Method for inhibiting formation of MCM complexes and method for screening for Anti-cancer compounds

Abstract

To provide a method for inhibiting the formation of MCM complexes, and to provide a method for screening for anti-cancer compounds.SOLUTION: A method for inhibiting the formation of functional MCM complexes from MCM subunits in normal and cancerous cells comprises the use of a compound selected from the group consisting of 17-beta-deacetyltanginin, 17-beta-neryifolin, and 17-beta-deacetyltanginin diol. A method for screening for anti-cancer compounds comprises the steps of (a) contacting a candidate compound with a population of cells for a period of time, and (b) determining in the cells an amount of functional MCM complexes formed from the subunits.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 61 / 644,442, filed May 9, 2012, the contents of which are incorporated herein by reference in their entirety.

[0002] (Technical field) The present invention relates to methods for treating cancer by using agents capable of inhibiting the functionality of the MCM complex (a heterohexameric ring formed from six subunits) in the process of DNA replication, and further relates to methods for screening for such agents by detecting the location and function of MCM subunits (e.g., hMcm2 and hMcm6) in cells treated with candidate compounds. [Background technology]

[0003] Cancerous cells are cells that divide and grow uncontrollably, invade nearby parts of the body, and may spread to other parts of the body via the lymphatic system and / or bloodstream. Cancer treatment typically involves the removal or destruction of cancerous cells, such as by surgery, chemotherapy, radiation therapy, or immunotherapy. However, one of the challenges in all of these forms of treatment is how to completely remove or destroy cancerous cells while simultaneously avoiding significant damage to normal or healthy cells and tissues. In the case of chemotherapy, screening for cytotoxic compounds has become a major focus of research and development for decades. Numerous chemical compounds have been shown to have cytotoxic and anticancer activity. As noted by Schwartsmann et al., by 1988, over 600,000 compounds had been screened, yet only approximately 40 of them had any clinical significance. This low success rate is primarily due to toxicity concerns regarding anticancer drugs. This is because anti-cancer drugs generally have a narrow therapeutic index, i.e., a small margin between the dose required for anti-cancer effect and the dose that causes unacceptable toxicity. The usefulness of discovering compounds that have an inhibitory effect on cell proliferation based on cytotoxicity is limited, and such discoveries are far from clinically relevant. What is needed, and more meaningful, is to find compounds that not only potently inhibit cell proliferation, but also do so with specificity for cancerous cells without causing significant damage to normal or healthy cells and tissues. Summary of the Invention [Means for solving the problem]

[0004] Therefore, one objective of the present invention is to provide a method for killing cancerous cells with high specificity without causing significant damage to normal cells. This objective is achieved by disrupting the formation of a functional MCM (minichromosome maintenance) complex from its subunits. The MCM complex is known to play an essential role in pre-RC (i.e., pre-replication complex) assembly (also known as replication licensing) and DNA replication elongation. A functional MCM complex requires all six MCM subunits (Mcm2-Mcm7) to form a heterohexamer ring that mounts on replication origins with the help of Orcl-6, Noc3, Ipil-3, Cdtl, Cdc6, and possibly several other proteins. As a member of the AAA+ (ATPase associated with various activities) family of proteins, the MCM complex is thought to co-migrate with replication forks and presumably act as a replicative helicase to unwind the DNA duplex. Previous studies by the present inventors, as disclosed in U.S. Patent Nos. 7,393,950 and 8,318,922, have demonstrated that antisense oligonucleotides targeting MCM subunit genes have cell growth inhibitory effects, the contents of which are hereby incorporated by reference.

[0005] Because MCM proteins must form intact complexes with the ring structures to be functional, disruption of their interaction (i.e., rendering the MCM proteins unable to form functional complexes) would inhibit DNA replication and induce apoptosis, and more importantly, the effects of disrupting MCM functionality would only be severe and permanent on cancerous cells, but not on normal and healthy cells.

[0006] Such great specificity is the essence of the present invention. Without wishing to be bound by theory, the great specificity of the present invention is believed to lie in the difference between normal cells, which have intact checkpoints that arrest the cell cycle in the G1 phase to avoid cell death, and cancer cells, which lack checkpoint control and will enter abortive S phase. In other words, normal cells have the ability to sense whether the MCM complex is formed and functional and will only enter S phase when they realize that the MCM complex is ready to fulfill its role in DNA replication. If not, the normal cell will be temporarily arrested in G1 phase. To use a metaphor, this is like a moving car with functioning brakes. The driver can stop the car when he notices that the bridge over the river ahead is broken. On the other hand, cancerous cells are like a car with malfunctioning brakes; the car will not be able to stop before reaching the broken bridge and will continue on its course (i.e., will continue moving and enter abortive S phase) until it falls into the river.

[0007] Another object of the present invention is to provide a method for screening for anti-cancer drugs with high specificity that kill cancerous cells while not causing significant damage to normal cells. This object is achieved by a process for identifying compounds that impair the formation of functional MCM complexes (heterohexamer ring structures) from subunits that become trapped in the cytoplasm and cannot be transported to the nucleus.

[0008] Preferably, the method comprises (a) contacting a population of cells with a number of candidate compounds over a period of time and (b) detecting the level of functional MCM complexes in the cells treated with the candidate compounds. More preferably, step (b) is performed indirectly by detecting a portion of MCM subunits located in the nucleus compared with a portion located in the cytoplasm. Because only functional MCM complexes can be located in the nucleus, the fewer MCM subunits located in the nucleus, the stronger the disruptive effect of the candidate compound on the formation of functional MCM complexes. Even more preferably, step (b) is performed by an indirect fluorescent method (immunostaining), in which a fluorescently labeled secondary antibody that recognizes a primary antibody against one or more endogenous MCM proteins allows visualization of the intracellular location of endogenous MCM proteins after exposure to the candidate compound for a specific duration. Alternatively, step (b) may be performed by a direct fluorescence method in which cells are transfected with a plasmid capable of expressing one or more MCM subunits fused to a fluorescent protein (e.g., hMcm2-GFP and / or hMcm6-GFP, etc.), thereby allowing detection of the location of the fluorescent MCM fusion protein after treatment of the cells with a candidate compound. Other methods for step (b) include detecting the physical interaction of MCM subunits or measuring the amount of MCM proteins bound to chromatin where various MCM proteins normally perform their functions.

