhibitor minoxidil of PLOD2 in tumor treatment, and the chemical name of the minoxidil is 6-(1-piperidyl)-2, 4-pyrimidinediamine, 3-oxide. The invention specifically relates to a second indication of a small molecule inhibitor minoxidil, namely a therapeutic effect on colorectal cancer by inhibiting PLOD2. According to the method, firstly,proteomics comparison is used for determining the colorectal cancer high-expression protein PLOD2; after partial knockout of a basic group encoding PLOD2, the proliferation, migration and invasion capabilities of colorectal cancer cells are reduced, and the possibility that PLOD2 is used as a colorectal cancer treatment target is proposed. An inhibitor minoxidil of PLOD2 protein is used for inhibiting PLOD2 in a colorectal cancer cell line, the malignant ability of tumor cells is reduced, and it is verified on a mouse model that minoxidil can inhibit growth of intestinal cancer xenograft tumors with high expression of PLOD2.

Description

technical field [0001] The invention belongs to the field of medicine, relates to the application of minoxidil, a small molecule inhibitor of PLOD2, in tumor treatment, and relates to the application of minoxidil, a small molecule inhibitor of PLOD2, in preparing tumor treatment drugs. The discovery of the second indication of the small molecule inhibitor minoxidil—the potential therapeutic effect on colorectal cancer by discovering the inhibition of PLOD2. Background technique [0002] Colorectal cancer is the most common malignant tumor of the digestive tract in clinical practice, and its morbidity and mortality rank among the top three in the world. At present, surgery is still the most important and effective method for the treatment of colorectal cancer, and radiotherapy and chemotherapy, as traditional treatment methods, are still an important part of colorectal cancer. At the same time, new treatments such as targeted therapy and immunotherapy have been more and more...

Claims

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Application Information

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IPC IPC(8): A61K31/506A61P35/00A61P35/04
CPCA61K31/506A61P35/00A61P35/04
Inventor 郑树郭天南徐凯伦邵营宽郑希周碧婷余捷凯
Owner ZHEJIANG UNIV
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