WO2003033016A1

WO2003033016A1 - Use of neurotrophic factors for treating neurodegenerative diseases and cancer

Info

Publication number: WO2003033016A1
Application number: PCT/CA2002/001536
Authority: WO
Inventors: Jenny Phipps; Jennifer Arnold; Brian Van Adel; Laurie Doering
Original assignee: Jenny Phipps; Jennifer Arnold; Brian Van Adel; Laurie Doering
Priority date: 2001-10-16
Filing date: 2002-10-16
Publication date: 2003-04-24

Abstract

A method of increasing connexin 43(Cx43) protein expression in a cell, as well as increasing intercellular gap junction communication is described. The method comprises comprising providing to the cell, a neurotrophic factor e.g. ciliary neurotrophic factor (CNTF) or a neuroprotective fragment thereof, alone, or in combination with a PKA activator. This method can be used to enhance intercellular communication, providing a novel therapeutic approach to treating neurodegenerative diseases and cancer, and enhancing the neuroprotective effects associated with CNTF and Cx43.

Description

EUROTROPHIC FACTORS FOR TREATING NEURODEGENERATIVE DISEASES AND CANCER

Field of the Invention

[0001] This invention relates to a method for increasing connexin 43(Cx43) protein expression in a cell, and intercellular gap junction communication.

Background of the Invention

[0002] It has been established that in many chronic diseases, such as cancer, cardiac conditions, and central nervous system diseases, gap junction mediated intercellular communication is dysfunctional (see for example

Trosko J.E. et al. (2000), Biofactors, 12(1-4), 259-263; Cotrina M.L. et al. (1998), Journal of Neuroscience, 18(7), 2520-37; Johnson E.C. et al. (2000), Investigative Ophthalmology & Visual Sciences, 41(2), 349-51; Nagy J.I. et al. (1996), Brain Research, 717(1-2), 173-178; and Bani-Yaghoub M.,et al. (1999), Neuroreport, 10(18), 3843-3846).

[0003] A gap junction is a membrane structure detectable at points of close proximity between adjacent cells. Gap junctions serve as passageway between the interiors of contiguous cells, and mediate the intercellular passage of small molecules from the cytoplasm of one cell to that of the adjacent cell.

[0004] Gap junctions are composed of clusters of membrane proteins collectively termed connexins which form structures called connexons. The proteins are peripherally disposed to form a central channel. Gap junction transmembrane passages are formed when a connexon of one cell aligns with a connexon of an adjacent cell. In this way, transmembrane intercellular pathways are formed that permit the passage of molecules between coupled cells.

[0005] The diameter of the connexon channels is about 1.5 to 2 ran. This diameter allows only substances of less than approximately 1000 daltons, such as ions, sugars, nucleic acids, amino acids, fatty acids, and small peptides, to pass but not large molecules, such as proteins, complex lipids, polysaccharides and polynucleotides.

[0006] The protein subunits of connexons may vary from cell to cell.

The connexins (Cx), form a multi-gene family whose members are distinguished according to their predicted molecular mass in kDa (e.g. Cx32, Cx43). Connexins are expressed in a cell-, tissue-, and developmentally- specific manner. See Beyer et al., /. Membr. Biol, 116:187-194 (1990); Dermietzel, R. et al., Anat. Embryol, 182:517-258 (1990); Warner, A., Seminars in Cell Biology, 3:81-91 (1992); Kumar, N. M. et al., Seminars in Cell Biology, 3:3- 16 (1992). For instance, Cx43(having a molecular mass of 43 kDa) is the predominant connexin expressed in cardiac muscle and in liver epithelial cells. In adult liver parenchymal cells, the predominant connexins are Cx32 and Cx26; non-parenchymal liver cells express other connexins. Each connexin forms channels with different conductance, regulatory, and permeability properties. In those tissues where more than one connexin is expressed, gap junctions may contain more than one connexin. However, it is not known whether individual connexons may be comprised of more than one connexin type.

