HK40016841B - Method and compounds for inhibiting the mcm protein complex and their application in cancer treatment - Google Patents

Description

Methods and compounds for inhibiting MCM protein complexes and their use in treating cancer

Cross Reference to Related Applications

This PATENT application is related to the PATENT application of the International PATENT Cooperation TREATY filed on 9/5/2013 (PATENT Cooperation TREATY; no. PCT/US 2013/040287) and the U.S. provisional PATENT application filed on 9/5/2012 (No. 61/644, 442), the entire contents of which are incorporated herein by reference.

The invention belongs to the field of the following:

the invention relates to the use of drugs to inhibit the function of a heterocyclic MCM protein complex containing 6 subunits in the DNA replication process in cancer cells to treat cancer, and to screening and identifying compounds by detecting the position and function of MCM protein subunits, such as hMcm2and hMcm6, in drug candidate treated cells.

Background

Cancer cells can grow out of control, divide, erode surrounding tissue, and can invade or spread to other parts of the body through the lymphatic and blood circulation systems. Conventional cancer treatments include the removal or destruction of cancerous cellular tissue, e.g., surgery, chemotherapy, radiation therapy, immunotherapy, and the like. Then, a common challenge faced by all therapies is how to destroy cancer cells while not causing serious damage to normal cells and tissues. In recent decades, the field of chemotherapy has been concerned with the development and investment of cytotoxic compounds. A large number of compounds have been shown to have cytotoxic and anti-tumor efficacy. Only about 40 of the over 600000 cytotoxic compounds are considered of some clinical interest (Schwartsmann et al, by 1988). The reason for this low success screening rate is that the toxicity of drugs causes them to often have a very narrow therapeutic window between the effective anticancer dose and the toxic dose. Thus, screening of antitumor drugs based on compounds having cytotoxicity is greatly limited, and has a large gap from the actual clinical efficacy. What is needed is a drug that has greater specificity for cancer cells, i.e., is resistant to tumors, while not causing significant damage to normal nuclear tissue.

Summary of the invention:

the invention aims to be as follows: a method and drug are provided that are capable of destroying tumor cells without causing significant damage to normal cells and tissues. This method is performed by disrupting the formation of an MCM (minichromosomal maintenance protein) complex, which plays a key role in the assembly of a pre-replication complex (this process is also called replication permissivity) and the extension process of DNA replication. Initiation of DNA replication requires a series of replication initiation proteins to form a pre-replication complex in order at each origin of replication. The activity of the MCM complex requires all six MCM subunits (MCM 2-MCM 7) to form a hetero-six-membered ring and to be assembled to the origin of replication with the help of ORC1-6, NOC3, IPI1-3, CDT1, cdc6 and possibly other proteins. As a member protein of the AAA + (ATPases associated with a variable of cellular activities) family, the MCM complex migrates along the replication fork, possibly acting as a replication helicase, unwinding the DNA double strand. Since all six MCM subunits are required in DNA replication, previous studies by the inventors are disclosed in US Pat. nos.7393950 and 8318922 (which patents are incorporated herein by reference), which indicate that: antisense oligonucleotide sequences capable of specifically binding to MCM subunits can inhibit cell proliferation.

Because all subunits of MCM proteins can fulfill their function by forming a complete cycle, the interaction between any two subunits that interrupt the heterotrimeric complex should disrupt the MCM ring structure and inactivate the MCM complex, inhibit DNA replication, and cause apoptosis in tumor cells without affecting normal cells. In the present invention, we screened and identified several compounds that inhibit the interaction and activity of human MCM proteins and block DNA replication and cell proliferation of tumor cells from plant extracts, component and compound libraries and artificially synthesized compounds. As an example of the present invention, several small molecules we have identified and invented can disrupt the interaction between hMcm2and hMcm6, impair the nuclear localization of MCM proteins and their binding to chromatin, and inhibit DNA replication. In addition, the compounds can induce tumor cell apoptosis without obvious cytotoxicity to normal cells, and show strong antitumor activity in a nude mouse transplantation tumor model without obvious toxic and side effects to nude mice.

The fundamental reasons for this specificity are: normal cells have an inspection system that can arrest the cells in the G1 phase, thereby avoiding apoptosis; cancer cells lack the checking system to control the process, so that normal cells can identify whether the MCM protein structure is complete or not and whether the function is complete or not, the MCM protein can enter an S phase to split only when the function is complete in the DNA replication process, and the MCM protein can stop in a G1 phase, so that a motor vehicle can brake because a front bridge is broken; the cancer cell can continue to advance to the S phase because the vehicle with the brake failure can not be stopped.

Another protection of this patent is: we have established a screening platform for screening anticancer drugs that enables screening of drugs that have a killing effect on cancer cells without affecting normal cells. This is accomplished by verifying whether the drug candidate disrupts the formation of the MCM protein complex, leaving the MCM subunits in the cytoplasm and not being transported to the nucleus to become dysfunctional.

