CN110841054B

CN110841054B - Combination of antibacterial peptide PFR and cytarabine and anticancer effect thereof

Info

Publication number: CN110841054B
Application number: CN201910759575.4A
Authority: CN
Inventors: 付彩云; 吕宇蝶; 邵罡; 张奇瑜; 蒙月明
Original assignee: Zhejiang Sci Tech University ZSTU
Current assignee: Zhejiang Sci Tech University ZSTU
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2023-11-24
Anticipated expiration: 2039-08-16
Also published as: CN110841054A

Abstract

The present application relates to a combination of the antibacterial peptide PFR and cytarabine and its anticancer effect. In particular, the application relates to a method of treating cancer in a subject, alleviating a symptom of cancer in a subject, or inhibiting proliferation of cancer cells comprising the antimicrobial peptide PFR in combination with cytarabine to a subject. Further, the present application relates to a medicament for the treatment of cancer comprising the antibacterial peptide PFR and cytarabine, and optionally a pharmaceutically acceptable carrier.

Description

Combination of antibacterial peptide PFR and cytarabine and anticancer effect thereof

Technical Field

The present application relates to an anticancer effect of antibacterial peptide PFR combined with cytarabine. In particular to: a method of treating cancer in a subject, alleviating a symptom of cancer in a subject, or inhibiting proliferation of cancer cells; and to two pharmaceutical products comprising their combined anticancer effect and instructions for their use in the treatment of neoplastic diseases.

Background

Cancer has become one of the major public health problems threatening human health. In 2018, 1810 ten thousand cancers are newly increased worldwide, 960 ten thousand cancers are dead world by world, wherein 43 ten thousand cancers are newly increased in leukemia, 30 ten thousand cancers are dead, the number is huge, and the number has rising trend, so that the research on leukemia mechanism and treatment is unprecedented ^[1] . Leukemia (Leukemia) is a disease caused by malignant proliferation of hematopoietic stem cells in blood, and its incidence and mortality in humans are both in the pre-cancerous state, with five-year survival rates of less than 50% ^[2] . Leukemia can be classified into Acute Leukemia (AL) and chronic leukemia (Chronic l eukemia, CL) according to the course and maturity of nature. Acute degreeLeukemia can be classified into acute lymphoblastic leukemia (Acute lymphob l ast ic l eukemia, ALL) and acute myeloblastic leukemia (Acute mye l oid l eukemia, AML), and AML incidence in China is about 1.62/10 ten thousand ^[3] ALL is about 0.69/10 ten thousand ^[2] 。

The treatment modes of leukemia are also very limited, mainly include bone marrow transplantation, radiotherapy, chemotherapy and the like, and have corresponding defects, the bone marrow transplantation is a mode with higher cure rate of leukemia, but the bone marrow transplantation is expensive and the matching type is difficult to find; radiotherapy kills cancer cells and simultaneously kills normal cells. The current standard approach for clinical treatment of AML is induction therapy. MrLozek demonstrates that nordaunorubicin can achieve higher remission and survival rates in AML patients, particularly in primary treatment of acute myeloid leukemia patients ^[4] . The Cozzio et al study found that combination of daunorubicin with albuterol for the treatment of AML significantly improved prognosis in patients under 50 years of age, but this regimen was often accompanied by serious side effects ^[5] . Serious toxic side effects are the biggest obstacle of chemotherapeutic drugs in the treatment of acute myeloid leukemia at large doses. Therefore, the combination of the chemotherapeutic medicine and other medicines is expected to reduce toxic and side effects and improve the treatment effect.

Human lactoferrin is an iron-binding glycoprotein of about 80kDa, can be isolated from breast milk, lacrimal water and the like, has antibacterial, antiviral and antitumor activities, and can enhance the immune activity of the organism. Lactoferrin can exert antitumor effect by removing iron, enhancing host defense, blocking cell cycle, inducing apoptosis, inhibiting angiogenesis, regulating and controlling cancerogenic metabolizing enzyme, etc ^[6] . Human lactoferrin is N-terminally an alpha-helical peptide fragment (LF 11 peptide) containing 11 polypeptides, which fragment has proliferation-inhibiting activity against gram-negative and gram-positive bacteria. The PFR peptide is a small molecular antibacterial peptide designed and synthesized according to the structure and activity relation of human lactoferrin LF11 peptide, and only contains 9 amino acids (LF 11-322: PFWRI RIRR) ^[7] . PFR peptides by inducing necrosis and blocking the cell cycle,a series of physiological reactions such as intracellular protein changes and the like, and can inhibit the proliferation of leukemia cells ^[8] .