[0007] Gap junctions do not necessarily remain open. Elevation of the intracellular level of calcium ions , among other factors, leads to a graded closure of gap junctions. In healthy, normal cells, these channels are fully open when the calcium ion level is less than 10^"7 M and are shut when the level of this ion is higher than 5xl0^'5 M. As the concentration of calcium ion increases in this range, the effective diameter of gap junctions decreases so that they become impermeable first to larger molecules. When the normally very low (lO^M) intracellular calcium levels rise, the gap junction proteins undergo conformational changes that close the gap. [0008] The ability of adjacent cells to form gap junctions that link them may depend on a number of factors including the ability of cells to interact with their neighbours (reportedly requiring the presence of compatible cell adhesion molecules), the level of connexin expressed, whether the connexon proteins formed in one cell are capable of linking with a connexon of a second cell to form a functional channel, and whether a molecule, such as a carcinogen, is present, that interferes with the normal function of any cellular protein that mediates cell to cell gap junctional communication. For instance, in certain diseases, the gap junction gating is dysfunctional resulting in numerous problems.

[0009] Many solid cancer tumours, for example, have deficient gap junctions, a condition that prevents the communication between the cancer cells and healthy surrounding cells leading to an alteration in the intracellular levels of calcium, and other small molecules. This leads to an upset in normal tissue homeostasis and favours undesirable cell proliferation.

[0010] By controlling gap junctions, communication between cells can be established. As a consequence, a diseased tissue or organ may regain normal function and regain growth homeostasis. For instance, in the case of tumours, re-establishment of intercellular communication and /or increase in cell coupling restores the normal phenotype of malignant cells and leads to growth arrest. In the case of stroke, gap junction communication may be maximized to prevent the secondary expansion of ischemic lesions from a stroke core. In the case of neurodegenerative diseases, restoration of gap junction communication may minimize neuronal cell loss.

[0011] There are several available compounds that are capable of uncoupling gap junctions, such as 18-alpha-glycyrrhetinic acid and carbenoxolone, but little is known about compounds which are reliable at increasing both connexin expression and functional coupling. [0012] It is, thus desirable to provide a method for enhancing gap junction communication in order to treat chronic diseases or increase "bystander effects". Examples of such diseases are cancer, such as neuroblastoma and other malignant solid tumours, cardiac conditions, such as arrhythmia, and central nervous system diseases.

[0013] Protein kinases are a group of enzymes which modulate the activities of a variety of proteins in different cells by phosphorylating them. Protein kinase A (PKA) is a cAMP-dependent kinase that initiates the transfer of an ATP phosphate group onto either a serine or threonine group on the target protein. PICA activity regulates many cellular processes including cell growth, cell differentiation, ion-channel conductivity, gene transcription, synaptic release of neurotransmitters, and memory. PKA activators, such as cAMP analogues can increase gap junctions (Holder, J. W. et al. (1993), Cancer Research 53, 3475-3485). cAMP analogues, such as 8-bromo cAMP and forskolin are known in the art.

[0014] Ciliary neurotrophic factor(CNTF) is best characterized for its neuroprotective ability on neurons (Sendtner, M., Schmalbruch, H., Stockli, K.A., Carroll, P., Kreutzberg, G.W., & Thoenen, H. (1992), Nature, 358, 502- 504). The exact mechanism of this neuroprotective effect has not been elucidated. Other neurotrophic factors, such as brain derived neurotrophic factor(BDNF)

Summary of the Invention

[0015] The Applicant has discovered that neurotrophic factors e.g.

CNTF work in one aspect by increasing Cx43 expression in cells, and further that the CNTF, alone, or in combination with PKA activators work to increase functional cell to cell coupling and neuroprotection. The combination of CNTF, or a neuroprotective fragment thereof, with a PKA activator, has a synergistic effect in increasing Cx43 expression and increasing functional cell to cell coupling. [0016] Thus, in one aspect, the invention provides a method of increasing Cx43 protein expression in a cell, as well as increasing intercellular gap junction communication, comprising providing to a cell a neurotrophic factor e.g. CNTF or a neuroprotective fragment thereof, alone, or in combination with a PKA activator, such as a cAMP analogue or forskolin.

[0017] This method can be used to enhance cell to cell communication, providing a novel therapeutic approach to treating cancer in mammals and enhancing the neuroprotective effects associated with CNTF and Cx43. This provides a new target for treatment of neurodegeneration by manipulating Cx43 expression via CNTF.

