CN114699416A - Application of cucurbitacin B in resisting membrane melanoma - Google Patents

Abstract

The invention provides application of cucurbitacin B in resisting membrane melanoma, and particularly provides application of cucurbitacin B shown in a formula (I) or pharmaceutically acceptable salts thereof, or solvates or prodrugs thereof; the cucurbitacin B is used for preparing a pharmaceutical composition, and the pharmaceutical composition is used for the purpose selected from the group consisting of: treating and/or alleviating conjunctival melanoma, or inhibiting conjunctival melanoma cell proliferation.

Description

Application of cucurbitacin B in resisting membrane melanoma

Technical Field

The invention belongs to the fields of life science and medicine, and particularly relates to an action mechanism and application of cucurbitacin B in resisting a membrane melanoma.

Background

Conjunctival melanoma is a rare but severely harmful and potentially fatal ocular tumor accounting for 2% of all ocular malignancies with annual incidence rates of 0.2-0.5/million in western countries and 0.15/million in asians. Conjunctival melanoma is usually caused by malignant change of pigmented nevus and primary acquired melanosis of conjunctiva, can occur in any part of conjunctiva of eyes, rapidly transfers to other structures of eyes, and can also transfer and infiltrate into ears, nose, neck, lung, liver, skin and even brain. Conjunctival melanoma is usually treated by local operation in clinic and then is supplemented with cryotherapy, radiotherapy and chemotherapy of mitomycin C and recombinant human interferon alpha-2B, but eye part, which is a special focal tissue, has serious difficulty in drug delivery, so that the prognosis of drug treatment is poor, and the 5-year recurrence rate is as high as 26-61%.

The conjunctival melanoma patients have few groups, extremely complex pathogenesis and long latent period, which causes difficulty in clinical diagnosis and treatment. Unfortunately, despite some similarities to skin and mucosal melanoma, there are currently no biomarkers for predicting or diagnosing conjunctival melanoma and no effective clinical treatments or agreed-upon treatment regimens.

Therefore, development of new drug development of conjunctival melanoma is helpful for filling up the blank of the corresponding drug development field, and has significant therapeutic value and social significance.

Disclosure of Invention

The invention aims to provide a compound for inhibiting conjunctival melanoma or a preparation containing the compound and an action mechanism of the compound.

Specifically, the invention provides an action mechanism and application of cucurbitacin B (CuB) shown in a general formula (I), or pharmaceutically acceptable salt thereof, or solvate thereof, or prodrug thereof in resisting membrane melanoma. The cucurbitacin B directly targets GRP78/Bip protein, so that FOXM1-PLK1-KIF20A signal path is inhibited, CDK1-cyclinB1 cyclin downstream of the signal path is down-regulated, tumor cell cycle G2/M phase block is induced, growth of conjunctival melanoma is inhibited, and a novel mechanism and method are provided for treatment of conjunctival melanoma.

In a first aspect of the invention, the invention provides a cucurbitacin B shown in formula (I), or pharmaceutically acceptable salt thereof, or solvate thereof or prodrug thereof;

characterized in that it is used for the preparation of a pharmaceutical composition and the use of said pharmaceutical composition is selected from the group consisting of: treating and/or alleviating conjunctival melanoma, or inhibiting proliferation of conjunctival melanoma cells.

In another preferred embodiment, the pharmaceutical composition is also used for regulating the FOXM1-PLK1-KIF20A signal pathway.

In another preferred embodiment, the pharmaceutical composition is further used for down-regulating the activity or expression amount of FOXM 1.

In another preferred embodiment, the pharmaceutical composition is also used for down-regulating the activity or expression amount of PLK 1.

In another preferred embodiment, the pharmaceutical composition is further used for down-regulating the activity or expression amount of KIF 20A.

In another preferred embodiment, the pharmaceutical composition is further used for regulating the cycle-related protein CDK1-CyclinB 1.

In another preferred embodiment, the pharmaceutical composition is further used for down-regulating the expression of CDK1-cyclin b1 cyclin downstream of the signaling pathway.

In another preferred embodiment, the pharmaceutical composition is also used for inducing the G2/M phase block of the tumor cell cycle.

In a second aspect of the invention, the invention provides a use of cucurbitacin B shown in formula (I), or pharmaceutically acceptable salt thereof, or solvate or prodrug thereof;

the method is characterized by being used for preparing a pharmaceutical composition, and the pharmaceutical composition is used for down-regulating the activity or expression quantity of GRP78 protein.