Cytarabine has been the most common chemotherapeutic agent for acute myeloid leukemia since the 60 s of the 20 th century ^[9-11] . Cytarabine is a structural analogue of DNA components, can replace deoxycytidine to enter DNA, and only after cytarabine enters the body, cytarabine is converted into cytarabine triphosphate, so that the cytarabine has anti-tumor activity and has a killing effect on cancer cells with relatively high proliferation speed. Cytarabine triphosphate can irreversibly stop DNA synthesis, thereby inhibiting proliferation of leukemia cells, having high sensitivity and achieving anticancer enhancing effect ^[12,13] . High-dose cytarabine treatment has better effect than the conventional long-term treatment, but has toxic and side effects on blood, nervous system and organs, and the survival rate within five years is not obviously improved ^[14,15] . The toxic and side effects of cytarabine and the dosage of cytarabine are positively correlated, and the clinical use of cytarabine is limited.

Disclosure of Invention

In order to provide a new approach to the treatment of cancer or to provide a broader choice for cancer patients, the present inventors have studied to find that the antibacterial peptide PFR has a remarkable antitumor effect in combination with cytarabine.

Accordingly, in one aspect the application provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibacterial peptide PFR in combination with cytarabine.

In another aspect the application provides the use of an antibacterial peptide PFR in combination with cytarabine in a medicament or agent for the treatment of cancer.

The application also provides a medicament or pharmaceutical composition comprising the antibacterial peptide PFR and cytarabine.

Drawings

FIG. 1 is a representative photograph of an antibacterial peptide PFR treated HL60 cells in combination with cytarabine, with the scale indicated at 50 μm.

Fig. 2 shows the effect of the antibacterial peptide PFR in combination with cytarabine on HL60 cell proliferation activity, with significant differences (< P < 0.001) in inhibition of HL60 cell viability in the combination compared to the dosing group alone.

FIG. 3 is MTT data for the effect of the antibacterial peptide PFR in combination with cytarabine on HL60 cells, wherein: (A) Inhibition of HL60 cells at 48 hours and 72 hours by 120 μm PFR peptide in combination with 0.4 μm cytarabine; (B) Inhibition of HL60 cells at 48 hours and 72 hours was achieved by 140 μm PFR peptide in combination with 0.4 μm cytarabine. The statistical analysis of the increase in the necrosis ratio of the combination dosing group was significantly different compared to the single dosing group (< P < 0.001).

FIG. 4 is a graph showing the detection of apoptosis necrosis of HL60 cells by the antibacterial peptide PFR in combination with cytarabine, wherein: (A) After the different dosing groups are treated for 24 hours, detecting the distribution condition of the fluorescence of HL60 cells annexin V-FITC and PI by a flow cytometer; (B) And under the treatment of different dosing groups, counting the proportion of necrotic cells. The statistical analysis of the increase in the necrosis ratio of the combination dosing group was significantly different compared to the single dosing group (< P < 0.001).

FIG. 5 shows the detection of the uptake of HL60 cell PI by the antibacterial peptide PFR in combination with cytarabine, wherein: (A) PI staining was photographed under a microscope for the different dosing groups. (B) And under the treatment of different dosing groups, the proportion of PI staining of the different dosing groups is counted. The statistical analysis of cellular PI staining of the combination dosing group had significant differences (< P < 0.001) compared to the dosing group alone. The scale bar in the figure represents 50 μm.

Fig. 6 shows the effect of the antibacterial peptide PFR in combination with cytarabine on HL60 cell LDH release with significant differences in statistical analysis of cell LDH release at different time points in the combination compared to the alone dosing group (< 0.05, < 0.001).

Fig. 7 is a photograph of PFR peptide in combination with cytarabine in treatment of tumor bearing nude mice.

Fig. 8 is a graph showing tumor volume increase and weight change in PFR peptide in combination with cytarabine in tumor-bearing nude mice, wherein: (A) The tumor volume of nude mice varies with time (P <0.05, statistical analysis has significant differences) under different drug treatments; (B) Change of nude mice body weight over time with different drug treatments.

FIG. 9 shows the size and weight of tumor-bearing nude mice treated with PFR peptide in combination with cytarabine, and the size and weight of tumor-bearing nude mice treated with PFR peptide in combination with cytarabine. (A) after dissection of nude mice, tumor comparison is taken out; (B) The tumor weights taken were counted (< P < 0.05), with significant differences in statistical analysis).

