WO2012122101A1 - Methods for inhibiting proliferation and inducing apoptosis of cancer cells expressing the g protein-coupled estrogen receptor gpr30 - Google Patents

Abstract

Methods for increasing the amount of calcium in the cytoplasm and the mitochondria of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30 comprise agonizing the GPR30. In some aspects, the methods further comprise inhibiting the biologic activity of Bcl-2 and Bcl-xL. The increased concentration of calcium in the cytoplasm and the mitochondria is sustained and is sufficient to inhibit cell proliferation and/or inhibit cell growth and/or induce apoptosis.

Description

METHODS FOR INHIBITING PROLIFERATION AND INDUCING APOPTOSIS OF CANCER CELLS EXPRESSING THE G PROTEIN-COUPLED ESTROGEN RECEPTOR GPR30

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/449,310, filed on March 4, 2011, the contents of which are incorporated by reference herein, in their entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

The inventions described herein were made, in part, with funds obtained from the National Cancer Institute, Grant Nos. HL090804 and H L090804-01A2S109. The U.S.

government may have certain rights in these inventions.

FIELD OF THE INVENTION

The invention relates generally to the field of cancer treatment. More particularly, the invention relates to methods for inhibiting the proliferation of and for inducing apoptosis in cancer cells expressing the G Protein-Coupled Estrogen Receptor GPR30 (a.k.a GPER).

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.

Ovarian cancer remains the most frequently fatal gynecological malignancy and the fifth most likely cause of death from cancer in women. A commonly used first-line adjuvant chemotherapy in ovarian cancer is platinum-based, such as cisplatin or carboplatin. Despite initial high response rates, more than 60% of ovarian cancer patients given such

chemotherapy relapse with incurable platinum-resistant disease and have a median survival of only approximately 2 years. There is a need for additional treatments for drug-resistant cancers, including platinum-resistant cancers.

The G protein-coupled receptor GPR30 (a.k.a. GPER) is a 7-pass transmembrane protein that binds 17P-estradiol (E2), and is distinct from ERct and ER . GPR30 mediates multiple non-genomic E2 signaling events including intracellular calcium (Ca²⁺) mobilization. GPR30 drug discovery efforts have yielded the synthetic agonist G-1 (Bologa CG et al. (2006) Nat. Chem. Biol. 2:207-12) and the structurally related antagonists G-15 (Dennis MK et al. l of 32 (2009) Nat. Chem. Biol. 5:421-27) and G36 (Dennis MK et ol. (2011) J. Steroid Biochem. Mol. Biol. 127:358-66). G-l shows specificity for GPR30 as it does not significantly bind to ERct, E , or 25 other G-protein coupled receptors (Blasko E et ol. (2009) J. Neuroimmunol. 214:67-77).

Calcium is a second messenger involved in a variety of cellular functions, including proliferation, but can also induce apoptosis. Transitory increases in cellular cytosolic Ca²⁺ are realized by Ca²⁺ influx across the plasma membrane through voltage- and ligand- sensitive Ca^z+ channels and by Ca²⁺ release from internal stores. The endoplasmic reticulum is the main internal Ca²⁺ store in the great majority of the cells types; lysosomes, mitochondria, Golgi apparatus, and the nucleus may also serve as Ca²⁺ stores. Cytosolic Ca²⁺ signals are often generated by agonist-evoked increases in intracellular messengers and the subsequent activation of intracellular Ca ⁺ channels. The best characterized intracellular Ca²⁺ release channels are inositol 1,4,5-trisphosphate (IP₃)-gated, referred to as IP₃ receptors (IP3RS), and Ca^2+"-gated, also known as ryanodine receptors (RyRs). Both IP₃Rs and RyR localize to the endoplasmic reticulum. IP₃ is the most ubiquitous of the Ca²⁺ messengers and is generated in response to a wide-range of stimuli that include hormones, growth factors and neurotransmitters.

Transitory/oscillatory increases in cytoplasmic Ca ⁺ [Ca²⁺]_c, mitochondrial [Ca²⁺]_m, and nuclear [Ca²⁺]_n represent commonly used intracellular signals. Under normal conditions, the mitochondrial Ca²⁺ uniporter mediates influx of Ca²⁺ into this organelle, and the mitochondrial Na⁺/Ca²⁺ exchanger mediates efflux. Ca²⁺ uptake by mitochondria helps for vital cell functions such as ATP synthesis by increasing efficiency of NADH production through Ca²⁺-sensitive dehydrogenases in the TCA cycle. Prolonged or sustained elevations in cytoplasmic Ca²⁺ concentrations, however, can lead to mitochondrial Ca²⁺ overload, which may result in cell death. In fact, even modest elevations in [Ca²⁺]m of 30-40% can also cause cell death when these elevations are sustained. Sustained mitochondrial Ca ⁺ overload, especially in the presence of reactive oxygen species (ROS; a by-product of increased ATP synthesis due to increased [Ca²⁺]), leads to formation of a permeability transition pore (PTP) between the inner and outer mitochondrial membranes (OMM) and loss of mitochondrial membrane potential (MMP; ΔΨηι). PTP opening also releases accumulated mitochondrial Ca²⁺ that is subsequently taken up by adjacent mitochondria and propagates mitochondrial dysfunction. Osmotic forces may then cause matrix swelling leading to eventual rupture of the OMM and release to the cytoplasm of apoptogenic factors including cytochrome C, Apaf-1, and Smac/Diablo, which then initiate the caspase cascade.

SUMMARY OF THE INVENTION

The invention features methods for increasing the level of calcium in the cytoplasm of a cell such as a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. Generally, the methods comprise agonizing the GPR30 of a cell expressing GPR30. The method may be carried out in vitro. Agonizing GPR30 may comprise contacting the GPR30 or the cell with an agent capable of agonizing the GPR30, or a composition comprising the agent, in an amount effective to increase the level of calcium in the cytoplasm of the cancer cell. The agent may be G-l, a compound of Formula I:

; or a pharmaceutically acceptable salt thereof. The agent may be capable of inhibiting viability of the cell at an IC₅₀ of about 4 μΜ or less, at an IC₅₀ of about 2.5 μΜ or less, at an IC₅₀ of about 1 μΜ or less, or an IC₅₀ of about 0.85 μΜ or less.

The cancer cell may be resistant to at least one platinum-based chemotherapeutic agent. The cancer cell may be sensitive to at least one platinum-based chemotherapeutic agent. The platinum-based chemotherapeutic agent may be cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

The increased level of calcium may be sustained for a period of time. The level of calcium may be increased to a concentration sufficient to substantially inhibit proliferation of the cancer cell. The level of calcium may be increased to a concentration sufficient to induce apoptosis of the cancer cell. The level of calcium may be increased to a

concentration sufficient to induce p53 expression in the cancer cell. The level of calcium may be increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 or Poly (ADP-ribose) polymerase in the cancer cell. The invention also features methods for increasing the level of calcium in the mitochondria of a cell such as a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. Generally, the methods comprise agonizing the GPR30 of a cell expressing GPR30. The methods may be carried out in vitro. Agonizing GPR30 may comprise contacting the GPR30 or the cell with an agent capable of agonizing the GPR30, or a composition comprising the agent, in an amount effective to increase the level of calcium in the mitochondria of the cancer cell. The agent may be a compound of Formula I or a pharmaceutically acceptable salt thereof. The agent may be capable of inhibiting viability of the cell at an IC₅₀ of about 4 μΜ or less, at an IC₅₀ of about 2.5 μΜ or less, at an IC₅₀ of about 1 μΜ or less, or an IC₅₀ of about 0.85 μΜ or less.