In another preferred embodiment, the pharmaceutical composition is also used for regulating the FOXM1-PLK1-KIF20A signal pathway.

In a third aspect of the present invention, there is provided a method of treating and/or ameliorating conjunctival melanoma, the method comprising the steps of: administering to a subject in need thereof a safe and effective amount of cucurbitacin B represented by formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof:

it is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is the structural formula of cucurbitacin B.

Fig. 2 is a flow cytometer analysis of the effect of cucurbitacin B (cub) and MEK162 on CRMM2 cell cycle and apoptosis, showing that cucurbitacin B is effective in causing G2/M phase arrest and apoptosis in cells.

FIG. 3 is an image of the inhibitory effect of cucurbitacin B (CuB) at different concentrations on FOXM1-PLK1-KIF20A signal pathways and cycle-related proteins of melanoma carcinoma cells CM-AS16, CRMM1, CRMM2, CM2005.1 and normal cell HL 7702.

Fig. 4A is a graph of the binding of GRP78 protein to cucurbitacin B at different concentrations.

FIG. 4B is a graph of absorbance values tested for GRP78 ATPase activity, indicating that cucurbitacin B (CuB) is capable of inhibiting GRP78 ATPase activity.

Fig. 5 is an image of the examination of the expression of the target proteins FOXM1, PLK1, KIF20A, CyclinB1 and CDK1, indicating that GRP78 knockdown reduces the expression of FOXM1, PLK1, KIF20A, CyclinB1 and CDK1 proteins.

Fig. 6A is a graph of the trend of tumor volume in mice over time after MEK162 (gavage) administration and cucurbitacin B (cub) injection administration, showing that cucurbitacin B administration significantly reduced the volume of conjunctival melanoma in NCG mice.

Fig. 6B is a graph of the trend of mouse body weight over time after MEK162 (gavage) administration and cucurbitacin (CuB) injection administration, showing that cucurbitacin B has no significant effect on mouse body weight.

Detailed Description

The inventor of the invention has made extensive and intensive studies, and has tried more than one hundred and four hundred kinds of known antitumor drugs, and has unexpectedly found for the first time a class of active ingredients which can effectively inhibit anti-membrane melanoma, namely cucurbitacin B shown in formula (I), or pharmaceutically acceptable salts, solvates or prodrugs thereof. Experiments show that cucurbitacin B directly targets GRP78/Bip protein, so that FOXM1-PLK1-KIF20A signal channel is inhibited, CDK1-cyclinB1 cyclin downstream of the signal channel is down-regulated, tumor cell cycle G2/M phase block is induced, and further growth of conjunctival melanoma is inhibited. The present invention has been completed based on this finding.

Active ingredient for treating conjunctival melanoma

In the present invention, an active ingredient that can inhibit conjunctival melanoma is provided. The active component is cucurbitacin B as shown in formula (I).

Experiments show that the cucurbitacin B serving as an active ingredient inhibits a FOXM1-PLK1-KIF20A signal channel by directly targeting GRP78/Bip protein, and down-regulates the expression of CDK1-cyclin B1 cyclin at the downstream of the signal channel, induces the G2/M phase block of a tumor cell cycle, and further inhibits the growth of subcutaneous transplantation tumor of a mouse.

As used herein, "active ingredient," "active compound of the invention," "active ingredient of the invention," are used interchangeably and refer to cucurbitacin B and structural analogs thereof.

Cucurbitacin B with molecular formula C₃₂H₄₆O₈Are naturally occurring tetracyclic triterpenoids. Terpenoids (terpenoids) are a generic term for compounds and their derivatives formed by isoprene as a basic structural unit and increasing in a side chain repeat manner, and are a very important family in plant secondary metabolic links. Triterpenes (triterpenoids) are an important class of terpenoids, widely distributed in nature, mainly in vines, fungi, monocotyledonous and dicotyledonous plants. The triterpenoid is used as a secondary metabolite of plants, and most of the triterpenoids have the effect of preventing diseases, pests or fungi from causing damage to the plants. Cucurbitacin B (cucurbitacin B) mainly exists in cucurbitaceae plants, is an important member in a cucurbitacin family, and has wide pharmacological activity.

It is to be understood that the active ingredient of the present invention includes tetracyclic triterpenoids of formula (I), or pharmaceutically acceptable salts, enantiomers, diastereomers or racemates thereof, or prodrugs thereof. It is to be understood that the active ingredients of the present invention also include crystalline, amorphous, and deuterated forms of the compounds of formula (I).

