CN111744001A - Application of drosophila Hsp22 protein in preparation of anti-tumor drugs - Google Patents
Application of drosophila Hsp22 protein in preparation of anti-tumor drugs Download PDFInfo
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Abstract
The invention belongs to the field of medicines, and particularly relates to application of drosophila Hsp22 protein in preparation of anti-tumor medicines. The pharmacological activity experiment of the invention partially aims at the treatment effect of the drosophila Hsp22 protein on a Ras tumor drosophila model and the treatment effect of a mouse tumor model, and finds that the drosophila Hsp22 protein has good anti-tumor activity. The drosophila Hsp22 protein can be used for developing novel anti-tumor drugs, and has important medical prospect and economic value.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of drosophila Hsp22 protein in preparation of anti-tumor medicines.
Background
Cancer is a malignant disease with extremely high mortality rate, has high treatment difficulty and brings heavy burden to patients and families. In recent years, the incidence of cancer in China has increased significantly, and cancer prevention and treatment face a severe situation. According to related research reports, in the 70 th century, the cancer of China is increased to 22.32% from 10.13%, and the death increasing rate is 82.11%. Cancer ranks first in urban deaths and second in rural areas. Especially, aging is getting more and more serious nowadays, and factors such as smoking, dietary structure change, microbial infection, obesity, reduced activity, bad work and rest are the main causes of cancer. At present, the first ten cancers in China are: lung cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, female breast cancer, pancreatic cancer, lymph cancer, bladder cancer and thyroid cancer. Lung cancer is a common disorder in urban males, and breast cancer is a common cancer in urban females; the cancer of the digestive system is the first disease of men and women in rural areas, and the lung cancer death rate is the highest.
Malignant tumor is a frequently encountered disease which is harmful to health and life, and the fatigue, pain and toxic and side effects of treatment brought by the malignant tumor seriously affect the physical and mental health and the life quality of patients. RAS oncogenes are activated in 30% of cancer types, leading to proliferation and transformation of tumor cells, and there is no effective inhibitor against this protein (encoded by RAS gene) at present, and thus there is an urgent need for anti-malignant drugs.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of drosophila Hsp22 protein in preparing anti-tumor drugs.
In order to achieve the purpose, the invention adopts the technical scheme that: an application of drosophila Hsp22 protein in preparing anti-tumor drugs, wherein the amino acid sequence of the drosophila Hsp22 protein is shown in SEQ ID NO. 1.
Heat shock protein 22/H11 kinase (Hsp22) is a stress-induced protein that responds to a variety of stress conditions that change in the tumor microenvironment, including ischemia.
Previous studies have shown that Hsp22 is localized in the mitochondrial and nuclear and cytoplasmic components. In addition, it has been shown in drosophila that the localization of Hsp22 in mitochondria is due to a translocation mechanism that is dependent on its N-terminal domain. The biological function of Hsp22 within mammalian mitochondria remains unknown. We found that the treatment of mouse liver cancer model with Drosophila Hsp22 protein could be achieved by down-regulating the transcription factor HIF2 α, resulting in down-regulation of oncogene RAS expression.
As a preferred embodiment of the use of the invention, the tumor is a Ras tumor or a liver cancer.
As a preferred embodiment of the use according to the invention, the drosophila Hsp22 protein is administered in the form of a solvate.
As a preferable embodiment of the application of the invention, the solvate is obtained by dissolving a freeze-dried product of drosophila Hsp22 protein in a solvent.
As a preferred embodiment of the application of the present invention, the solvent comprises at least one of water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.
As a preferred embodiment of the use according to the invention, the medicament is for subcutaneous, intravenous, intramuscular or nasal administration.
The invention also provides a pharmaceutical composition, which comprises drosophila Hsp22 protein and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the drosophila Hsp22 protein is shown in SEQ ID NO. 1.
The invention has the beneficial effects that:
the pharmacological activity experiment of the invention partially aims at the treatment effect of drosophila Hsp22 protein on Ras tumor drosophila model and the treatment effect of mouse tumor model, and finds that the drosophila Hsp22 protein has good antitumor activity.
Drawings
FIG. 1 shows fluorescence of the present inventionRespectively detecting the fluorescence intensity of drosophila larvae by using a body type microscope; wherein, A: a normal control group; b: rasV12；scrib-/-Larvae of fruit flies, tumors; c: hsp22 overexpressed RasV12；scrib-/-Tumor drosophila larvae.
FIG. 2 shows the RT-PCR detection of Ras with hsp22 overexpressionV12；scrib-/-Expression level of RAS gene in Drosophila melanogaster larvae.
FIG. 3 shows the RT-PCR detection of Ras with hsp22 overexpressionV12；scrib-/-Expression level of Myc gene in Drosophila melanogaster larvae.
FIG. 4 shows the RT-PCR detection of Ras with hsp22 overexpressionV12；scrib-/-Expression level of Hif1 α gene in drosophila larvae.