The cancer cell may be resistant to at least one platinum-based chemotherapeutic agent. The cancer cell may be sensitive to at least one platinum-based chemotherapeutic agent. The platinum-based chemotherapeutic agent may be cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

The increased level of calcium may be sustained for a period of time. The level of calcium may be increased to a concentration sufficient to substantially inhibit proliferation of the cancer cell. The level of calcium may be increased to a concentration sufficient to induce apoptosis of the cancer cell. The level of calcium may be increased to a

concentration sufficient to compromise the integrity of the mitochondria membrane. The level of calcium may be increased to a concentration sufficient to induce p53 expression in the cancer cell. The level of calcium may be increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 or Poly (ADP-ribose) polymerase in the cancer cell.

The invention also features methods for inhibiting the proliferation of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. In general, the methods comprise contacting the cell with an amount of a compound of Formula I, or a

pharmaceutically acceptable salt thereof, effective to inhibit proliferation of the cell. The compound may be in a composition comprising a carrier. The methods may be carried out in vitro. The cancer cell may be susceptible or resistant to at least one platinum-based chemotherapeutic agent such as cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell. The invention also features methods for inducing apoptosis in a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. In general, the methods comprise contacting the cell with an amount of a compound of Formula I, or a

pharmaceutically acceptable salt thereof, effective to induce apoptosis in the cell. The compound may be in a composition comprising a carrier. The methods may be carried out in vitro. The cancer cell may be susceptible or resistant to at least one platinum-based chemotherapeutic agent such as cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

The invention also features methods for enhancing the expression of the IP3 type 1 receptor in a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. In general, the methods comprise contacting the cell with an amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, effective to enhance the expression of the IP3 type 1 receptor in the cell. The compound may be in a composition comprising a carrier. The methods may be carried out in vitro. The cancer cell may be susceptible or resistant to at least one platinum-based chemotherapeutic agent such as cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

Any of the methods may further comprise inhibiting the biologic activity of one or more of B cell lymphoma 2 (Bcl-2) and B cell lymphoma extra large (Bcl-xL) in the cancer cell. Inhibiting the biologic activity of Bcl-2 and/or Bcl-xL may comprise contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL. The agent may comprise Navitoclax, Formula II,

or a pharmaceutically acceptable salt thereof. The agent may comprise a pharmaceutically acceptable carrier. The invention also features methods for inhibiting the growth of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. In general, the methods comprise inhibiting the biologic activity of one or more of B cell lymphoma 2 (Bcl-2) and B cell lymphoma extra large (Bcl-xL) in the cancer cell, and agonizing the GPR30 in the cancer cell. Inhibiting the biologic activity of Bcl-2 may comprise contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-2 in an amount effective to inhibit the biologic activity of Bcl-2. Inhibiting the biologic activity of Bcl-xL comprises contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-xL in an amount effective to inhibit the biologic activity of Bcl-xL. In some aspects, the biologic activity of both Bcl-2 and Bcl-xL is inhibited. The agent capable of inhibiting the biologic activity of Bcl-2 may be compound of Formula II, or a pharmaceutically acceptable salt thereof. The agent capable of inhibiting the biologic activity of Bcl-xL may be compound of Formula II, or a

pharmaceutically acceptable salt thereof. The compound may be in a composition comprising a carrier. The methods may be carried out in vitro. The cancer cell may be susceptible or resistant to at least one platinum-based chemotherapeutic agent such as cisplatin. The cancer cell is preferably an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows protein levels of the GPR30 51 kDa and 38 kDa isoforms by Western blotting using the LiCor Odyssey IR imaging system. Quantitation of GPR30 protein is shown. Fig. IB shows total GPR30 mRNA levels measured by quantitativer reverse- transcriptase polymerase chain reaction (qRT-PCR). CP70, C30 and C200 cells were selected from cisplatin-sensitive A2780 ovarian cancer cells for increasing resistance to cisplatin.

Fig. 2 shows that G-1 inhibits growth in cisplatin-sensitive and -resistant ovarian cancer cells. Fig. 2A shows cell viability in response to cisplatin for 3 days; Fig. 2B shows cell viability in response to G-1 for 3 days; Fig. 2C shows cell viability in response to G-1 for 6 days in isogenic A2780, CP70, C30, and C200 cells. Cells were treated with either cisplatin once or with G-1 daily. Viability was measured using the Cell Titer-Glo™ assay (Promega, Madison, Wl). Data shown represent the mean and SD of 5 replicates. Data were fit to a 4 point logistic equation to determine IC_50s.

Fig. 3 shows G-1 induces apoptosis in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Fig. 3A and Fig. 3B shows that G-1 induced apoptosis as determined by flow cytometric analysis of cells stained with DilCi(5) for MMP and Annexin V for externalized phosphatidylserine (PS). Cells were treated with or without 2.5 μΜ G-l every 24 h over 5 days, and apoptosis was measured daily. Fig. 3A shows representative flow cytometry cytograms taken from the day of maximal G-l-induced apoptosis in each cell line; A2780 (day 4), CP70 (day 5), C30 (day 5), C200 (day 4). Apoptotic cells were defined as MMP-negative and Annexin V-positive as indicated by the gate in the lower right corner of each cytogram, and the % apoptotic cells is indicated. At least 30,000 singlet events were collected per sample. Fig. 3B shows quantitation of the 5 day time course of G-l induced apoptosis. Each data point represents 3 replicates and error bars are SDs. The rank order of sensitivity to G-l induced apoptosis was A2780 > C30 > CP70 > C200. Fig. 3C shows G-l induced p53 and p53 up-regulated modulator of apoptosis (PUMA) protein expression, and cleavage of caspase-3 (CASP3) and poly (ADP-ribose) polymerase (PARP) proteins. Protein levels of p53, PUMA, and cleaved-CASP3 (C-CASP3) were determined after 48 h of 2.5 μΜ G- 1 exposure, and of cleaved-PARP (c-PARP) after 72 h of exposure by Western blotting as carried out in Fig. 1.