[0009] If desired, additional steps may be performed to complement step (b), or as a separate step, to examine DNA replication defects by various methods, e.g., BrdU incorporation assay, flow cytometry, etc. Further steps may also be taken to confirm that compounds identified by their ability to disrupt the formation of functional MCM complexes also have potent differential effects in terms of anti-proliferation and apoptosis induction between cancerous and normal cells.

[0010] Another object of the present invention is to provide specific compounds as anti-cancer agents with high specificity for implementing the therapeutic methods according to the present invention. Preferred compounds are of formula (I) containing a four-ring backbone structure: wherein R1 is H or is substituted by one or more sugar units; R2 is a 5- or 6-membered ring group in a beta configuration; R3 and R4 are each H or OH, or R3 and R4 are each a single O atom that forms a three-membered ring with the two C atoms to which R3 and R4 are each bonded; R5 is OH and R6 is H, or R5 and R6 are each a single O atom that forms a three-membered ring with the two C atoms to which R5 and R6 are each bonded.

[0011] The methods and compounds of the present invention are applicable to all forms of cancer that are sensitive to disruption of the MCM complex, for example, cervical cancer, prostate cancer, colon cancer, breast cancer, ovarian cancer, acute myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, pancreatic cancer, stomach cancer, skin cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, small cell lung cancer, follicular lymphoma, or non-small cell lung cancer.

[0012] In summary, the particular technical feature underlying the present invention involves an agent capable of selectively killing cancerous cells by preventing the formation of functional MCM complexes from their subunits.

[0013] The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its uses, reference should be made to the drawings and following descriptive matter in which preferred embodiments of the invention are illustrated and described. [Brief explanation of the drawings]

[0014] [Figure 1] The structures of the anti-cancer compounds (3-8) of the present invention are shown in comparison with the structures of the inactive isomers (1-2).

[0015] [Figure 2] Direct microscopy observations (A) and WST-1 (water-soluble tetrazolium-1) assay data (B-H) are shown, demonstrating that 17β-deacetyltanginine (A-C), 17β-nerifolin (D), and 17β-deacetyltangininediol (E), three representative examples of anticancer compounds of the present invention, possess potent anticancer activity without significant cytotoxicity to normal cells. For comparison, paclitaxel (Taxol) is more cytotoxic to normal cells than to cancer cells (F), and VP16 (etoposide phosphate) has little selectivity between normal and cancer cells (G and H).

[0016] [Figure 3] We show by co-immunoprecipitation that 17beta-deacetyltanginine (DAT) and 17beta-nerifolin (NRF) disrupt the interaction between MCM subunits, whereas 17beta-deacetyltangininediol (Diol) has a weaker activity.

[0017] [Figure 4] Figure 1 shows indirect immunofluorescence microscopy data demonstrating that 17beta-deacetyltanginine (DAT), 17beta-nerifolin (NRF), and 17beta-deacetyltangininediol (Diol) impair the nuclear localization of hMcm2 (h, human) and hMcm6.

[0018] [Figure 5] We present chromatin binding assay and flow cytometry data to demonstrate that 17beta-deacetyltanginine inhibits pre-replicative complex (pre-RC) assembly and induces apoptosis in cancer cells.

[0019] [Figure 6] Flow cytometry shows that 17beta-deacetyltanginine inhibits DNA replication and induces apoptosis in cancer cells.

[0020] [Figure 7] 1 shows BrdU incorporation assay data to demonstrate that 17beta-deacetyltanginine inhibits DNA replication.

[0021] [Figure 8] 1 shows flow cytometry and Annexin V staining data indicating that 17beta-deacetyltanginine (DAT), 17beta-nerifolin (NRF) and 17beta-deacetyltangininediol (Diol) can induce apoptosis in cancer cells.

[0022] [Figure 9] It has been shown that normal cells, but not cancer cells, are able to resume growth after 17beta-deacetyltanginine is withdrawn, as measured by the WST-1 assay.

[0023] [Figure 10] 1 shows the in vivo antitumor activity of 17beta-deacetyltanginine in a nude mouse xenograft model.

[0024] [Figure 11] 17beta-deacetyltanginine was shown to have no apparent toxicity in nude mice subjected to the experiment shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0025] Cell lines and plasmids Human Mcm6 and Mcm2 cDNA fragments were cloned into the pEGFP-C3 vector (Invitrogen) for localization detection. HeLa cells (cervical adenocarcinoma), HepG2 cells (hepatocellular carcinoma), Hep3B cells (hepatocellular carcinoma), HK1 (nasopharyngeal carcinoma), and C666-1 (nasopharyngeal carcinoma) were cultured in DMEM containing 10% FBS. L-02 cells (normal human liver cells) were cultured in RPMI1640 with 10% FBS. NP460 cells were cultured in 1:1 Keratinocyte-SFM (Invitrogen) and MEPI500CA with supplement S0125 (Cascade Biologics). All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0026] Antiproliferative activity assay The antiproliferative activity of the compounds was tested in human cell lines and IC 50 To calculate the α-threshold, we used cancer cells (4 × 10 5 cells / well) and normal L-02 cells (5 × 10 5 Cells (cells / well) were seeded in 100 μl of culture medium into a 96-well plate and incubated at 37°C for approximately 12 hours. Cells were treated with two-fold serial dilutions of drugs for 48 hours. The medium was removed, and 100 μl of culture medium containing 1 μM WST-1 (water-soluble tetrazolium-1) was added to each well. Cells were incubated for 2 hours, after which the absorbance at 405 nm (reference at 630 nm) was measured. Cell counts and OD 405 To construct a standard curve of the relationship between absorbance at 1000 kJ / mL, serial dilutions of cells with known cell numbers were seeded and incubated for 6 hours, after which measurement was performed using the WST-1 assay. Cell viability was expressed as the ratio of the number of viable cells treated with candidate compounds to the number of DMSO-treated cells.

