INDUCING CASPASE ACTIVITY

Abstract

Embodiments are directed towards methods of inducing caspase activity. The methods include contacting a cell with a treatment compound represented by the following Formula (I), where the treatment compound is a random copolymer, R.sup.1 is selected from hydrogen and a hydroxyl group, R.sup.2 is selected from hydrogen and a hydroxyl group, R.sup.1 is different than R.sup.2, and a sum of x and y is from 4 to 20,000.

##STR00001##

Claims

1. A method of inducing caspase activity, the method comprising contacting a cell with a treatment compound represented by the following Formula: ##STR00004## where the treatment compound is a random copolymer, R.sup.1 is selected from hydrogen and a hydroxyl group, R.sup.2 is selected from hydrogen and a hydroxyl group, R.sup.1 is different than R.sup.2, and a sum of x and y is from 4 to 20,000.

2. The method of claim 1, wherein the treatment compound has a number average molecular weight from 200 to 2,000,000 g/mol.

3. The method of claim 1, wherein the treatment compound has a 0.001 millimolar to 75 millimolar concentration in a treatment medium.

4. The method of claim 1, wherein the cell is a cancerous cell.

5. The method of claim 1, wherein the caspase is an effector caspase.

6. The method of claim 1, wherein the caspase is selected from caspase 3, caspase 6, caspase 7, or combinations thereof.

7. The method of claim 1, further comprising inciting apoptosis.

Description

EXAMPLES

[0029] In the Examples, various terms and designations for materials are used including, for instance, the following:

[0030] Poly(ethylene glycol ran-propylene glycol) (Mn of 2500 g/mol; obtained from Sigma-Aldrich);

[0031] Cells (human colon; colorectal adenocarcinoma; HT-29 (ATCC® HTB-3); obtained from ATCC);

[0032] McCoy's 5A (growth medium; obtained from ThermoFisher Scientific);

[0033] Fetal bovine serum (obtained from ATCC);

[0034] Dulbecco's Phosphate-Buffered Saline (GIBCO 14190-144; obtained from ThermoFisher Scientific);

[0035] Complete Growth Medium (ATCC® 30-2007; obtained from ATCC);

[0036] Trypsin-EDTA (GIBCO Trypsin-EDTA (0.25%); Catalog number 25200056; obtained from ThermoFisher Scientific);

[0037] Thiazolyl Blue Tetrazolium Bromide (obtained from ThermoFisher Scientific);

[0038] Dulbecco's Phosphate-Buffered Saline with calcium and magnesium (GIBCO 14040-133; obtained from ThermoFisher Scientific);

[0039] Caspase-Glo 3/7 Assay (luminescent assay; Catalog number G8093; obtained from Promega);

[0040] Dimethyl sulfoxide (Catalog number 276855; obtained from Sigma-Aldrich).

[0041] Culture Initiation and Maintenance

[0042] Culture initiation and maintenance was performed as follows. Culture initiation and maintenance was performed in accordance with “Thawing, Propagating, and Cryopreserving Protocol” NCI-PBCF-HTB38 (HT-29) Colon Adenocarcinoma (ATCC®HTB-38™); Feb. 27, 2012; Version 1.6.

[0043] HT-29 (ATCC® HTB-38™) cells (which contained approximately 1×10.sup.6 cells per mL) were initiated and seeded into a T-25 flask containing McCoy's 5A and fetal bovine serum (10% (v/v)). Then, ATCC® 30-2007 (warmed in 37° C. water bath for at least 15 minutes) was used to expand the HT-29 cells. The cells were grown in a humidified incubator (SANYO INCT-16-CMT; MCO-19AIC (UV)) maintained at 37° C. and 5% CO2. Then, the cells were rinsed with 1X Dulbecco's Phosphate-Buffered Saline and sub-cultured in T-75 flasks 1 to 3 times per week using 1X Trypsin-EDTA, applied for ≤5 minutes; enzymatic action of the trypsin-EDTA was stopped by adding complete growth medium to the detached cells. Then, upon reaching 80 to 90% confluency the cells were split into the following split ratio ranges: 1:5 to 1:16. Subculture and growth expansion activities were recorded, such as passage number, % confluency, % viability (only on experimental set-up day), and cell morphology throughout all phases. The cells were maintained in log-phase growth.

