CN114099689A

CN114099689A - Application of NKAP inhibitor and cisplatin in treatment of cancer

Info

Publication number: CN114099689A
Application number: CN202210090363.3A
Authority: CN
Inventors: 李腾; 夏晴; 韩秋颖; 李卫华; 陈亮; 陈媛; 周涛; 李爱玲
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2022-03-01
Anticipated expiration: 2042-01-26
Also published as: CN114099689B

Abstract

The invention belongs to the field of biomedicine, and particularly relates to an application of NKAP inhibitor and cisplatin in treatment of cancer. Specifically, the invention provides a pharmaceutical composition comprising a NKAP inhibitor and a chemotherapeutic agent. Preferably, the inhibitor is an agent used in a siRNA interference method and the chemotherapeutic agent is cisplatin.

Description

Application of NKAP inhibitor and cisplatin in treatment of cancer

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an application of NKAP inhibitor and cisplatin in treatment of cancer.

Background

Cisplatin, also known as cis-dichlorodiammineplatinum, is a platinum-containing anticancer drug in the form of orange yellow or yellow crystalline powder, which is sparingly soluble in water and readily soluble in dimethylformamide and can be gradually converted into trans form and hydrolyzed in aqueous solution. Cisplatin cross-links to DNA strands and exhibits cytotoxic effects. Because the concentration of intracellular chloride ions is low (4 mmol/L), the chloride ions are replaced by water, the charge is positive, the double-function group has the function similar to an alkylating agent, the double-function group can be combined with the basic group of DNA in a cell nucleus to form three forms of cross-linking, DNA damage is caused, DNA replication and transcription are damaged, and the synthesis of RNA and protein is inhibited at high concentration. Cisplatin has the advantages of wide anticancer spectrum, effectiveness of hypoxic cells, strong action and the like, is widely used for treating testicular cancer, ovarian cancer, uterine cancer, bladder cancer, neck cancer, prostatic cancer, brain cancer and the like, and has obvious curative effect. However, cisplatin has toxicity and side effects when used for treating cancer, so that analogs with low toxicity and similar clinical effects to cisplatin are continuously sought.

Chemotherapy is an important modality of tumor treatment. In order to improve the treatment effect of tumors, a chemotherapy scheme combining two or more medicaments is adopted clinically. However, the combination may affect the efficacy and toxicity of chemotherapy due to the interaction between drugs or the cycle specificity of the anticancer drugs, so that the correct sequence of administration not only increases the antitumor efficacy, but also reduces the toxic side effects.

From a pharmaceutical perspective, the order of administration of the chemotherapeutic regimen should follow the following three principles: interaction principle, cell dynamics principle, and stimulatory principle.

Disclosure of Invention

As used hereinafter, the terms "having," "including," or any grammatical variants thereof, are used in a non-exclusive manner. Thus, these terms may refer to the absence of other features in the entities described in this context than those introduced by these terms, and may refer to the presence of one or more other features than those presented.

Furthermore, as used hereinafter, the terms "preferably," "more preferably," "most preferred," "particularly," "more particularly," "specifically," "more specifically," or similar terms are used in conjunction with optional features, without limiting other possibilities.

The term "treating" refers to ameliorating the disease or disorder referred to herein or the symptoms associated therewith to a significant extent.

In order to provide a novel approach to the treatment of cancer, the present invention provides the use of a NKAP inhibitor in combination with a chemotherapeutic agent, said chemotherapeutic agent being cisplatin, in the manufacture of a medicament for the treatment of cancer. When the NKAP inhibitor and the cisplatin are used simultaneously, the NKAP inhibitor and the cisplatin can generate a synergistic effect, so that cancer cells are killed and killed more efficiently, and the purpose of treating cancers is achieved.

In one aspect, the present invention provides a pharmaceutical composition comprising a NKAP inhibitor and a chemotherapeutic agent.

Preferably, the inhibitor comprises an agent used in siRNA interference, CRISPR/cas9 method, homologous recombination, gene knockout, gene replacement, chemical drug method.

Preferably, the inhibitor used in the present invention is siRNA, that is, siRNA targeting NKAP, more specifically, the NKAP-targeting siRNA used in the present invention is product of Invitrogen with the product numbers HSS128451, HSS 188063.