FIG. 10 is the effect of PFR peptide in combination with cytarabine treatment of tumor-bearing nude mice on the organ index of nude mice, wherein: (A) after dissection of nude mice, taking out liver for comparison; (B) counting viscera index of the liver of the nude mice; (C) after dissection of the nude mice, taking out kidney comparison; and (D) counting the organ index of the kidney of the nude mice.

Detailed Description

The present inventors have conducted extensive studies to find that the combination of the antibacterial peptide PFR and cytarabine has excellent antitumor effect, but that the combination of the antibacterial peptide PFR and many other anticancer active ingredients does not have synergistic effect, for example, we have found that the combination of the antibacterial peptide PFR and doxorubicin has no synergistic effect in the treatment of leukemia.

Accordingly, in one aspect the application provides a method of treating cancer in a subject or alleviating symptoms of cancer in a subject comprising administering to the subject an effective amount of an antibacterial peptide PFR in combination with cytarabine. The application also provides a method of inhibiting proliferation of a cancer cell (e.g., a blood cancer cell) comprising the step of contacting the cancer cell with an antimicrobial peptide PFR in combination with cytarabine. In yet another aspect, the application provides a pharmaceutical composition comprising the antimicrobial peptide PFR and cytarabine; and instructions for including the product relating to the pharmaceutical composition.

For the subject of the application, it is preferably a mammal, more preferably a human. Although the application is illustrated with blood cancer cells, the term "cancer" may include, but is not limited to, primary melanoma, metastatic melanoma, adenocarcinoma, squamous cell carcinoma, thymoma, sarcoma, non-hodgkin's lymphoma, leukemia, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer, renal cancer, brain cancer, bone cancer, gastric cancer, esophageal cancer, gastric cancer, intestinal cancer (e.g., rectal cancer and duodenal cancer), liver cancer, pancreatic cancer, lung cancer, bone cancer, bladder cancer, ovarian cancer, lymphoma, blood cancer, breast cancer, head and neck cancer, cervical cancer, uterine cancer, melanoma, and various cancers and related cancer symptoms. Preferably, the cancer treated using the present application is leukemia and its associated symptoms.

For the pharmaceutical composition of the application, it may be any of the antibacterial peptide PFR and chemotherapeutic drug cytarabine capable of treating cancer in a subject or alleviating symptoms of cancer in a subject. As described in the background art, the PFR peptide is a small molecular antibacterial peptide designed and synthesized according to the structure and activity relation of human lactoferrin LF11 peptide, and only contains 9 amino acids, so that the PFR peptide has a killing effect on leukemia cells; cytarabine has been the most common chemotherapeutic agent for acute myeloid leukemia. The PFR peptide in combination with cytarabine is also possible to achieve the object of the application on the basis of the teaching of the present application.

The PFR peptide and cytarabine as active ingredients of the present application may be used together with a pharmaceutically acceptable carrier. In addition to the active ingredient, the methods, uses and products of the application may also comprise suitable agronomically acceptable carriers, including excipients and auxiliaries which facilitate processing of the active compounds into preparations.

Formulations suitable for injection or infusion include, for example, aqueous and non-aqueous sterile injectable solutions which optionally contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injection, immediately prior to use.

The active ingredients of the present application may optionally be combined with solid excipients and the resulting mixture optionally milled and, if desired, after the addition of suitable adjuvants, the mixture of granules processed to obtain the desired dosage form. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, for example crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate.

Illustratively, the active ingredients of the present application may be used at a dosage of 1-15mg/kg of PFR peptide, 1-10mg/kg of cytarabine, e.g., 2-12mg/kg of PFR peptide, 2-8mg/kg of cytarabine. Preferably, the PFR peptide is 10mg/kg and cytarabine is 5mg/kg.

According to the present application, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present application may be administered to a subject in any effective number of doses. Preferably, the pharmaceutical composition of the present application may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4 to 10 doses, most preferably about 6 doses. In a particularly preferred embodiment, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the application is administered to a subject, such as by injection, infusion or oral administration, at a frequency of about once every three weeks during administration. In a particularly preferred embodiment, the administration is by injection.

It will be appreciated that the pharmaceutical compositions of the present application may be formulated in any suitable manner for administration by any suitable route.

