FUSED POLYPEPTIDE AND USE THEREOF

Abstract

The present invention discloses a fused polypeptide with multifunctional activity and use thereof, relating to the field of biopharmaceuticals. In the fused polypeptide with multifunctional activity, the polypeptide contains the following domains: N-Acetyl-Ser-Asp-Lys-Pro, Ser-Asp-Lys-Pro, Thr-Ser-Leu-Asp-Ala-Ser-Ile-Ile-Trp-Ala-Met-Met-Gln-Asn, and Leu-Ser-Lys-Leu, or domains in which any amino acid in the foregoing domains is mutated. The fused polypeptide can treat various fibrotic diseases including pulmonary fibrosis, hepatic fibrosis, skin fibrosis, renal fibrosis, and myocardial fibrosis, and has activity of inhibiting a plurality of types of human tumor cells.

Claims

1. A fused polypeptide with multifunctional activity, wherein the polypeptide comprises the following domains: N-Acetyl-Ser-Asp-Lys-Pro (SEQ ID NO: 7), Ser-Asp-Lys-Pro (SEQ ID NO: 7), Thr-Ser-Leu-Asp-Ala-Ser-Ile-Ile-Trp-Ala-Met-Met-Gln-Asn (SEQ ID NO: 8), and Leu-Ser-Lys-Leu (SEQ ID NO: 9), or domains in which any amino acid in the foregoing domains is mutated.

2. The fused polypeptide with multifunctional activity according to claim 1, wherein the fused polypeptide is linked by a linker, and the linker is a flexible linker composed of Gly-Gly-Gly-Gly (SEQ ID NO: 10), Ser-Ser-Ser or other amino acids.

3. The fused polypeptide with multifunctional activity according to claim 2, wherein an amino acid sequence of the fused polypeptide is the following sequence or a sequence with 80% homology therewith: TABLE-US-00011 polypeptide I: (SEQ ID NO: 1) N-Acetyl-Ser-Asp-Lys-Pro-Gly-Gly-Gly-Gly-Leu-Ser- Lys-Leu-Gly-Gly-Gly-Gly-Thr-Ser-Leu-Asp-Ala-Ser- Ile-Ile-Trp-Ala-Met-Met-Gln-Asn; polypeptide II: (SEQ ID NO: 2) N-Acetyl-Ser-Asp-Lys-Pro-Gly-Gly-Gly-Gly-Thr-Ser- Leu-Asp-Ala-Ser-Ile-Ile-Trp-Ala-Met-Met-Gln-Asn- Gly-Gly-Gly-Gly-Leu-Ser-Lys-Leu; polypeptide III: (SEQ ID NO: 3) Thr-Ser-Leu-Asp-Ala-Ser-Ile-Ile-Trp-Ala-Met-Met- Gln-Asn-Gly-Gly-Gly-Gly-Ser-Asp-Lys-Pro-Gly-Gly- Gly-Gly-Leu-Ser-Lys-Leu; polypeptide IV: (SEQ ID NO: 4) Thr-Ser-Leu-Asp-Ala-Ser-Ile-Ile-Trp-Ala-Met-Met- Gln-Asn-Gly-Gly-Gly-Gly-Leu-Ser-Lys-Leu-Gly-Gly- Gly-Gly-Ser-Asp-Lys-Pro; polypeptide V: (SEQ ID NO: 5) Leu-Ser-Lys-Leu-Gly-Gly-Gly-Gly-Ser-Asp-Lys-Pro- Gly-Gly-Gly-Gly-Thr-Ser-Leu-Asp-Ala-Ser-Ile-Ile- Trp-Ala-Met-Met-Gln-Asn; and polypeptide VI: (SEQ ID NO: 6) Leu-Ser-Lys-Leu-Gly-Gly-Gly-Gly-Thr-Ser-Leu-Asp- Ala-Ser-Ile-Ile-Trp-Ala-Met-Met-Gln-Asn-Gly-Gly- Gly-Gly-Ser-Asp-Lys-Pro.