Fig. 4 shows that G-l induces Ca²⁺ mobilization in the cytoplasm leading to Ca²⁺ uptake by mitochondria in parental cisplatin-sensitive A2780 and isogenic cisplatin-resistant CP70, C30 and C200 ovarian cancer cells. Fig. 4A and Fig. 4B show measurements of cytosolic Ca²⁺ concentration [Ca²⁺]c, in response to G-l using the cytosolic Ca²⁺ indicator, Fura-2 AM, which is a ratiometric indicator that allows absolute quantification of [Ca²⁺]c. Fig. 4A shows fluorescent images illustrating the basal [Ca²⁺]c (top panels) and the [Ca²⁺]c after administration of 2.5 μΜ G-l (bottom panels). Fig 4B shows quantification of [Ca²⁺]c: Number of cells quantitated per group; A2780 (n = 21), CP70 (n = 37), C30 (n = 29), C200 (n = 46). Comparison of G-l-induced maximum increase in [Ca²⁺]c: C200 > C30 > A2780 > CP70. Fig. 4C and Fig. 4D show measurements of concomitant cytosolic and mitochondrial Ca²⁺ levels in response to G-l using the cytosolic Ca²⁺ indicator, Fluo-4 and the mitochondrial Ca²⁺ indicator, Rhod-2. Fig. 4C shows an Illustration of changes in fluorescence before (top panels) and after administration of 2.5 μΜ G-l (bottom panels). Fig. 4D shows tracings of relative changes in cytosolic Ca2+ (cyto Ca²⁺) and mitochondrial Ca²⁺ levels (mito Ca²⁺) in response to 2.5 μΜ G-l. To achieve concomitant measurements in real-time, cells were simultaneously loaded with Rhod-2 and Fluo-4. Fluo-4 used in (Fig. 4C-D) is known to be unstable for measurements > 5 minutes, explaining the small decrease in cytosolic Ca²⁺ after 8-10 minutes, whereas G-l-induced Ca ⁺ increases measured by Fura-2 in (Fig. 4A-B) were sustained. Also, Fluo-4 is not a ratiometric dye, therefore quantification of [Ca²⁺]c is relative rather than absolute as with Fura-2. Number of cells quantitated per group; A2780 (n = 49), CP70 (n = 86), C30 (n = 62), C200 (n = 73). The rank order of G-l-induced increases in cytosolic Ca²⁺ levels was C200 > C30 > CP70 > A2780 cells, whereas mitochondrial Ca²⁺ levels were increased in A2780 > C30 > CP70 > C200 cells.

Fig 5 show cisplatin-resistant ovarian carcinoma cells differentially express IP3R and RyR Ca²⁺ release channels, and over-express anti-apoptotic proteins Bcl-2 and Bcl-xL. Fig. 5A shows protein expression of IP3 receptor types 1, 2, and 3 (IP3R1, IP3R2, and IP3R3, respectively) and Fig. 5C shows the anti-apoptotic proteins Bcl-2 and Bcl-xL were measured by Western blotting as in Fig. 1. Fig. 5B shows mRNA expression of ryanodine receptor type 1, 2, and 3 (RYR1, RYR2, and RYR3, respectively) measured by qRT-PCR. IP3R2 and RYR1 were significantly expressed in A2780 cells, whereas IP3R1, IP3R3, RYR2 and RYR3 were significantly expressed in cisplatin-resistant CP70, C30, and C200 cells. Bcl-2 and Bcl-xL were overexpressed at similar levels in the cisplatin-resistant CP70, C30 and C200 cells compared to cisplatin-sensitive A2780 cells.

Fig. 6 shows that knock-down of GPR30 in A2780 cells dramatically blocks G-l- induced Ca²⁺ mobilization and apoptosis. GPR30 shRNA (GPR30 sh) and as a control, enhanced green fluorescent protein shRNA (eGFP sh) expression plasmids were stably transfected into A2780 cells. Fig. 6A shows the level of GPR30 knock-down by qRT-PCR. Each group was measured in triplicate and error bars represent SDs. Fig. 6B shows G-l- evoked increases in cytoplasmic Ca²⁺ levels using Fura-2 as carried out in Fig. 4. Number of cells quantitated per group: eGFP shRNA cells at 1 μΜ G-l (n = 74) and at 2.5 μΜ G-l (n = 62), GPR30 shRNA cells at 1 μΜ G-l (n = 83) and at 2.5 μΜ G-l (n = 97). Fig. 6C shows G-l- induced apoptosis according to the percentage of MMP-negative/Annexin V-positive cells as determined in Fig. 3 using flow cytometric analysis.

Fig. 7 shows the Bcl-2 -family inhibitor Navitoclax (ABT-263) sensitizes cisplatin- resistant CP70, C30 and C200 ovarian cancer cells to G-l -induced apoptosis. Fig. 7 shows cell viability resulting from treating cells using a 10 x 6 matrix of G-l and Navitoclax concentrations, respectively. Cells were treated daily for 48 h (A2780 and CP70) or 72 h (C30 and C200). Cell viability was assessed using the Cell Titer-Glo™ assay. Each data point represents 3 replicates and associated SEMs. Each data series was fit to a 4 point logistic curve.

DETAILED DESCRIPTION OF THE INVENTION

Various terms relating to aspects of the invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

As used herein, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise.

Formula I:

Pharmaceutically acceptable salts of Forumla I or Formula II may be acid or base salts. Non-limiting examples of pharmaceutically acceptable salts include sulfates, methosulfates, methanesulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, besylates, phosphates, monohydrogenphosphates, dihydrogenphosphates,

metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, dioates, benzoates, chlorobenzoates, methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, toluenesulfonates, xylenesulfonates, pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y- hydroxybutyrates, glycollates, tartrates, methanesulfonates, propanesulfonates, mandelates, and other salts customarily used or otherwise FDA-approved.

It has been observed in accordance with the invention that GPR30 is expressed in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells, and that the synthetic GPR30 agonist G-1 inhibited growth and induced apoptosis in these cells. It has been further observed that increased expression of the G Protein-Coupled Estrogen Receptor GPR30 on cancer cells can be exploited by pharmacological super-activation to cause calcium overload and induce apoptosis in the cells. Pharmacologic super-activation of GPR30 using the synthetic agonist G-1 (Formula I) exhibited therapeutic potential against isogenic platinum- sensitive and -resistant ovarian cancer cell lines. In addition, it was observed that combining G-1 with the Bcl-2-family inhibitor Navitoclax (ABT-263) (Formula II) could inhibit the growth of these cells. Accordingly, the invention features methods that comprise agonizing the GPR30 in cancer cells. Any of the methods may be carried out in vitro, in vivo, or in situ.

In some aspects, the methods comprise increasing the level of calcium in the cytoplasm of a cell expressing GPR30. In general, increasing the level of calcium in the cytoplasm comprises agonizing the GPR30 of the cell. Agonizing the GPR30 may comprise contacting the GPR30, or the cell expressing the GPR30, with an agent capable of agonizing GPR30. The agent is preferably used at an amount or concentration effective to increase the level of calcium in the cytoplasm of the cell. In some aspects, the level of calcium is preferably increased to a concentration sufficient to substantially inhibit proliferation of the cell. In some aspects, the level of calcium is preferably increased to a concentration sufficient to induce apoptosis of the cell. In some aspects, the level of calcium is increased to a concentration sufficient to induce p53 expression in the cell. In some aspects, the level of calcium is increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 (CASP3) or Poly (ADP-ribose) polymerase (PARP) in the cell.

In some aspects, the methods comprise increasing the level of calcium in the mitochondria of a cell expressing GPR30. In general, increasing the level of calcium in the mitochondria comprises agonizing the GPR30 of the cell. Agonizing the GPR30 may comprise contacting the GPR30, or the cell expressing the GPR30, with an agent capable of agonizing the GPR30. The agent is preferably used at an amount or concentration effective to increase the level of calcium in the mitochondria of the cell. In some aspects, the level of calcium is preferably increased to a concentration sufficient to substantially inhibit proliferation of the cell. In some aspects, the level of calcium is preferably increased to a concentration sufficient to induce apoptosis of the cell. In some aspects, the level of calcium is increased to a concentration sufficient to destabilize or otherwise compromise the integrity of the mitochondria membrane, as determined by any suitable measurement in the art such as membrane potential measurements. In some aspects, the level of calcium is increased to a concentration sufficient to induce p53 expression in the cell. In some aspects, the level of calcium is increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 or Poly (ADP-ribose) polymerase in the cell.

