AU2014100884A4 - Method for treating apoptosis resistant cancer - Google Patents

Abstract

Abstract The present invention provides a SERCA inhibitor for use in inducing cell death in apoptosis-resistant cells or cancer / tissues having the apoptosis-resistant cells. In particular, the present invention uses celastrol as the SERCA inhibitor for inducing cell death in the apoptosis-resistant cells of cancer in order to treat said cancer. 00 Cr)fN II0 17t~ C 0 0-l -

Description

METHOD FOR TREATING APOPTOSIS RESISTANT CANCER FIELD OF INVENTION The present invention relates to a method of inducing cell death in apoptosis-resistant cell. More particularly, the present invention relates to a method of inhibiting SERCA to induce cell death in apoptosis-resistant cell for use in treating cancer. BACKGROUND OF INVENTION Heterogeneous expression of therapeutic target proteins in cancer cells often lead to drug resistance, which is the major obstacle in the treatment of cancer via target therapy [']. Clinical strategies to tackle this heterogeneous problem are limited and therefore, small-molecules that retain effectiveness against drug-resistant cancers are highly desired. Targeting of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump protein would be a promising strategy to overcome this problem because the continued expression and calcium transport function of SERCA are critical to the survival of all cancer cell types . Previous studies indicated that inhibition of SERCA could disrupt the calcium homeostasis in cancer cells, thereby activates endoplasmic reticulum (ER)-stress response and induces permanent mitochondrial damage by Ca 2 + overload, leading to cell death induction . Most importantly, SERCA inhibitors exhibit potent anti-cancer effect toward Bax- and Bak-deficient apoptosis-resistant tumors and other multi-drug resistance (MDR) cancer cells [35], which further highlight the remarkable application of SERCA inhibitors as promising anti-cancer agents for the treatment of apoptosis resistant tumors, as well as MDR cancer cells. Therefore, there exists a need for cancer treatment strategies that can overcome apoptosis resistance and multi-drug resistance in cancers. 1 SUMMARY OF INVENTION It is an objective of the present invention to provide a SERCA inhibitor for use in inducing cell death in apoptosis-resistant cells. In particular, it is an objective of the present invention to provide a SERCA inhibitor for use in inducing cell death in cancer cells, leading to treatment for the cancer. The present invention relates to identification of novel SERCA inhibitor, a compound having structure of Formula I: HO 0 Formula I Compound of Formula I is also known as celastrol. The present invention is first to disclose the SERCA inhibition activity of celastrol. The present invention also discloses selective cytotoxic activity of celastrol towards cancer cells. In particular, the cancer cells are human cancer cells. The compound of formula I can be chemically synthesized or isolated from Tripterygium Wilfordi (Thunder of God vine) and Celastrus Regelii. In a first aspect, the present invention relates to a method of inducing cell death in cells comprises exposing the cells to celastrol that inhibits SERCA activity in said cells. Said induction of cell death includes an apoptosis, autophagic cell death and necrosis. Said cells comprise apoptosis-resistant cells or cancer cells. In one embodiment, said apoptosis-resistant 2 cells or cancer cells are originated from human or mouse. In another embodiment, the present method of inducing cell death further comprises selectively targeting apoptosis-resistant cells or cancer cells only. In a second aspect, the present invention provides a composition for use in the treatment of cancer comprising a compound of Formula I, wherein said composition is administered to a subject in need thereof to inhibit SERCA activity in cancer cells of said cancer. Inhibition of SERCA activity leads to cell death in said cancer. Said subject includes human