[0027] Natural product screening and bioactivity-guided isolation of anticancer compounds The general protocol for preparing chemical samples from natural sources for anticancer drug screening assays was as follows: 10–100 grams of herbal material (whole plant, root, stem, leaf, or fruit) was extracted with methanol three times at room temperature. Each total extract was suspended in water and then sequentially partitioned with EtO, EtOAc, and n-BuOH to obtain four fractions: EtO fraction, EtOAc fraction, n-BuOH fraction, and HO fraction. Each total extract or fraction was dissolved in DMSO as a 10 mg / ml stock solution for the screening assay. Purified single compounds were each prepared as 1 mg / ml stock solutions.

[0028] Using the above screening method, we identified one fraction with potent MCM complex disruption and anticancer activity. This fraction was the EtO fraction of extracts of dried leaves or young brach of Cerebra manhas and Cerebra odollam. This fraction was subsequently subjected to activity-guided fractionation using a combination of different column chromatographies: SiO2, MCI Gel CHP 20P (75-150 m, Mitsubishi Chemical Corporation, Japan), Chromatorex ODS (100-200 mesh, Fuji Silysia Chemical Ltd., Japan), and Toyopearl HW-40F (Tosoh Corporation, Japan). 17β-deacetyltanginine was isolated as the lead compound responsible for the activity of the active fractions of dried leaves and young brach of Cerebra odollam and Cerebra manhas.

[0029] Structural identification of 17beta-deacetyltanginine The structure of 17beta-deacetyltanginine was characterized based on spectroscopic evidence. NMR spectra were recorded using a Varian-400 spectrometer. Coupling constants are given in Hz, and chemical shifts are expressed in ppm relative to Me4Si as an internal standard. HR-ESI-MS was performed on a Q-TOF mass spectrometer (Bruker Daltonics, MA, USA).

[0030] High resolution ESI-MS (cation mode): m / z 549.3077[M+H] + (Calculated value, C 30 H 45 O9:549.3064). 1 H-NMR (400 MHz, pyridine-d5): δ 6.31 (1H, s, H-22), 5.25 (1H, d, J = 2.9 Hz, H-1'), 5.20 (1H, m, H-21), 5.02 (1H, dd, J = 18.0, 1.4 Hz, H-21), 4.33 (1H, m, H-5'), 4.12 (1H, brs, H-3), 4.09 (1H, dd, J = 9.0, 4.0 Hz, 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). 13C-NMR (100 MHz, pyridine-d5): δ 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), 13.0 (C-18), 25.0 (C-19), 175.9 (C-20), 74.4 (C-21), 113.4 (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).

[0031] 17 beta-deacetyltanginine, C 30 H 44 Its high-resolution ESI-MS corresponds to the molecular formula of O9, showing characteristic signals derived from cardenolides. 1 The H-NMR spectrum [methylene proton at C-21 (δ 5.20, m; 5.02, dd, J = 18.0, 1.4 Hz) and olefinic proton at C-22 (δ 6.31, s)] and the anomeric proton signals of the sugar moiety (δ 5.25, d, J = 2.9 Hz) confirmed the identity of this compound as a cardenolide monoglycoside. The presence of epoxy groups at C-7 and C-8 positions was suggested by the large downfield shifts of the C-7 and C-8 signals compared with those of neriforrin (the major cardenolide from the leaves of Cerbera manghas), and further supported by chemical shift comparison with those of cardenolides bearing 7,8-epoxy groups. The configuration of C-17 is confirmed by the H-17 signal (δ 2.82, dd, J = 9.0, 5.0 Hz) and the C-12 signal (δ C 41.4), it has been established that the aglycone is β. 13By comparing the C-NMR data with published data, the sugar moiety was identified as 3β-hydroxy-7β,8β-epoxy-14β-hydroxy-card-20(22)-enolide. By comparing its proton and carbon signals with those reported in the literature, the sugar moiety was identified as α-L-thevetose (3-O-methyl-6-deoxy-α-L-glucopyranosyl). Based on the above evidence, the structure of HMG-17β-deacetyltanginine was characterized as 3β-O-(3-O-methyl-6-deoxy-α-L-glucopyranosyl)-7β,8β-epoxy-14β-hydroxy-card-20(22)-enolide (17β-deacetyltanginine). [ka]

[0032] Immunostaining assay To study the effects of anti-cancer compounds on the interaction between hMcm2 and hMcm6 in cells, immunostaining was performed to detect the intracellular localization of the proteins. HeLa cells grown on coverslips (coated with poly-D-lysine) with or without drug treatment were fixed with 4% PFA in PBS for 20 minutes at room temperature. After permeabilization with 0.1% Triton X-100 and 1% BSA in PBS for 20 minutes, the cells were blocked with 1% BSA and then incubated with rabbit anti-hMcm6 primary antibody (Santa Cruz; 1:500) and mouse anti-hMcm2 primary antibody (Becton Dickinson, 1:500) at room temperature for 1 hour. The cells were then incubated with Alexa Fluor 488-conjugated donkey anti-goat antibody and Alexa Fluor 594-conjugated donkey anti-mouse antibody (Invitrogen, 1:500) at room temperature for 1 hour. After each antibody incubation, the cells were washed three times with PBS. Then, for nuclear staining, the cells were incubated with Hochest 33852 (Sigma Chemical Company; 1 μg / ml) at room temperature for 15 minutes, and then washed three times with PBS. Finally, the cells were mounted on slides and observed under a Nikon TE2000E fluorescence microscope.