In some preferred aspects, the increased level of calcium in the cytoplasm or the increased level of calcium in the mitochondria is sustained over a period of time, for example, a period of time sufficient to mobilize calcium into the mitochondria of the cell, a period of time sufficient to destabilize or otherwise compromise the integrity of the mitochondria membrane, a period of time sufficient to inhibit proliferation of the cell, and/or a period of time sufficient to induce apoptosis in the cell. The period of time may be about one minute, at least about two minutes, at least about three minutes, at least about four minutes, at least about 6 minutes, at least about 8 minutes, at least about 10 minutes, or at least about 12 minutes. Longer periods of time are possible, including at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about one hour, or longer than one hour. In general, the period of sustained increased calcium levels is longer than the normal and physiological transient mobilization of calcium in the cell cytoplasm or mitochondria.

The agent may be a chemical compound, or may be a biomolecule such as an antibody that specifically binds to an antigen on the GPR30 and that agonizes GPR30 by its binding. The chemical compound may be a compound of Formula I, also referred to herein as G-l (Bologa CG, et al. (2006) Nat. Chem. Biol., 2:207-12), or a pharmaceutically acceptable salt thereof. Other agonists that may be used include beta-estradiol, the phytoestrogens genistein (Vivacqua A et al. (2006) Mol. Pharmacol. 70:1414-23), and quercetin (Maggiolini M et al. (2004) J. Biol. Chem. 279:27008-16), 7α,17β-[9-[(4,4,5,5,5- Pentafluoropentyl)sulfinyl]nonyl]estra-l,3,5(10)-triene-3,17-diol, and tamoxifen citrate - (2)- 2-[4-(l,2-Diphenyl-l-butenyl)phenoxy]-N,N-dimethylethanamine citrate, or

pharmaceutically acceptable salt thereof. The tamoxifen analogue STX (Lin BC et al. (2009) Cancer Res. 69:5415-23) binds and activates GPR30 but not estrogen receptors (ERs). Other estrogen receptor agonists and antagonist that also bind GPR30 including the selective estrogen receptor modulator (SERM) tamoxifen (TAM), and the complete antiestrogen fulvestrant, but these ligands act as agonists of GPR30 (Thomas P et al. (2005) Endocrinology 146:624-32). The TAM active metabolite 4-hydroxytamoxifen (40HT) also activates GPR30- dependent signaling (Vivacqua A et al. (2006); and Filardo EJ et al. (2002) Mol. Endocrinol. 16:70-84).

The agent may be formulated as a composition, for example, with a carrier. Thus, the GPR30 or cell expressing GPR30 may be contacted with a composition comprising the agent, for example, G-1 or a pharmaceutically acceptable salt thereof. The carrier is preferably a pharmaceutically acceptable carrier. Preferred pharmaceutically acceptable carriers include nonaqueous vehicles such as nonpolar alcohols and oils, including plant or vegetable-derived oils such as olive oil, cottonseed oil, corn oil, canola oil, sesame oil, and other non-toxic oils. The compositions may comprise one or more pharmaceutically acceptable excipients, particularly excipients that enhance the water solubility of the compound of G-1. The carrier may comprise dimethyl sulfoxide. The carrier may comprise a micelle.

An effective amount may comprise from about 0.01 μΜ to about 1 mM of the agent, for example G-1 (Formula I) or pharmaceutically acceptable salt thereof, or Navitoclax (Formula II) or pharmaceutically acceptable salt thereof. An effective amount may comprise from about 0.1 μΜ to about 500 μΜ. An effective amount may comprise from about 0.1 μΜ to about 100 μΜ. An effective amount may comprise from about 0.1 μΜ to about 50 μΜ. An effective amount may comprise from about 0.1 μΜ to about 5 μΜ. An effective amount may comprise from about 0.1 μΜ to about 3 μΜ.

In preferred aspects, an effective amount of the agent, for example G-1 (Formula I) or pharmaceutically acceptable salt thereof, or Navitoclax (Formula II) or pharmaceutically acceptable salt thereof, inhibits the viability of the cell at an IC₅o of about 10 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 5 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 4 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC_S0 of about 3.8 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 3 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 2 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 1.9 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 1 μΜ or less. An effective amount of the agent may inhibit the viability of the cell at an IC₅₀ of about 0.85 μΜ or less.

The methods may be used with any cell expressing GPR30, and preferably the cell expressing GPR30 is a cancer cell or a precancerous cell. The cell may be a cell stably transformed with a nucleic acid encoding GPR30. The cell may be a cell line. The cancer cell may be resistant to at least one platinum-based chemotherapeutic agent such as cisplatin, or the cancer cell may be susceptible to at least one platinum-based chemotherapeutic agent such as cisplatin. The cancer cell may be an ovarian cancer cell, a breast cancer cell, an endometrial cancer cell, a prostate cancer cell, an urothelial cancer cell, or a thyroid cancer cell. Ovarian cancer cells are highly preferred.

In some aspects, the methods may further comprise inhibiting the biologic activity of the B cell lymphoma 2 (Bcl-2) protein. In some aspects, the methods may further comprise inhibiting the biologic activity of the B cell lymphoma extra large (Bcl-xL) protein. In some aspects, the methods may further comprise inhibiting the biologic activity of both the Bcl-2 and the Bcl-xL proteins. The biologic activity may comprise modulation of mitochondrial uptake of calcium in the cell.

Inhibiting the biologic activity of Bcl-2 and/or Bcl-xL may comprise contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL. The agent may comprise a compound, a biomolecule such as an antibody that specifically binds to Bcl-2 or an antibody that specifically binds to Bcl-xL, or a polypeptide that inhibits the biologic activity of Bcl-2 and/or Bcl-xL. A non-limiting example of a compound that inhibits the biologic activity of Bcl-2 and/or Bcl-xL is Natitoclax, also referred to as ABT-263, which has Formula II. The agent may comprise a composition, including the active agent and a pharmaceutically acceptable carrier. For example, a composition may comprise a compound of Formula II and a pharmaceutically acceptable carrier.

The invention also features methods for inhibiting the growth of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30. In general, the methods comprise inhibiting the biologic activity of one or more of B cell lymphoma 2 (Bcl-2) and B cell lymphoma extra large (Bcl-xL) in the cancer cell, and agonizing the GPR30 in the cancer cell. Inhibiting the biologic activity of Bcl-2 may comprise contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-2 in an amount effective to inhibit the biologic activity of Bcl-2. Inhibiting the biologic activity of Bcl-xL comprises contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-xL in an amount effective to inhibit the biologic activity of Bcl-xL. The agent may comprise a compound of Formula II or a pharmaceutically acceptable salt thereof.