The method comprises the following steps: 1) Adding the candidate compound to the cell culture medium and treating the cells for a period of time; 2) Detecting the level of functional MCM protein in the cells to realize the screening of the compound with anticancer function. The level of functional MCM protein may be measured by measuring the proportion of MCM subunits distributed between the nucleus and cytoplasm, since only functionally intact protein complexes can be present in the nucleus, and thus the more destructive the compound is to MCM protein complex formation, the fewer MCM subunits can be localized in the nucleus. The distribution of MCM subunit in the cell can be detected by an indirect fluorescence detection method, and after the cell is treated by the compound, a secondary antibody with fluorescence label can identify and combine with endogenous MCM protein primary antibody, thereby determining the position of MCM protein in the cell. Alternatively, direct observation of fluorescence can be performed by the method: the fluorescent protein-fused MCM subunits may be detected after treatment of the cells with the compound by transfecting the cells with a plasmid capable of expressing one or more fluorescent protein-fused MCM subunits, such as hMcm2-GFP and/or hMcm 6-GFP.

In addition, screening for anti-cancer drugs can also be achieved by detecting DNA replication by methods such as BrdU nuclide incorporation, flow cytometry, etc., and whether the compounds have different cell proliferation-inhibiting abilities in normal nuclear cancer cells, in addition to disrupting MCM structure, can also be detected by other procedures.

Another object of the patent is to provide highly specific compounds against tumors by this mechanism. Compounds having this function generally have the following structural rules: main structure I (4 ring main structure)

Wherein R1 is H or one to several glycosyl groups; r2 is a5 or 6 membered ring in B (. Beta.) configuration; r3 and R4 are H or OH; r3 and R4 may also be an O atom and form a 3-membered ring with the C atom to which R3 and R4 are attached; r5 is OH, R6 is H, R5 and R6 may also be an O atom and form a 3-membered ring with the C atom to which R5 and R6 are attached.

All compounds and related methods protected in the current patent are applicable to all cancer species that are sensitive to MCM protein inhibitory drugs, including cervical, prostate, colon, breast, ovarian, acute myeloid, chronic lymphocytic, non-hodgkin and hodgkin lymphomas, acute lymphocytic leukemias, pancreatic, gastric, skin, bladder, esophageal, nasopharyngeal, small cell lung, and non-small cell lung cancers, among others.

To summarize: the technical core and the protection focus of the patent are drugs capable of selectively destroying cancer cells by destroying MCM proteins.

The various points of innovation in this patent are set forth with particularity in the content of the protection sought. To facilitate an understanding of this patent disclosure, the operational advantages, objects of use, references, etc., of the present invention are set forth in the following description.

Illustration of the drawings

FIG. 1 shows that: the antitumor compounds of the present invention have the structure (3-8) and inactive isomers (1-2)

In FIG. 2, A: direct microscopic observation; B-H: WST-1 (water soluble tetrazolium-1) measurement data; 17beta-deacetyl cerberenin (17 beta-deacetyl cerberenin; see A-C), 17 beta-oleandrin (17 beta-Neriifolin; see D) and 17beta-deacetyl cerberenin diol (17 beta-deacetyl cerberenin diol; see E) are representative of three compounds having anticancer activity in the present application, and these drugs have high selectivity to cancer cells and low specificity to normal cells. The control drugs Paclitaxel (Paclitaxel; taxol; see F) and etoposide carbonate (VP 16; see G and H) are very toxic to normal cells and have poor selectivity to normal and cancer cells.

FIG. 3 shows: it can be observed by immunofluorescence that 17beta-deacetyl cerberenin and 17 beta-oleandrin can destroy the binding between MCM subunits, and the effect of 17beta-deacetyl cerberenin diol is weaker than that of the former two.

FIG. 4 shows that: it was observed by immunofluorescence that 17 β -deacetyl ceriferin, 17 β -oleandrin and 17 β -deacetyl ceriferin diol all disrupted the localization of hMcm2and hMcm6 in the nucleus.

FIG. 5 shows that: chromatin immunoprecipitation experiments and flow cytometry methods showed: 17beta-deacetyl cerberenin is capable of inhibiting the pre-replication complex (pre-RC) assembly, leading to apoptosis of cancer cells.

FIG. 6 shows that: the flow cytometer method can observe: the 17beta-deacetyl cerbera mangiferin can inhibit DNA replication and guide cancer cell apoptosis.

FIG. 7 shows that: it was observed that 17 β -deacetyl cerberenin can inhibit DNA replication by BrdU nuclide incorporation.

FIG. 8 shows that: flow cytometry and annexin V staining methods can be observed: 17beta-deacetyl cerberenin, 17-oleandrin and 17-deacetyl cerberenin diol can all cause apoptosis of cancer cells.