[0033] Cell synchronization To arrest cells in M ​​phase, HeLa cells were presynchronized with 2 μM thymidine for 18 hours, released into fresh medium for 6 hours, and then arrested in early M phase with 0.1 μg / ml nocodozole for 6 hours. HeLa cells were arrested at the G1 / S phase boundary by treatment with 0.5 mM mimosine for 20 hours. An aliquot of G1 / S phase cells was then released into hydroxyurea-containing medium for 4 hours to obtain early S phase cells.

[0034] BrdU incorporation assay HeLa cells grown on poly-D-lysine-coated coverslips were treated with 17β-deacetyltanginine for 24 hours and then incubated with 50 μM BrdU (Sigma) for 1 hour at 37°C. The cells were then fixed with 4% PFA in PBS for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 and 1% BSA in PBS for 20 minutes, and then sequentially incubated with anti-BrdU (Sigma Chemical Company; 1:500) and anti-mouse IgG-FITC conjugate (Sigma Chemical Company; 1:500) for 1 hour each at 37°C. Three washes with PBS were performed after each antibody incubation. BrdU signals were observed under a fluorescence microscope (Nikon TE2000E).

[0035] Chromatin binding assay Cells were harvested by trypsinization and washed twice with cold PBS. Extraction buffer (EB; approximately 20 μl / 10 6The cells were resuspended in 100 mM KCl, 50 mM HEPES-KOH pH 7.5, 2.5 mM MgCl, 50 mM NaF, 5 mM NaP0, 0.1 mM NaVO, 0.5% Triton X-100, 1 mM PMSF, 2 μg / ml pepstatin A, 20 μg / ml leupeptin, 20 μg / ml aprotinin, 0.2 mM Pefabloc, 2 mM benzamidine HCl, and 0.2 mg / ml bacitracin, and lysed by pipetting. The cells were placed on ice for 10 minutes and gently mixed by tapping every 2–3 minutes during the incubation period. A volume of 30% ice-cold sucrose equal to the volume of EB containing protease inhibitors was added to the bottom of the tube. The tube was centrifuged at maximum speed in a microcentrifuge for 10 minutes to separate the chromatin and free protein. The supernatant was transferred to a new tube and kept on ice. The pellet was washed with an equal volume of EB by gently tapping the tube to dislodge the pellet from the wall of the tube and resuspended by gentle vortexing. The suspension was again centrifuged at maximum speed for 5 minutes. The two supernatants were combined. The pellet was resuspended in EB equal to half the volume of the supernatant. The supernatant and pellet fractions were finally processed for immunoblotting.

[0036] Flow cytometry (FACS analysis) Both floating and adherent cells were collected and washed once with PBS. Cells were fixed in 70% ethanol for 1 hour to overnight at -20°C, thoroughly washed with PBS, and then stained with 50 μg / ml RNase A, 0.1% Triton X-100, 0.1 mM EDTA (pH 7.4), and 50 μg / ml propidium iodide for 30 minutes at 4°C. Samples were analyzed using a FACSort instrument (Becton Dickinson).

[0037] Identifying antiproliferative agents with high specificity between normal and cancer cells After screening hundreds of samples of plant-derived compounds, fractions, or crude extracts, as well as synthetic compounds, we identified several candidates capable of inhibiting human MCM proteins and DNA replication. Among these candidates, a small compound called 17β-deacetyltanginine (Figure 1) was isolated and identified after several rounds of activity-guided fractionation, purification, and testing of plant extracts, fractions, and compounds. Several other compounds structurally related to 17β-deacetyltanginine and its chemical derivative (7β-deacetyltanginine diol) were also found to have activity similar to 17β-deacetyltanginine (Figure 1 (3)–(8)). Meanwhile, their 17α-isomers (Figure 1 (1) and (2)) were found to be inactive.

[0038] A pair of human cell lines, L-02 (normal liver cells) and HepG2 (liver cancer cell line), were treated with 17β-deacetyltanginine for 48 hours. Direct observation of cell density and morphology under microscopy showed that 17β-deacetyltanginine could efficiently inhibit the growth of cancer cells (HepG2) in culture, while having much lower activity against normal cells (L-02) (Figure 2A). Therefore, 17β-deacetyltanginine was identified as a highly active antiproliferative agent with little cytotoxicity against normal cells. Quantification of viable cell numbers was performed using a WST-1 (water-soluble tetrazolium-1) assay (Figures 2B–H).

[0039] To further test 17β-deacetyltanginine and determine its IC50 value, we treated Hep3B cells (another p53-negative liver cancer cell line), HeLa cells (cervical cancer cell line), HK1 cells and C666-1 cells (nasopharyngeal carcinoma), as well as an hTert-immortalized normal nasopharyngeal cell line (NP460) with 17β-deacetyltanginine for 48 hours, and determined relative cell viability using the WST-1 assay (Figures 2B and 2C). Using these and other cancer cell lines (including lung cancer, data not shown), we demonstrated that 17β-deacetyltanginine can kill a wide range of cancer cells. Growth of all tested cancer cell lines was significantly inhibited by 17β-deacetyltanginine. Although IC50 values ​​varied slightly among various cancer cell lines, the average IC50 of 17β-deacetyltanginine against cancer cell lines was approximately 0.1 μg / ml (0.2 μM), while the IC50 for normal cells was much higher (>4 μg / ml). Similar anticancer activity and selectivity between cancer and normal cells were obtained for several structurally related compounds: for example, 17β-neryifolin (Figure 2D), 17β-deacetyltangininediol, a novel chemical derivative of 17β-deacetyltanginine synthesized by the present inventors (which had lower anticancer activity than 17β-deacetyltanginine; Figure 2E), bufalin, resibufogenin, and cinobufagin (Table below). On the other hand, 17alpha-deacetyltanginine and 17alpha-neryifolin have little antiproliferative activity (Table below), indicating that the 17beta configuration is very important for the antiproliferative activity of these compounds. [Table 1]

[0040] For comparison, the clinical anticancer drugs paclitaxel (Taxol; Figure 2F) and VP16 (etoposide phosphate; Figures 2G and 2H) did not show significant selectivity between cancer and normal cells (they are cytotoxic to normal cells as well as cancer cells; in fact, paclitaxel is more toxic to normal liver cells than to liver cancer cells).