Agonizing the GPR30 may comprise contacting the GPR30, or the cell expressing the GPR30, with an agent capable of agonizing the GPR30. The agent may comprise a compound of Formula I or a pharmaceutically acceptable salt thereof.

Inhibiting the biologic activity of one or more of Bcl-2 and Bcl-xL may be carried out substantially at the same time as agonizing the GPR30, and preferably is carried out before agonizing the GPR30. For example, in some aspects, the cancer cell is contacted with an effective amount of the agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL, and after a sufficient period of time for the biologic activity of Bcl-2 and/or Bcl-xL to be inhibited, the cancer cell is contacted with an effective amount of the agent that agonizes the GPR30. A sufficient period of time may comprise seconds, minutes, or hours.

The growth of any cancer cell expressing GPR30 may be inhibited according to the methods. The cancer cell may also be a precancerous cell. The cell may be a cell line. The cancer cell may be resistant to at least one platinum-based chemotherapeutic agent such as cisplatin, or the cancer cell may be susceptible to at least one platinum-based

chemotherapeutic agent such as cisplatin. The cancer cell may be an ovarian cancer cell, a breast cancer cell, an endometrial cancer cell, a prostate cancer cell, an urothelial cancer cell, or a thyroid cancer cell. Ovarian cancer cells are highly preferred.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

Example 1

Cisplatin-Resistant Cells Show Progressive Increases in Protein Expression of the 51 kDa

GPR30 Isoform

GPR30 protein levels were measured by Western blotting in a panel of cisplatin- sensitive A2780 and isogenic cisplatin-resistant CP70, C30, and C200 cells (Fig 1). A2780 ovarian cancer cells were derived from a patient prior to therapy and exhibit sensitivity to cisplatin. A2780 cells were repeatedly exposed to incrementally higher concentrations of cisplatin to derive the resistant stable clones CP70, C30 and C200. GPR30 protein is expressed as two isoforms, one at its predicted molecular weight of 38 kDa, and one at 51 kDa which is believed to be due to heavy glycosylation predicted by sequence analysis. The 51 kDa GPR30 isoform was detected with an N-terminal antibody (LS-A1183; MBL

International) and the 38 kDa GPR30 with an independent antibody directed against the third extracellular loop of GPR30 (LS-A4271; MBL International, Woburn MA). GPR30 detected with either the N-terminal antibody (LS-A1183) antibody or the third extracellular loop antibody (LS-A4271) have been reported to be depleted by GPR30-targeting siRNA or shRNA methodology. The major GPR30 isoform expressed in cisplatin-sensitive A2780 cells was the 38 kDa variant (Fig. 1A). Whereas in cisplatin-resistant cells, the 38 kDa isoform decreased by approximately 50%, expression of the 51 kDa isoform increased progressively with increasing cisplatin resistance (Fig. 1A). GPR30 mRNA expression was determined by qRT-PCR using a primer set that recognized all three GPR30 transcripts and thus measured total GPR30 mRNA levels (Fig. IB). GPR30 mRNA levels, relative to A2780 cells, was decreased to 63% in CP70 cells and increased to 2-fold and 3.8-fold in the C30 and C200 cells, respectively. Hence, the total expression of both GPR30 protein isoforms was consistent with total GPR30 mRNA expression levels.

Example 2

G-1 Blocks Proliferation Of Cisplatin-Sensitive And -Resistant Ovarian Cancer Cells

The growth effects of cisplatin and G-1 were determined in the ovarian cell panel. Cells were exposed to varying concentrations of cisplatin for 3 days (Fig. 2A), and to G-1 for 3 (Fig. 2B) and 6 days (Fig. 2C), followed by assessment of viability using Cell Titer-Glo™ assays (Promega). Comparison of the cisplatin IC₅₀s validates that A2780 cells were sensitive to cisplatin with an IC₅₀ of 0.8 μΜ, while CP70, C30, and C200 cells were 14-fold, 70-fold, and 157-fold resistant, respectively (Fig. 2A). Comparison of the G-1 IC₅₀s following 3 days of treatment showed that A2780, CP70, C30 and C200 cells exhibited the same rank order of G-1 sensitivity as for cisplatin sensitivity (Fig. 2B). Comparison of cells treated for 6 days vs. 3 days with G-1 showed improved inhibition of cell growth with longer treatment time and a decrease in the G-1 IC₅₀s (Fig. 2C). Importantly, after 6 days of G-1, all of the A2780-derived cisplatin-resistant cells exhibited sub-micromolar G-1 IC₅₀s, whereas the cisplatin IC₅₀s for these cells ranged from 11 - 126 μΜ. GPR30 antagonists G-15 and G-36 were also evaluated, but these compounds did not significantly affect growth at concentrations up to 10 μΜ (data not shown).

Example 3

G-1 Induces Apoptosis In Cisplatin-Sensitive And -Resistant Cells

The potential for G-1 to induce apoptosis was examined by treating the ovarian cancer cell line panel with 2.5 μΜ G-1 every 24 h over 5 days. Apoptosis was determined daily by flow cytometric analysis of cells stained for loss of mitochondrial membrane potential (MMP) using the indicator DilCi(5), and for loss of membrane asymmetry by externalization of phosphatidyl serine using Annexin V staining (Fig. 3A-B). Representative examples of the flow cytograms are shown in Fig. 3A, and quantitation of apoptosis in the cell panel across the 5 day time course is shown in Fig. 3B. G-1 led to loss of MMP in all cell lines, implicating mitochondrial dysfunction in the process of G-1 -induced apoptosis. The rank order of G-l-induced apoptosis was: A2780 (50.8%, day 4) > C30 (43.6%, day 5) > CP70 (28.6%, day 5) > C200 (25.0%, day 4)(Fig. 3B).

Example 4

G-1 Induces Expression Of p53 And PUMA (p53 Up-Regulated Modulator Of Apoptosis), And Cleavage Of Caspase-3 (CASP3) And PARP (Poly (ADP-Ribose) Polymerase)

To characterize G-1 induced apoptosis, several molecular markers were examined (Fig. 3C). The A2780 series of cells were treated with 0, 0.5, 1, and 2.5 μΜ G-1 for up to 72 h. G-1 induced expression of the DNA damage sensor p53 in a concentration dependent manner (48 h) in A2780, CP70, C30, and C200 cells. Downstream of p53, PUMA was also induced in a concentration-dependent manner, except in CP70 cells in which p53 has been reported to be expressed in a transcriptionally inactive conformation. G-1 also induced cleavage of the apoptotic markers CASP3 (48 h) and PARP (72 h) in all cells (Fig. 3C).