The dosage units of the pharmaceutical compositions of the application are based on conventional administration to a subject. For example, a dosage unit may be administered more than once daily, once weekly, once monthly, etc. The dosage units may be administered on a twice/week basis, i.e. twice weekly, for example once every three days.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any expression herein of "comprising", especially when describing a method, use or product of the application, is to be understood to include those products, methods and uses consisting essentially of and consisting of the recited components or elements or steps. The application illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The instructions contained in the pharmaceutical product of the present application that relate to the pharmaceutical product may contain the following: indications (e.g. blood cancers), administered dosages (e.g. as exemplified above), side effects that may occur, etc.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the application claimed. Therefore, it should be understood that although the present application has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this application as defined by the appended claims.

For a clearer description of the present application, reference will now be made in detail to the following examples, which are illustrative of the present application and are not to be construed as limiting the application.

Examples

1. Materials and methods

1.1 materials, reagents and instruments

1.1.1 materials

Human acute myeloid leukemia cells HL60 were donated by a first hospital affiliated with the university of Zhejiang medical college. Antibacterial peptide PFR was purchased from the institute of biotechnology (Shanghai) stock, cytarabine was purchased from beijing solibao technologies.

1.1.2 major reagents

1.1.3 reagent preparation

(1) Phosphate buffer (Phosphate buffer sa l i ne, PBS): accurately weighing 0.2g of potassium chloride, 8g of sodium chloride, 0.24g of monopotassium phosphate and 3.63g of disodium hydrogen phosphate dodecahydrate by using an electronic balance, adding a rotor cleaned by secondary water, dissolving in the secondary water by using a magnetic stirrer, adjusting the pH value to 7.4, fixing the volume to 1000mL, subpackaging, sterilizing at the high temperature and high pressure under the conditions of 121 ℃ for 20 minutes, and preserving at the later 4 ℃.

(2) Antibacterial peptide PFR: 10mM stock solution was prepared with PBS and stored at-20 ℃.

(3) Cytarabine: preparing 40mM of storage solution by using PBS, preserving the storage solution in a refrigerator at-80 ℃, diluting the storage solution into 1mM of working solution or diluting the storage solution into 0.1mM of working solution according to requirements, preserving the storage solution in the refrigerator at-20 ℃, and thawing the storage solution at normal temperature before using the storage solution.

(4) MTT 250mg MTT (Th i azo l y l B l ue Tetrazo l i um Bromi de) powder is accurately weighed by an electronic balance, is firstly dissolved in sterile PBS for internal use in a beaker, is added with a rotor and is wrapped with tinfoil paper to avoid light, is dissolved for 4-6 hours in a magnetic stirrer to avoid light, and is then fixed to 50mL. In an ultra clean bench, the mixture was filtered through a sterilized 0.22. Mu.M aqueous syringe filter, packaged in 1.5mL centrifuge tubes, wrapped with tinfoil, and stored at-20 ℃.

(5) P I: the powder was dissolved in sterile PBS to 10mg/mL stock and stored at-20 ℃. The stock solution was diluted to 1mg/mL with sterile PBS and stored at-20deg.C.

(6) Cyclophosphamide: a proper amount of cyclophosphamide is weighed into a 1.5mL centrifuge tube, a certain amount of sterile PBS solution is added, and the solution is blown by a gun and oscillated uniformly to prepare cyclophosphamide working solution with the concentration of 10 mg/mL.

1.1.4 Main instruments

1.2 Experimental methods

1.2.1 cell culture

The state of the cells, whether the cells are polluted or not and how much the cells are dense are observed by an inverted microscope every day, and when the confluency of the cells reaches 70-80%, the cells can be passaged. At passage of a 100mm diameter cell culture dish, because of the suspension of cells, the culture medium and cells in the dish were first blown evenly and placed in a sterile 15mL centrifuge tube and centrifuged at 1000rpm for 5 minutes.

The old medium was aspirated, 1mL of fresh medium was added to the cell pellet, the cells were gently resuspended, a portion of the cell suspension was discarded according to the cell proliferation rate, and the remaining cell suspension was added to the petri dish to adjust to the appropriate cell concentration. The culture dish was replenished with fresh medium to keep the total volume 10mL, and was blown with a gun to uniformity. The cell culture dish was then returned to the cell incubator for stationary culture.

