CN108295085B - Application of protodioscin in preparation of drug-resistant osteosarcoma drug - Google Patents

Abstract

The invention discloses application of protodioscin in preparation of a drug-resistant bone tumor medicament, wherein the drug-resistant bone tumor is an osteosarcoma cell line with drug resistance to common anti-tumor medicaments, and particularly U2OS tumor cells with drug resistance to methotrexate, adriamycin and cisplatin. Meanwhile, as a natural plant extract medicine, the protodioscin has wide application prospect in the field of antineoplastic drug therapy.

Description

Application of protodioscin in preparation of drug-resistant osteosarcoma drug

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of protodioscin in preparation of a medicine for resisting drug-resistant bone tumors.

Background

Primary osteosarcoma is a common malignant bone tumor, and the onset age is mainly in adolescence. It is usually found in middle and late stages because it has hidden and advanced. Therefore, the operation treatment effect is poor, and the radiotherapy and the chemotherapy have obvious adverse reactions, so that the osteosarcoma patient has poor quality of life and short life cycle. There is a need for effective and safe antitumor drugs for the treatment of osteosarcoma.

Under normal physiological conditions, the proliferation of cells is strictly regulated and cells can be metabolized by programmed death, i.e., apoptosis occurs. However, when related molecular events such as oncogene activation, oncogene inactivation, functional deletion of repair-related genes, loss of apoptosis mechanism, telomerase overexpression, disturbance of signal transduction regulation mechanism, etc. occur, cells are excessively proliferated and accompanied by apoptosis abnormality, resulting in occurrence of malignant tumor. Inhibition of tumor cell proliferation and induction of apoptosis are the most desirable means for achieving tumor therapy.

In recent years, a comprehensive treatment means mainly based on traditional Chinese medicines can effectively control the disease development of osteosarcoma patients, improve the life quality and prolong the life cycle, and the natural antitumor medicines have obvious effects and almost have no adverse reaction.

Chinese patent CN1224392C finds that diosgenin extracted from dioscorea nipponica makino and other plants can be used for treating gastric cancer, breast cancer, lung cancer, colon cancer, glioma, erythroleukemia, melanoma, cervical cancer, liver cancer and the like, and can promote tumor cell death and induce apoptosis thereof.

The Chinese patent with application number of 201310325252.7 introduces amino straight chain fatty acid, amino acid residue, carbonyl and C at diosgenin-3-position₁-C₁₈The alkyl, phenyl or acceptable salt, etc., can be used for treating colon cancer, hepatocarcinoma, and breast cancer.

Hongquan et al report that 100 μmol/L diosgenin acted for 32h, and then acted on osteosarcoma U-2OS cell (cell number 10)⁴) The growth inhibition rate of the cells is 89.8 percent (Hongshengqiang, et al. transmission electron microscope observation of diosgenin induced human osteosarcoma U-2OS cell apoptosis [ J)]Fujian Chinese medicine, 2009,40(4): 39-41).

However, tumor cell lines resistant to anti-tumor drugs exist in bone tumor cells, which greatly reduces the therapeutic effect of traditional drugs, and diosgenin does not show significant inhibitory effect on such cells.

The diosgenin has structure shown in formula I, and molecular weight of 414 Da. The protodioscin has a structure different from diosgenin, as shown in formula II, and has a molecular weight of 1049Da, which is in R of diosgenin₁A glycosyl functional group is added at the position R₂Sugar ring opening glycosylation of diosgenin, followed by cyclopentyl and rightThe flanking molecular conformations are also rearranged and their functions are different from that of diosgenin. At present, the effects of protodioscin on inhibiting the proliferation and inducing the apoptosis of osteosarcoma cells are not reported, and particularly, the research on drug-resistant osteosarcoma cells is not reported.

Disclosure of Invention

Aiming at the defect that the existing medicine (including diosgenin) has poor treatment effect on drug-resistant osteosarcoma, the invention aims to provide the application of protodioscin in preparing a drug for resisting drug-resistant bone tumors.

The purpose of the invention is realized by the following technical scheme:

the protodioscin can effectively inhibit the proliferation of osteosarcoma cells and induce the apoptosis of the osteosarcoma cells;

the drug-resistant bone tumor is an osteosarcoma cell line with drug resistance to one or more common anti-tumor drugs.

The anti-tumor drug comprises one or more than one of methotrexate, adriamycin and cisplatin.

The osteosarcoma cell line with drug resistance is preferably U2OS/MTX 300.

The protodioscin also has good effects of inhibiting proliferation and inducing apoptosis on human osteosarcoma cell lines U2OS, HOS, MNNGHOS, 143B, MG63, SJSA1 and G292 and primary culture osteosarcoma cells ZOS and ZOSM.

The effective concentration of the protodioscin in the anti-osteosarcoma application is 0.01-200 mu mol/L.

The medicine also comprises acceptable auxiliary materials and other effective components which play a compatible and synergistic effect;

the medicine can be in various dosage forms, such as capsules, tablets, powder, wine, suspension, emulsion, syrup, aerosol, injection, and the like.