FIG. 9 shows that: detected by the method of WST-1: by treating the cells with 17 β -deacetyl cerberrin and then removing the drug, normal cells can resume growth, while cancer cells cannot.

FIG. 10 shows: the antitumor effect of 17 β -deacetyl ceriferin was observed in nude mice.

FIG. 11 shows that: in the experiment shown in fig. 10, 17 β -deacetyl cerberenin was not significantly toxic to nude mice. Detailed description of specific embodiments of the invention

Cell lines and plasmids

The human MCM6 and MCM2 cDNA fragments were cloned into pEGFP-C3 (Invitrogen) for intracellular localization detection, respectively. HeLa cells (cervical adenocarcinoma), HK1 (nasopharyngeal carcinoma), C666-1 (nasopharyngeal carcinoma), hepG2 cells (liver cancer) and Hep3B cells (liver cancer) were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS). L-02 cells (human normal hepatocytes, from the institute of biochemistry and cell research of the Shanghai Life sciences of the Chinese academy of sciences) were cultured in RPMI 1640 medium at 10% FBS. NP460 cells were cultured in 1. All cell lines were cultured in an incubator at 37C,5% carbon dioxide.

Determination of antiproliferative Activity

To test the effect of drugs on human cell lines and to calculate the semi-lethal dose (IC) for each cell line ₅₀ ) Tumor cells such as HepG2 cells, heLa cells and Hep3B cells (about 4X 105 cells/well) and human normal liver cells L-02 (about 5X 105 cells/well) were seeded in 96-well plates, respectively, and incubated in 100. Mu.l of medium and at 37 ℃ for at least 12 hours. The drug was then treated with 2-fold gradient dilutions for 48 hours. The medium was removed and 100. Mu.l of fresh medium containing water-soluble tetrazolium-1 (WST-1, 1. Mu.M) was added. The cells were returned to the incubator for 2 hours. Absorbance at 405nm (630 nm reference) was measured. Six hours before addition of WST-1, a known number of cells were seeded into a 96-well plate to obtain a gradient of cell number and its absorbance OD ₄₀₅ The standard curve of (2). Cells of the test sampleThe amount can be calculated from this curve. Cell viability was expressed as the percentage of the number of drug-treated cells relative to the number of DMSO-treated control cells.

Lead compound 17beta-deacetyl cerbera mangifera (17 beta- Deacetyltandhin) separation

Samples for activity testing were prepared as follows: 10-100 g of plant sample (whole plant, root, rhizome, stem, leaf or fruit, etc.) was extracted with methanol at room temperature for 3 times to obtain total extract of each sample. Suspending the total extract in water, and sequentially extracting with diethyl ether, ethyl acetate and n-butanol to obtain 4 fractions, i.e. diethyl ether, ethyl acetate, n-butanol and water fraction. The total extract and four parts of each plant sample are respectively dissolved in DMSO to prepare a stock solution of 10mg/ml, and the purified monomer is prepared into a stock solution of 1 mg/ml. The activity screening was performed using our reverse phase two hybrid yeast system.

By adopting the method, 1 sample with stronger activity, namely the ether part of the mango (Cerebra manghas and Cerebra odorlam) branch and leaf extract, is screened out. Then adopting separation strategy under the guidance of activity, and applying various column chromatography methods including silica gel (SiO) ₂ ) MCI-gel CHP 20P (Mitsubish Chemical Corporation, japan), chromatex ODS (Fuji Silysia Chemical Ltd., japan) and Toyopearl (Tosoh Corporation, japan) from which an active ingredient, the lead compound 17. Beta. -deacetylhamartonin, was isolated.

17βStructural identification of-deacetylceriferin

The structure of the lead compound 17beta-deacetyl ceriferin is determined according to the spectral data. Nuclear Magnetic Resonance (NMR) data were measured using a Varian-400 NMR spectrometer. Chemical Shift value [ relative to internal Standard Tetramethylsilane (Me) ₄ Si)]Expressed in ppm. High resolution electrospray mass spectrometry (HR-ESI-MS) was performed on a Q-TOF mass spectrometer (Bruker Daltonics, MA, U.S.A.). The spectral data are as follows:

high resolution electrospray mass spectrum (positive ion mode) m/z 549.3077[ m + H ]] ⁺ (the calculated molecular formula is C ₃₀ H ₄₅ O ₉ :549.3064). ¹ H-NMR(400MHz,pyridine-d ₅ ):6.31(1H,s,H-22),5.25(1H,d,J＝2.9Hz,H-1'),5.20(1H,m,H-21),5.02(1H,dd,J＝18.0,1.4Hz,H-21),4.33(1H,m,H-5'),4.12(1H,brs,H-3),4.09(1H,dd,J＝9.0,4.0Hz,H-2'),4.03(1H,t,J＝9.5Hz,H-3'),3.85(3H,s,3'-OMe),3.69(1H,m,H-4'),3.41(1H,d,J＝5.8Hz,H-7),2.82(1H,dd,J＝9.0,5.0Hz,H-17),1.66(1H,d,J＝6.2Hz,H-6'),1.06(3H,s,H-19),0.99(3H,s,H-18). ¹³ C-NMR(100MHz,pyridine-d5):32.7(C-1),28.0(C-2),73.7(C-3),33.5(C-4),34.8(C-5),28.9(C-6),51.9(C-7),65.1(C-8),32.5(C-9),34.4(C-10),21.5(C-11),41.4(C-12),53.2(C-13),82.4(C-14),35.9(C-15),29.3(C-16),51.5(C-17),18.0(C-18),25.0(C-19),175.9(C-20),74.4(C-21),118.4(C-22),175.1(C-23),99.6(C-1'),74.0(C-2'),86.0(C-3'),77.2(C-4'),69.6(C-5'),19.2(C-6'),61.2(C-3'-OMe).

Deducing the molecular formula of the lead compound as C according to the high-resolution mass spectrum ₃₀ H ₄₄ O ₉ In combination with hydrogen spectrum ( ¹ H-NMR), i.e. a methylene signal at C-21 (5.20, m;5.02,dd, j =18.0,1.4 hz) and the olefinic proton signal at C-22 (6.31,s), and the terminal glycosyl hydrogen signal (5.25,d, j =2.9 hz), which are presumed to be a cardiac glycoside component. Compared with the main cardiac glycoside nerriforlin in Cerbera manghas (Cerbera manghas) leaves, the C-7 and C-8 signals of the lead compound are obviously shifted to low fields, which indicates that the position is possibly connected with an epoxy group, and the existence of the 7,8-epoxy group can be determined by comparing with the cardiac lactone compound with the 7,8-epoxy group. C-17 was presumed to be configurational from the H-17 signal (2.82, dd, J =9.0,5.0 Hz), and further by the C-12 signal under the lactone ring shielding effect (C-12) _c 41.4 ) was confirmed. The aglycone structure can be deduced from the above information, and the aglycone structure is confirmed to be 3-hydroxy-7, 8-epoxy-14-hydroxy-cardiotonic-20 (22) -enolide [3-hydroxy-7,8-epoxy-14-hydroxy-card-20 (22) -enolide by comparing with data reported in the literature]. Part of the sugar was deduced to be-L-thevetose (-3-O-methyl-6-deoxy-L-glucopyranose) from comparison with hydrogen and carbon spectra data reported in the literature. According to the above-mentioned evidences,the structure of the lead compound is presumed to be 3-O- (3-O-methyl-6-deoxidation- -L-glucopyranose) -7, 8-epoxy-14-hydroxyl-cardiotonic-20 (22) -alkene lactone [3-O- (3-O-methyl-6-deoxy-L-glucopyranosyl) -7,8-epoxy-14-hydroxy-card-20 (22) -enolide](17-deacetylceriferin; 17-deacetyltanngin).

Immunohistochemical analysis

Immunohistochemistry measures the effect of a drug on the localization of hMcm2and hMcm6 in vivo within cells. HeLa cells grown on coverslips (coated with poly-D-lysine) were treated differently and fixed with 4% PFA in PBS for 20 min at room temperature. After 20 minutes of 0.1% Triton X-100 permeabilization, cells were blocked with 1% bsa for half an hour and then incubated with goat anti-hMcm 6 (Santa Cruz,1 500) and mouse anti-hMcm 2 (Becton Dickinson, 1, 500) for 1 hour at room temperature. The coverslips were washed three times with PBS for 10 minutes each, and then incubated with Alexa Fluor 488-labeled donkey anti-goat antibody and Alexa Fluor 594-labeled donkey anti-mouse antibody (Invitrogen; 1. Nuclei were stained with Hochest 33852 (Sigma Chemical Company,1 μ g/ml) for 15 minutes and washed 3 times with PBS. Finally, the cells were fixed with a fixative (Biosciences) and observed under a fluorescent microscope (nikon TE 2000E).

Cell synchronization experiment

HeLa cells were pretreated with thymine (2. Mu.M) for 18 hours, released in fresh medium for 6 hours, and then added with thiaurethane-pyridazole (nocodazole; 0.1. Mu.g/ml) for 6 hours to give early M-stage synchronized cells. HeLa cells were treated with 0.5 mmol/l mimosine for 20 hours, with cell synchronization at the boundary of the G1/S phase. Cells in the G1/S phase were synchronized and released into hydroxyurea-containing medium for 4 hours to obtain early S phase cells.