1.2.2 cell proliferation Activity experiments

(1) Cell plating and drug addition treatment

Cells in the logarithmic growth phase were collected, centrifuged, resuspended in a sterile dosing bowl with fresh medium, then mixed well and 20. Mu.L of cells were counted. 96-well plates were spread, and the number of HL60 cells in each well was 4×10 ⁴ The system was 200. Mu.L. The stock was diluted to 4X 10 according to the number of cells per well ⁵ The diluted cell suspension was homogenized at each mL, and 100. Mu.L of the diluent was added to each well, 6 wells each.

(2) According to the final concentration of the medicines added in each hole, the final concentration of cytarabine is 1 mu m, the final concentration of PFR peptide is 120 mu m, the volume of the medicines added in each hole is calculated, then the needed culture medium is added, and the medicines are added and supplemented to 200 mu L. After plating, the plate is gently rocked to make the cells and the medicines uniform, then the cell culture plate is placed in a cell culture box for static culture for 24 hours, cell activity statistics is carried out, and the experiment is repeated at least three times.

1.2.3 MTT assay

(1) 96-well plates were spread, and the number of HL60 cells in each well was 5×10 ³ The final concentration of cytarabine was 0.4. Mu.m, and the final concentrations of PFR peptide were 120. Mu.m and 140. Mu.m.

(2) At two time points, 48 hours and 72 hours after dosing, 10. Mu.L of 5mg/mL MTT was added and incubated for 4 hours at 37℃in an incubator. After 4 hours, the medium was centrifuged at 1000 revolutions for 5 minutes, carefully discarded in 96-well plates, 150. Mu.L of DMSO was added, and incubated in an incubator for 30 minutes.

After 30 minutes, each DMSO-added well was blown well with a gun in an ultra clean bench, transferred to another external 96-well plate, and ready for detection on an microplate reader. The enzyme label instrument needs to be started and preheated half an hour in advance, and engineering files are newly built during detection, and holes to be detected are named. Absorbance was measured at 490nm and 570nm and the file was saved.

(3) After at least three replicates, data statistics were performed. And opening the stored appearance l table, respectively averaging each group, calculating the inhibition rate according to the average value, creating an appearance l working table, and counting the inhibition rate of three repetitions in the table. Pasting the data into a GraphPad Pri sm for drawing, using SPSS for significance analysis, beautifying the picture, saving the engineering file, and outputting the picture.

1.2.4 apoptosis and necrosis detection

(1) Plating in 6-well plates, 1X 10 per well ⁶ Individual cells. Two plates were laid, 4 groups of three wells each. The final concentration of PFR peptide is 120 μm and the final concentration of cytarabine is 1.8 μm.

(2) After 24 hours of cell treatment, 4 15mL centrifuge tubes were taken, cells in the well plate were blown evenly, and the cells were collected and centrifuged at 1000rpm for 5 minutes, respectively, to collect cell pellets. The cell pellet was washed 2 times with clean PBS solution and centrifuged at 1000rpm for 5 minutes. The supernatant PBS was aspirated and the cells were resuspended at 500. Mu. L Bi ndi ng buffer in an apoptosis detection kit. The cells were filtered to prevent clumping of cells.

Each tube of sample was added to 5. Mu.L of annexin V-FI TC in the kit, mixed upside down, incubated for five minutes and then added to 5. Mu. L Propi d i um I odide solution. The cell suspension was mixed upside down, incubated at room temperature for 30 minutes in the absence of light, and detected on a cell assay instrument C6.

(3) The FL1/FL2 channel was selected to collect the detected cells, where FL1 represents the FITC green fluorescent channel and FL2 represents the PI red fluorescent channel. A small amount of cells was collected first. A cell scatter plot or cell density plot is shown with the X/Y axis parameters set to FSC-A/SSC-A. The gates were adjusted according to the cell population collected as needed.

The assay was started and 1×104 cells were collected per cell sample in the selected gate. And c6, after the detection is finished, storing and exporting the data file in the format of c 6.

(4) Experiments were repeated at least three times. Opening the c6 file in CF low P i us software, adjusting the coordinate axis range to be suitable, adjusting the position of the normal cell group to the position of the lower left corner of the graph, circling the cell group, adding a quadrant gate according to the circled cell group, and dividing the image into four parts of Q1/Q2/Q3/Q4. Wherein the upper left quadrant represents cells that can be stained with PI and cannot be stained with fytc; the upper right quadrant represents cells that can be stained with P I and fITC simultaneously; the lower left quadrant represents normal cells that cannot be stained by PI and fitc; the lower right quadrant represents cells that cannot be stained by P I, can be stained by fITC, and are generally apoptotic cells, and the percentage of cells corresponding to each quadrant is known by the numbers on the graph. An appearance l worksheet is created and three repeated results are counted in the sheet. The GraphPad Pr i sm software was opened, plotted in the software, and the results were counted.