[0018] It will be appreciated by those skilled in the art that any long- term mammalian neurodegenerative disease could be potentially treated by such a method. Examples of such diseases include Alzheimer's disease, Parkinson disease, Amyotrophic lateral sclerosis (also called ALS or Lou Gehrig's disease), glaucoma, and stroke. Epilepsy is also contemplated as a disease which could be treated using the method of the invention.

[0019] Other features and advantages of the invention will become apparent from the following description and from the claims.

Brief Description of the Drawings [0020] The invention will now be explained by way of example only and with reference to the attached drawing in which:

Figure 1 shows the expression of CNTF (A) and CNTFR-alpha (B) in C6 cells,

Figure 2 shows the expression of Cx43 in C6 cells, Figure 3 shows the effect of exogeneous rrCNTF on CNTFR-alpha (A) and Cx43 (B) expression,

Figure 4 shows the effect of glutamate, in the absence (A) and presence (B) of exogeneous rrCNTF, on C6 cells, and Figure 5 shows the effect of glutamate, in the absence (A) and presence (B) of rrCNTF and 8-bromo-cAMP in neuroblastoma cells.

Detailed Description of the Preferred Embodiments

[0021] It has been shown in a related application (U.S. 60/298,102, incorporated by reference herein), that 8-bromo-cAMP can enhance the transcription of intracellular Cx43 and that it can, in certain cell systems, induce the transfer of Cx43 from around the nucleus to the cell membrane.

[0022] CNTF or a neuroprotective fragment thereof can be introduced into cells or an animal using techniques known in the art, including in pure form, in recombinant form, as a transfected viral adenovirus, or by means of cells modified to overexpress the CNTF protein or a neuroprotective fragment thereof.

Examples

[0023] Materials: Four rat glioma cell lines were used. The first is the C6 parental (C6-P) cell line that has not been manipulated or transfected in any way. The second is the same C6 cell line that has undergone lipofectin- mediated transfection of cDNA to induce the over-expression of connexin 43 (C6-Cx43). The two remaining cell lines are C6 cells which have been transfected with non-secretory (C6-NS) and secretory (C6-S) CNTF expression constructs; the 600-bp full length rat CNTF cDNA was introduced under the control of a GFAP promoter which leads to the over expression of CNTF that is sequestered in the cytosol (C6-NS) or is secreted in large quantities (C6-S). The natural location of CNTF is in the cytosol. CNTF is normally secreted by cells only under conditions requiring neuroprotection (Inoue, M., Nakayama, C, & Noguchi, H. (1996), Molecular Neurobiology, 12(3), 195-209).

[0024] Effect of CNTF expression on levels of Cx43: C6 cell lines (C6-P, C6-

Cx43, C6-NS and C6-S) were examined for constitutive expression of several proteins, including Cx43, CNTF, and CNTFR-alpha, one subunit of the three- subunit receptor for CNTF, using densitometric analysis of Western blots. 30 micrograms of protein obtained from cell extracts (2xl0⁶) was separated by 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were probed with goat anti-CNTF (1:200, R&D Systems), goat-anti CNTFR- alpha (1:200, R&D Systems), or rabbit anti-Cx43 (1:1000, Zymed), and then alkaline phosphatase conjugated donkey anti-goat IgG or goat anti-rabbit IgG (1:4000, Jackson Laboratories), followed by colourimetric detection by BCIP/NBT (Sigma). Densitometry values were obtained using a GelPro Analyzer.

[0025] Referring to Figures 1 and 2, it is shown that the over-expression of CNTF increases the expression of Cx43 in glioma cells. In C6-P, CNTF was present at very low levels, as was its high-affinity receptor, CNTFR-alpha. C6-Cx43 over-expressing cells had a significantly higher level of CNTF in comparison to the C6-P cell line. This is an important point because the only difference between these two cell lines is the transfection by a vector containing Cx43. The level of CNTFR-alpha was equivalent to the C6-P cells in the C6-Cx43 cells. C6-NS cells had a very high level of CNTF expression with a slighter higher level of CNTFR-alpha in comparison to the C6-P and C6-Cx43 cells. Cx43 was roughly equivalent to the level of C6-P cells. In the C6-S cells, CNTF was expressed at high levels but was lower than the C6-NS cell line. However, C6-S had the highest level of CNTFR-alpha expression which was expected given that transcription of the receptor is ligand driven. Cx43 was expressed at high levels, significantly higher than both the C6-P and the C6-NS cell lines. In comparison to the C6-P cells, C6-S cells show a significantly elevated level of Cx43 protein expression.