4. Use of the fused polypeptide with multifunctional activity according to claim 1 in the preparation of anti-fibrosis drugs.

5. Use of the fused polypeptide with multifunctional activity according to claim 1 in the preparation of antitumor drugs.

6. The use of the fused polypeptide with multifunctional activity in the preparation of anti-fibrosis drugs according to claim 4, wherein the fibrosis comprises pulmonary fibrosis, hepatic fibrosis, renal fibrosis, myocardial fibrosis, and skin fibrosis.

7. The use of the fused polypeptide with multifunctional activity in the preparation of antitumor drugs according to claim 5, wherein the tumors originated from human head and neck, brain, thyroid, esophagus, pancreas, liver, lung, stomach, breast, kidney, colon or rectum, ovary, cervix, uterus, prostate, melanoma, hemangioma, or sarcoma.

8. The use of the fused polypeptide with multifunctional activity in the preparation of anti-fibrosis drugs according to claim 4, wherein the fused polypeptide is a polypeptide or a pharmaceutically acceptable salt thereof, and a dosage form thereof is an injection, capsule, tablet, pill, nasal spray or aerosol of the polypeptide or the salt thereof.

9. The use of the fused polypeptide with multifunctional activity in the preparation of antitumor drugs according to claim 5, wherein the fused polypeptide is a polypeptide or a pharmaceutically acceptable salt thereof, and a dosage form thereof is an injection, capsule, tablet, pill, nasal spray or aerosol of the polypeptide or the salt thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 is a diagram of HE staining of pulmonary fibrosis treated with fused polypeptides I, II, III, IV, V, and VI according to the present invention;

[0041] FIG. 2 is a diagram of Masson staining of pulmonary fibrosis treated with the fused polypeptides I, II, III, IV, V and VI according to the present invention;

[0042] FIG. 3 shows that fused polypeptides I, II, III, IV, V and VI according to the present invention inhibit the expression content of TGF-β1 in a renal fibrosis model;

[0043] FIG. 4 shows that fused polypeptides I, II, III, IV, V and VI according to the present invention inhibit the expression content of HYP in a skin fibrosis model; and

[0044] FIG. 5 shows inhibitory effects of the fused polypeptides I, II, III, 1V, V and VI according to the present invention on the growth of different types of tumors.

DETAILED DESCRIPTION

[0045] The polypeptides I, II, III, IV, V, and VI were synthesized by GenScript (Nanjing) Co., Ltd.

Example 1 Pulmonary Fibrosis Animal Model

[0046] Experimental Animals and Materials:

[0047] 1. Experimental Animals:

[0048] Source and strain: clean SD rats, provided by Comparative Medicine Center of Yangzhou University (laboratory animal production license: SCXK (Su) 2012-0004); Laboratory Animal Use License: SYXK (Su) 2012-0035).

[0049] Weight: 180-200 g at the time of purchase and 190-210 g at the beginning of modeling.

[0050] Gender: Male.

[0051] 2. Experimental Materials:

TABLE-US-00002 Bleomycin Manufacturer: Han Hui Pharmaceutical Co., Ltd. Normal saline Manufacturer: Anhui Double-Crane Pharmaceutical Co., Ltd. Chloral hydrate Manufacturer: Sinopharm Chemical Reagent Co., Ltd. BIBF1120 (Nintedanib) Manufacturer: Jinan Synovel Chemical Co., Ltd. Tissue fixative Manufacturer: Wuhan servicebio Co., Ltd.