FIG. 5 is a graph showing the tumor volume of mice on different days of administration according to the present invention; wherein, A: a time-varying line plot of tumor volume in mice; b: a mouse tumor volume real object map; c: the tumor volume histograms of the model group and the administration group mice. (Note: significant differences are present p < 0.05;. p < 0.01;. p <0.001)
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the concentrations of the reagents in the present invention are mass concentrations; unless otherwise specified, the experimental methods in the present invention are all conventional methods; unless otherwise specified, the reagents, materials, cells, experimental animals, etc. of the present invention are commercially available or otherwise publicly available.
Example 1 treatment of drosophila Hsp22 protein with Ras tumor drosophila model
(1) Construction of Drosophila tumor model
Firstly, constructing RasV 12; scrip-/-fruit fly tumor model (oncogenic protein Ras overexpression and cancer suppressor gene scrip-/-invisible homozygous mutation)
The Drosophila melanogaster is hybridized with RasV12 Drosophila melanogaster with GFP fluorescent marker by using a promoter scrib-GAL4 which is selectively expressed specifically on eye disc and abdominal nerve cord of Drosophila melanogaster larvae (Drosophila melanogaster is given by Schroer professor of the institute of Life sciences of Shanghai university), and the obtained F1 generation is RasV 12; scrib-/-tumor drosophila (oncogenic protein Ras overexpression and scrib-stealth homozygous mutation).
② constructing RasV12 overexpressed by hsp 22; scrib-/-tumor drosophila
Hybridizing hsp22 overexpression Drosophila melanogaster (genotype: +/cyo; hsp22 OE/TM2) with RasV12 Drosophila melanogaster (genotype: RasV 12/sco; TM3/+) and screening RasV12/cyo in F1 generation; hsp22 OE/TM3 fruit fly (the fruit fly has and only has the characters of rolling wings and short setae), culturing the fruit fly strain for a period of time, and picking RasV12 without characters; hybridizing hsp22 OE with scrip-GAL 4 Drosophila, wherein the filial generation is RasV12 overexpressed by hsp 22; scrib-/-tumor drosophila
(2) Detection of fruit fly tumor model larva GFP fluorescence intensity
The drosophila Ras tumor model is provided with a GFP marker, fluorescence is observed under a body type fluorescence microscope, and the tumor size can be directly observed under the fluorescence microscope.
Detection of normal control group (FIG. 1A) and Ras with fluorescent microscopeV12；scrib-/-Tumor Drosophila melanogaster (FIG. 1B) and hsp22 overexpressed RasV12；scrib-/-Tumor (FIG. 1C) fluorescence intensity of Drosophila larvae, the results showed that hsp22 overexpression reduced the tumor size and improved the tumor status.
(3) RT-PCR detection of Ras overexpressed by hsp22V12；scrib-/-Expression level of tumor-associated gene in Drosophila melanogaster larvae
Collecting the Ras overexpressed by hsp22 constructed in step (1)V12；scrib-/-10 tumor drosophila larvae, extracting RNA, respectively detecting the expression quantity of tumor-related genes Ras, Myc and Hif1 α by using fluorescent quantitative PCR, and respectively detecting the expression quantity of the tumor-related genes Ras, Myc and Hif1 α with RasV12；scrib-/-Comparison was made with the tumor Drosophila larvae.
As shown in FIGS. 2-4, Ras tumor group (i.e., Ras)V12；scrib-/-Drosophila tumor model) the expression of cancer-associated genes was greatly increased compared to the normal group when hsp22 was overexpressed (i.e., hsp22 overexpressed Ras)V12；scrib-/-Fruit fly tumor), RAS gene, tumorigenesis and outcome of tumorThe expression of the related MyC gene and hypoxia inducible gene (HIF-1 α) was significantly reduced.
Example 2 treatment of mouse liver cancer with drosophila Hsp22 protein
(1) Construction of monoclonal bacteria
The whole gene synthesizes the gene sequence (the amino acid sequence is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2) of the drosophila Hsp22 protein, the gene sequence is cut by BamHI and XhoI, and then inserted into a pET28a vector to obtain a recombinant plasmid, and after the sequencing is correct, the recombinant plasmid is transformed into host bacterium escherichia coli to obtain a monoclonal bacterium.
(2) Drosophila Hsp22 protein expression
Preparing a culture medium, sterilizing, coating the monoclonal bacteria obtained in the step (1) on a Kanamycin (KA) plate at night, and standing overnight. The next day, the monoclonal bacteria were selected to KA (50ug/ml) liquid medium 10ml, 250rpm37 ℃ overnight. Transferring 1% overnight strain to 1000ml liquid culture medium on the third day, expanding the strain at 37 deg.C and 250rpm for 3h to logarithmic phase, adding IPTG (0.5mM) at 8 o 'clock, shaking the strain overnight at 20 deg.C and 250rpm to the next day at 8 o' clock. And centrifuging at 7000rpm on the fourth day for 20min to collect thalli, washing once with PBS, resuspending with a bacteria breaking solution, performing ultrasonic treatment at 60-70% frequency for 10s at intervals for 30min totally until the bacteria solution is clear. Packaging into 1.5ml EP tube, centrifuging at 12000rpm for 20min, collecting supernatant, and passing through chromatography column (BDTL 0011-K, Beijing Boolong Ni layer medium gravity prepacked column). SDS-PAGE (15%) was performed on day five, with hsp22 in the 26kD position. Protein concentration was measured at BCA on the sixth day, and the tubes were ultrafiltered at 10kd for desalting, freeze-dried and stored at-80 ℃. And dissolving the drosophila Hsp22 protein freeze-dried powder in sterile physiological saline on the eighth day, and detecting the concentration again by using the BCA to obtain the final concentration.