[0044] Cell Culture Plating (Day 0)

[0045] Cell culture plating was performed as follows. A cell suspension from a single 80 to 90% confluent T-75 flask was harvested with trypsin-EDTA and complete growth medium. To obtain cell concentration and viability, cell counts were obtained using a COUNTESS automated cell counter (INVITROGEN C10227; CNTR-7-CMT) in which 2 chambers of each slide were provided with 10 μL each of 1:1, 0.4% trypan-blue dye (INVITROGEN T10282) and cell suspension. Cell counts and percent viability were averaged from both chambers of a single slide. Then, viable cells (defined as viability≥90%) containing complete growth medium were plated onto sterile 96-well plates using a multi-channel pipette. Per cell density, between 5000 and 6000 cells per well (40,000 to 48,000 cells per mL) were added to each well, except for wells that were utilized as ‘saline only’ no cell control wells; equal volumes each of 125 μL of cell suspension were added per well beginning with row A to H on the plate. The plates used for each of 2 endpoints, apoptosis and cytotoxicity, were solid white plates and clear plates, respectively. The cells were incubated for 24 ±2 hours to allow attachment.

[0046] Poly(ethylene glycol-ran-propylene glycol) Stock Preparation

[0047] Stock solutions were prepared at respective target concentrations of poly(ethylene glycol-ran-propylene glycol) in sterile saline. For the assays, based on the solubility limits due to high molecular weight, adjustments to lower stock concentration preparations (w/v) to generate either a solution or a pipettable suspension were made if necessary, or solubilization was achieved by adding small increments of saline, continuous mixing, vortexing, sonicating, or stirring prior to use in assay. If necessary for solubilization, the saline was pre-heated to 37° C. prior to mixing with the poly(ethylene glycol-ran-propylene glycol). Total volumes of 10 mL were prepared per tested substance on the day of cell suspension plating (Day 0).

[0048] Cytotoxicity Reagent Preparation

[0049] Thiazolyl blue tetrazolium bromide was prepared at 5 mg/mL in Dulbecco's Phosphate-Buffered Saline with calcium and magnesium. Total volumes of 30 mL were prepared (w/v) per set-up day (Day 0) and stored at 4° C. until use.

[0050] Dosing Solution Preparation (Day 0)

[0051] Dosing solutions/suspensions of each test substance stock were prepared in a total of 15 mL each of McCoy's 5A and 1% fetal bovine serum. Various amounts of dosing stock were utilized to achieve dosing solutions/suspensions from 0.0015 to 60 mM. The dosing solutions/suspensions were prepared in sterile reservoirs and repeatedly mixed with a pipette until visible uniformity was achieved. Using a 2 mL capacity sterile 96-deep well block, 2 mL of dosing solution/suspension was added to each of 6 replicate wells for the treatment groups and each of 12 wells for the saline only cell controls and saline only ‘no cell’ background correction controls. The plates were established following a semi-randomized statistical design. Each test substance was identified numerically and via a color code used for identifying wells to be treated. The blocks were covered with sealing tape, plate lid and placed into a 4 ° C. lab refrigerator (Fischer Scientific, 135B1; RFR-22-CMT) overnight.

[0052] Treatment (Day 1)

[0053] All 96-deep well blocks containing dosing solutions/suspensions were removed from the refrigerator and placed in a 37° C. bead bath for a minimum of 30 minutes. Approximately 24 hours after plating, well plates were removed from the incubator and treated one at a time. All wells from a cell plate were aspirated using a 6-well aspirating device starting with row A to H. Using a multi-channel pipette, 100 each of dosing solution/suspension (from the blocks) was added to each well of the 96-well cell treatment plate; starting from row A to row H (same order). All wells were aspirated and treated 2 rows at a time to prevent well drying and maintain cell attachment and viability; pipette tips were changed per row. All plates were placed into the incubator and allowed to treat for 24 ±2 or 48 ±2 hours prior to harvest.

[0054] Harvest (Day 2 and Day 3)

[0055] Apoptosis

[0056] Assessment of apoptosis was performed as follows. Apoptosis was performed in accordance with “Caspase-Glo 3/7 Assay” 4.B. Standard Protocol for Cells in a 96-Well Plate (Promega). The Caspase-Glo 3/7 Assay components were pre-warmed to room temperature for approximately 60 minutes. White plates were removed (one at a time) from the incubator and the treatment medium was aspirated. Using a multi-channel pipette, 100 μL of 1X Dulbecco's Phosphate-Buffered Saline was added to each well of the 96-well plate. The Assay reagents (buffer and substrate) were manually mixed and added to a reagent reservoir; using a multi-channel pipette, 100 μL of the Assay reagent mixture was added to each well of the 96-well plate. The plate(s) (protected with foil from light) were placed on a plate shaker and allowed to rotate for 5 minutes at approximately 800 rpm at room temperature. The plates were then incubated at room temperature for an additional 25 minutes prior to analysis. Luminescence was recorded in terms of Relative Light Units (RLU) for each plate on a FLUOstar Omega Plate Reader.