[0052] 3. Experimental method:

[0053] SD rats were anesthetized by intraperitoneal injection of 1 mL/100 g 4% chloral hydrate. After anesthesia, the rats were fixed and their necks were disinfected by using cotton with 75% alcohol. The skin of the rat neck was longitudinally cut with scissors, and the fascia and muscle were longitudinally bluntly torn with tweezers to expose the trachea. A syringe was inserted into the trachea to inject 5 mg/kg bleomycin, while a blank group was injected with an equal amount of normal saline. Then a rat plate was quickly erected and rotated, the rats' breathing was observed, the neck wound was sterilized after rotation and was sewn, and an amoxicillin anti-inflammatory drug was sprinkled on the suture. After the operation, the rats were put back into a dry and clean cage for resting, waiting was performed for awakening. The rats were awakened after about 1-2 hours, and then fed normally. On the 7.sup.th day after modeling, modeling group animals randomly fell into a model group, a Nintedanib positive drug group, polypeptide I, II, III, IV, V, VI dosage groups, and a normal control group, and the groups were administered separately for an administration cycle of 14 days. Living situations of rats were observed every day and their weights were weighed. After administration for 14 days, the SD rats were dissected, the lung tissue was taken, and the right lung tissue was placed in a tissue fixative only for fixation, and HE staining and Masson staining and slice analysis were performed.

[0054] 4. Experimental Grouping and Dosage Setting

TABLE-US-00003 TABLE 1 Experimental grouping and dosage regimen Administration Administration Group Drug Dosage mode frequency Quantity Blank group Normal saline 0.5 mL/200 g Subcutaneous injection Twice a day 10 Model group Normal saline 0.5 mL/200 g Subcutaneous injection Twice a day 10 Positive drug Nintedanib 25 mg/kg Intragastric administration Once a day 10 Test drug (1) Polypeptide I 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (2) Polypeptide II 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (3) Polypeptide III 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (4) Polypeptide IV 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (5) Polypeptide V 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (6) Polypeptide VI 10 mg/kg Subcutaneous injection Twice a day 10

[0055] 4. Experimental Results

[0056] (1) Impact of a Polypeptide on the Survival Rate of SD Rats Induced by Bleomycin

[0057] As shown in Table 2, compared with the survival rate (50%) of SD rats in the model group, the survival rate of SD rats in each test dnig group was higher than that of the model group, and each test drug could significantly increase the survival rate of SD rats, and the survival rate of the polypeptide I group was equivalent to that of the positive drug group.

TABLE-US-00004 TABLE 2 Impact of a polypeptide on survival rate (%) of SD rats with bleomycin-induced pulmonary fibrosis Number of animals Number Dosage at the of animals Survival Group (mg/kg) beginning at the end rate (%) Blank group — 10 10 100 Model group — 10  5  50 Positive drug group 10 10  9  90 Polypeptide I 10 10  9  90 Polypeptide II 10 10  8  80 Polypeptide III 10 10  8  80 Polypeptide IV 10 10  8  80 Polypeptide V 10 10  7  70 Polypeptide VI 10 10  7  70

[0058] 2. Pathological Analysis of a Polypeptide on Bleomycin-Induced Pulmonary Fibrosis in SD Rats

[0059] Research results showed that a pulmonary fibrosis model in SD rats was successfully established in this study. Main manifestations of lung tissue lesions are fibroblast proliferation and collagen fiber formation in the alveolar wall and mesenchyme around intrapulmonary bronchi and vascular branches. Masson staining showed blue-green staining reaction, and inflammatory cell infiltration, congestion in the alveolar wall, cell degeneration disorder and other lesions occurred. After administration, the degree of pulmonary fibrosis and other lesions were less than those in the model group. See FIG. 1 and FIG. 2 for HE staining and Masson staining.

Example 2 In Vitro Hepatic Fibrosis Model

[0060] 1. Experimental Method

[0061] The inhibitory effect of a polypeptide on LX-2 hepatic stellate cells was detected by MTT assay. Cells were cultured in a 1640 medium containing 10% of FBS, the cytoplasm was made into 4×10.sup.5/mL cell suspension, and 100 μL per well was inoculated into a 96-well plate. After the cells adhered to the wall, the medium was replaced with a serum-free 1640 medium, and the serum-free medium was discarded after 24 hours. The cells were cultured with different polypeptides of 1 μmol/L, and 5 multiple wells were set for each concentration. After 12, 24 and 48 hours separately, 10 μL of MTT was added to each well. After 4 hours, MTT was sucked out, and 150 μL of DMSO was added to each well. After reaction for 5 min, an OD value was measured at 570 nm by a microplate reader.