The method comprises the steps of performing conventional culture on osteosarcoma cell lines U2OS, HOS, MNNGHOS, 143B, ZOS, ZOSM, MG63, SJSA1 and G292, performing continuous culture on U2OS/MTX300 cell lines with Methotrexate (MTX) drug resistance by using a culture medium containing 300 mug/L MTX, and detecting the influence of protodioscin with different concentrations on the vitality of the osteosarcoma cells U2OS, U2OS/MTX300, HOS, MNNGHOS, 143B, ZOS, ZOSM, MG63, SJSA1 and G292 by adopting MTT; detecting the influence of proliferation capacity of U2OS, U2OS/MTX300, ZOS and 143B cells after protodioscin treatment by a clone formation test; observing Hoechst staining through an inverted microscope to detect the apoptosis condition of U2OS and 143B cells after protodioscin treatment; detecting the apoptosis conditions of U2OS, U2OS/MTX300, ZOS and 143B cells after protodioscin treatment by a flow cytometer. Detecting the change of apoptosis related indexes after protodioscin treatment by a western blot technology; the treatment effect of protodioscin on osteosarcoma in animal bodies is detected through a nude mouse animal experiment. The experimental result shows that: the protodioscin can inhibit proliferation of U2OS/MTX300 and induce apoptosis, and has good effects of inhibiting proliferation and inducing apoptosis for other osteosarcoma cell lines.

Compared with the prior art, the invention has the following advantages and effects:

(1) the protodioscin has obvious effects of inhibiting cell proliferation and inducing apoptosis on drug-resistant bone tumor cells.

(2) The lower concentration of protodioscin (5 mu mol/L) can play a role in inhibiting U2OS by high concentration of diosgenin (100 mu mol/L).

(3) Under the same administration concentration, the diosgenin has no inhibitory activity to a drug-resistant U2OS/MTX300 cell line, while the protodioscin shows good inhibitory activity.

(4) As a natural plant extract medicine, the protodioscin has wide application prospect in the field of antineoplastic drug therapy.

Drawings

FIG. 1 is a graph showing the effect of various concentrations of protodioscin on the viability of osteosarcoma cells.

FIG. 2 is a graph showing the effect of protodioscin on the proliferative capacity of osteosarcoma cells.

FIG. 3 is a Hoechst staining pattern of protodioscin after acting on osteosarcoma cells.

FIG. 4 is a flow cytometry detection diagram of protodioscin after acting on osteosarcoma cells.

FIG. 5 shows the change of apoptosis-related indexes after western blot detection of protodioscin treatment.

FIG. 6 shows the therapeutic effect of protodioscin on osteosarcoma in animal body; wherein, A-tumor volume change curve, B-tumor size actual map, C-mouse body weight change curve and D-tumor mass change curve.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Examples

Application of protodioscin in inhibiting osteosarcoma cell proliferation and inducing osteosarcoma cell apoptosis

Human osteosarcoma U2OS, HOS, MNNGHOS, 143B, MG63, SJSA1 and G292 cell lines used in the invention are purchased from Shanghai cell bank of Chinese academy of sciences; the U2OS/MTX300 cell line was purchased from the RIZZOLI osteomalacia research center, Italy; ZOS and ZOSM are human osteosarcoma primary cell lines constructed by the department of Oncology of the first hospital affiliated to Zhongshan university; protodioscin was purchased from Anbang biosciences, Guangzhou, Hoechst 33258 from Thermo, USA; DMEM high glucose medium and fetal bovine serum were purchased from Invitrogen; MTT kit was purchased from Sigma. Nude mice were purchased from Nanjing university-Nanjing biomedical research institute.

The experimental method comprises the following steps:

1. cell culture and drug formulation

U2OS, HOS, MNNGHOS, 143B, ZOS, ZOSM, MG63, SJSA1, G292 cell culture fluid is high sugar complete culture fluid containing 10% fetal calf serum, 100MG/L streptomycin and 100MG/L penicillin; MTX resistant cell line U2OS/MTX300 was cultured continuously in complete high-sugar medium containing 300. mu.g/L MTX, 10% fetal bovine serum, 100mg/L streptomycin and 100mg/L penicillin. The environment of the incubator is 37 ℃ and 5 percent CO₂And saturated humidity.

The protodioscin is completely dissolved in DMSO and diluted by using a complete culture medium. The animal experiment adopts the original dioscin suspension, and the preparation concentration is 60 mg/kg.

2. Effect of protodioscin on cell proliferation ability

After osteosarcoma cells are respectively treated by protodioscin (0-20 mu mol/L) with different concentrations for 72 hours, the absorbance (A) value (OD) is read under the wavelength of 450nm by a multifunctional microplate reader, and the cell proliferation inhibition rate (%) (1-average A value of dosing holes/average A value of control holes) x 100%) and IC50 value are calculated. The result is shown in figure 1, and the determination shows that the cell proliferation has an inhibition effect and is dose-time-dependent, and the IC50 value of the protodioscin acting on osteosarcoma cells for 72h is 0.6551-2.5800 [ mu ] mol/L (specifically shown in table 1), and the protodioscin has an inhibition activity.