The term "chemotherapeutic agent" includes alkylating agents, antimetabolites, antitumor antibiotics, antitumor botanicals and miscellaneous.

Preferably, the heteroclasses include, but are not limited to, cisplatin, carboplatin, platinum oxalate, nedaplatin, oxaliplatin, asparaginase.

Preferably, the chemotherapeutic agent is cisplatin.

Preferably, the pharmaceutical composition is a pharmaceutically compatible combined preparation.

The term "pharmaceutical composition" as used herein comprises the NKAP inhibitor and cisplatin according to the present invention, optionally together with one or more pharmaceutically acceptable carriers or diluents.

It will be understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend upon the amount of active ingredient combined therewith, the route of administration, and other well-known variables.

Preferably, the pharmaceutical carrier used may be, for example, a solid, gel or liquid.

Illustrative examples of such solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, degradable polymers such as PLGA, and the like.

Illustratively, the liquid carrier is a phosphate buffered saline solution, a syrup, an oil such as peanut oil and olive oil, water, an emulsion, various types of wetting agents, a sterile solution, or the like.

Preferably, the diluent is selected so as not to affect the biological activity of the NKAP inhibitor and/or cisplatin. Examples of such diluents are distilled water, physiological saline, Ringer's solution, glucose solution and Hank's solution.

Preferably, the pharmaceutical composition may also contain other non-toxic, non-therapeutic, non-immunogenic stabilizers, active oxygen scavengers, and the like.

The pharmaceutical composition of the present invention can be formulated into various dosage forms as required, and can be administered at a dose that is determined by a physician in consideration of the kind, age, weight and general condition of a patient, administration mode, and the like. The pharmaceutical composition may be in any dosage form and administered in any manner. The preparation can be prepared into tablets, capsules, granules, oral liquid, suspensions, injections, microspheres, liposomes and the like according to a conventional method. The use mode comprises oral administration or parenteral administration, wherein the parenteral mode is intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection and the like.

In another aspect of the invention, the NKAP inhibitor and the chemotherapeutic agent, and the application of the pharmaceutical composition in preparing the product for treating cancer are also provided.

Preferably, the cancer comprises any one of: acute lymphocytic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal carcinoma, appendiceal carcinoma, astrocytoma, atypical teratoma, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, brain stem glioma, breast carcinoma, Burkitt's lymphoma (Burkitt's lymphoma), carcinoid tumor, cerebellar astrocytoma, cervical carcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon carcinoma, colorectal carcinoma, craniopharyngioma, endometrial carcinoma, ependymoma, esophageal carcinoma, extracranial germ cell tumor, gonadal germ cell tumor, extrahepatic bile duct carcinoma, gallbladder carcinoma, gastric carcinoma, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck carcinoma, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal carcinoma, hypothalamic and visual pathway glioma, melanoma, kaposi sarcoma (kaposi sarcoma), laryngeal carcinoma, medulloblastoma, melanoma, merkelell carcinosoma, mesothelioma, oral cavity cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignancy potential tumor, papillomatosis, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, nasopharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymus cancer, thymoma, thyroid cancer, urinary tract cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Fahrenheit macroglobulinemia (macroglobulinemia), and Wilms' tumor (wilms tumor).

Preferably, the cancer is cervical cancer, more preferably, a hela (hela) cell line.

Finally, the present invention also provides a method of treating cancer comprising the use of an NKAP inhibitor and a chemotherapeutic agent as defined above, or the use of a pharmaceutical composition as defined above.

Preferably, the cancer is cervical cancer, such as the HELA cell line used in embodiments of the present invention.

Preferably, the treatment can also be used in combination with other treatment modalities such as radiation therapy, surgical therapy, and the like.

Preferably, the NKAP inhibitor and the aforementioned chemotherapeutic agent are administered separately or in combination.

As used herein, "separate administration" may also be referred to as "separate administration," specifically referring to: modes of administration of the NKAP inhibitor and the aforementioned chemotherapeutic agents administered via different routes at different sites of the subject's body. For example, NKAP inhibitors are administered by enteral administration (e.g., oral), while the aforementioned chemotherapeutic agents are administered by parenteral administration (e.g., intravenous).