subject and said human subject either has drug resistance to conventional therapeutic agents which induce cell death in cancer cells or tends to have said drug resistance. Said cells include apoptosis-resistant cells or cancer cells. Said compound or composition may only target said apoptosis-resistant cells or cancer cells in said subject in order to provide a cancer-specific treatment. BRIEF DESCRIPTION OF FIGURES FIG. 1 shows cell cytotoxicity of celastrol towards various cancer cells and normal cells. FIG. 2 shows 3D schematic representation (ribbon diagram) illustrating celastrol binds and suppresses the SERCA pump (FIG. 2A) and, percentage of Ca2+ ATPase activity of SERCA in the presence of celastrol (FIG. 2B) FIG. 3 shows flow cytometry graph of HeLa cells stained with fluo-3 having treated with DMSO (Control; FIG. 3A); 1 [tM Celastrol (FIG. 3B) and 5 tM Celastrol (FIG. 3C), and percentage of intracellular calcium level in HeLa cells having treated with or without Celastrol (FIG. 3D) FIG. 4 shows IC 50 of celastrol in various apoptosis-resistant wild types and mutants mouse embryonic fibroblasts. FIG. 5 shows flow cytometry plot of Bax-Bak double knock out (DKO) mouse embryonic fibroblasts in the absence of celastrol and 1 tM of celastrol (FIG. 5A), with 2 pM celastrol, and 2 [tM celastrol and 10 pM BAPTA/AM (FIG. 5B) 3 FIG. 6 shows percentage of apoptosis of Bax-Bak double knock out mouse embryonic fibroblasts having treated with or without celastrol or BAPTA/AM DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. The first aspect of the present invention provides a method of inducing cell death in cells comprises exposing the cells to a composition comprising a compound of Formula I to inhibit SERCA activity in said cells. In one embodiment, the present method comprises selectively inducing cell death in apoptosis-resistant cells or cancer cells. Said selectively inducing cell death in apoptosis-resistant cells or cancer cells of the present method may refer to substantially no induction or little induction of cell death in normal cells which undergo apoptosis under normal physiological conditions. Said apoptosis-resistant cells or cancer cells are originated from human or mouse. Said apoptosis-resistance cells or cancer cells are cells that do not undergo apoptosis under normal physiological conditions. The present invention is capable to induce cell death in said apoptosis-resistance cells and cancer cells. In another embodiment, the cell death induced by the present method includes apoptosis, autophagic cell death and necrosis. In yet another embodiment, the apoptosis-resistant cells or cancer cells comprise cervical, breast, liver, lung, and prostate type human and mouse cancer cells. The second aspect of the present invention provides a composition for use in the treatment of cancer comprises a compound of Formula I, wherein said composition is administered to a subject in need thereof to inhibit SERCA activity in cancer cells of said cancer. Inhibition of SERCA activity of the compound leads to cell death in said cancer cells. In one embodiment, the 4 present method comprises selectively inhibiting SERCA activity in apoptosis-resistant cells or cancer cells or tissues. Said selectively inhibiting SERCA activity in apoptosis-resistant cells or cancer cells or tissues of the present method may refer to substantially no inhibition or little inhibition of SERCA activity in normal or healthy cells/tissues which undergo apoptosis under normal physiological conditions. Said apoptosis-resistant cells, cancer cells or tissues are either mouse or human cells or tissues. Said apoptosis-resistance cells or cancer cells are cells that do not undergo apoptosis under normal physiological conditions. The present invention is capable to induce cell death in said apoptosis-resistance cells and cancer cells. In one embodiment, the cell death induced by the present method includes apoptosis, autophagic cell death and necrosis. In another embodiment, cancers treated by the present