Example 5

G-1 Evokes Sustained Increases In Cytosolic And Mitochondrial Ca²⁺ Levels In Cisplatin- Sensitive And -Resistant Ovarian Cancer Cells

G-l-induced cytosolic Ca²⁺ mobilization responses were examined at the single cell level using the fluorescent Ca²⁺ indicator Fura-2 and microscopy (Brailoiu E et al. (2009) J. Cell Biol. 186:201-9)(Fig. 4A-B). G-1 produced a robust and sustained increase in [Ca²⁺]_c instead of oscillations. G-1 increased [Ca²⁺]_c by ~200 nM in A2780 and CP70 cells, by ~1 μΜ in C30 cells, and by ~1.4 μΜ in C200 cells (Fig. 4B). Notably, an increase of 200 nM in [Ca ]_c is sufficient to induce apoptosis in other models. Next, cytoplasmic [Ca²⁺]_c and

mitochondrial [Ca²⁺]_m were concomitantly measured using the fluorescent indicators Fluo-4 and Rhod-2, respectively, in real-time by microscopy (Fig. 4C-D). G-l caused elevations in mitochondrial Ca²⁺ levels after rises in cytosolic Ca²⁺ levels. The rank order of G-l-induced increases in mitochondrial Ca²⁺ levels was A2780 > C30 > CP70 > C200 cells (Fig. 4D). In contrast to physiologic oscillatory Ca²⁺ elevations, these rises in mitochondrial Ca²⁺ levels were sustained, and therefore likely to lead to apoptosis. In the A2780 series, the rank order of G-l-induced apoptosis was the same as that for G-l -evoked elevations in mitochondrial Ca²⁺ levels.

Example 6

IP3R1 and RYR2 May Modulate Cytoplasmic Ca²⁺ Responses Evoked By G-l

GPR30 signals Ca²⁺ mobilization through inositol 1,4,5-trisphosphate (ip₃) -gated receptors (IP3RS), and Ca^2+"-gated ryanodine receptors (RyRs). Hence expression of these receptors was determined. Protein levels of the three IP₃Rs (IP3R1, IP3R2, and IP3R3) were measured by Western blotting and RNA levels of the three RyRs (RYR1, RYR2, and RYR3) by qRT-PCR. The relative affinity of IP₃ for IP₃Rs is: IP3R2 > IP3R1 > IP3R3. A2780 cells expressed IP3R2, the highest affinity IP₃R, while CP70, C30 and C200 switched to expressing lower affinity IP3R1 and IP3R3. IP3R1 showed approximately 2.4-fold increased expression in the cisplatin-resistant cells (Fig. 5A). Considering RyRs (Fig. 5B), RYR1 was expressed in A2780 cells (100% reference) and decreased to 29% - 58% in the cisplatin-resistant cells. RYR2 exhibited large increases in expression that associated with increasing cisplatin resistance with a rank order of A2780 (100% reference) < CP70 (7.7-fold) < C30 (139-fold) < C200 (163-fold). RYR3 was not detected in A2780 cells, but was expressed at its highest levels in CP70 cells, and at ~50% of that in C30 and C200 cells. These results suggest that the major Ca²⁺ release channels were IP3R2 and RYR1 in A2780 cells, IP3R1 and RYR3 in CP70 cells, and IP3R1 and RYR2 in C30 and C200 cells.

The rank order of G-l-evoked cytoplasmic Ca²⁺ mobilization was the same as total GPR30 expression, except in CP70 cells, which showed a similar magnitude in cytoplasmic Ca²⁺ mobilization as A2780 cells despite expressing lower GPR30 levels (compare Fig. 4B with Fig. 1A-B). It is believed that this higher than predicted cytoplasmic Ca²⁺ mobilization in CP70 cells was likely due to the increased expression of IP3R1, IP3R3, RYR2, and RYR3 in CP70 relative to A2780 cells (compare Fig. 5A-B with Fig. 4B). Similarly, it is believed that the increased IP3R1, IP3R3, RYR2, and RYR3 in conjunction with increased GPR30 expression in C30 and C200 cells likely explains the very large G-l-evoked cytoplasmic Ca²⁺ mobilization in these cells compared to A2780 cells (compare Fig. 5A-B with Fig. 4B). It is believed that taken together, GPR30-coupled Ca²⁺-release channels likely modulate G-l-evoked cytoplasmic Ca²⁺ responses.

Example 7

Bcl-2 and Bcl-xL Modulate Mitochondrial Ca²⁺ Uptake Evoked By G-l

The rank order of G-l-evoked mitochondrial Ca²⁺ levels changed from that of cytosolic Ca²⁺ increases (Fig. 4D), e.g., C200 cells showed the largest increase in cytosolic Ca²⁺ levels but the lowest increase in mitochondrial Ca²⁺ levels. Meanwhile, the opposite was true for A2780 cells, which exhibited relatively small G-l-induced increases in cytosolic Ca²⁺ but the largest increases in mitochondrial Ca²⁺ levels. B cell lymphoma 2 (Bcl-2) and B cell lymphoma extra large (Bcl-xL) suppress mitochondrial Ca²⁺ uptake, therefore their expression was measured. Bcl-2 and Bcl-xL were expressed at low levels in A2780 cells (100% reference), and at 2.8 to 4.8 -fold in CP70, C30, and C200 cells (Fig. 5B). It is believed that low Bcl-2/Bcl-xL expression in A2780 cells facilitated mitochondrial Ca²⁺ uptake, whereas high Bcl-2/ Bcl-xL levels suppressed mitochondrial Ca²⁺ uptake in CP70, C30 and C200 cells. It is believed that additional factors may also contribute to the modulation of mitochondrial Ca²⁺ uptake.

Example 8

G-l induced Ca²⁺ mobilization and apoptosis is dependent on GPR30 in A2780 cells

To demonstrate that G-l effects were mediated by GPR30, A2780 cells were stably- transfected with a GPR30-targeting short hairpin RNA (shRNA) expression plasmid previously reported to knock-down GPR30 expression, and as a control, stably-transfected with an enhanced green fluorescent protein (eGFP)-targeting shRNA expression plasmid. Cells were selected for stable shRNA expression using puromycin, and clones were generated by limiting serial dilution. Clones were screened for GPR30 knockdown at the RNA level by real-time PCR.

A representative GPR30 shRNA-transfected (GPR30 sh) clone showed 85% knockdown of the target mRNA relative to a control eGFP shRNA-transfected (eGFP sh) clone (Fig. 6A). G-l induced cytoplasmic Ca²⁺ mobilization was examined (Fig. 6B) using the indicator Fura-2 and microscopy as in Figs. 4A-B. In control eGFP shRNA cells, 1 μΜ and 2.5 μΜ G-1 stimulated a 187 and 221 nM rise in [Ca²⁺]_c, respectively. In contrast, in GPR30 shRNA cells, 1 μΜ caused a non-significant rise of 26 nM in [Ca²⁺]_c (P-value = 0.65) and 2.5 μΜ G-1 only a marginally significant rise of 52 nM in [Ca²⁺]_c (P-value = 0.038) (Fig. 6B).

G-l-induced apoptosis was evaluated (Fig. 6C) by flow cytometric analysis of cells stained with DilCi(5) and Annexin V as in Figs. 3A-B. G-1 at 1 μΜ induced apoptosis in 42% of eGFP shRNA cells but in only 10.9% of GPR30 shRNA cells. Similarly, 2.5 μΜ G-1 induced a very high 75% rate of apoptosis in eGFP shRNA cells but a dramatically lower rate of only 27% of GPR30 shRNA cells (Fig. 6C). Taken together, depletion of GPR30 in A2780 cells prevented Ca²⁺ mobilization and apoptosis induced by G-1.

