Patent Application
20150290160
1. Field of the Invention
The present invention relates to a method for assaying the spermidine/spermine N1-acetyltransferase (SSAT) activity of mRNA up-regulated cancer cells and, in particular, to the use of SSAT substrates as anti-cancer drug compounds and in anti-cancer treatments.
2. Description of the Related Art
U.S. Pat. No. 6,811,967 which issued to Sitar et al. on Nov. 4, 2004, and the full disclosure of which is incorporated herein by reference, discloses a method for assaying activity of the enzyme SSAT using SSAT substrates by detecting acetylated forms of the SSAT substrates. The SSAT substrates may include amantadine wherein metabolism of amantadine occurs in part by the action of the inducible enzyme SSAT to produce the acetylated metabolite N-acetylamantadine. Disclosed also is the correlation of SSAT activity to pathological conditions.
SSAT is an important enzyme in polyamine metabolism. Polyamines, including spermidine and spermine, are essential for cell survival and SSAT is a rate-limiting enzyme in the catabolic pathway which converts spermidine and spermine into acetylpolyamines to maintain intracellular polyamine homeostasis. It has been reported that in certain cancer cell lines a high expression of SSAT mRNA have been detected. See, for example, Chen et al. Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase. Biochemical Journal. (2003), Volume 373, 661-667, the full disclosure of which is incorporated herein by reference.
It has also been reported that SSAT expression and enzymatic activity may be elevated following chemotherapy or treatment with spermidine analogues. In vitro cell line studies have further positively correlated SSAT expression and enzymatic activity with levels of cytotoxicity of new drug candidates. A number of anti-proliferative agents and polyamine analogues have accordingly been developed to prevent cancer cell proliferation via SSAT induction. See for example, Wallace, H. M. et al. A perspective of polyamine metabolism. Biochemical Journal. (2003), Volume 376, 1-14, the full disclosure of which is incorporated herein by reference.
It is an object of the present invention to provide an improved anti-cancer drug compounds and anti-cancer treatment.
Certain cancer cells have high expressed spermidine/spermine N1-acetyltransferase (SSAT) mRNA which can be treated with SSAT substrates to inhibit the acetylation of the polyamines by SSAT and catabolized biochemically. SSAT substrates are effective anti-cancer agents against the cancer cells with high expression of SSAT mRNA. The method allows a cancer type screening and identifies an effective anti-cancer drug treatment to enhance the cancer treatment efficacy. The method disclosed herein also allows for assaying the SSAT mRNA up-regulated cancer cells and use of SSAT substrates in anti-cancer treatments.
There is accordingly provided an anti-cancer drug compound comprising an SSAT substrate. The SSAT substrate may be a monoamine. The SSAT substrate may be amantadine, rimantadine, dopamine or L-DOPA. There is also provided a method comprising the use of an SSAT substrate to treat cancer. The SSAT substrate may be a monoamine. The SSAT substrate may be amantadine, rimantadine, dopamine or L-DOPA.
The invention will be more readily understood from the following description of the embodiments thereof given, by way of example only, with reference to the accompanying drawings, in which:
A method of using spermidine/spermine N1-acetyltransferase (SSAT) substrates as anti-cancer drug compounds is disclosed herein.
The relative SSAT expression levels in human tumor cell lines, HEK-293, Malme-3M, HeLa, PC-3 and US-02 cell lines were determined by a reverse transcription—quantitative polymerase chain reaction assay (RT-qPCR assay) and, as shown in
Referring now to
It was subsequently shown that the SSAT substrates including amantadine, rimantadine, dopamine and L-DOPA will exhibit a selective and relative high level of cytotoxicity in human tumor cell lines over-expressing SSAT.
The following four monoamine test drugs were evaluated for cytotoxicity against human cancer cell lines.
Identity: Amantadine
Identity: Rimantadine
Identity: Dopamine
Identity: L-DOPA
Spermidine being a polyamine substrate for SSAT was used as a positive control test drug.
Identity: Spermidine
Three cell lines from each of lung, breast and prostate cancers have been selected based on literature SSAT expression data and used for potential cytotoxicity screening in the MTT assay. The SSAT non-expressing human osteosarcoma cell line U2-OS was used as the negative control cell line. The following human cell lines were used:
1American Type Culture Collection
Each human cancer cell line was incubated with each of the four monoamine test drugs at a range of testing concentrations. Cytotoxicity expressed as half maximal inhibitory concentration or IC50 was determined based on a (dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide or MTT assay. In parallel, the expression levels of SSAT in these cell lines were measured using a RT-qPCR assay.
Cells of the ten cell lines were harvested from their established adherent cultures with trypsin EDTA, pelleted by centrifugation, and resuspended in the appropriate medium to yield a suspension of cells for each cell line.
Each of the monoamine test drugs (amantadine, rimantadine, dopamine and L-DOPA) and the positive control (spermidine) was accurately weighed, dissolved and further diluted with sterile water into a series of solutions at 100× of their target incubation concentrations. The target incubation concentrations for amantadine, rimantadine, and dopamine were 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300 and 1000 μM. The target incubation concentrations for L-DOPA and the positive control, spermidine, were 0.1, 0.3, 1, 3, 10, 30, 100, 300 and 1000 μM.