[0026] Effect of exogenously applied CNTF on levels ofCx43: Densitometric analysis of Western blots probed for ciliary neurotrophic factor receptor (CNTFR-alpha) and connexin 43 (Cx43) was done by treating cells (lxlO⁶) with 50 micrograms/ml of rat recombinant CNTF (rrCNTF) for 24 or 48hrs prior to cell extraction. 30 micrograms of protein was separated by 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were probed with goat anti-CNTFR-alpha (1:200, R&D Systems) or rabbit anti-Cx43 (1:1000, Zymed), then alkaline phosphatase conjugated donkey anti-goat or donkey anti-rabbit IgG, respectively (1:4000, Jackson Laboratories), followed by colourimetric detection by BCIP/NBT (Sigma). Densitometry values were obtained by the GelPro Analyzer. Control cells were grown in equivalent number and medium but received no rrCNTF. Figure 3A shows the expression of CNTFR-alpha in the C6 parental and C6 Cx43 over-expressing cell lines following 24 and 48hrs of exposure to rrCNTF. Figure 3B shows the expression of Cx43 in the C6 parental and C6 Cx43 over-expressing cell lines following 24 and 48hrs of exposure to rrCNTF. Application of exogenous rrCNTF for 24hrs and 48hrs resulted in significant changes in both C6-P and C6-Cx43 cell lines, as shown in Figure 3. At the 24 hr time point there was an increase in CNTFR-alpha expression in the C6-Cx43 cells and an increase by the 48 hr time point in the C6-P cells. Increased Cx43 protein expression was seen in the C6-P cells at the 24 hr time point. There were no detectable differences between control and rrCNTF treated cells in the level of CNTF expression.

[0027] Effect of exogenously applied CNTF on functional coupling of gap junctions: A standard dye-coupling technique was used to determine that this increase in Cx43 protein expression is functional. All four C6 cell lines were grown on glass coverslips to near confluence and then examined for Lucifer Yellow dye transfer by scrape-loading. The amount of dye flow into neighbouring cells provides an estimate of the level of functional gap junction coupling, as described in Dowling-Warriner, C.V. & Trosko, J.E. (2000), Induction of gap junctional communication, connexin 43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway, Neuroscience, 95(3), 859-868. Untreated C6 cells (1.5xl0⁵) were grown on glass coverslips for 48hrs. Those cells being treated with rrCNTF were then subjected to 50 microgram/ml of rrCNTF for 24hrs; whereas the other cells remained untreated. All cells were then rinsed twice in PBS, scratched with a 26 gauge needle, incubated for 3 minutes in 2.5% Lucifer Yellow in PBS, rinsed four times in PBS then examined for levels of dye-coupling.

[0028] Control levels of Lucifer Yellow dye flow varied across the four cell lines. C6-P cells had minimal to no detectable dye coupling. The C6-NS cells had a slightly higher level of dye coupling than the C6-P and the C6-S cells had a significantly higher level of dye-coupling in comparison to both the C6-P and the C6-NS cell lines. The C6-Cx43 cells had the highest control level of dye-coupling.

[0029] Application of rrCNTF for 24hrs resulted in an increase in dye coupling in both the C6-P and C6-Cx43 cells. Both cell lines exhibited an increase by 24hrs. The treated C6-P cells, previously deficient in gap junction intercellular communication (GJIC), almost reached the same level of dye coupling seen in the untreated C6-Cx43 cells. In comparison, the dye- coupling in the treated C6-Cx43 cells was very extensive and far-reaching.