1.2.5 PI absorption experiment

(1) Plating in 96-well plates, 4X 10 per well ⁴ Individual cells. A plate was laid out, 4 groups of 6 wells each. The final concentration of PFR peptide is 120 μm and the final concentration of Ara-c is 1. Mu.μm.

(2) After the 48 hour arrival at the drug treatment time point, 10. Mu.L of PI working solution was added and incubated in an incubator for 30 minutes. A representative field of view was found by photographing with an inverted fluorescence microscope, and a 200-fold image was taken, preferably about 200 cells in the field of view. And (3) adjusting the focal length until the cell morphology is clear, taking a picture under the white light condition, switching the picture into red light without changing the position, and taking the picture again. 1 shot per well, 6 duplicate wells per concentration.

(3) Experiments were repeated at least three times. The shooting software is used for exporting the shooting picture format into JPG pictures, two white and red light pictures with the same visual field are simultaneously opened in Photoshop and put into the same picture file, and the mixed mode is set to be a 'color' mode, so that PI dyeing conditions are overlapped by cells in a white light image, and statistics is convenient. The total cell number and the number of cells absorbed P I in each photograph were counted using a counting tool in Photoshop. The counts were saved in Exce l, data plotted using GraphPad Pri sm, and significance analyzed using SPSS.

1.2.6 LDH release detection experiment

(1) Plates were plated in 96-well plates, 2.5X10 each ⁴ Individual cells. A plate was laid out, 6 groups of 6 wells each. The final concentration of PFR peptide is 120 μm and the final concentration of cytarabine is 0.6 μm.

(2) After 48 and 72 hours of drug addition, the cell culture plates were centrifuged at 400g for 5 minutes, the supernatant was aspirated off, 150 μl LDH releasing reagent was added, and incubated in an incubator for 1 hour. After incubation, the cell culture plates were centrifuged at 400g for 5 min, 120 μl of fresh plates were removed from each well, 60 μl of LDH assay working fluid was added to each well, and incubated in a decolorizing shaker at room temperature in the absence of light for 30 min. Absorbance per well was measured at 490nm and 600nm and the absorbance size was proportional to intracellular LDH content.

(3) The experiment is repeated at least three times, the light absorption value of the non-dosing control group is set to be the total LDH content in the cells, namely 100%, the LDH content released by the cells under each concentration is converted, and the analysis data is used as a statistical chart to carry out significance analysis.

1.2.7 Experimental method for tumor formation of HL60 cell nude mice by PFR peptide combined with cytarabine

(1) Nude mouse nodulation

Four-week-old 12 nude mice were randomly divided into 4 cages, and recovered in a sterile environment for two days, and 200. Mu.L of 10mg/mL cyclophosphamide was intraperitoneally injected into each nude mice each day after two days, followed by 2 consecutive days of injection. HL60 cells in the logarithmic growth phase were collected, washed three times with sterile PBS, centrifuged at 1000rpm for 5 minutes, and 20 μl of cell suspension was counted before the last centrifugation, and 1×10 each mouse was injected ⁷ Each BALB/c nude mouse was injected subcutaneously on the back with 200L of cell suspension.

The back of the nude mice was observed daily for neoplasia, and the nude mice were padded and water was changed every three days. When the tumor body of nude mice grows to 100-150mm ³ At the time of (1), the nude mice were randomly grouped.

(2) Treatment of nude mice with drug

Nude mice were divided into four groups, namely control group, PFR peptide group, cytarabine group, and combination group, and the nude mice were numbered.

Once every two days, the PFR peptide is administered at 10mg/kg, cytarabine is administered at 5mg/kg, the control group is intraperitoneally injected with PBS, cytarabine is intraperitoneally injected with 100L cytarabine diluent, the PFR peptide group is intratumorally injected with 100L PFR peptide diluent, and the combination group is intraperitoneally administered with 100 2L cytarabine diluent and intratumorally injected with 100L PFR peptide diluent. The nude mice were weighed simultaneously with the measurement of tumor volume of the nude mice, and each mouse was photographed.

When the tumor volume of nude mice exceeds 1600mm ³ I.e. stopping the experiment, all nude mice were sacrificed humanizedly. Tumor bodies of the nude mice were peeled off, and livers and kidneys of the nude mice were taken out. The peeled tumor and viscera were weighed and counted.