The pharmaceutically acceptable salt is a conventional non-toxic salt formed by reacting the compound of the formula (I) with an inorganic acid or an organic acid. For example, conventional non-toxic salts can be prepared by reacting a compound of formula (I) with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of formula (I) and propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid after forming ester and then forming inorganic base; or the methylamine, ethylamine or ethanolamine salt of a compound of formula (I) with an organic base; or the compound of the formula (I) forms ester with lysine, arginine and ornithine and then forms corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid or forms corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

Pharmaceutical composition and application

The invention also provides application of the tetracyclic triterpenoid shown in the formula (I) or one or more of pharmaceutically acceptable salts, enantiomers, diastereomers or racemates and prodrugs thereof as an active ingredient in preparing a medicament for treating and/or preventing and relieving conjunctival melanoma and other related diseases.

The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of active ingredient, preferably 0.1-90 wt% of the compound of formula (I) as active ingredient, the rest being pharmaceutically acceptable carrier, diluent or solution or salt solution.

If necessary, one or more pharmaceutically acceptable carriers can be added to the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.

The compounds and pharmaceutical compositions provided herein may be in a variety of forms such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and the like, and may be presented in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or instillation.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the preparation formulation thereof usually contains 0.05-400mg of the compound of formula (I), preferably 1-500 mg of the compound of formula (I).

The compounds and pharmaceutical compositions of the present invention may be administered to mammals in the clinical setting, including humans and animals, by oral, nasal, dermal, pulmonary or gastrointestinal routes of administration. Most preferably oral. Most preferably, the daily dose is 0.01-400mg/kg body weight, and is administered once or in portions of 0.01-200mg/kg body weight. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.

The drug or inhibitor of the present invention can be administered by a variety of different means, e.g., by injection, spray, nasal drop, eye drop, osmotic, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed with other materials or encapsulated and introduced into body.

The advantages of the invention include:

(1) the compound cucurbitacin B can effectively inhibit the proliferation of various conjunctival melanoma cells at an extremely low concentration, and inhibits IC (integrated circuit) of the conjunctival melanoma cells CM-AS16, CRMM1, CRMM2 and CM2005.1₅₀The values were 0.08. mu.M, 0.24. mu.M, 0.15. mu.M and 0.38. mu.M, respectively.

(2) Experiments show that cucurbitacin B directly targets GRP78/Bip protein, so that FOXM1-PLK1-KIF20A signal channel is inhibited, CDK1-cyclinB1 cyclin at the downstream of the signal channel is down-regulated, tumor cell cycle G2/M phase block is induced, and growth of subcutaneous transplantation tumor of mice is inhibited.

(3) The compound has low toxic and side effects and good pharmacy.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1: proliferation inhibiting effect of cucurbitacin B on membrane melanoma cells CM-AS16, CRMM1, CRMM2 and CM2005.1

The Han's conjunctival melanoma CM-AS16 cells used in the experiment were obtained from the ninth national hospital of Shanghai university of transportation, the conjunctival melanoma cell lines CRMM1, CRMM2 and CM2005.1 were obtained from the medical center of Layton university in the Netherlands, and the normal cells HL7702 were purchased from the cell bank of the Chinese academy of sciences. Cucurbitacin B was obtained from a laboratory aged drug depot, MEK162 from MCE, CCK-8 from TargetMol, usa. Cells grown in log phase were collected at 1X 10⁴The density of each hole is inoculated in a 96-hole plate, and the medicine is added after the cells adhere to the wall. Drugs were added to 96-well plates to final concentrations of 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M, 0.625. mu.M, 0.312. mu.M, 0.156. mu.M, 0.078. mu.M and 0.039. mu.M, respectively. A positive control drug MEK162 group and a blank control group were set. After 72 hours of culture, the drug-containing medium was aspirated, 10% CCK-8 solution was added, incubation was performed for 1 to 4 hours in the dark, absorbance was measured at 450nm using a microplate reader, and the growth inhibition rate at different concentrations was calculated, where (%) (average value of Control group-average value of administration group)/average value of Control group ═ 100%. The concentration of the compound with the cell growth inhibition rate of 50 percent is the IC₅₀The value is obtained. Experimental data are expressed as mean ± SD values and data analysis is performed using Graphpad 7.0.

The results are shown in the table below, and cucurbitacin B has inhibition effect on proliferation of each conjunctival melanoma cell, and IC₅₀The values are 0.15. mu.M, 0.08. mu.M, 0.24. mu.M and 0.38. mu.M, respectively, and are nontoxic to normal cell HL 7702.