[0030] Use of CNTF and increased Cx43 protein expression as neuroprotective: Subjecting cells to glutamate acts as a stressor on the cells. Moderate levels of glutamate, such as 0.1M or 0.25M cause the cells to undergo apoptosis. Very high levels of glutamate, on the other hand, favour necrosis. Neurodegeneration is a form of uncontrolled apoptosis. Thus, subjecting cells to glutamate, simulates neurodegeneration. Increased Cx43, that results in functional coupling, is able to buffer the effects of glutamate- induced excitotoxicity and provide some degree of neuroprotection.

[0031] Figure 4A shows the effect of glutamate on C6 cells (4xl0³) were seeded in 96 well plates and left overnight. The next day cells were treated with 0.05M, 0.1M, or 0.25M L-glutamic acid for 48hrs. Control cells received a medium change and no treatment. Following the 48hr treatment, a Hoechst assay was performed to calculate cell survival. Spectrofluorometric data was calculated as a percentage of cell survival relative to the control cells. The effect of CNTF on glutamate toxicity in C6 cells is shown in Figure 4B, and was examined by treating C6 cells after seeding and leaving overnight, as above, with lOOmicrograms/ml rrCNTF and left for 24hrs. Cells were then treated with 0.1M L-glutamic acid for 48hrs as above, followed by a Hoechst assay to calculate cell survival. Control cells received an equivalent number of medium changes and no treatment. Thus, the 0.1M GLUT cells received no CNTF pre-treatment, and the CNTF group received a 24 hr CNTF pre- treatment followed by 48hrs of exposure to 0.1M L-glutamic acid.

[0032] C6-S cells and C6-Cx43 cells were able to withstand the effects of glutamate toxicity and this effect was enhanced by the addition of exogenous rrCNTF. The survival capacity of the C6-NS cells was significantly improved by the addition of rrCNTF. It is important to note that the three resistant C6 cell lines all express a significantly higher level of CNTF and Cx43 than the C6-P cells which were unable to survive the glutamate toxicity even with the addition of exogenous CNTF.

[0033] The glutamate toxicity model was first tested on the four C6 cell lines without adding any exogenous rrCNTF. Following 48hrs of exposure to glutamate, the C6-S cells had the highest level of cell survival (54%) followed by the C6-Cx43 cells (50%), then the C6-NS cells (45%) and the C6-P cells had the lowest level of cell survival at 26%.

[0034] The addition of rrCNTF had a dramatic impact on cell survival. The C6-S cells improved from 54% to 94%, the C6-Cx43 cells from 50% to 81%, the C6-NS cells from 45% to 89%, and the C6-P cells showed little improvement in cell survival going from 26% to 39%.

[0035] The neuroprotective effects of CNTF on a neural model was tested. Two neuroblastoma cell lines were chosen (IMR32 and SH-SY5Y) because the tripartite receptor for CNTF is present (Kuroda, H., Sugimoto, T., Horii, Y., Sawada, T. (2001), Medical and Pediatric Oncology, 36, 118-121) and it has been established that there is a deficit in GJIC in these cell lines (Carystinos G.D., Alaoui-Jamali M.A., Phipps J., Yen L., Batist G. (2001), Cancer Chemotherapy & Pharmacology, 47(2), 126-32).

[0036] Shown in Figure 5 is the effect of glutamate toxicity on neuroblastoma cells. Neuroblastoma cells (4xl0³) were seeded in 96 well plates and the same procedure as described above for glioma cells was used. The results are shown in Figure 5 A. The effect of CNTF and 8-Bromo-cAMP on glutamate toxicity in neuroblastoma cells was also examined and the results are given in Figure 5B. 8-Bromo-cAMP causes an increase in Cx43 expression and a re-localization of Cx43 to the cell membrane as described in US 60/298,102, incorporated by reference herein. Neuroblastoma cells (lxlO³) were seeded in 96 well plates and left overnight. The next day cells were treated with ImM 8-Bromo-cAMP for 48hrs, after which time cells were washed and treated with 100 micrograms/ml rrCNTF for 24hrs. Cells were then treated with 0.1M L-glutamic acid for 48hrs followed by a Hoechst assay to calculate cell survival. Spectrofluorometric data was calculated as a percentage of cell survival relative to the control cells. Control cells received an equivalent number of medium changes and no treatment, 0.1M GLUT cells received no 8-Bromo-cAMP or rrCNTF pre-treatments, the CNTF group received a 24 hr rrCNTF pre-treatment followed by 48hrs of exposure to 0.1M L-glutamic acid, and the cAMP/CNTF group received both the 8-Bromo- cAMP and rrCNTF treatments prior to exposure to 0.1M L-glutamic acid.