2 experimental results

2.1 PFR peptide and cytarabine can obviously synergistically inhibit HL60 cell proliferation

HL60 cells were treated with 120 μm PFR peptide and 1 μm cytarabine alone or in combination for 24 hours and the cell status was directly observed from an inverted microscope. The cell edge of the control group is smooth, the light transmittance is good, the cell density is high, the cells of the dosing group shrink and crush, the cell edge presents an irregular shape, and the cell density is low. From microscopic observations, it was observed that the combined treatment of PFR peptide and cytarabine may have a synergistic effect in inhibiting HL60 cell proliferation.

Cell proliferation activity statistics were performed on four groups of 24 hours after dosing. Based on the control, the control has 100% of cell activity, the cell activity of the dosing group is reduced, wherein the cell activity of the 120 mu M PFR peptide dosing group is 47.94 +/-1.90%, the cell activity of the 1 mu M cytarabine dosing group is 28.81+/-0.60%, and the cell activity of the combined dosing group is only 14.67+/-0.78%.

The inhibition ratio of the MTT test was calculated in the dosing group based on the inhibition ratio of the control group being 0. HL60 cells were treated with 120 μm PFR peptide and 0.4 μm cytarabine with a 48-hour inhibition of 33.39 ±1.49% for the PFR peptide group, 44.93 ±0.97% for the cytarabine group, 69.87 ±0.98% for the combination group, 39.66 ±1.10% for the PFR peptide group, 58.62±0.96% for the cytarabine group, and 73.70±0.54% for the combination group. The inhibition rate of the PFR peptide group at 140. Mu.M and cytarabine at 0.4. Mu.M was 39.22.+ -. 0.62%, 45.55.+ -. 0.54%, 73.06.+ -. 1.50% and 44.47.+ -. 0.88%, 58.53.+ -. 0.43% and 80.67.+ -. 0.33% for the combination group in the 140. Mu.M PFR peptide and 0.4. Mu.M cytarabine treated HL60 cells.

Fig. 1, 2 and 3 intuitively demonstrate the combined inhibitory effect of the antibacterial peptide PFR and cytarabine on HL 60.

2.2 Induction of HL60 cell necrosis by PFR peptide combined with cytarabine

Flow cytometry was performed using a P I and Annexi n V-FI TC double staining method. The Q4 quadrant is a normal cell population not infected with PI and annexin V-FI TC, the Q3 quadrant is an Annex I n V-FI TC single-staining region, the Q1 quadrant is a necrosis quadrant of the PI single-staining region, the Q2 quadrant is a P I and Annex I n V-FI TC double-staining region, and if the early apoptosis phenomenon of the Q3 phenomenon is not detected, the cells of the Q1 and Q2 regions can be considered as necrotic cells. The PFR peptide combined with cytarabine treated HL60 cells, no cell population was seen in early apoptosis quadrant Q3, and a distinct cell population was seen in q1+q2, demonstrating that PFR peptide combined with cytarabine treated HL60 cells induced necrosis but not apoptosis.

The control group had a necrosis ratio of 3.7.+ -. 0.32%, a 120. Mu.M PFR peptide treatment of 26.82.+ -. 0.47%, a 1.8. Mu.M cytarabine treatment of 16.2.+ -. 1.44% and a combination group treatment of 51.93.+ -. 0.40%. Treatment of HL60 cells with PFR peptide in combination with cytarabine induced necrosis, which significantly increased the proportion of HL60 cells necrotic compared to the group administered alone.

Analysis of the necrotic data row PFR peptide in combination with cytarabine treatment of HL60 cells induced necrosis, which significantly increased the necrotic proportion of HL60 cells compared to the dosing group alone.

Fig. 4 intuitively demonstrates that PFR peptide and cytarabine in combination have synergistic HL60 cell necrosis inducing effects.

2.3 Effect of PFR peptide in combination with Cytarabine on HL60 cell PI uptake

The PI staining conditions of different dosing groups are counted, the PI staining rate of a control group is 3.34+/-0.57%, the PI staining rate of a 120 mu M PFR peptide-alone treatment group is 10.22+/-0.45%, the PI staining rate of a 1 mu M cytarabine-alone treatment group is 21.26+/-1.19%, and the PI staining rate of the PFR peptide combined cytarabine is 42.52 +/-0.66%. From the statistical figures, it can be seen that there is a significant difference between PI staining rates of PFR peptide and cytarabine alone and in combination.