[0041] Disruption of MCM complex formation and nuclear localization of MCM proteins To test whether 17β-deacetyltanginine targets hMcm2 and hMcm6 proteins in human cells, we examined the possible co-immunoprecipitation (co-IP) of these two proteins in human cell extracts obtained from cells treated with 17β-deacetyltanginine. In Figure 3, asynchronous HeLa cells were treated with DMSO or 17β-deacetyltanginine (DAT) (Figure 3A), or with DMSO, 17β-nerifolin (NRF), or 17β-deacetyltangininediol (Diol) (Figure 3B). Whole cell extracts were also prepared and further incubated with compounds, and then used for co-IP in the presence of compounds. The immunoprecipitates were then immunoblotted with anti-hMcm6, anti-hMcm2, anti-hMcm4, and anti-hMcm7 antibodies. The results showed that 17beta-deacetyltanginine disrupted the interaction between hMcm2 and hMcm6 as well as the interactions between other MCM subunits (Figure 3A). Similarly, 17beta-nerifolin was also able to disrupt the hMcm2-hMcm6 interaction, whereas 17beta-deacetyltangininediol showed weaker activity (Figure 3B).

[0042] Because pairwise interactions between MCM subunits are required for the MCM heterohexamer ring structure and are essential for their import into the nucleus, disruption of the interaction between hMcm2 and hMcm6 would disrupt this hexamer and result in the failure of nuclear localization of MCM proteins. To test this, we used both indirect fluorescence microscopy (immunostaining) using antibodies against endogenous MCM proteins and direct fluorescence microscopy after transfection with plasmids to express hMcm2-GFP and hMcm6-GFP in cells.

[0043] In immunostaining, HeLa cells were treated with 17beta-deacetyltanginine for 24 hours, and endogenous hMcm2 and hMcm6 were detected using specific antibodies against these proteins. The results showed that the nuclear localization of hMcm2 and hMcm6 was impaired by 17beta-deacetyltanginine (Figure 4A). In direct fluorescence microscopy, HeLa cells expressing hMcm2-GFP and hMcm6-GFP were treated with 17beta-deacetyltanginine for 36 hours, starting 4 hours after transfection with plasmids expressing hMcm2-GFP and hMcm6-GFP. The results showed that a portion of the expressed hMcm2-GFP and hMcm6-GFP was located in the cytoplasm, whereas in untreated control cells, almost all of the expressed hMcm2-GFP and hMcm6-GFP was located in the nucleus (Figure 4B). To exclude the possibility that the cytoplasmic localization of some MCM proteins was due to cell cycle arrest caused by 17beta-deacetyltanginine, we used mimosine to arrest cells in late G1, a phase in which all MCM proteins must be nuclear if uninhibited. We found that the nuclear localization of hMcm2 and hMcm6 was still prevented by 17beta-deacetyltanginine (data not shown).

[0044] Taken together, these data indicate that 17β-deacetyltanginine can specifically block the nuclear localization of MCMs. We also found that 17β-neriifoline, another compound isolated from Cerebrum manhas and structurally related to 17β-deacetyltanginine, can disrupt the nuclear localization of MCMs as efficiently as 17β-deacetyltanginine, whereas a chemical derivative of 17β-deacetyltanginine, 17β-deacetyltangininediol, has weaker activity (Figure 4C).

[0045] Inhibition of pre-RC assembly As a component of the pre-PC, the MCM complex plays a central role in the licensing of DNA replication. Because 17β-deacetyltanginine can disrupt the interaction of hMcm2 and hMcm6 and prevent their nuclear localization, it is likely that 17β-deacetyltanginine inhibits the chromatin association of MCM proteins, indicating the failure of pre-RC assembly (replication licensing). To test this, we performed a chromatin-binding assay to detect chromatin-associated proteins. As shown in Figures 5A and 5B, asynchronous HeLa cells were treated with 17β-deacetyltanginine for 24 hours and analyzed by chromatin-binding assay (Figure 5A). Untreated cells, cells treated with the solvent DMSO, and cells treated with 0.2, 0.4, or 0.8 μg / ml of 17β-deacetyltanginine were analyzed for pre-RC components in the chromatin and supernatant fractions by immunoblotting (Figure 5A). Beta-actin was used as a loading control. Each cell sample was also analyzed for cell cycle distribution by flow cytometry (Figure 5B). The experiments shown in Figures 5C and 5D were similar to those in Figures 5A and 5B, except that cells were synchronized in M ​​phase using nocodazole (Noc.) and then released into fresh medium containing DMSO or 17beta-deacetyltanginine (DAT), as indicated.

[0046] Consistent with predictions, both hMcm2 and hMcm6 were significantly reduced in the chromatin fraction by 17beta-deacetyltanginine in a dose-dependent manner (Figure 5A). These results suggest that pre-RCs were unable to assemble in the presence of 17beta-deacetyltanginine. Moreover, cells underwent apoptosis, as determined by flow cytometry using aliquots of cells obtained from the same experiment (Figure 5B).