method comprise cervical, breast, liver, lung, prostate cancers. Example 1 In Vitro Cytotoxicity Test Of Celastrol In A Panel Of Human Or Mouse Cancer And Normal Cells Cell culture and cytotoxicity assay: Celastrol is dissolved in DMSO at a final concentration of 100 mmol/L and stored at -20 'C. Cytotoxicity is assessed using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay. 4000-8000 HeLa (human cervical cancer), MCF-7 (human breast cancer), HepG2 and Hep3B (human liver cancer), H1299 and A549 (human lung cancer), PC3 and LNCap (human prostate cancer), LLC-1 (mouse Lewis lung carcinoma) and L02 (human normal liver) cells are seeded on 96-well plates per well. After overnight pre-incubation, the cells are exposed to different concentrations of celastrol (0.039 - 100 mol/L) for 3 days. Subsequently, 10 tL of MTT reagents is added to each well and incubated at 37 C for 4 hours followed by the addition of 100 [tL solubilization buffer (10% SDS in 0.01 mol/L HCl) and overnight incubation. Absorbance at 585 nm is determined from each well the next day. The percentage of cell viability is calculated using the following formula: Cell viability (%) Cells 5 number treated / Cells number DMSO control x 100. Data are obtained from three independent experiments. There is significant cell cytotoxicity with mean IC 5 o value ranging from 0.432-1.96ptM observed in a panel of human and mouse cancer cells treated with celastrol for 72 hours, which is revealed by MTT assay (FIG. 1). Celastrol indicates 2.5 to 10 fold less cytotoxic effect in human normal liver cells than the panel of human cancer cells, revealing that Celastrol exhibits potent and specific cytotoxicity toward a panel of human and mouse cancer cells. Example 2 Inhibitory Effect Of Celastrol On SERCA, A Calcium Pump, And Induction Of Cytosolic Calcium Released By Celastrol (A) Molecular computational docking. The 3D structure of celastrol is obtained from the PubChem (http://pubchem.ncbi.nlm.nih.gov). The compound is preprocessed by the LigPrep [7] which uses OPLS-2005 force field [8] to obtain the corresponding low energy 3D conformers. The ionized state is assigned by using Epik3 at a target pH value of 7.0 ± 2.0. The 3D crystal structure of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is used in molecular docking. The 3D structure of SERCA is retrieved from the Protein Data Bank (PDB ID code 2AGV) [9]. The Protein Preparation Wizard is used to remove crystallographic water molecules, add hydrogen atoms, and assign partial charges based on OPLS-2005 force field [101. Energy minimization is also performed and is terminated when the root-mean-square deviation (RMSD) reached a maximum value of 0.3 A. The celastrol is docked into the thapsigargin (TG) binding site of the SERCA using Glide program I"l with the extra precision (XP) scoring mode. The docking grid box is defined by centering on TG in the SERCA. (B) Measurement of SERCA activity. Purified Ca2+ ATPase (SERCA1A) is prepared from female rabbit hind leg muscle [12]. ATPase activity is determined using the enzyme-coupled 6 method utilizing pyruvate kinase and lactate dehydrogenase as previously described in Michelangeli et al. (1990). All SERCA inhibition data are fitted to the allosteric dose versus effect equation using Fig P (Biosoft): Activity = minimum activity + (maximum activity - minimum activity) / (1 + ([I]/ICso)P). (C) Flow cytometry analysis of cytosolic calcium level. Intracellular free calcium is measured by a fluorescent dye, Fluo-3. Briefly, HeLa cells are washed twice with MEM media after Celastrol treatment (1 tM or 5ptM) for 4h. Then cell suspensions are incubated with 5 [M Fluo-3 at 37'C for 30 min. Then the cells are washed twice with HBSS. After re-suspended cell samples are subjected to FACS analysis. At least 10,000 events are analyzed. In molecular docking, 5000 poses are generated during the initial phase of the docking calculation, out of which best 1000 poses are chosen for energy minimization by 1000 steps of conjugate gradient