BrdU incorporation experiments

Hela cells grown on poly-D-lysine-coated coverslips were drug-treated for 24 hours and then incubated with 50. Mu.M BrdU (Sigma) in a 37-incubator for 1 hour. The cells were then fixed with 4% PFA in PBS for 20 minutes at room temperature, after permeabilization with 0.1% Triton X-100 for 20 minutes, the cells were denatured with 4M hydrochloric acid for 10 minutes, 1% BSA was blocked for half an hour, and then incubated with anti-BrdU (Sigma; 1; 500) and anti-mouse IgG-FITC conjugate antibody (Sigma; 1. Finally the BrdU signal was observed under a fluorescence microscope (Nikon TE 2000E).

Chromatin binding assays

HeLa cells grown in six-well plates were collected by trypsinization after drug treatment. Washed twice with cold PBS. According to about 20ul/10 ⁶ Amount of cells E.B. buffer (100mM KCl,50mM HEPES-KOH pH7.5,2.5mM MgCl) ₂ ,50mM NaF,5mM Na ₄ P ₂ O ₇ ,0.1mM NaVO ₃ 0.5% Triton X-100,1mM PMSF, 2. Mu.g/ml Pepstatin A, 20. Mu.g/ml Leuteptin, 20. Mu.g/ml Aprotetin, 0.2mM Pefabloc,2mM Benzamidine HCl and 0.2mg/ml Bacitracin) suspension and lysis of the cells. The cell lysate was pipetted several times every 2-3 minutes in an ice bath for 10 minutes. An equal volume of 30% ice cold sucrose solution (containing protease inhibitor) was added to the bottom of the tube in e.b. buffer. Cryocentrifugation was performed at the highest speed for 10 min to separate chromatin and free protein. The supernatant was transferred to a new tube and stored on ice. The precipitate was washed with an equal volume of e.b. and flicked against the vessel wall or briefly vortexed. The suspension was centrifuged again at the highest speed for 5 minutes. The supernatants from both centrifugations were combined. The pellet was suspended with half the volume of the supernatant e.b. The supernatant and pellet were used for immunoblotting experiments.

Flow cytometry (FACS) analysis

The suspended and adherent cells were collected and washed once with PBS. Fix with 70% ethanol and leave at-20 for 1 hour to overnight. The cells were collected by low-temperature centrifugation at 6,000 rpm for ten minutes. The cells were washed well with ice-cold PBS and collected by centrifugation (same as above). Then stained in P.I. solution (50. Mu.g/ml RNase A,0.1% Triton X-100,0.1mM EDTA (pH 7.4), and 50. Mu.g/ml propidium iodide) for 30 minutes, and finally analyzed by flow cytometry (Becton Dickinson Co.)

Identification of antiproliferative agents with high recognition specificity for cancer cells versus normal cells

Through preliminary screening of hundreds of samples consisting of plant extracts, components, compounds and chemically synthesized compounds, we identified several candidates that could inhibit human MCM protein and DNA replication. After separation, purification and detection by using activity as an index, we screened a small molecule compound named 17 β -deacetyl ceriferin (17 β -deacetyl ceriferin; DAT, figure 1), and verified that several other compounds and a chemical derivative with similar structures all have the activity of inhibiting human MCM protein and DNA replication (figure 1).

A pair of human cell lines, L-02 (normal liver cells) and HepG2 (liver cancer cells), were treated with 17 β -desacetylceriferin for 48 hours, and the density and morphology of the cells were directly observed under a microscope, and it was found that 17 β -desacetylceriferin could effectively inhibit the proliferation of tumor cells (HepG 2) and kill them, but it was also effective in inhibiting the proliferation of normal cells (L-02) but has little killing power (FIG. 2A). Thus, 17 β -deacetyl ceriferin was identified as a highly active antiproliferative compound with minimal toxicity to normal cells. The number of viable cells was measured by the method of WST-1 (Water-soluble tetrazolium-1) in FIGS. 2B-H.

To further test 17 β -deacetylceriferin, its IC was determined ₅₀ In addition to L-02 and HepG2 cells, we also treated Hep3B cells (p 53-negative hepatoma cell line), heLa cells (cervical cancer cell line), HK1 and C666-1 cells (nasopharyngeal cancer cell line), A549 cells (lung cancer cell line) and a normal nasopharyngeal cell line (NP 460) immortalized with hTERT gene with 17 β -deacetyl ceriferin for 48 hours, and determined the cell viability by the WST-1 method (FIG. 2B, C). Experiments show that the 17beta-deacetyl cerberrin is a compound capable of killing various cancersThe broad-spectrum anticancer candidate drug of the cell has obvious inhibition effect on all tested cancer cell strains, and has less damage to normal cells. Although 17beta-deacetyl cerberenin IC in different cancer cell lines ₅₀ Some difference in value, IC of all cancer cells tested ₅₀ Average of 0.1. Mu.g/ml (. About.0.2. Mu. Mol), and IC in normal cells ₅₀ The values were significantly higher (over 4. Mu.g/ml). In addition, 17 β -oleandrin (FIG. 2D), 17 β -desacetylhamiltonindiol (less active against cancer than 17 β -desacetylhamiltonidin; FIG. 2E), bufalin, resibufogenin and cinobufagin, which possess similar structures, also exhibited anticancer activities and selectivity to cancer cells similar to 17 β -desacetylhamiltonidin (see Table below). On the other hand, the antiproliferative activity of 17 α -deacetyl ceriferin and 17 α -oleandrin was low (see table below), indicating that the antiproliferative activity of the 17 β structure is crucial.