[0047] To clarify the effect of 17β-deacetyltanginine on synchronized cells, HeLa cells were first synchronized in late G1 / early S phase with thymidine and then arrested in M ​​phase with nocodozole. The cells were then released into fresh medium in the presence of 17β-deacetyltanginine. Control cells, including DMSO-treated and untreated cells, progressed through M and G1 phases and entered S phase (Figure 5D). However, 17β-deacetyltanginine-treated cells only entered G1 phase, and most of the cells did not enter S phase (Figure 5D). Immunoblotting analysis showed that 17β-deacetyltanginine significantly disrupted MCM loading onto chromatin (Figure 5C). These results indicate that 17β-deacetyltanginine can disrupt pre-RC assembly in human cells.

[0048] Inhibition of DNA replication accompanying apoptosis in cancer cells Because 17β-deacetyltanginine disrupts the interaction between hMcm2 and hMcm6 and inhibits the association of MCM proteins with chromatin, it is likely that 17β-deacetyltanginine blocks DNA replication. To confirm this, we treated HeLa cells with 17β-deacetyltanginine for 24 hours and then labeled the cells with BrdU for 1 hour. Incorporated BrdU in cellular DNA was detected with an anti-brdU antibody followed by an FITC-anti-mouse secondary antibody visualized under a fluorescent microscope (Figure 6A). DAPI was used to stain nuclei (Figure 6A), and the percentage of BrdU-positive cells was quantified (Figure 6B). Significant inhibition of DNA replication was observed in 17beta-deacetyltanginine-treated HeLa cells, as BrdU signal was almost completely absent above 0.2 μg / ml of 17beta-deacetyltanginine, whereas approximately 30% of DMSO-treated and untreated cells were BrdU positive, as expected (Figure 6A, B).

[0049] Furthermore, we were able to demonstrate the inhibition of DNA replication and subsequent induction of apoptosis by 17β-deacetyltanginine using flow cytometry. In Figure 7, HeLa cells were arrested in M ​​phase by nocodazole (Noc; Figure 7A), at the G1 / S transition by mimosine (MMS; Figure 7B), or in early S phase by hydroxyurea (HU; Figure 7C), and then released into fresh medium in the presence of 17β-deacetyltanginine (DAT). Cells at various time points after release were analyzed by flow cytometry. Ayn. indicates asynchronous cells. Untreated cells and cells treated with the solvent DMSO were able to complete M, G1, and S phases after release (Figure 7A). Cells treated with 17β-deacetyltanginine were able to complete mitosis but failed to enter S phase after release from nocodazole arrest in M ​​phase, and apoptosis was initiated with prolonged treatment with 17β-deacetyltanginine (Figure 7A). Similarly, cells released from mimosine (MMS) arrest (at the G1 / S transition) (Figure 7B) or hydroxyurea (HU) arrest (in early S phase) (Figure 7C) failed to complete S phase in the presence of 17β-deacetyltanginine. These results are consistent with inhibition of MCM function in both the initiation and elongation of DNA replication.

[0050] 17β-deacetyltanginine also induced apoptotic cell death in cancer cells. A sub-G1 population of cancer cells (indicating apoptosis) was detected by flow cytometry after treatment with 17β-deacetyltanginine (Figures 5B, 7, and 8A), whereas normal L-02 cells were mostly arrested in the G1 phase with a reduced G2 / M population (Figure 8A). In Figure 8A, flow cytometry was performed to analyze DNA content in HepG2 and L-02 cells treated with various concentrations of 17β-deacetyltanginine for 24 hours. In Figure 8B, HeLa cells were treated with 17β-deacetyltanginine for 24 hours and labeled with Annexin V-Cy3 (Arm.Cy3) for 20 minutes. Mitoxantrone (MTX), a clinical anticancer drug that can induce apoptosis in cancer cells, was used as a positive control. The results show that 17beta-deacetyltanginine-treated cancer cells could be stained with Annexin V (Fig. 8B), supporting the idea that apoptosis was induced by 17beta-deacetyltanginine.

[0051] As described above, 17β-deacetyltanginine inhibited DNA replication (as determined by BrdU incorporation assay) in asynchronous HeLa cells (Fig. 6). Furthermore, as judged by flow cytometry, most synchronized cells released from M phase entered G1 phase, but apparently did not enter S phase in the presence of 17β-deacetyltanginine (Fig. 7A). Longer incubation of cancer cells with 17β-deacetyltanginine could induce apoptosis, as evidenced by the sub-G1 population in flow cytometry profiles (Fig. 7A) and Annexin V staining (Fig. 8B). To clarify the cause of cell death in cancer cells treated with 17β-deacetyltanginine, we arrested HeLa cells at the G1 / S transition with mimosine and then pretreated the cells with 17β-deacetyltanginine for 12 hours. Cells were then released from mimosine block in the presence of 17β-deacetyltanginine, collected at various time points after release, and analyzed by flow cytometry and BrdU incorporation assay. BrdU incorporation results showed that approximately 40% of cells treated with 17β-deacetyltanginine were BrdU-positive, compared with approximately 100% of untreated and DMSO-treated cells; however, the BrdU signal intensity in 17β-deacetyltanginine-treated cells was much lower than that in untreated and DMSO-treated cells (Figure 8C). This suggests that at least some 17β-deacetyltanginine-treated cells underwent reduced DNA replication, which was likely due to incomplete inhibition of the MCM complex in 17β-deacetyltanginine-treated cells, resulting in reduced activation of some replication origins and limited elongation of DNA replication. As such, abortive partial genome duplication most likely caused DNA damage, thereby leading to apoptosis.

[0052] In addition to 17beta-deacetyltanginine, a number of structurally related compounds, such as 17beta-neryifolin and 17beta-deacetyltangininediol, were also found to be able to induce apoptosis in cancer cells, as indicated by cells in the sub-G1 population in flow cytometry analysis (Figure 8D).