Comparative example 1: proliferation inhibiting effect of conventional antitumor drug on conjunctival melanoma cells CM-AS16 and CRMM2

Used Chinese conjunctival melanoma CM-AS16 cellsFrom the ninth national hospital of the university of transportation, shanghai, the conjunctival melanoma cell line CRMM2 was from the university of leiton, netherlands medical center. The conventional antitumor drugs of cisplatin, dacarbazine and thalidomide are from old drug libraries in laboratories. Cells grown in log phase were collected at 1X 10⁴The density of each hole is inoculated in a 96-hole plate, and the medicine is added after the cells adhere to the wall. The final concentrations of drug added to the 96-well plate were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M, 0.78. mu.M and 0.39. mu.M, respectively. A positive control drug MEK162 group and a blank control group were set. After 72h of incubation, the drug-containing medium was aspirated, 10% CCK solution was added, and the cells were incubated for 1 to 4h in the dark, and the absorbance was measured at 450nm using a microplate reader to calculate the growth inhibition rate at different concentrations, where (%) growth inhibition rate (average of Control group-average of administration group)/average of Control group ═ 100%. The concentration of the compound at which the inhibition of cell growth was 50% was the IC50 value. Experimental data are expressed as mean ± SD values and data analysis is performed using Graphpad 7.0.

As shown in the following table, conventional antitumor agents such as cisplatin, dacarbazine, and thalidomide have poor inhibitory effects on conjunctival melanoma cell proliferation (IC thereof)₅₀Values all greater than 20 μ M). This may be related to the specificity of the conjunctival melanoma tumor species, which is different from the common tumors in terms of both its prognostic site and genetic characteristics.

Example 2: effect of cucurbitacin B on cell cycle and apoptosis

The cell cycle and apoptosis detection kit and the cell apoptosis kit used in the experiment are purchased from Biyuntian biotechnology company. CRMM2 cells grown in log phase were collected at 1X 10⁵The density of each cell/well is inoculated in a 6-well plate, and the medicine is added after the cells adhere to the wall. The final concentrations of drug added to the 6-well plates were 0.1. mu.M, 0.2. mu.M, respectively. A positive control drug MEK162 group and a blank control group were set. After 24h of culture, cells were collected according to kit instructions and incubated and stained in the dark, and a detection cycle was performed using a flow cytometerAnd apoptotic changes.

As shown in FIG. 2, cucurbitacin B can effectively cause the G2/M phase block of cells and cause the apoptosis of the cells.

Example 3: inhibition of FOXM1-PLK1-KIF20A signaling pathway by cucurbitacin B Primary antibodies used include FOXM1 antibody, PLK1 antibody, KIF20A antibody, CDK1 antibody, CyclinB1 and GAPDH antibody purchased from Abcam company. The secondary antibodies included rabbit and murine antibodies, purchased from Saint Hipposhu Biotech, Inc. BCA kit, RIPA lysate and ECL developer were purchased from Shi next Biotech Ltd. Cells grown in log phase (CRMM2, CM-AS16, CRMM1, CM2005.1, HL7702) were harvested at 1X 10⁵The density of each cell/well is inoculated in a 6-well plate, and the medicine is added after the cells adhere to the wall. The final concentrations of drug added to the 6-well plates were 0, 0.1. mu.M, 0.2. mu.M, respectively. After 24h of culture, cells were collected, lysed thoroughly with RIPA lysate and centrifuged to take the supernatant. The BCA kit quantitated the total protein concentration. Protein separation was performed by SDS-PAGE, followed by PVDF membrane, BSA blocking, addition of specific antibody (primary antibody) for immunoreaction, and overnight incubation at 4 ℃. Incubation with HRP-labeled secondary antibody for 1h at room temperature, and final detection by ECL chemiluminescence, scanning with Tanon-4600SF apparatus.

The results are shown in FIG. 3, and cucurbitacin B can inhibit FOXM1-PLK1-KIF20A signaling pathway and cycle-related protein CDK1-cyclinB1 of conjunctival melanoma cells.