Fig. 5 shows intuitively that PFR peptide in combination with cytarabine significantly increases PI staining rate, indicating that PFR peptide in combination with cytarabine damages cell membranes during cell death.

2.4 PFR peptide combined with cytarabine to promote LDH release

LDH release was detected for different dosing groups using LDH detection kit. LDH release in the dosing group was calculated based on LDH release in the control group being 0. At 48 hours, the LDH release of 120 μm PFR peptide-dosed group was 23.16±0.33%, the LDH release of 0.6 μm cytarabine-dosed group was 36.71±1.23%, and the LDH release of the combined-dosed group was 55.70 ±0.88%; at 72 hours, the LDH release of the 120 μm PFR peptide-dosed group was 11.25±2.84%, the LDH release of the 0.6 μm cytarabine-dosed group was 53.36 ±2.38%, and the LDH release of the combined dosed group was 77.71±0.86%.

Fig. 6 intuitively demonstrates that LDH release was significantly higher in the combination dosing group than in the PFR peptide and cytarabine alone dosing group, and that LDH release was more pronounced with increasing time. The PFR peptide in combination with cytarabine disrupts the cell membrane of HL60 cells.

2.5 Synergistic therapeutic effect of PFR peptide combined with cytarabine on tumor-bearing nude mice

The back of the mice is injected with cells subcutaneously to form solid tumors, the nude mice in the control group grow most rapidly after being treated by different drug treatment groups, the tumor volume is also the largest, compared with the control group, the nude mice in the PFR peptide treatment group and the cytarabine treatment group have certain relieving effect, the tumor growth of the combined group is the slowest, and the tumor volume is the smallest. There was no significant difference in weight gain between the groups. Tumor mass weighing statistics of stripped nude mice show that the control group has the largest tumor volume, no significant difference exists between the PFR peptide treatment group and the cytarabine independent treatment group, and the tumor significance of the combined group is smaller than that of the control group and the independent treatment group. The weight of the nude mouse tumor of the control group is 1.35+/-0.13 g, the weight of the nude mouse tumor of the PFR peptide treatment group is 0.97+/-0.06 g, the weight of the nude mouse tumor of the cytarabine treatment group is 0.92+/-0.13 g, and the weight of the nude mouse tumor of the combined group is 0.61+/-0.10 g.

The liver index of the control group is 5.29+/-0.10%, the kidney index is 1.28+/-0.10%, the liver index of the PFR peptide-added group is 5.59+/-0.47%, the kidney index is 1.67+/-0.04%, the liver index of the cytarabine-added group is 5.83+/-0.11%, the kidney index is 1.78+/-0.05%, the liver index of the combined group is 6.20+/-0.09%, and the kidney index is 1.66+/-0.07%. In the process of drug treatment, the liver and the kidney of the nude mice are not significantly changed, which indicates that PFR peptide combined with cytarabine does not generate obvious toxic and side effects on the nude mice.

Fig. 7, 8, 9 and 10 intuitively demonstrate that PFR peptide in combination with cytarabine significantly inhibited tumor increase in mice in animal experiments.

Taken together, the antibacterial peptide PFR in combination with cytarabine has synergistic inhibitory effects on HL60 cells both in vivo and in vitro. In cell experiments, the PFR peptide and cytarabine can obviously inhibit proliferation of HL60 cells and induce necrosis of the HL60 cells, and the PFR peptide and cytarabine can damage HL60 cell membranes and obviously increase PI absorption and LDH release. In animal experiments, the PFR peptide and cytarabine combined treatment of nude mice can obviously inhibit the growth of HL60 tumor cells, and simultaneously has no obvious toxic or side effect.

It will be appreciated by those of ordinary skill in the art that methods and materials, other than those specifically exemplified, etc., may be employed in the practice of the present application without undue experimentation. All functional equivalents known in the art of any such methods and starting materials, etc., are intended to be included in the present application. It will also be understood by those skilled in the art that various changes and modifications may be made to the application as described in the specification and claims, and that the application includes all such changes and modifications. The application also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or concurrently, and any and all combinations of any 2 or more of the steps or features.

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Claims

1. use of the antibacterial peptide PFR in combination with cytarabine for the manufacture of a medicament for treating leukemia in a subject.

2. The use of claim 1, wherein the subject is a mammal.

3. The use according to claim 2, wherein the mammal is a human.