[0053] Further testing of the specificity of antiproliferative compounds against cancerous cells The data in Figure 2 and Figure 8A show that the antiproliferative compounds of the present invention can specifically kill cancer cells without causing significant cytotoxicity to normal cells.To test whether normal cells and / or cancer cells can resume cell growth after removing 17β-deacetyltanginine, L-02 (normal liver) cells and HepG2 (liver cancer) cells were incubated with 17β-deacetyltanginine or DMSO for 1 day, and then 17β-deacetyltanginine or DMSO was removed, and the cells were further incubated with fresh growth medium for 3 days.The number of viable cells was monitored daily by WST-1 assay. In Figure 9, L + DAT indicates L-02 cells treated with 17β-deacetyltanginine; L + D indicates L-02 cells treated with DMSO; H + DAT indicates HepG2 cells treated with 17β-deacetyltanginine; H + D indicates HepG2 cells treated with DMSO and then released into fresh medium. The results showed that normal cells, but not cancer cells, resumed growth after removing 17β-deacetyltanginine (Figure 9). This is consistent with our finding that the majority of normal cells remained in the G1 phase when treated with 17β-deacetyltanginine, whereas cancer cells entered abortive S phase and died under the same treatment (Figure 8A).

[0054] In vivo anti-cancer activity in nude mouse xenograft models In vivo anticancer activity studies were performed in nude mouse xenograft models by inoculating nude mice with HeLa cells into both the left and right flanks. After randomization, nude mice were treated intraperitoneally with 17β-deacetyltanginine or vehicle (30% propylene glycol in PBS). In the first study, 3 days after tumor inoculation, when small tumors began to form, two groups of nude mice were treated with 3.5 mg drug / kg body weight and 7.0 mg drug / kg body weight of 17β-deacetyltanginine on days 1-3 and 6-10, respectively, while another group of control mice was treated with the same volume of vehicle (Figure 10A). In Figure 10A, tumor volume data, represented by small data points connected by thin lines, were obtained on days when both tumor size measurements and drug injections were performed, while tumor volume data, represented by large data points connected by thick lines, were obtained on days without drug injections. Each tumor size was the average of 10 tumors in 5 mice in each group. The results showed that 17β-deacetyltanginine significantly inhibited tumor growth, with 90% inhibition at the high dose (7.0 mg / kg) and 70% inhibition at the low dose (3.5 mg / kg). In fact, at the high dose, tumor size even decreased after the last five consecutive injections of 17β-deacetyltanginine. This suggests that 17β-deacetyltanginine induced tumor cell death in mice.

[0055] Comparative studies and in vivo toxicity evaluation with Taxol Next, a further set of animal experiments was conducted over a longer period, during which 15 drug injections at 5.0 mg / kg were administered to tumors with a size of 0.05-0.1 cm. 3The data were collected one week after tumor inoculation, when tumors reached 17β-deacetyltanginine (Figure 10B). Drug injections and tumor size measurements were performed on the days indicated by the data points, and the photographs in Figure 10C were taken on day 20. The results again showed that tumor growth was significantly suppressed; tumors in mice treated with 17β-deacetyltanginine were over 80% smaller than those in vehicle-treated control mice (Figures 10B and 10C). For comparison, paclitaxel (Taxol) at 10 mg / kg per injection showed much less antitumor activity than 17β-deacetyltanginine in the first 10 days, and these mice died on day 10 due to Taxol toxicity (Figure 10B).

[0056] When drug treatment as described in Figure 10B was terminated, no obvious weight loss was observed in nude mice after 20 days of intraperitoneal administration of 5.0 mg / kg 17β-deacetyltanginine (Figure 11A). In Figure 11, S indicates vehicle-treated mice without tumor inoculation; S + T indicates vehicle-treated mice with tumor inoculation; DAT indicates 17β-deacetyltanginine-treated mice without tumor inoculation; DAT + T indicates 17β-deacetyltanginine-treated mice with tumor inoculation; and P + T indicates paelitaxel-treated mice with tumor inoculation. Three of the five mice with intermediate tumor size in each group were selected for physiological parameter examination. Tumors (T) and internal organs, including the liver (L), heart (H), and kidneys (K), were excised from each mouse. The tumor size (Figure 11B) was consistent with the tumor volume measurements shown in Figure 10B. All organs appeared normal. For example, neither swelling nor abnormal coloration was observed (FIG. 11B), and organ weight relative to body weight was not significantly altered (FIG. 11C).

[0057] In addition, blood from each mouse was also collected for testing of ALT (alanine aminotransferase) and LDH (lactate dehydrogenase) activities. ATL levels reveal liver damage, while LDH levels generally reflect tissue damage, regardless of the tissue. The results of both tests were expressed as the values ​​of variously treated mice relative to untreated mice. As shown in Figure 11D, 17beta-deacetyltanginine did not induce a significant increase in blood ATL or LDH levels. Taken together, these data strongly suggest that 17beta-deacetyltanginine has significant antitumor activity in mice with little toxicity.

[0058] While the essential novel features of the invention as applied to preferred embodiments thereof have been described and pointed out, it will be understood that various omissions and substitutions and changes may be made by those skilled in the art in the form and details of the illustrated embodiments without departing from the spirit of the invention. The present invention is not limited to the above-described embodiments, which are given by way of example only, but can be modified in various ways within the scope of protection defined by the appended claims.