Preferably, the aforementioned pharmaceutical compositions administered separately comprise at least two physically separate formulations, wherein each formulation contains at least one pharmaceutically active compound.

In contrast, "combined administration" as used herein relates to the administration of the pharmaceutically active compounds of the present invention via the same route, e.g., simultaneously orally, simultaneously intravenously, etc.

Further, preferably, the aforementioned pharmaceutical compositions are for simultaneous or sequential administration.

As used herein, "simultaneous administration" may be the simultaneous administration of the NKAP inhibitor and the aforementioned chemotherapeutic agent, preferably the NKAP inhibitor and the aforementioned chemotherapeutic agent are administered within a time interval of less than 15 minutes, more preferably within a time interval of less than 5 minutes.

"sequential administration" may be administration of the NKAP inhibitor before or after the aforementioned chemotherapeutic agent, preferably, administration of the NKAP inhibitor and the aforementioned chemotherapeutic agent are separated by at least 1 day, 2 days, 3 days, 7 days, 30 days, or longer.

As used in particular embodiments of the invention, the NKAP inhibitor is administered prior to the chemotherapeutic agent. NKAP inhibitors may further enhance the therapeutic effect of chemotherapeutic agents.

In another aspect of the invention, there is also provided a method of killing cancer cells in vitro, characterized in that a NKAP inhibitor and a chemotherapeutic agent are administered to the cancer cells.

Preferably, the chemotherapeutic agent is cisplatin.

Preferably, the method comprises the step of introducing the siRNA into the cell.

Preferably, the introduction into the cell may take into account any method known in the art for delivering genetic material into the cell. Non-limiting examples include viral transduction, electroporation transfection, liposome delivery (lipofection), polymeric carriers, chemical carriers, lipid complexes, polymeric complexes, dendrimers, nanoparticles, emulsions, natural endocytic or phagocytic pathways, cell penetrating peptides, microinjection, microneedle delivery, particle bombardment, and the like. Methods of lipofection are used.

Preferably, the introduction into the cells uses a method of liposome delivery. More particularly, transfection methods as used in the embodiments of the present invention.

Drawings

FIG. 1 is a graph showing the results of protein detection and cell detection after interference with NKAP gene, wherein A is protein detection and B is cell detection.

FIG. 2 shows the therapeutic effect of NKAP in combination with different chemotherapeutic agents on cells, wherein A is in combination with cisplatin and B is in combination with paclitaxel.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

The apparatus and reagents used in the present invention are shown in table 1 below:

TABLE 1 instruments or reagents used in the present invention

Note: the control siRNA is targeted against Photinus pyralis luciferase gene, target sequence 5'-GGAUUUCGAGUCGUCUUAAUGUAUA-3';

NKAP siRNA #1 target sequence 5'-GAAGAGTCC GAAGCCCAGCAAATCT-3';

NKAP siRNA #2 target sequence 5'-GACAAGTGAAGAAATTGCATCATTT-3'.

Example 1 promotion of DNA damage and apoptosis in tumor cells after intervention of NKAP Gene in tumor cells

The experimental process comprises the following steps:

1. the day before transfection, cervical cancer tumor cells (HeLa cells) were inoculated into 24-well plates, and each well was inoculated with 2.5X10⁵Cells were in 500. mu.l DMEM medium (10% fetal bovine serum). Cells were replaced to 0.4ml complete medium before transfection.

2. siRNA-NKAP liposome-transfected cells: (1) adding Control and NKAP siRNA into 50 μ l of Opti-MEM culture medium corresponding to each well, wherein the final concentration is 100 uM; mu.l Lipofectamine RNAiMAX per well was added to 50. mu.l Opti-MEM, gently mixed and incubated at room temperature for 5 min.

3. Then, the siRNA dilutions were mixed with Lipofectamine RNAiMAX dilutions (total volume 100. mu.l) and gently mixed. Incubate at room temperature for 20 min. After 20min, 100. mu.l of the mixture was added to the culture wells containing 0.5ml of cultured cells, and the culture was gently shaken and mixed. The cells were returned to the 37 ℃ incubator for further culture.

4. 72 hours after transfection, half of the cells were collected for western blot detection and half for flow cytometry detection.