[0057] Cytotoxicity

[0058] Assessment of cytotoxicity was performed as follows. Cytotoxicity reagent (5 mg/mL), as previously described, was pre-warmed to room temperature for approximately 30 minutes and then diluted into 1X Dulbecco's Phosphate-Buffered Saline with calcium and magnesium to provide a concentration of 0.675 mg/mL (final). Clear plates were removed from the incubator (one at a time) and the treatment medium was aspirated. Using a multi-channel pipette, 200 μL of the cytotoxicity reagent (final) was added to each well of the 96-well plate; then the plates were covered with sealing tape and incubated in a humidified 37 ° C. incubator for 4 hours. Following incubation, the supernatant was aspirated and dimethyl sulfoxide (200 μL) was added to each well. Following thorough mixing by repeat pipetting, the cell lysate was transferred to a new clear 96-well plate and absorbance was quantified at 600 and 630 nm on a FLUOstar Omega Plate Reader.

[0059] Analysis

[0060] Relative caspase activity was calculated as follows:

[0061] (RLU.sub.Foreground)−(RLU.sub.Saline Only ‘no cell’ control)=(RLU.sub.Background corrected);

[0062] ((RLU.sub.Background corrected) of each test substance containing well)/(Average (RLU.sub.Foreground) of 12 saline only control wells)=relative caspase activity; where RLU=relative light unit.

[0063] Relative caspase activity of each test substance containing well/6 replicates=Average relative caspase activity. The results are reported in Table for the various utilized concentrations.

[0064] Cell viability was calculated as follows:

[0065] (Abs.sub.600 Foreground)−(Abs.sub.600 Saline Only ‘no cell’ control)=(Abs.sub.600 Background corrected)

[0066] (Abs.sub.630 Foreground)−(Abs.sub.630 Saline Only ‘no cell’ control)=(Abs.sub.630 Background corrected)

[0067] (Abs.sub.600 Background corrected)−(Abs.sub.630 Background corrected)=(Abs.sub.600-630)

[0068] ((Abs.sub.600-630) of each test substance containing well)/(Average (Abs.sub.600-630) of 12 saline only control wells)=% Cell Viability

[0069] % Cell Viability of each test substance containing well/6 replicates=Average % Cell Viability. The results are reported in Table 2 for the various utilized concentrations.

TABLE-US-00001 TABLE 1 Poly(ethylene Poly(ethylene Poly(ethylene glycol-ran- glycol-ran- glycol-ran- propylene propylene propylene glycol) glycol) glycol) [15 mM] [30 mM] [60 mM] Average relative 1.35 1.83 2.98 caspase activity (Run 1) Standard deviation 0.13 0.16 0.42 relative caspase activity (Run 1) Average relative 1.36 1.44 1.64 caspase activity (Run 2) Standard deviation 0.09 0.11 0.27 relative caspase activity (Run 2) Average relative 1.30 1.51 2.03 caspase activity (Run 3) Standard deviation 0.17 0.07 0.46 relative caspase activity (Run 3) Average relative 1.34 1.59 2.22 caspase activity (Runs 1, 2, 3) Standard deviation 0.03 0.21 0.69 relative caspase activity (Runs 1, 2, 3)

[0070] The data of Table 1 illustrate that advantageous relative caspase activities, i.e. average relative caspase activity>1, were provided when cells were exposed to 15, 30, and 60 mM concentrations of poly(ethylene glycol-ran-propylene glycol), as indicted by the respective average relative caspase activity (Runs 1, 2, 3) values.

TABLE-US-00002 TABLE 2 Poly(ethylene Poly(ethylene Poly(ethylene glycol-ran- glycol-ran- glycol-ran- propylene propylene propylene glycol) glycol) glycol) [15 mM] [30 mM] [60 mM] Average viability % 129 101 66 (Run 1) Standard deviation  14  38 20 viability % (Run 1) Average viability % 155 158 54 (Run 2) Standard deviation  42  29 45 viability % (Run 2) Average viability %  97  90 37 (Run 3) Standard deviation  20  85 13 viability % (Run 3) Average viability % 127 116 52 (Runs 1, 2, 3) Standard deviation  29  37 15 viability % (Runs 1, 2, 3)

[0071] The data of Table 2 illustrate that desirable adequate viability, i.e. average viability % of 50% or greater for (Runs 1, 2, 3) were provided after 24 hours when cells were exposed to 15, 30, and 60 mM concentrations of poly(ethylene glycol-ran-propylene glycol).