[0059] (Addendum) (Appendix 1) A method for treating cancer in a patient, comprising the steps of: (a) selecting an anti-cancer agent for its inhibitory effect on human MCM complexes in cancerous cells; and (b) administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising the selected anti-cancer agent. (Appendix 2) 2. The method of claim 1, wherein the anticancer agent is further selected for its ability to cause cancerous cells to enter abortive S phase and substantially arrest normal cells in G1 phase. (Appendix 3) The method of claim 2, wherein the inhibitory effect is achieved by disrupting the formation of a functional MCM complex from MCM subunits. (Appendix 4) The method of claim 3, wherein the functional MCM complex is a heterohexamer ring structure that can translocate into the nucleus and is required for DNA replication. (Appendix 5) The method of claim 3, wherein the selected anti-cancer agent disrupts the formation of the functional MCM complex by interfering with the interaction between hMcm2 and hMcm6. (Appendix 6) The anticancer agent is a compound represented by the formula (I): (wherein R1 is H or is substituted by one or more sugar units; R2 is a 5-membered or 6-membered ring group in a beta configuration; R3 and R4 are each H or OH, or R3 and R4 are each a single O atom that forms a three-membered ring with the two C atoms to which R3 and R4 are each bonded; R5 is OH and R6 is H, or R5 and R6 are each a single O atom that forms a three-membered ring with the two C atoms to which R5 and R6 are each bonded). (Appendix 7) 7. The method of claim 6, wherein the anticancer agent is selected from the group consisting of 17beta-deacetyltanginine, 17beta-nerifolin, bufalin, 17beta-deacetyltangininediol, resibufogenin, and cinobufagin. (Appendix 8) 8. The method of claim 7, wherein the anticancer agent is 17beta-deacetyltanginine. (Appendix 9) 8. The method of claim 7, wherein the anticancer agent is 17beta-deacetyltanginine diol. (Appendix 10) 8. The method of claim 7, wherein the anticancer drug is bufalin, cinobufagin, or resibufogenin. (Appendix 11) A method for screening for anti-cancer compounds, comprising: (a) contacting a candidate compound with a population of cells for a period of time; and (b) determining in the cells the amount of functional MCM complex formed from its subunits. (Appendix 12) 12. The method of claim 11, wherein step (b) is carried out by detecting a portion of the MCM subunit located in the nucleus compared to a portion located in the cytoplasm. (Appendix 13) The method described in Appendix 12, wherein an indirect fluorescent process (immunostaining) is performed in step (b) using one or more primary antibodies against one or more endogenous MCM subunits and a fluorescently labeled secondary antibody that recognizes the primary antibodies to visualize the location of the MCM proteins after the cells have been treated with the candidate compound for a specific duration. (Appendix 14) The method described in Appendix 12, wherein the direct fluorescence process is carried out in step (b) in which the cells are transfected with one or more plasmids capable of expressing one or more MCM subunits fused to a fluorescent protein, and then the location of the fluorescently labeled MCM subunits is detected after the cells are treated with the candidate compound for a specific duration. (Appendix 15) The method of claim 12, wherein the MCM subunits include one or more selected from the group consisting of hMcm2, hMcm3, hMcm4, hMcm5, hMcm6 and hMcm7. (Appendix 16) The method of claim 11, wherein step (b) is carried out by detecting physical interactions of the MCM subunits or by measuring the amount of MCM protein bound to chromatin. (Appendix 17) The method of claim 11, wherein step (b) is carried out by measuring the enzymatic function (e.g., ATPase activity and / or helicase activity) of the MCM protein. (Appendix 18) 12. The method of claim 11, further comprising a confirmation step of determining the anti-proliferative and apoptosis-inducing effects of the candidate compound on the cells. (Appendix 19) 18. The method of claim 17, wherein the confirming step is carried out by using a BrdU incorporation assay and / or flow cytometry. (Appendix 20) A method for treating cancer in a patient, comprising administering to the patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a combination thereof (wherein R1 is H or is substituted by one or more sugar units; R2 is a 5-membered or 6-membered ring group in a beta configuration; R3 and R4 are each H or OH, or R3 and R4 are each only one O atom that forms a three-membered ring with the two C atoms to which they are each bonded, provided that when R1 is substituted by one or more sugar units, R3 and R4 must be only one O atom, thereby forming a three-membered ring; R5 is OH and R6 is H, or R5 and R6 are each only one O atom that forms a three-membered ring with the two C atoms to which they are each bonded). (Appendix 21) 20. The method of claim 19, wherein the compound is 17beta-deacetyltanginine.

Claims

1. Use of a compound capable of inhibiting the formation of a functional MCM complex from MCM subunits in cancerous cells for the manufacture of a medicament for the treatment of cervical cancer, nasopharyngeal cancer or liver cancer, comprising: The use of a compound, wherein said compound is selected from the group consisting of 17beta-deacetyltanginine, 17beta-neryifolin and 17beta-deacetyltangininediol.

2. 2. The use of claim 1, wherein the compound is further selected for its ability to cause cancerous cells to enter abortive S phase, but arrest normal cells in G1 phase.

3. The use according to claim 1, wherein the functional MCM complex is a heterohexamer ring structure that can translocate into the nucleus and is required for DNA replication.

4. The use according to claim 1, wherein inhibition of the formation of functional MCM complexes from MCM subunits in cancerous cells is achieved by disrupting the interaction between hMcm2 and hMcm6.

5. A method for screening for anti-cancer compounds having an inhibitory effect on human MCM complexes, comprising: (a) contacting a candidate compound with a population of cells for a certain period of time; and (b) determining in the cells the amount of functional MCM complexes formed from the subunits, wherein step (b) is carried out by detecting a portion of MCM subunits located in the nucleus compared to a portion located in the cytoplasm, and wherein the inhibitory effect is brought about by disrupting the formation of functional MCM complexes by disrupting the interaction between MCM subunits.

6. The method of claim 5, wherein inhibiting the formation of functional MCM complexes from MCM subunits in cancerous cells is achieved by disrupting the interaction between hMcm2 and hMcm6.

7. The method of claim 5, wherein an indirect fluorescent process (immunostaining) is performed in step (b) in which one or more primary antibodies against one or more endogenous MCM subunits and a fluorescently labeled secondary antibody that recognizes the primary antibodies are used to visualize the location of the MCM proteins after the cells have been treated with the candidate compound for a specific duration.

8. The method of claim 5, wherein the direct fluorescence process is carried out in step (b) in which the cells are transfected with one or more plasmids capable of expressing one or more MCM subunits fused to fluorescent proteins, and then the location of the fluorescently labeled MCM subunits is detected after the cells are treated with the candidate compound for a specific duration.

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