5. Western blot detection: each group of cells was lysed with a lysis solution of RIPA (50 mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.1% SDS). SDS-PAGE electrophoresis was performed after adding an electrophoretic loading buffer, and then protein transfer was performed to a PVDF membrane. After blocking with 5% BSA at 1h at room temperature, primary antibody was added and incubated overnight at 4 deg.C, and after washing with TBST for 3 times, secondary antibody was added and incubated for 1 hour at room temperature. After washing 3 times with TBST, ECL coloration was carried out.

6. Detecting apoptosis by flow cytometry: firstly, digesting cells to 1.5ml of EP tube by using pancreatin, centrifuging for 5min at 1000g, and collecting the cells; PBS resuspend and wash cells once; cell count about 5-10 ten thousand cells per group 195. mu.l Annexin V-FITC binding solution was added to gently resuspend the cells; then 5. mu.l Annexin V-FITC and 10. mu.l Propidium Iodide (PI) were added and mixed gently, incubated at room temperature for 10-20 minutes in the absence of light, and then placed in an ice bath. The samples were immediately flow cytometric tested and analyzed.

Results of the experiment

As shown in FIG. 1, Western Blot detection shows that the interference of NKAP not only causes the aggravation of DNA damage, but also increases apoptosis. The flow cytometer utilizes apoptosis marker-Annexin V staining to prove that the tumor apoptosis is obviously increased after NKAP interference.

Example 2 inhibition of NKAP expression potentiates the apoptotic effects of cisplatin on HeLa tumor cells without potentiating the apoptotic effects of other chemotherapeutic agents on HeLa tumor cells

Experimental procedure

The highest efficiency of #1 NKAP siRNA knockdown was achieved, and further the next step was verified using #1 NKAP siRNA: 48 hours after transfection with siRNA according to the transfection method described in example 1, cells were treated with cisplatin at various concentrations (0, 2, 4, 8, 10. mu.M) for 4 hours in different wells, washed away, and then cultured in an incubator for 24 hours with the complete medium replaced. In addition, siRNA was transfected 24 hours later according to the transfection method described in example 1, and cells were treated with paclitaxel at different concentrations (0.1, 1, 10, 100, 800 nM) for 48 hours in different wells.

Subsequently, MTT experiments were performed: the medium was first aspirated from each well, 100. mu.l of lysate containing 1mg/ml MTT RIPA was added, and incubation was carried out at 37 ℃ for 60 min. The supernatant was then aspirated off and 100. mu.l DMSO was added to dissolve the dye. Student's t test is adopted in the statistical analysis between the two groups; p < 0.05.

The absorbance was measured by a microplate reader at a wavelength of 590nm, and a cell viability curve was prepared.

Results of the experiment

As shown in FIG. 2, the results indicate that #1 NKAP siRNA can increase the killing effect of cisplatin on tumor cells after interfering with NKAP. After #1 NKAP siRNA interferes with NKAP, the killing effect of paclitaxel on tumor cells is not obviously changed.

Therefore, the NKAP inhibitor can specifically enhance the curative effect of the cisplatin, especially in the treatment of gynecological tumors (especially cervical cancer tumor cells).

Claims

Use of a combination of a NKAP inhibitor and cisplatin for the manufacture of a product for the treatment of cancer.
2. The use of claim 1, wherein said inhibitor comprises an agent used in any one of siRNA interference, CRISPR/cas9 method, homologous recombination, gene knock-out, gene replacement, and chemical pharmaceutical method.
3. The use of claim 2, wherein the inhibitor is an agent used in a method of siRNA interference.
4. The use of claim 1, wherein the cancer comprises cervical cancer.
5. The use of claim 1, wherein the treatment comprises enhancing apoptosis of cancer cells.
6. The use of claim 6, wherein the cancer cell is a HeLa cell line.
7. A method of killing cancer cells in vitro comprising administering a NKAP inhibitor and cisplatin to the cancer cells.
8. The method of claim 7, wherein the inhibitor is an agent used in a method of siRNA interference comprising the step of introducing siRNA into the cell.
9. The method of claim 8, wherein said introducing into the cell is by liposome delivery.
10. The method of claim 9, wherein said cisplatin is administered after administration of the NKAP inhibitor.