Example 4: cucurbitacin B binds to GRP78 protein and inhibits its function

GRP78 protein used for the experiments was purchased from Abcam, RED-tris-NTA protein labeling kit was purchased from NanoTemper, and malachite green phosphate detection kit was purchased from Cayman. GRP78 protein was dissolved in 1 XPBST and RED-tris-NTA was added to label the protein at a concentration of 100 nM. The marked GRP78 protein and cucurbitacin B with different concentrations are incubated according to the volume ratio of 1:1, the maximum concentration of the drug is 100 mu M, and 2-fold gradient dilution is carried out for 12 gradients. The protein-drug solution was aspirated by capillary and placed in Monolith NT for detection, and the binding curve is shown in FIG. 4A. For the GRP78 ATPase activity assay, 20mM Tris (pH 7.5), 50mM KCl, and 1.5mM MgCl were used₂The buffer solution is used for preparing the cucurbitacin BDifferent concentrations of 0-100 μ M are reserved, and 0.25 μ M GRP78 protein is added into the liquid medicine and incubated for 3h at 37 ℃. Adding a detection reagent of the working kit into the malachite green phosphate detection kit according to the instruction of the malachite green phosphate detection kit, and detecting the light absorption value at the wavelength of 620 nm.

As a result, as shown in FIG. 4A, cucurbitacin B was able to bind to GRP78 protein with a Kd value of 0.11. mu.M; as shown in FIG. 4B, cucurbitacin B can further inhibit ATPase activity of the protein.

Example 5: use of Gene knockdown to influence the biological function of a target protein

Cells grown in log phase (CRMM2, CM-AS16, CRMM1, CM2005.1) were seeded into six-well plates at a density of 1.0X 10⁵Individual cells/well were cultured for 24h prior to shGRP78 adenoviral transfection (using the gro construct and experimentally confirmed GRP78 knock-down of the adenoviral-packaged gene). After the cells are infected for 24h, removing the adenovirus and continuing to culture for 24h, extracting proteins in the cells to perform a subsequent Western blot experiment, and detecting the expression conditions of target proteins FOXM1, PLK1, KIF20A, CyclinB1 and CDK 1.

As a result, as shown in fig. 5, GRP78 knockdown reduced expression of FOXM1, PLK1, KIF20A, CyclinB1, and CDK1 proteins.

Example 6: tumor bearing experiment in mice

The CMMC2 cells grown logarithmically were prepared at a concentration of 1X 10⁷The cells were suspended at a volume/mL and inoculated subcutaneously into the forelimb axilla of a 6-7 week-old NCG male mouse, and the inoculated mice were randomly divided into a treatment group and a control group (7 mice per group). The size of the tumor mass is about 50mm³At the beginning of the administration, different administration modes were set at the time of administration, and control groups of different doses:

(1) MEK162 (gavage) dosing: administered at a dose of 10 mg/kg/day;

(2) intraperitoneal injection of CuB: CuB is injected at 1mg/kg daily.

The administration was continued for 5 weeks, and the change in body weight and tumor volume of the mice was observed 2 or 3 times per week, and a trend graph of the change in tumor volume with time and a trend graph of the change in body weight of the mice with time were plotted.

The results are shown in fig. 6A, and cucurbitacin B can inhibit the growth of mouse graft tumor, and as shown in fig. 6B, cucurbitacin B has no significant effect on the body weight of mice.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims (10)

1. Use of cucurbitacin B shown in formula (I), or pharmaceutically acceptable salt thereof, or solvate or prodrug thereof;

characterized in that it is used for the preparation of a pharmaceutical composition and the use of said pharmaceutical composition is selected from the group consisting of: treating and/or alleviating conjunctival melanoma, or inhibiting conjunctival melanoma cell proliferation.

2. The use of claim 1, wherein said pharmaceutical composition is further used to modulate the FOXM1-PLK1-KIF20A signaling pathway.

3. The use of claim 1, wherein said pharmaceutical composition is further used to down-regulate the activity or expression of FOXM 1.

4. The use of claim 1, wherein said pharmaceutical composition is further used to down-regulate the activity or expression of PLK 1.

5. The use of claim 1, wherein said pharmaceutical composition is further used to down-regulate the activity or expression of KIF 20A.

6. The use according to claim 1, wherein the pharmaceutical composition is further used for modulating the cycle-related protein CDK 1-cyclenb 1.

7. The use of claim 1, wherein the pharmaceutical composition is further used to down-regulate the expression of CDK1-cyclin b1 cyclin downstream of the signaling pathway.

8. The use of claim 1, wherein said pharmaceutical composition is further used to induce a G2/M phase block in the tumor cell cycle.

9. Use of cucurbitacin B shown in formula (I), or pharmaceutically acceptable salt thereof, or solvate or prodrug thereof;

the method is characterized by being used for preparing a pharmaceutical composition, and the pharmaceutical composition is used for down-regulating the activity or expression quantity of GRP78 protein.

10. A method of treating and/or ameliorating conjunctival melanoma comprising the steps of: administering to a subject in need thereof a safe and effective amount of cucurbitacin B represented by formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof:

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