[0037] The neuroprotective effect of CNTF on neuroblastoma cells was significantly improved by the increased expression and membrane localization of Cx43. The glutamate toxicity model was first tested on the two neuroblastoma cell lines without adding any exogenous rrCNTF. Following 48hrs of exposure to glutamate, IMR32 cells had a 27% survival rate and the SH-SY5Y cells were significantly higher at 41%. The addition of rrCNTF had a dramatic impact on cell survival. IMR32 cells improved from 27% survival to 48% survival and the SH-SH5Y cells from 41% to 68%. These values reflect the neuroprotective effects of CNTF alone without the benefit of functional Cx43 due to the abnormal localization of Cx43 in these two neuroblastoma cell lines.

[0038] To test the synergistic effect of exogenous rrCNTF and increased

Cx43 expression, IMR32 and SH-SY5Y cells were exposed to 8-bromo-cAMP for 48hrs prior to beginning the glutamate study. The 8-bromo-cAMP increased Cx43 expression and caused a re-localization of Cx43 to the cell membrane as well as doubling the number of functionally coupled cells. There was a significant improvement in both cell lines that received 8-bromo- cAMP pretreatment and rrCNTF. IMR32 cell survival improved from 48% to 64% and SH-SH5Y cell survival improved from 70% to 88%.

[0039] All documents mentioned herein are incorporated by reference.

Claims

What is claimed is:

1. A method of increasing connexin 43 (Cx43) protein expression in a cell, comprising providing the cell with a neurotrophic factor or a neuroprotective fragment thereof.

2. A method of increasing connexin 43 (Cx43) protein expression in a cell, comprising providing the cell with a neurotrophic factor or a neuroprotective fragment thereof, and providing the cell with a PKA activator.

3. The method of Claim 1 or 2, wherein the neurotrophic factor is ciliary neurotrophic factor(CNTF).

4. The method of Claim 2 or 3, wherein the PKA activator is cAMP, a cAMP analogue or forskolin.

5. The method of Claim 2 or 3, wherein the PKA activator is 8-bromo- cAMP.

6. A method of increasing gap junction communication in a cell, comprising providing the cell with a neurotrophic factor or a neuroprotective fragment thereof.

7. A method of increasing intercellular gap junction communication, comprising providing a cell with a neurotrophic factor or a neuroprotective fragment thereof, and providing the cell with a PKA activator.

8. The method of Claim 6 or 7, wherein the neurotrophic factor is ciliary , neurotrphic factor(CNTF).

9. The method of claim 7 or 8, wherein the PKA activator is cAMP, a cAMP analogue or forskolin.

10. The method of Claim 7 or 8, wherein the PKA activator is 8-bromo- cAMP.

11. A method of treating a neurodegenerative disease in mammals, comprising administering to a mammal in need thereof, an effective amount of a neurotrophic factor or a neuroprotective fragment thereof, together with a pharmaceutically acceptable PKA activator.

12. A method of treating a cancer in mammals, comprising administering to a mammal in need thereof, an effective amount of a neurotrophic factor or a neuroprotective fragment thereof.

13. A method of treating a cancer in mammals, comprising administering to a mammal in need thereof, an effective amount of a neurotrophic factor or a neuroprotective fragment thereof, together with a pharmaceutically acceptable PKA activator.

14. The method of Claim 11, 12 or 13, wherein the neurotrophic factor is ciliary neurotrophic factor (CNTF).

15. The method of Claim 11, 13 or 14, wherein the PKA activator is cAMP, a cAMP analogue or forskolin.

16. The method of Claim 11, 13 or 14, wherein the PKA activator is 8- bromo-cAMP.

17. The method of Claim 12 or 13, wherein the cancer is neuroblastoma.

18. The method of any of the preceding Claims, wherein the CNTF or neuroprotective fragment thereof is introduced into the cell or mammal in pure form, in recombinant form, as a transfected viral adenovirus, or by means modified to overexpress the CNTF protein or fragment thereof.