As reference, the clinical anticancer drugs Paclitaxel (Paclitaxel; taxol; FIG. 2F) and VP16 (Etoposide phosphate; FIG. 2G, H) did not show selectivity to cancer cells and normal cells in the experiment (they were cytotoxic to both normal and cancer cells).

Interference with MCM complex formation and nuclear localization of MCM proteins

We treated human cells with 17 β -desacetylcerberrin and detected co-immunoprecipitation of hMcm2and hMcm6 proteins in the total extracted protein to examine whether 17 β -desacetylcerberrin has been targeted to hMcm2and hMcm6 proteins in human cells. The results show that: 17 β -deacetylhamnobuterol blocked the interaction between hMcm2and hMcm6, as well as other MCM subunits (fig. 3A). Similarly, 17 β -oleandrin also blocked the interaction between hMcm2and hMcm6, whereas 17 β -deacetylhamiltin diol had low activity (fig. 3B).

Since the MCM hetero-hexamer ring structure requires interaction between two MCM subunits, whereas the hexamer ring structure is essential for MCM entry into the nucleus, blocking the interaction of hMcm2and hMcm6 should disrupt the hexamer and nuclear localization of MCM proteins. To demonstrate this, we performed direct immunostaining using antibodies against endogenous MCM proteins and indirect fluorescent microscopy after transfection of cells with expression plasmids for hMcm2-GFP and hMcm 6-GFP.

In immunostaining, heLa cells were treated with 17 β -desacetylceriferin for 24 hours and then detected with specific antibodies against endogenous hMcm2and hMcm 6. The results show that 17 β -deacetylhamiltin reduces the nuclear localization of hMcm2and hMcm6 (fig. 4A). In direct fluorescence microscopy HeLa cells were treated with 17 β -desacetylceriferin for 36 hours starting 4 hours after transfection of hMcm2-GFP and hMcm6-GFP expression plasmids. The results showed that some of the expressed hMcm2-GFP and hMcm6-GFP were localized in the cytoplasm, whereas in untreated control cells almost all of the expressed hMcm2-GFP and hMcm6-GFP were localized in the nucleus (FIG. 4B). To rule out the possibility of indirect cytoplasmic localization of some MCM proteins due to cell cycle arrest by 17 β -deacetyl cerberenin, we arrested the cells in the G1 late phase with mimosine, at which time all MCM proteins should be in the nucleus in the absence of inhibitors, but we found that 17 β -deacetyl cerberenin also prevented the nucleation of hMcm2and hMcm6 (data not shown).

Taken together, these data strongly suggest that 17 β -deacetyl cerberenin can specifically block nuclear localization of MCM. We have also found that another compound isolated from cerbera mangifera, 17 β -oleandrin, which is structurally similar to 17 β -deacetyl cerbera, also interferes effectively with the nuclear localization of MCM's like 17 β -deacetyl cerbera mangifera, a chemical derivative of 17 β -deacetyl cerbera mangifera, 17 β -deacetyl cerbera diol, has a weaker activity (fig. 4C).

Inhibition of pre-replicative complex assembly

As a component of the pre-replication complex, the MCM complex plays a central role in the permissivity of DNA replication. Since 17 β -desacetyl ceriferin disrupts the interaction between hMcm2and hMcm6, preventing its nucleation, 17 β -desacetyl ceriferin should inhibit the binding of MCM proteins to chromatin, leading to failure of the assembly of the pre-replication complex. To demonstrate this, we performed chromatin binding experiments to detect proteins bound to chromatin. Asynchronous HeLa cells were treated with 17 β -desacetylceriferin for 24 hours, chromatin was extracted and chromatin-binding proteins were detected by immunoblotting.

Consistent with the prediction, 17 β -deacetylhamiltin caused a significant decrease in chromatin for hMcm2and hMcm6 in a dose-dependent manner (fig. 5A). These results indicate that the pre-replicative complex failed to assemble in the presence of 17 β -deacetyl ceriferin. Furthermore, flow cytometry analysis confirmed that the cells of the above experiment were apoptotic (fig. 5B).