minimizations. The performance of molecular docking is evaluated by comparing the docked pose with the experimental structure for the celastrol in the X-ray co-crystallized complex. TG in the X-ray co-crystallized complexes is re-docked into the binding sites and the RMSD for re-docked result of TG is 1.78 A. Comparison of the docking pose of celastrol (XP score: -4.16) with known SERCA inhibitor thapsigargin (XP score: -7.23) indicates that the two compounds are located in the space within the SERCA binding pocket (FIG. 2A). To ascertain whether the SERCA pump is suppressed by celastrol, celastrol's SERCA inhibitory effect is quantified using purified rabbit skeletal muscle sarcoplasmic reticulum (SR) membranes to measure the expression of the SERCA1A isoform by the SR membrances [. Most of existing SERCA inhibitors show similar inhibitory effect in SERCA isoform [1.16] The SERCAlA pump (from rabbit skeletal muscle SR) is inhibited by celastrol in a dose-dependent manner (FIG. 2B), which is fitted to an allosteric dose versus effect equation. Inhibition of SERCA will contribute to the cell cytosolic calcium accumulation. To address whether celastrol treatment will affect the 7 calcium homeostasis, the calcium imaging dye, fluo-3 is used to determine the calcium release by flow cytometry. Results show that celastrol dose-dependently increases the calcium signal in HeLa cells (FIGs. 3A-3D), indicating the calcium is released in cytosolic region upon the celastrol treatment. Celastrol is confirmed to bind and suppress SERCA activity, leading to the release of cytosolic calcium in cells. Example 3 Cytotoxic Effect Of Celastrol On Various Apoptosis-Resistant Cells Cell culture and cytotoxicity assay: The present compound of celastrol is dissolved in DMSO at a final concentration of 100 mmol/L and stored at -20 'C. Cytotoxicity is assessed using 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay in Wong et al. (2009) [6]. 2500 of caspase wild-type, caspase-3 deficient, caspase-7 deficient, caspase-3/-7 deficient, caspase-8 deficient, Bax-Bak wild-type and Bax-Bak double knock out (DKO) mouse embryonic fibroblasts (MEFs) are seeded on 96-well plates per well. After overnight pre-incubation, the cells are exposed to different concentrations of celastrol (0.039 - 100pmol/L) for 3 days. Subsequently, 10 pL of MTT reagents is added to each well and incubated at 37'C for 4 hours followed by the addition of 100 pL solubilization buffer (10% SDS in 0.01 mol/L HCl) and overnight incubation. Absorbance at 585 nm is determined from each well the next day. The percentage of cell viability is calculated using the following formula: Cell viability (%) = Cells number treated / Cells number DMSO control x 100. Data are obtained from three independent experiments. Celastrol is found to exhibit similar cytotoxic effect on both wild-type and apoptosis-resistant cells, i.e. caspase-3-/-, caspase-7-/-, caspase-3-/-/7-/- and caspase-8-/- compared to the caspase 8 wild-type MEFs (FIG. 4). In addition, it also shows similar cytotoxicity in Bax-Bak DKO apoptosis-resistant cells compared to and Bax-Bak wild-type MEFs is shown (FIG. 4), indicating that celastrol is able to induce cell death in apoptosis-resistant cells. Example 3 demonstrates that celastrol is capable to induce cell death in apoptosis-resistant cells or apoptosis-resistant cancer cells. Example 4 Celastrol induces cell death in apoptosis-resistant cells through SERCA inhibition and calcium mobilization. Flow cytometry analysis. Cell apoptosis is measured using an annexin V staining kit (BD Biosciences, San Jose, CA, USA). Briefly, Bax-Bak DKO MEFs are treated with 1 pM or 2pM celastrol (Cel) in the presence or absence of 10pM BAPTA/AM for 24 h. Cells are then harvested and analysed by multiparametric flow cytometry using FITC-Annexin V and Propidium iodide staining (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Flow cytometry is then carried out using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Data acquisition and analysis is performed