[0062] 2. Experimental Results

[0063] At 24 hours and 48 hours, polypeptides I, II, III, IV, V and VI could inhibit the proliferation of cardiac fibroblasts of rats at 1 μmol/L. The results are shown in Table 3:

TABLE-US-00005 TABLE 3 Impact of a polypeptide on the proliferation of LX-2 hepatic stellate cells Optical density values at different time points Group 12 h 24 h 48 h Blank group 0.456 ± 0.012 0.548 ± 0.01  0.812 ± 0.016 Polypeptide I 0.452 ± 0.008 0.542 ± 0.03     0.680 ± 0.014*** (1 μmol/L) Polypeptide II 0.463 ± 0.012    0.394 ± 0.005***    0.578 ± 0.005*** (1 μmol/L) Polypeptide III 0.455 ± 0.002   0.435 ± 0.013**  0.642 ± 0.018* (1 μmol/L) Polypeptide IV 0.478 ± 0.018  0.472 ± 0.03**    0.580 ± 0.012*** (1 μmol/L) Polypeptide V 0.462 ± 0.004   0.477 ± 0.015**    0.618 ± 0.015*** (1 μmol/L) Polypeptide VI 0.453 ± 0.021  0.502 ± 0.013*  0.652 ± 0.018* (1 μmol/L) ***P < 0.001, **P < 0.01, *P < 0.05 VS control.

Example 3 Establishment of a Renal Fibrosis Model

[0064] 1. Experimental Animals

[0065] Clean grade male SD rats, purchased from Nanjing Qinglong Mountain Animal Farm, and weighed 180-200 g at the time of purchase, 190-210 g at the beginning of modeling, and 180-200 g at the beginning of administration.

[0066] 2. Experimental Materials:

[0067] Normal saline Manufacturer: Anhui Double-Crane Pharmaceutical Co., Ltd.

[0068] Rat TGF-β1 ELISA kit Manufacturer: Tianjin Annuo Ruikang Biotechnology Co., Ltd.

[0069] 3. Experimental Method

[0070] A renal fibrosis animal model was established. SD rats were anesthetized with 4% chloral hydrate, injected with 1 mL/100 g intraperitoneally, fixed to an operation board, and sterilized in an operation area for later use. The abdominal cavity was cut open about 3-4 mm to the left of the ventrimeson, left kidney ureter was separated in an operation group, the ureter was ligated and separated close to the ureter near the lower pole of the inferior pole of kidney, and the ureter was cut short between two ligations after the double ligations. Muscular layers and abdominal walls were sewed layer by layer, the suture was disinfected with alcohol. After SD rats woke up, the rats were put into a cage for feeding. In the blank group, ureter was not ligated, and other steps were the same.

[0071] Then, the animals fell into a blank group, a model group, and polypeptide administration groups, with 10 animals in each group, and the administration was started on the second day after the operation, twice a day for 14 days. After administration for 14 days, blood was taken and supernatant was taken to detect the content of TGF-β1 in serum.