To determine the effect of 17 β -deacetyl ceriferin on synchronized cells, heLa cells were pre-arrested in late G1/early S with thymidine and later in early M with thiabendazole. The cells were then released into fresh medium containing 17 β -deacetyl cerberenin. Control cells, including DMSO-treated and untreated cells, were able to pass through G1 phase, enter S phase, and complete one cell cycle within 36 hours (fig. 5C). However, 17 β -deacetyl ceriferin-treated cells entered G1 phase, but did not appear to enter S phase. Longer incubation with 17 β -desacetylceriferin induced apoptosis (fig. 5C). Immunoblot analysis showed that loading of MCM into chromatin was prevented (fig. 5D). These results indicate that 17 β -deacetylhamiltin prevents the assembly of the pre-replicative complex in human cells.

Inhibition of DNA replication and induction of apoptosis in cancer cells

Since 17 β -desacetyl ceriferin disrupts the interaction between hMcm2and hMcm6, inhibiting the binding of MCM proteins to chromatin, 17 β -desacetyl ceriferin should prevent DNA replication. To confirm this, we treated HeLa cells with 17 β -desacetyl ceriferin for 24 hours and then labeled with BrdU for 1 hour. BrdU incorporated into DNA was detected by anti-BrdU antibodies. The secondary antibody was observed under a fluorescence microscope using an FITC-labeled anti-mouse antibody, DAPI for nuclear staining (FIG. 6A), and BrdU-positive-reacting cells were quantified (FIG. 6B). 17 β -desacetylcerberlin significantly inhibited DNA replication in treated HeLa cells, since little BrdU signal was observed above 0.2mg/ml for 17 β -desacetylcerberlin, whereas BrdU positive cells were approximately 30% as expected in DMSO-treated and untreated cells (FIG. 6A, B).

In addition, cells treated with 17 β -desacetyl ceriferin were unable to enter S phase following the synchronous re-release of nocodazole (M phase) and started to apoptosis following longer treatment with 17 β -desacetyl ceriferin (fig. 7A). Similarly, cells released after synchronization with mimosine (MMS) (at the G1/S transition point; FIG. 7B) or Hydroxyurea (HU) (at early S phase; FIG. 7C) failed to complete S phase in the presence of 17 β -deacetyl ceriferin. These results are consistent with the inhibition of function of MCM, whether at the initiation or extension of DNA replication.

17beta-deacetyl cerberrin can also induce apoptosis of cancer cells. As shown in the sub-G1 cell population in flow cytometry analysis (FIGS. 5B,7 and 8A), 17 β -desacetylcerberlin induced cell death in tumor cells, whereas normal L-02 cells arrested primarily in the G1 phase and the amount of G2/M cells decreased (FIG. 8A). In FIG. 8A, DNA content in HepG 2and L-02 cells after 24 hours of 17 β -deacetyl ceriferin treatment was analyzed by flow cytometry. In FIG. 8B, hela cells were labeled with annexin V-Cy3 for 20 minutes and then treated with 17 β -desacetyl hamiltonin for 24h. Mitoxantrone (Mitoxantrone) was used as a positive control anticancer drug. The results show that: 17 β -deacetylhamiltin-treated cancer cells were stained by annexin V (fig. 8B), supporting the conclusion that 17 β -deacetylhamiltin induced apoptosis.

As described above, 17 β -desacetylceriferin inhibited DNA replication in non-synchronized HeLa cells (BrdU incorporation assay), while flow cytometry analysis also showed that 7-desacetylceriferin treated cells entered G1 phase after release from M phase, but did not appear to enter S phase. sub-G1 cell populations in flow cytometry analysis also showed that 7-deacetylhamononen induced apoptosis of cancer cells under prolonged treatment. To determine the cause of 17 β -deacetyl cerberenin induced cancer cell death, we used mimosine to arrest HeLa cells at the G1/S transition point and pre-treated the cells for 12 hours with 17 β -deacetyl cerberenin in the culture medium. Cells were then released from mimosine stagnation points into 17 β -deacetyl ceriferin-containing medium, harvested at various time points after release, and analyzed by flow cytometry and BrdU incorporation experiments. BrdU incorporation results indicated that-40% of cells were BrdU positive; however, the signal intensity was much lower compared to control cells (fig. 8C), indicating that the cells underwent a low degree of DNA replication. This is due to the incomplete inhibition of the MCM complex by 17 β -deacetyl ceriferin, and thus some origins of replication are activated resulting in a limited extension of DNA replication. The partial genome replication of the premature death is likely to cause DNA damage, leading to apoptosis.

17 beta-oleander saponins and 17beta-deacetyl ceriferin diol can also induce cancer cell apoptosis

We also found that 17 β -oleandrin and 17 β -desacetylceriferin diol can also induce apoptosis of tumor cells as shown by sub-G1 cell population in flow cytometry analysis (fig. 8D).