Example 9

Navitoclax Sensitizes CP70, C30, And C200 Cells To G-l-lnduced Growth Inhibition

Since Bcl-2 and Bcl-xL suppress mitochondrial Ca²⁺ uptake it was hypothesized that blocking their activity using the Bcl-2 family inhibitor Navitoclax would facilitate greater levels of mitochondrial Ca²⁺ accumulation in response to G-1 and thereby potentiate apoptosis. The effects of the Bcl2-family inhibitor Navitoclax on G-l-mediated growth inhibition were tested by cell viability assays (Fig. 7).

G-1 and Navitoclax were combined in a 10 x 6 concentration matrix, respectively, including no G-1 or no Navitoclax controls. Cells were treated daily for 48 h (A2780 and CP70) or 72 h (C30 and C200). Navitoclax, at concentrations which as a single agent did not significantly decrease viability but when combined with G-1, potentiated growth inhibitory effects of G-1 by ~2-fold in CP70, C30, and C300 cells but not in A2780 cells. For instance, G- 1 alone vs. combined with 4 μΜ Navitoclax resulted in a decrease of minimum viability from 30% to 11% in CP70, from 38% to 19% in C30 cells, and from 55% to 26% in C200 cells, all with <10% decrease in viability by 4 μΜ Navitoclax alone (Fig 7 and Table 1). Statistical analysis was conducted of the shift in growth inhibition between no (0 μΜ) and increasing Navitoclax concentrations (1 - 16 μΜ), all in the presence of increasing G-1, by comparing the top and bottom of the logistic regression curves (Table 1). This analysis showed that G- 1-inhibited growth was potentiated by Navitoclax at concentrations up to 4 μΜ in CP70, C30 and C200 cells (P-values = 2 x 10^"8, 0.0004, and 0.00001, respectively). These data indicate Navitoclax and G-1 interacted to block growth and therefore that G-1 and Navitoclax likely acted on the same pathway to induce apoptosis; G-1 evokes Ca²⁺ mobilization, while inhibiting Bcl-2/Bcl-xL facilitates greater mitochondrial Ca ⁺ uptake in response to G-1, thereby potentiating mitochondrial dysfunction and decreased viability.

Table 1 shows the mean maximum (top of curve) and minimum (bottom of curve) cell viability for each Navitoclax concentration in the presence increasing G-1 concentrations as determined by the logistic curve. The starred (*) concentrations of Navitoclax indicate which concentrations significantly sensitize cells to G-l-mediated growth inhibition with <10% toxicity due to Navitoclax alone.

Table 1. Effects of Navitoclax on G-l-mediated growth inhibition (as shown in Fig. 7).

CP70 cells

% Viability (in presence of increasing G-1)

Navitoclax Top (± SE) Bottom (+ SE) Bottom - Top Shift in Bottom P-value

(μΜ) - Top

0 105 (± 1.4) 30 (± 1.3) -75 n.a. n.a.

1* 104 (± 1.3) 16 (± 1.5) -88 -13 1 x 10^"6

2* 101 (± 1.4) 13 (± 1.5) -88 -13 1 x 10^"6

4* 98 (± 0.7) 11 (± 0.7) -87 -12 2 x 10^'s

8 81 (± 1.0) 11 (± 1.1) -70 6 0.99

16 16 (± 0.3) 6 (± 0.3) -9 66 1

C30 cells

% Viability (in presence of increasing G-1)

Navitoclax Top (± SE) Bottom (± SE) Bottom - Top Shift in Bottom P-value

(μΜ) - Top

0 114 (± 2.1) 38 (± 1.0) -76 n.a. n.a.

1* 114 (± 2.5) 32 (± 1.4) -83 -7 0.031

2 110 (± 2.8) 29 (± 1.5) -81 -5 0.082

4* 105 (± 2.1) 19 (± 1.0) -87 -11 0.0004

8 86 (± 1.6) 13( ± 1.0) -74 2 0.76

16 10 (± 0.5) 2 (± 0.3) -7 68 1

Example 10

Summary

The majority of ovarian cancer patients are treated with platinum-based

chemotherapies but eventually relapse with incurable disease. G-1, a synthetic and specific agonist of the G protein-coupled estrogen receptor GPR30 (also GPER) was found to be a potential therapeutic agent in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. GPR30 stimulates Ca²⁺ mobilization responses, and Ca²⁺ signaling coordinates multiple cellular activities crucial in survival and proliferation, including production of ATP in mitochondria. However, high and sustained Ca²⁺ elevations can activate the Ca²⁺-mediated mitochondrial-dependent apoptosis pathway. It was found that (i) GPR30 was expressed in a panel of cisplatin-sensitive (A2780) and -resistant ovarian cancer cell lines (CP70, C30, C200), and that expression of the GPR30 51 kDa isoform progressively increased with increasing cisplatin resistance, (ii) G-1 inhibited growth and induced apoptosis by loss of mitochondrial membrane potential (MMP), (iii) G-1 elicited large and sustained increases in cytoplasmic Ca²⁺ concentrations preceding sustained rises in mitochondrial Ca²⁺ levels in these cells, and (iv) GPR30 depletion by shRNA methodology prevented G-l-induced apoptosis in A2780 cells. It was further demonstrated that inhibiting the Bcl-2 family with Navitoclax (ABT-263) sensitized CP70, C30 and C200 cells to apoptotic effects of G-1 by doubling G-l's growth inhibitory effects at concentrations of Navitoclax that when used alone did not significantly decrease viability. It is believed that sensitization by Natitoclax was likely due to blocking Bcl-2 and Bcl-xL activity, resulting in enhanced mitochondrial Ca⁺² uptake.

The invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

Claims

We claim:

1. A method for increasing the level of calcium in the cytoplasm of a cancer cell

expressing the G Protein-Coupled Estrogen Receptor GPR30, comprising agonizing the GPR30 of a cancer cell expressing the GPR30.

2. The method of claim 1, wherein the increased level of calcium is sustained over time.

3. The method of claim 1, wherein agonizing the GPR30 comprises contacting the cell with an agent capable of agonizing the GPR30 in an amount effective to increase the level of calcium in the cytoplasm of the cancer cell.

4. The method of claim 3, wherein the agent is a compound of Formula I:

; or a pharmaceutically acceptable salt thereof.

5. The method of claim 4, wherein the agent is present in a composition comprising a nonpolar carrier.

6. The method of claim 3, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 4 μΜ or less.

7. The method of claim 3, wherein the agent is capable of inhibiting viability of the cell at an IC₅o of about 2.5 μΜ or less.

8. The method of claim 3, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 1 μΜ or less.

9. The method of claim 3, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 0.85 μΜ or less.

10. The method of claim 1, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

11. The method of claim 10, wherein the platinum-based chemotherapeutic agent is cisplatin.

12. The method of claim 1, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

13. The method of claim 12, wherein the platinum-based chemotherapeutic agent is cisplatin.

14. The method of claim 1, wherein the level of calcium is increased to a concentration sufficient to substantially inhibit proliferation of the cancer cell.

15. The method of claim 1, wherein the level of calcium is increased to a concentration sufficient to induce apoptosis of the cancer cell.

16. The method of claim 1, wherein the level of calcium is increased to a concentration sufficient to induce p53 expression in the cancer cell.

17. The method of claim 1, wherein the level of calcium is increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 or Poly (ADP-ribose) polymerase in the cancer cell.