(3) Establishment of mouse liver cancer abdominal transplantation tumor model
After freeze-drying the drosophila Hsp22 protein, the drosophila Hsp22 protein is prepared into solutions with different concentrations with the solvent of the invention for administration.
Male C57BL/6 mice, 6-8 weeks old, 20 + -2 g in body weight, inoculated with H22 liver cancer cell strain suspension (containing about 1 × 10 cells) subcutaneously10One) followed by continuous observation, i.e. intraperitoneal injection of 4mg/kg of drosophila Hsp22 protein aqueous solution every 1 day after 1 week of inoculation, respectively, as well asThe tumor volume of each group was measured and the survival rate of each group of mice after molding was counted.
After the Hsp22 liver cancer cells are inoculated subcutaneously in the abdominal cavity, the living and mental states, diet conditions and weight changes of the mice are observed, obvious irregular protrusions appear on the backs of the mice after successful inoculation, the mental states are not good, the diet is reduced, the weight is reduced or increased slowly, and the immunity is reduced.
One trial cycle was eighteen days, and dosing was initiated on the eighth day of trial. hsp22 group, tumor group + hsp22 group were given hsp22(4mg/kg) normal group and tumor group were given equal volumes of physiological saline. Once every other day, after the experiment was terminated, the mice were sacrificed by cutting the marrow, the tumor body was stripped, the surface adipose tissue was removed, the tumor body was weighed, and placed on a plate to measure the length (L) and width (W) of the tumor with a vernier caliper, and the tumor volume was calculated.
Tumor volume measurement formula V ═ length × width2)/2。
(2) Results of the experiment
As shown in fig. 5A, a significant difference occurred at the sixth day of administration, and the tumor volume of the mice in the 4mg/kg Hsp 22-treated group increased significantly slower than the control group.
The experimental results were obtained by taking the materials at the end of one experimental period (i.e., eighteen days), as shown in fig. 5B, with the passage of time, the difference appeared after the fourth day of administration, the tumor volume of the drosophila Hsp22 protein group mice at 4mg/kg increased significantly slower than that of the control group mice, the tumor volume of the control group increased faster after the fourth day, the mental state of the mice improved, the body weight increased significantly, and the feeding became gradually stable and normal.
As shown in fig. 5C, administration started on day eight, was every other day, and after completion of administration, tumor volume and weight were measured by dissection on day eighteenth, it was found that drosophila Hsp22 protein was able to inhibit tumor growth in a mouse model, and that drosophila Hsp22 protein at 4mg/kg was able to reduce tumor volume by 50%.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Guilin medical college subsidiary hospital
<120> application of drosophila Hsp22 protein in preparation of antitumor drugs
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<170>PatentIn version 3.3
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Claims (7)
1. The application of drosophila Hsp22 protein in preparing anti-tumor drugs is characterized in that the amino acid sequence of the drosophila Hsp22 protein is shown in SEQ ID NO. 1.
2. The use of claim 1, wherein the tumor is a Ras tumor or a liver cancer.
3. The use of any one of claims 1-2, wherein the drosophila Hsp22 protein is administered in the form of a solvate.
4. The use of claim 3, wherein the solvate is a lyophilizate of Drosophila Hsp22 protein dissolved in a solvent.
5. The use according to claim 4, wherein the solvent comprises at least one of water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.
6. The use according to claim 1, wherein the medicament is for subcutaneous, intravenous, intramuscular or nasal administration.
7. A pharmaceutical composition, which comprises drosophila Hsp22 protein and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the drosophila Hsp22 protein is shown as SEQ ID NO. 1.
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| CN202010495941.2A CN111744001B (en) | 2020-06-03 | 2020-06-03 | Application of drosophila Hsp22 protein in preparation of anti-tumor drugs |
| PCT/CN2020/100557 WO2021243797A1 (en) | 2020-06-03 | 2020-07-07 | Use of drosophila melanogaster hsp22 protein in preparation of anti-tumor drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113456800A (en) * | 2021-07-26 | 2021-10-01 | 桂林医学院附属医院 | Application of human CRYAB protein in preparation of anti-tumor drugs |
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- 2020-06-03 CN CN202010495941.2A patent/CN111744001B/en active Active
- 2020-07-07 WO PCT/CN2020/100557 patent/WO2021243797A1/en active Application Filing
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113456800A (en) * | 2021-07-26 | 2021-10-01 | 桂林医学院附属医院 | Application of human CRYAB protein in preparation of anti-tumor drugs |
| CN113456800B (en) * | 2021-07-26 | 2022-09-06 | 桂林医学院附属医院 | Application of human CRYAB protein in preparation of anti-tumor drugs |
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| WO2021243797A1 (en) | 2021-12-09 |
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