with CellQuest (BD Biosciences, San Jose, CA, USA). Data are obtained from three independent experiments. FIGs. 5A and 5B are the flow cytometry plot of Bax-Bak DKO cells having treated 1 pM or 2 pM celastrol in the presence or absence of 10 pM BAPTA/AM and results of FIG. 5A and FIG. 5B are converted quantitatively in FIG. 6 as shown as percentage of apoptosis. Annexin V staining shows that the celastrol-induced cell death in Bax-Bak DKO cells. In the presence of calcium chelator, BAPTA/AM, the celastrol-induced cell death diminishes (FIG. 6). It is confirmed that celastrol induces cell death in apoptosis-resistant cells or apoptosis-resistant cancer cells via direction inhibition of SERCA and calcium release. 9 10 References 1 Denmeade SR, Isaacs JT. The SERCA pump as a therapeutic target: making a "smart bomb" for prostate cancer. Cancer Biol Ther 2005; 4 (1): 14-22. eng. 2 Wong VK, Li T, Law BY, Ma ED, Yip NC, Michelangeli F, et al. Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells. Cell Death Dis; 4: e720. eng. 3 Das SG, Hermanson DL, Bleeker N, Lowman X, Li Y, Kelekar A, et al. Ethyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy. ACS Chem Biol; 8 (2): 327-35. eng. 4 Tian F, Schrodl K, Kiefl R, Huber RM, Bergner A. The hedgehog pathway inhibitor GDC-0449 alters intracellular Ca2+ homeostasis and inhibits cell growth in cisplatin-resistant lung cancer cells. Anticancer Res; 32 (1): 89-94. eng. 5 Janssen K, Horn S, Niemann MT, Daniel PT, Schulze-Osthoff K, Fischer U. Inhibition of the ER Ca2+ pump forces multidrug-resistant cells deficient in Bak and Bax into necrosis. J Cell Sci 2009; 122 (Pt 24): 4481-91. eng. 6 Wong VK, Zhou H, Cheung SS, Li T, Liu L. Mechanistic study of saikosaponin-d (Ssd) on suppression of murine T lymphocyte activation. J Cell Biochem 2009; 107 (2): 303-15. eng. 7 Schr6dinger L, New York, NY LigPrep, version 2.3. 2009. 8 Kaminski GAF RAT-R, J.; Jorgensen, W. L. Evaluation and reparametrization of the OPLS-AA force field for proteins via comparison with accurate quantum chemical calculations on peptides. JPhys Chem B 2001; 105. 9 Obara K, Miyashita N, Xu C, Toyoshima I, Sugita Y, Inesi G, et al. Structural role of countertransport revealed in Ca(2+) pump crystal structure in the absence of Ca(2+). Proc Natl Acad Sci U S A 2005; 102 (41): 14489-96. eng. 10 Epik, version 2.0, Schrudinger, LLC, New York, NY,. 2009. 11 Glide, version 5.5, Schr6dinger, LLC, New York, NY 2009. 12 Michelangeli F, Munkonge FM. Methods of reconstitution of the purified sarcoplasmic reticulum (Ca(2+)-Mg2+)-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles. Anal Biochem 1991; 194 (2): 231-6. eng. 13 Michelangeli F, Colyer J, East JM, Lee AG. Effect of pH on the activity of the Ca2+ + Mg2(+)-activated ATPase of sarcoplasmic reticulum. Biochem J 1990; 267 (2): 423-9. eng. 14 Wu KD, Lee WS, Wey J, Bungard D, Lytton J. Localization and quantification of endoplasmic reticulum Ca(2+)-ATPase isoform transcripts. Am J Physiol 1995; 269 (3 Pt 1): C775-84. eng. 15 Michelangeli F, East JM. A diversity of SERCA Ca2+ pump inhibitors. Biochem Soc Trans; 39 (3): 789-97. eng. 11 16 East JM, Michelangeli F. Recent advances in Membrane Biochemistry. Biochem Soc Trans; 39 (3): 703-6. eng. 12

Claims (5)

1. A method of inducing cell death selectively in apoptosis-resistant cells comprising exposing said apoptosis-resistant cells to a composition comprising celastrol to inhibit SERCA activity in said cells.

2. A method of treating cancer comprising administering a composition comprising celastrol to a subject in need thereof to selectively induce cell death in apoptosis-resistant cells in said cancer.

3. The method of claim 1 or 2, wherein the cell death induced includes apoptosis, autophagic cell death and necrosis.

4. The method of claim 1, wherein the cells are cancer cells or apoptosis-resistant cancer cells.

5. The method of claim 1, 2 or 4, wherein the cancer cells or cancer comprises cervical cancer, breast cancer, liver cancer, lung cancer and prostate cancer. 13