[0072] 4. Experimental Grouping and Dosage Setting

TABLE-US-00006 TABLE 4 Experimental grouping and dosage regimen Administration Administration Group Drug Dosage mode frequency Quantity Blank group Normal saline 0.5 mL/200 g Subcutaneous injection Once a day 10 Model group Normal saline 0.5 mL/200 g Subcutaneous injection Once a day 10 Test drug (1) Polypeptide I 7.5 mg/kg Subcutaneous injection Twice a day 10 Test drug (2) Polypeptide II 7.5 mg/kg Subcutaneous injection Twice a day 10 Test drug (3) Polypeptide III 7.5 mg/kg Subcutaneous injection Twice a day 10 Test drug (4) Polypeptide IV 7.5 mg/kg Subcutaneous injection Twice a day 10 Test drug (5) Polypeptide V 7.5 mg/kg Subcutaneous injection Twice a day 10 Test drug (6) Polypeptide VI 7.5 mg/kg Subcutaneous injection Twice a day 10

[0073] 5. Experimental Results

[0074] (1) Impact of a polypeptide on the content of TGF-β1 in serum of SD rats with renal fibrosis TGF-β1 is the most important fibrogenic factor. In renal fibrosis, the expression of TGF-β1 was significantly increased. The result is shown in FIG. 3, and there was a highly significant difference between the model group and the blank group (***p<0.001). After administration, all groups could significantly reduce the content of TGF-β1 in serum, and the polypeptide I group, the polypeptide II group and the polypeptide IV group were highly significantly different from the model group (***P<0.001), and the polypeptide III group, the polypeptide V group and the polypeptide VI group were highly significantly different from the model group (**P<0.01).

Example 4 Establishment of a Myocardial Fibrosis Model

[0075] 1. Experimental Method

[0076] The inhibitory effect of a polypeptide on cardiac fibroblasts of rats was detected by MTT assay. Cells were cultured in a DMEM medium containing 10% of FBS, the cytoplasm was made into 1×10.sup.5/mL cell suspension, and 100 μL per well was inoculated into a 96-well plate. After the cells adhered to the wall, the medium was replaced with a serum-free DMEM medium, and the serum-free medium was discarded after 24 hours. The cells were cultured with different polypeptides of 1 μmol/L, and 5 multiple wells were set for each concentration. After 12, 24 and 48 hours separately, 10 μL of MTT was added to each well. After 4 hours, MTT was sucked out, and 150 μL of DMSO was added to each well. After reaction for 5 min, an OD value was measured at 570 nm by a microplate reader.

[0077] 2. Experimental Results

[0078] At 24 hours and 48 hours, polypeptides I, II, III, IV, V, and VI could inhibit the proliferation of cardiac fibroblasts of rats at 1 μmol/L. The results are shown in Table 5.

TABLE-US-00007 TABLE 5 Impact of a polypeptide on the proliferation of cardiac fibroblasts of rats Optical density values at different time points Group 12 h 24 h 48 h Blank group 0.353 ± 0.001 0.464 ± 0.018 0.896 ± 0.001 Polypeptide I 0.362 ± 0.006  0.402 ± 0.002*   0.678 ± 0.002** (1 μmol/L) Polypeptide II 0.352 ± 0.004    0.367 ± 0.016***    0.568 ± 0.013*** (1 μmol/L) Polypeptide III 0.349 ± 0.012  0.413 ± 0.003*   0.612 ± 0.018** (1 μmol/L) Polypeptide IV 0.362 ± 0.015    0.392 ± 0.008***    0.583 ± 0.012*** (1 μmol/L) Polypeptide V 0.357 ± 0.024    0.397 ± 0.015***    0.588 ± 0.019*** (1 μmol/L) Polypeptide VI 0.340 ± 0.012  0.412 ± 0.005*  0.622 ± 0.007* (1 μmol/L) ***P < 0.001, **P < 0.01, *P < 0.05 VS control.

Example 6 Establishment of a Skin Fibrosis Model

[0079] 1. Experimental Animals

[0080] Male C57/BL black mice aged 6-8 weeks, purchased from Nanjing Qinglong Mountain Animal Fai in.