TABLE 1

Cell lines	IC50(μM)
		U2OS	2.1710
U2OS/MTX300	2.4570
		HOS	0.7005
MNNGHOS	0.8308
		143B	0.7606
MG63	1.6370
		ZOS	1.6300
ZOSM	2.5800
		SJSA1	0.6551
G292	0.7478

Detection of diosgenin on human osteosarcoma methotrexate resistant cell line by same method

The results of the inhibitory activity of U2OS/MTX300 are shown in Table 2, diosgenin has no inhibitory activity to U2OS/MTX300, and protodioscin has better inhibitory activity to human osteosarcoma methotrexate resistant cell line U2OS/MTX 300.

TABLE 2

Compound (I)	IC50(μM)
		Protodioscin	2.4570
Diosgenin	——

In addition, osteosarcoma cells U2OS, U2OS/MTX300, ZOS and 143B are uniformly paved in a six-well plate, 1000 cells/well, the cells are treated with 2.5 mu mol/L protodioscin for 48h, and then are continuously cultured in an incubator for 8-10 days (PBS is added to a control group; the same is applied below), the cells are washed twice by PBS, methanol is added for fixation for 30min, 1% crystal violet is added for staining for 30min, and the crystals are washed and photographed. The results are shown in fig. 2, the clone number of osteosarcoma cells in the drug-treated group is greatly reduced, and the protodioscin is proved to have an inhibition effect on the proliferation of the cells.

3. Effect of protodioscin on apoptosis of osteosarcoma cells

Taking U2OS and 143B cells in logarithmic phase, adding protodioscin with final concentration of 0 and 2.5 mu mol/L respectively, culturing for 24h, washing twice with PBS, adding 4% paraformaldehyde for fixation, adding Hoechst 33258 dye solution, incubating at 37 deg.C in a dark state for 45min, observing change of cell nucleus under a fluorescence microscope, and taking pictures.

The morphological observation result is shown in fig. 3, the cells after the action of the protodioscin are in an obvious apoptosis state, the cell nucleus is shrunk and brightened, the nucleosome fragments are increased, and the osteosarcoma cell apoptosis is obviously induced.

In addition, osteosarcoma cells U2OS, U2OS/MTX300, ZOS and 143B were cultured by adding protodioscin to final concentrations of 0 and 2.5. mu. mol/L for 48h, and apoptosis was detected by flow cytometry. The results are shown in fig. 4, and the protodioscin can induce osteosarcoma cells to generate obvious apoptosis.

Further, after U2OS and 143B cells are treated by protodioscin with different concentrations for 48 hours, the cells are cracked by RIPA cell lysate, total cell protein is extracted, and apoptosis related indexes are detected by a western blot technology. The results are shown in fig. 5, after the treatment of the protodioscin, the apoptosis index cleared PARP increases along with the increase of the drug concentration, and the anti-apoptosis indexes Bcl2 and Bcl-xL decrease along with the increase of the drug concentration, which indicates that the protodioscin can induce the tumor cells to undergo apoptosis.

4. Therapeutic effect of protodioscin on osteosarcoma in animal body

Inoculating osteosarcoma cells 143B under skin of 4-week-old female nude mice, and randomly grouping the nude mice 1 week later into control group, protodioscin group, and ADM group. Performing intragastric administration with distilled water for 1 time per day; performing intragastric administration of protodioscin suspension with concentration of 60mg/kg for 1 time per day; ADM was administered intraperitoneally at a concentration of 6mg/kg 1 time per week. Animal body weight and tumor volume were measured every 4 days.

The experimental result is shown in fig. 6, from the changes of the volume and the weight of the tumor and the body weight of the mouse, the protodioscin suspension has the treatment effect on osteosarcoma in the animal body, the effect is more obvious than that of ADM, and no obvious toxic or side effect of the protodioscin in the animal body is observed.

The experimental results prove that the protodioscin can inhibit the proliferation and induce the apoptosis of drug-resistant osteosarcoma cells and is suitable for being used as a medicine for inhibiting the proliferation and inducing the apoptosis of the osteosarcoma cells.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (3)

1. The application of protodioscin in preparing drug-resistant bone tumor medicines is characterized in that: the drug-resistant bone tumor is an osteosarcoma cell line with drug resistance to methotrexate;

the osteosarcoma cell line resistant to methotrexate is U2OS/MTX 300.

2. Use according to claim 1, characterized in that: the medicine contains acceptable auxiliary materials and other effective components which play a compatible and synergistic effect.

3. Use according to claim 1, characterized in that: the medicine is capsule, tablet, powder, medicinal liquor, suspension, emulsion, syrup, aerosol or injection.

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