Further testing the specificity of 17beta-deacetyl cerberenin for inhibition of cancer cell growth

The data in fig. 2and 8A show that: the antitumor drug can specifically kill cancer cells, has no obvious effect on normal, and in order to check whether normal and/or tumor cells can recover cell growth after 17beta-deacetyl cerberenin is removed, normal L-02 liver cells and HepG2 liver cancer cells are treated by the 17beta-deacetyl cerberenin for one day, and a fresh growth culture medium without the 17beta-deacetyl cerberenin is replaced. Viable cell number was monitored by WST-1 method daily for 4 days. The results show that normal cells, but not cancer cells, increase recoverably after removal of 17 β -deacetyl cerberenin (fig. 9), consistent with our other experimental results: most normal cells treated with 17 β -deacetyl cerberrin stayed in G1 phase, while similarly treated cancer cells entered an apoptotic S phase.

Antitumor activity in nude mouse animal model

To determine the anticancer activity of 17 β -deacetyl ceriferin in and its possible toxicity in animal models, we performed nude mice inoculated tumor model animal experiments, inoculated left and right lateral sides in nude mice with HeLa cells. After random grouping, nude mice were intraperitoneally injected with 17 β -deacetylceriferin (dissolved in PBS containing 30% propylene glycol) and solvent, respectively. In the first experiment, on day 3 after inoculation and starting to form tumors, two groups of nude mice were injected with 3.5 and 7.0 mg/kg body weight of 17 β -desacetylhamonolide, respectively, while control group of nude mice were injected with the same volume of solvent (fig. 10A). 17 beta-deacetylceriferin significantly inhibited tumor growth, at 90% at high doses (7.0 mg/kg) and 70% at low doses (3.5 mg/kg). In fact, at high doses, the tumor size was even reduced in the last 5 consecutive injections of 17 β -desacetylceriferin, indicating that 17 β -desacetylceriferin induced tumor cell death in nude mice.

In vivo vs. paclitaxel experimental toxicity assessment

We performed another set of longer experiments, beginning 15 injections with 5.0 mg/kg of 17 β -desacetylceriferin one week after tumor inoculation and tumor size reached 0.05-0.1 cc. The growth of the tumor was again significantly inhibited (FIG. 10B, C). In the last five injections, we reduced the frequency of injections (every two days). The results show that tumor size was reduced by more than 80% in nude mice injected with 17 β -deacetyl ceriferin compared to the solvent control group. For comparison, paclitaxel had less anti-tumor activity than 17 β -desacetylhamiltonin for the first 10 days of each injection at 10 mg/kg, and then nude mice died due to the toxicity of paclitaxel (fig. 10B).

At the end of drug treatment, there was no significant weight loss in nude mice after 20 days of intraperitoneal injection of 5.0 mg/kg of 17 β -desacetylceriferin (fig. 11A). In 5 nude mice of each group, 3 mice with intermediate tumor sizes were selected for physiological parameter examination. Each nude mouse was dissected for organs including liver, heart and kidney. All organs looked normal, e.g., neither enlarged nor produced abnormal color (fig. 11B).

Also, there was no significant change in organ weight compared to body weight (FIG. 11C). In addition, blood was collected from each nude mouse, and ALT (alanine aminotransferase) and LDH (lactate dehydrogenase) activities were measured. ATL levels reveal liver function impairment, while LDH levels reflect impairment of all tissues. The results of these two tests are expressed as the relative values of the drug injected and the solvent injected nude mice. As shown in FIG. 11D, 17 β -deacetyl cerberenin did not cause a significant increase in the level of ATL or LDH in the blood. Taken together, these data strongly suggest that 17 β -deacetyl ceriferin has significant antitumor activity with little toxicity to mice.

While there have been described what are believed to be the essential novel features of the invention as applied to preferred embodiments thereof, it will be understood that various omissions and substitutions and changes in the form and details of the embodiments illustrated herein may be made by those skilled in the art without departing from the spirit of the invention. The patent is not limited to these embodiments, since they are presented by way of example only, but can be modified in various ways within the scope of protection defined by the patent claims.

Claims (3)

1. Use of a compound in the manufacture of a medicament for treating and/or alleviating cervical adenocarcinoma, nasopharyngeal carcinoma and/or liver cancer capable of inhibiting an MCM protein complex in a cancer cell, wherein the compound is selected from any one of 17 β -desacetyl ceriferin, 17 β -desacetyl ceriferin diol, 17 β -oleandrin, the compound being effective in inhibiting proliferation of a tumor cell and effective in killing a tumor cell, but having little killing power on normal cells; wherein the compound disrupts the formation and structure of the MCM protein complex by interfering with the interaction of hMcm2and hMcm6 in the MCM protein complex.

2. The use of claim 1, wherein the compound is capable of interfering with the assembly of MCM subunits into MCM protein complexes.

3. The use as claimed in claim 1 wherein the MCM protein complex is a fully functional heterohexatomic ring protein complex and is capable of entering the nucleus and participating in DNA replication.