Incubation was performed in triplicate at each drug concentration for each cell line tested. In parallel to each test drug treatment, each cell line was also treated with spermidine as positive controls.
An aliquot of the cell suspension was added into each well of 96-well culture plates and the plates were incubated overnight at 37° C. with a highly humidified atmosphere of 95% air and 5% carbon. On the following day each well was replaced with fresh medium and an aliquot of the appropriate test drug solution was added at 1% of the cell culture volume to achieve the target testing concentration. Blank culture media were used in lieu of the substrate solutions to prepare the vehicle controls (i.e. 0 μM substrate). The plates were then returned to incubation for three days at 37° C. with a highly humidified atmosphere of 95% air and 5% carbon.
After three days of incubation with substrates, an aliquot of 5 mg/mL MTT was added to each well and then incubated for 1 to 3 hours. Following incubation the medium was replaced with DMSO to dissolve the formazan. An aliquot from each well was measured for absorbance at 550 nm or 555 nm on a 96-well flat bottom plate with a microplate reader and DMSO for background absorbance correction.
RNA extraction was performed using a QIAshredder™ Kit and RNeasy™ Mini Kit both which are available from Qiagen, Inc. having an address at Suite 200-27220 Turnberry Lane, Valencia, Calif., United States of America. Re-suspended cells from each of the cell lines tested were lysed with RNeasy™ lysis buffer. RNA was then extracted from the lysate using RNeasy™ Mini Spin columns. Sufficient total RNA concentration in each extracted sample was confirmed by Nanodrop spectrophotometric measurement.
RT-qPCR for SSAT was performed using a QuantiTect SYBR Green RT-PCR Kit also available from Qiagen, Inc. The reaction mixture for each sample consisted of QuantiTect SYBR Green RT-PCR master mix, QuantiTect RT mix, RNase-free water, SSAT PCR primers, and the extracted RNA from each sample.
Expression levels of the house-keeping genes GAPDH and HPRT1 were measured in parallel for each sample as outlined above using the corresponding PCR primers for these genes. SSAT expression levels were normalized with GAPDH or HPRT1 as the internal reference, and expressed as fold difference relative to that of A549.
Denaturation: 15 sec at 94° C.; 20° C./sec
Annealing: 20 sec at 55° C.; 20° C./sec
Extension: 30 sec at 72° C.; 20° C./sec
A summary of the cytotoxicity expressed as IC50 for each SSAT monoamine test drug for each human cancer cell line tested is presented in
From the data, the IC50 values of the four monoamine test drugs ranged from 34.1 μM to 1605 μM across all the cell lines evaluated, with a majority between 100 μM and 500 μM. Overall, the most potent monoamine test drug was dopamine acting on the negative control U2-OS osteosarcoma cell line with an IC50 value of 34.1 μM. This is followed by dopamine acting on the breast cancer cell line BT-549 with an IC50 value of 52.0 μM. The least potent test drug was amantadine with IC50 values of 1605 μM and 1158 μM when acting on NCI-H23 (lung cancer) and BT-549 (breast cancer), respectively.
The IC50 values of the four monoamine test drugs were relatively higher than the positive control spermidine, ranging from 3.72 μM to 32.7 μM, reflecting that the monoamine test drugs were lower in cytotoxic potency compared with polyamine spermidine. For each cell line, it was noted that in general the rank-ordering of IC50 (i.e. cytotoxicity) of the monoamine test drugs appeared to correlate with that of the polyamine spermidine positive control. This apparently similar rank-order of cytotoxicity may suggest a common mode of mechanism between the monoamine and polyamine drugs.
A summary of the relative SSAT expression levels in the human cell lines tested is presented in
From the results, LNCaP was observed to have the highest relative expression level of SSAT with approximately 5-fold more than that of A549 when normalized with GAPDH, and 3-fold more when normalized with HPRT1. T-47D had the second highest expression level with approximately 2-fold difference of SSAT expression relative to A549. The SSAT non-expressing cell line U2-OS (negative control) had the lowest SSAT expression level as anticipated.
RT-qPCR results were compared against the IC50 values of each test drug for each cell line in an attempt to correlate SSAT expression level with cytotoxicity of the monoamine test drugs. From correlation of SSAT expression against potency expressed as 1/IC50, shown in
The monoamine test drugs amantadine, rimantadine, dopamine and L-DOPA were evaluated for cytotoxicity against three SSAT over-expressing human cancer cell lines from each of lung, breast and prostate cancers. Across all nine tumor cell lines tested, the cytotoxic potency of the monoamine test drugs were observed to be lower compared with spermidine which was a polyamine positive control. In general, the rank-ordering of cytotoxicity of the monoamine test drugs appeared to correlate with that of the polyamine spermidine, suggesting a common mode of mechanism between the monoamine and polyamine drugs. It is accordingly concluded that the monoamine test drugs and other SSAT substrates may be used as anti-cancer drug compounds and in anti-cancer treatment.
It will be understood by a person skilled in the art that many of the details provided above are by way of example only, and are not intended to limit the scope of the invention which is to be determined with reference to the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2013/050873 | 11/14/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61726534 | Nov 2012 | US |