18. The method of claim 1, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

19. The method of claim 1, wherein the method is carried out in vitro.

20. A method for increasing the level of calcium in the mitochondria of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30, comprising agonizing the GPR30 of a cancer cell expressing the GPR30.

21. The method of claim 20, wherein the increased level of calcium is sustained over time.

22. The method of claim 20, wherein agonizing the GPR30 comprises contacting the cell with an agent capable of agonizing the GPR30 in an amount effective to increase the level of calcium in the mitochondria of the cancer cell.

23. The method of claim 22, wherein the agent is a compound of Formula I:

; or a pharmaceutically acceptable salt thereof.

24. The method of claim 23, wherein the agent is present in a composition comprising a nonpolar carrier.

25. The method of claim 22, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 4 μΜ or less.

26. The method of claim 22, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 2.5 μΜ or less.

27. The method of claim 22, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 1 μΜ or less.

28. The method of claim 22, wherein the agent is capable of inhibiting viability of the cell at an IC₅₀ of about 0.85 μΜ or less.

29. The method of claim 20, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

30. The method of claim 29, wherein the platinum-based chemotherapeutic agent is cisplatin.

31. The method of claim 20, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

32. The method of claim 31, wherein the platinum-based chemotherapeutic agent is cisplatin.

33. The method of claim 20, wherein the level of calcium is increased to a concentration sufficient to substantially inhibit proliferation of the cancer cell.

34. The method of claim 20, wherein the level of calcium is increased to a concentration sufficient to induce apoptosis of the cancer cell.

35. The method of claim 20, wherein the level of calcium is increased to a concentration sufficient to induce p53 expression in the cancer cell.

36. The method of claim 20, wherein the level of calcium is increased to a concentration sufficient to induce cleavage of one or more of Caspase-3 or Poly (ADP-ribose) polymerase in the cancer cell.

37. The method of claim 20, wherein the level of calcium is increased to a concentration sufficient to compromise the integrity of the membrane of the mitochondria.

38. The method of claim 20, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

39. The method of claim 20, wherein the method is carried out in vitro.

40. A method for inhibiting the proliferation of a cancer cell expressing the G Protein- Coupled Estrogen Receptor GPR30, comprising contacting the cell with an amount of a compound of Formula I:

; or a pharmaceutically acceptable salt thereof, effective to inhibit proliferation of the cell.

41. The method of claim 40, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

42. The method of claim 41, wherein the platinum-based chemotherapeutic agent is cisplatin.

43. The method of claim 40, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

44. The method of claim 43, wherein the platinum-based chemotherapeutic agent is cisplatin.

45. The method of claim 40, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

46. The method of claim 40, wherein the compound is present in a composition comprising a nonpolar carrier.

47. The method of claim 40, wherein the method is carried out in vitro.

48. A method for inducing apoptosis in a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30, comprising contacting the cell with an amount of a compound of Formula I:

; or a pharmaceutically acceptable salt thereof, effective to induce apoptosis in the cell.

49. The method of claim 48, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

50. The method of claim 49, wherein the platinum-based chemotherapeutic agent is cisplatin.

51. The method of claim 48, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

52. The method of claim 51, wherein the platinum-based chemotherapeutic agent is cisplatin.

53. The method of claim 48, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

54. The method of claim 48, wherein the compound is present in a composition

comprising a nonpolar carrier.

55. The method of claim 48, wherein the method is carried out in vitro.

56. A composition comprising a compound of Formula I:

; or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, nonpolar carrier.

57. The composition of claim 56, wherein the carrier comprises dimethyl sulfoxide.

58. The composition of claim 56, wherein the carrier is a micelle.

59. A method for enhancing the expression of the IP₃ type 1 receptor in a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30, comprising contacting the cell with an amount of a compound of Formula I:

; or a pharmaceutically acceptable salt thereof, effective to enhance the expression of the IP₃ type 1 receptor in the cell.

60. The method of claim 59, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

61. The method of claim 60, wherein the platinum-based chemotherapeutic agent is cisplatin.

62. The method of claim 59, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

63. The method of claim 62, wherein the platinum-based chemotherapeutic agent is cisplatin.

64. The method of claim 59, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

65. The method of claim 59, wherein the compound is present in a composition

comprising a nonpolar carrier.

66. The method of claim 59, wherein the method is carried out in vitro.

67. A method for inhibiting the growth of a cancer cell expressing the G Protein-Coupled Estrogen Receptor GPR30, comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) or B cell lymphoma extra large (Bcl-xL) in the cancer cell, and agonizing the GPR30 in the cancer cell.

68. The method of claim 67, wherein inhibiting the biologic activity of Bcl-2 comprises contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-2 in an amount effective to inhibit the biologic activity of Bcl-2.

69. The method of claim 67, wherein inhibiting the biologic activity of Bcl-xL comprises contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-xL in an amount effective to inhibit the biologic activity of Bcl-xL.

70. The method of claim 67, wherein the method comprises inhibiting the biologic

activity of Bcl-2 and Bcl-xL in the cancer cell.

71. The method of claim 70, wherein inhibiting the biologic activity of Bcl-2 and Bcl-xL comprises contacting the cell with an agent capable of inhibiting the biologic activity of Bcl-2 and Bcl-xL in an amount effective to inhibit the biologic activity of Bcl-2 and Bcl-XL.

72. The method of any of claims 68, 69, or 71, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.

73. The method of claim 67, wherein agonizing the GPR30 comprises contacting the cell with an agent capable of agonizing the GPR30.

74. The method of claim 73, wherein the agent capable of agaonizing the GPR30 is a compound of Formula I:

; or a pharmaceutically acceptable salt thereof.

75. The method of claim 67, wherein the cancer cell is resistant to at least one platinum- based chemotherapeutic agent.

76. The method of claim 75, wherein the platinum-based chemotherapeutic agent is cisplatin.

77. The method of claim 67, wherein the cancer cell is sensitive to at least one platinum- based chemotherapeutic agent.

78. The method of claim 77, wherein the platinum-based chemotherapeutic agent is cisplatin.

79. The method of claim 67, wherein the cancer cell is an ovarian cancer cell, a breast cancer cell, or an endometrial cancer cell.

80. The method of claim 67, wherein the method is carried out in vitro.

81. The method of claim 1, further comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) and/or B cell lymphoma extra large (Bcl-xL) in the cell by contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL.

82. The method of claim 81, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.

83. The method of claim 20, further comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) and/or B cell lymphoma extra large (Bcl-xL) in the cell by contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL.

84. The method of claim 83, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.

85. The method of claim 40, further comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) and/or B cell lymphoma extra large (Bcl-xL) in the cell by contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL.

86. The method of claim 85, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.

87. The method of claim 48, further comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) and/or B cell lymphoma extra large (Bcl-xL) in the cell by contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL.

88. The method of claim 87, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.

89. The method of claim 59, further comprising inhibiting the biologic activity of B cell lymphoma 2 (Bcl-2) and/or B cell lymphoma extra large (Bcl-xL) in the cell by contacting the cell with an effective amount of an agent that inhibits the biologic activity of Bcl-2 and/or Bcl-xL.

90. The method of claim 89, wherein the agent is a compound of Formula II:

; or a pharmaceutically acceptable salt thereof.