[0081] 2. Experimental Materials

TABLE-US-00008 Bleomycin Manufacturer: Han Hui Pharmaceutical Co., Ltd. Normal saline Manufacturer: Anhui Double-Crane Pharmaceutical Co., Ltd. Rat TGF-β1 ELISA kit Manufacturer: Tianjin Annuo Ruikang Biotechnology Co., Ltd. Alkaline HYP kit Manufacturer: Nanjing Jiancheng Bioengineering Institute

[0082] 3. Modeling Method

[0083] Bleomycin (10 μg/mL) was injected subcutaneously every day for 28 days to form skin fibrosis. During the modeling period, the administration groups were given polypeptide drugs twice a day for treatment. After modeling, the mice were killed on the next day, and the skin tissue of the mouse back was taken to detect the content of HYP in the skin tissue.

[0084] 4. Experimental Grouping and Dosage Regimen

TABLE-US-00009 TABLE 6 Experimental grouping and dosage regimen Administration Administration Group Drug Dosage mode frequency Quantity Blank group Normal saline 0.2 mL Subcutaneous injection Twice a day 10 Model group Normal saline 0.2 mL Subcutaneous injection Twice a day 10 Test drug (1) Polypeptide I 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (2) Polypeptide II 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (3) Polypeptide III 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (4) Polypeptide IV 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (5) Polypeptide V 10 mg/kg Subcutaneous injection Twice a day 10 Test drug (6) Polypeptide VI 10 mg/kg Subcutaneous injection Twice a day 10

[0085] 5. Experimental Results

[0086] (1) Expression of HYP Content in the Skin Tissue of Each Group of Mice

[0087] The content of hydroxyproline in the skin tissue of the mouse back was detected. As the characteristic protein of collagen, hydroxyproline can reflect the content of collagen in the skin tissue from the side. As shown in FIG. 4, each polypeptide group could reduce the expression of HYP in the skin tissue. The polypeptide II group, the polypeptide IV group and the polypeptide VI group could significantly reduce the expression of HYP in the lung tissue, and were highly significantly different from the model group (***P<0.001). The polypeptide I group, the polypeptide III group and the polypeptide V group could reduce the content of HYP in the lung tissue of SD rats, and were highly significantly different from the model group (*P<0.05).

Example 7 Inhibitory Effect of a Polypeptide According to the Present Invention on the Growth of Tumor Cells from a Plurality of Sources Detected by Using MITT Assay

[0088] A plurality of types of human tumor cells were cultured in a 5% CO.sub.2 incubator at 37° C. and digested with trypsin when the density was 90% or above. The cells were resuspended in a culture solution and counted, and the cell concentration was adjusted to 2×10.sup.4 cells/mL. The cell suspension was inoculated into a 96-well plate with 100 μL per well, and then cultured overnight in a 5% CO.sub.2 incubator at 37° C. After the cells completely adhered to the wall, each polypeptide according to the present invention was added as an administration group, and the culture solution without any drug was used as a blank control group. The solutions were diluted to 1 μmol/L by using a diluent. Each diluent was separately added to the 96-well plate with 100 per well, and the cells continued to be cultured in a 5% CO.sub.2 incubator for 48 hours at 37° C. Then 20 μL of MTT was added, and the cells continued to be cultured for 4 hours. The medium was sucked, and 100 μL of DMSO was added to each well for dissolution. Absorbance was measured by a microplate reader at a detection wavelength of 570 nm and a reference wavelength of 630 nm, and the growth inhibition rate was calculated. The formula was as follows: tumor growth inhibition rate (%)=(1−absorbance of the administration group/absorbance of the blank group)*100%. The experiment was repeated independently for 3 times. Experimental results were expressed by mean±standard deviation, and the tumor growth inhibition rate of the blank group was 0. Results in Table 8 showed that the polypeptide according to the present invention had a significant inhibitory effect on the growth of a plurality of types of tumors (FIG. 5).

TABLE-US-00010 TABLE 7 Inhibitory effect (%) of a polypeptide according to the present invention on the growth of a plurality of types of tumors detected by MTT assay Polypeptide Polypeptide Polypeptide Polypeptide Polypeptide Polypeptide Tumor type I II III IV V VI Docetaxel Head and neck cancer 54.48 ± 12.59 59.48 ± 2.98  61.48 ± 3.99 49.68 ± 13.16 67.68 ± 10.66 47.48 ± 5.81  62.48 ± 2.12  Brain cancer 60.13 ± 20.12 65.13 ± 19.36  67.13 ± 16.15 55.33 ± 23.49 73.33 ± 14.34 53.13 ± 16.94 68.13 ± 10.26 Esophageal cancer 56.33 ± 10.53 61.33 ± 9.75  63.33 ± 6.54 51.53 ± 13.88 69.53 ± 4.75  49.33 ± 7.35  64.39 ± 8.06  Pancreatic cancer 48.79 ± 11.54 53.79 ± 10.76 55.79 ± 7.55 43.99 ± 14.89 61.99 ± 5.76  41.79 ± 8.36  76.74 ± 10.09 Thyroid cancer 65.26 ± 20.71 70.26 ± 19.93  72.26 ± 16.72 60.46 ± 24.06 78.46 ± 14.93 58.26 ± 17.53 73.21 ± 19.26 Liver cancer 73.42 ± 18.21 78.42 ± 17.43  80.42 ± 14.22 68.62 ± 21.56 86.62 ± 12.43 66.42 ± 15.03 74.22 ± 11.71 Breast cancer 52.15 ± 13.36 65.35 ± 12.58 59.15 ± 9.37 87.38 ± 16.71 65.38 ± 7.58  85.18 ± 10.18 65.12 ± 10.66 Gastric cancer 68.14 ± 9.86  73.14 ± 9.08  75.14 ± 5.87 63.34 ± 13.21 81.34 ± 4.08  61.14 ± 6.68  74.16 ± 6.38  Kidney cancer 87.48 ± 22.39 92.48 ± 21.61 94.48 ± 18.4 82.68 ± 25.74 85.68 ± 16.61 80.48 ± 19.21 75.48 ± 10.23 Colorectal cancer 65.55 ± 11.54 70.55 ± 10.76 72.55 ± 7.55 60.75 ± 14.89 78.7 ± 5.76 58.55 ± 8.36  53.55 ± 10.41 Ovarian cancer 74.75 ± 24.12 79.75 ± 23.34  81.75 ± 20.13 69.95 ± 27.47 87.95 ± 18.34 67.75 ± 20.94 62.75 ± 20.23 Cervical cancer 68.47 ± 15.31 73.47 ± 14.53  75.47 ± 11.32 63.67 ± 18.66 81.67 ± 9.53  61.47 ± 12.13 66.56 ± 11.31 Uterus cancer  57.2 ± 17.76  62.2 ± 16.98  64.2 ± 13.77  52.4 ± 21.11  70.4 ± 11.98  50.2 ± 14.58 57.24 ± 12.28 Prostate cancer  60.4 ± 15.12 65.4 ± 5.53  67.4 ± 6.54  55.6 ± 15.71  73.6 ± 13.21 53.4 ± 8.36 78.4 ± 4.21 Melanoma 54.48 ± 6.54  59.48 ± 19.12  61.48 ± 10.31 49.68 ± 12.76 67.68 ± 20.32 47.48 ± 13.32 68.42 ± 6.23  Hemangioma 58.98 ± 16.59 63.98 ± 6.98  65.98 ± 7.99 54.18 ± 17.16 72.18 ± 14.66 51.98 ± 9.81  78.76 ± 6.16  Sarcoma 62.15 ± 5.54  67.15 ± 14.12 69.15 ± 5.31 57.35 ± 7.76  75.35 ± 10.86 55.15 ± 12.32 62.51 ± 8.75  Lung cancer 68.15 ± 12.21 68.15 ± 12.21 63.42 ± 3.51 64.57 ± 6.77  76.45 ± 8.06  60.87 ± 3.12  73.32 ± 7.03