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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Lorus Therapeutics Inc., Toronto, Ontario, Canada
Received September 5, 2003; accepted October 29, 2003.
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Abstract |
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). Nonsmall cell lung cancer (NSCLC) represents approximately 80% of all lung cancers, and no curative treatment is available for the advanced stages of these diseases. Current treatment of NSCLC involves surgical resection coupled with the use of cytotoxic drugs. The surgical procedures are highly invasive and are applicable to only a small number of patients. Standard chemotherapy is broadly used but offers only a limited survival benefit at the expense of substantial toxicity, drug resistance, and poor target specificity. Thus, there is a need for innovative strategies that effectively treat patients, maintain patient quality of life, and exhibit more favorable safety profiles in advanced NSCLC. Deregulation of genes involved in the cell cycle is a hallmark of a variety of human cancers, including NSCLC, breast, and colorectal cancer, B-lymphoma, and prostate cancer (Buolamwini, 2000). Therefore, the use of compounds capable of targeting aberrantly expressed cell cycle-related proteins may be a promising approach to treatment of these types of cancers.
The G1-S cell cycle checkpoint controls the passage of eukaryotic cells from G1 phase into the DNA synthesis S phase. This process is dependent on the activities of CDKs that are sequentially regulated by cyclins D, E, and A (Pines, 1994; Sherr, 1996). Cyclin D associates with CDK4 and CDK6, and the catalytic activities of the assembled holoenzymes are first manifested by mid-G1, increase to a maximum at the G1-S transition, and contribute to G1 exit (Sherr, 1996). CDK2 associates with either cyclin E or cyclin A and the resultant kinase activities increase at the G1/S transition or in the early S phase, respectively (Matsushime et al., 1991; Pines, 1994). CDK 4/6-cyclin D, CDK2-cyclin E, and the transcription complex that includes pRb and E2F are pivotal in controlling progression through the late G1 restriction point. The phosphorylation of pRb late in G1 initially triggered by CDK4/6 and later accelerated by CDK2 induces pRb to dissociate from E2F, which can transactivate S-phase genes encoding for proteins that amplify the G1-to-S phase switch and are required for DNA replication (Sherr, 1996).
Clotrimazole (CLT), an antifungal synthetic imidazole derivative, inhibited in vitro proliferation of human cancer cells by a unique mechanism involving Ca2+ store-mediated inhibition of translation initiation and demonstrated antimeta-static properties in an in vivo mouse model (Benzaquen et al., 1995). Further studies demonstrated that CLT blocks the cell cycle specifically in G1 by reducing synthesis and expression of G1 cyclins, thereby inhibiting the associated CDK activity required for progression into S phase (Aktas et al., 1998). Specifically, CLT induces the release of Ca2+ from endoplasmic reticulum intracellular stores and blocks the influx of extracellular Ca2+ through Ca2+ store-regulated Ca2+ channels. The sustained depletion of intracellular Ca2+ stores activates RNA-dependent protein kinase R, resulting in phosphorylation of eukaryotic translation initiation factor (eIF2
) at serine 51 and its concomitant inactivation. Inactivation of eIF2 inhibits the formation of ternary complex between Met-tRNA, eIF2, and GTP, a rate-limiting step in translation initiation. This results in an abrogation of synthesis of growth promoting proteins such as cyclin D, E, and A at the level of translation initiation, leading to growth arrest in early G1. Furthermore, CLT was also shown to be a potent inhibitor of angiogenesis in an animal model (Takahashi et al., 1998).
Systemic use of CLT as an anticancer agent is severely limited by hepatotoxicity associated with the imidazole moiety (Tettenborn, 1974). In an attempt to develop CLT analogs that lack the imidazole moiety while retaining cell cycle-modulatory activity, we designed and synthesized a series of derivatives that approximated the spatial configuration of CLT without the imidazole ring (Al-Qawasmeh et al., 2004). In the current study, one of these compounds, designated as NC381 (Fig. 1), was found to inhibit NSCLC cell growth in vitro via a mechanism similar to that of CLT. More importantly, NC381 also exhibited significant inhibitory effects on the growth of human lung tumors in a mouse xenograft model. The fact that NC381 can alter the activities of cell cycle-related proteins provides support for development of NC381 as an NSCLC therapeutic.
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Materials and Methods |
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-minimal essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin (100 units/ml) and streptomycin (10 units/ml) (HyClone Laboratories, Logan, UT) in a humidified 37°C incubator with 5% CO2 in air. Antibodies and NC381. Antibodies used for Western blotting and immunoprecipitation analyses were purchased from the following vendors: Santa Cruz Biotechnology (Santa Cruz, CA) for anti-cyclin B, anti-CDK 4, anti-p16Ink4, anti-p27Kip1, and anti-VEGF-R1 antibodies; Cell Signaling Technology (Mississauga, ON, Canada) for anti-pRb and anti-phosphorylated form of pRb (ppRb) antibodies; Neomarkers (Fremont, CA) for anti-Cyclin D1 antibody; and Biodesign International (Saco, ME) for anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. NC381 (chemical structure shown in Fig. 1) was synthesized by Torcan Chemical Ltd. (Aurora, ON, Canada) using a synthetic scheme described elsewhere (R. A. Al-Qawasmeh, Y. Lee, M.-Y. Cao, S. Viau, X. Gu, J. A. Wright, and A. Young, manuscript in preparation).
Determination of Antiproliferative Activity. NC381 was submitted to the National Cancer Institute, National Institutes of Health, in Rockwell, MD, and its antiproliferative activity was evaluated in the National Cancer Institute's revised anticancer screen. The standard 48/72-h 60-cell line assay and in vitro time-course assay were as described previously (Alley et al., 1988). In the standard 60-cell line assay, a minimum of five NC381 concentrations at 10-fold dilutions were tested against 60 cell lines and the cell growth, determined at 48 and 72 h, was assayed using a sulforhodamine B assay. For the time-course analysis, tumor cells were treated with the drug for the indicated times as described in the text and then washed and grown in drug-free medium until the end of the experiment at 144 h. This assay used 20% FBS to better approximate the minimum concentrations and times drug exposure conditions that were required to achieve activity in vivo. Cell growth was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay (Twentyman and Luscombe, 1987), and the drug concentration required for growth inhibition was determined. The inhibitory effects of the compound were obtained as GI50 values, which represent the molar drug concentrations required to cause 50% growth inhibition. [Note: The National Cancer Institute has renamed the IC50 value, the concentration that causes 50% growth inhibition or 50% cell kill, the GI50 value to emphasize the correction for the cell count at time 0; thus, GI50 is the concentration of test drug where 100 x (T –T0)/(C –T0) = 50. The optical density of the test well after a 48-h period of exposure to test drug is T, the optical density at time 0 is T0, and the control optical density is C]. The GI50 measures the growth inhibitory power of the test agent. The graph was plotted from the original results of representative determinations.
Cell Cycle Analysis. Alterations in cell cycle were determined using flow cytometric analyses. NCI-H460 cells were synchronized by plating in medium containing 0.5% FBS for 24 h followed by culturing in FBS-free medium for 48 h. The cells were then released into complete medium containing 0.1% DMSO (vehicle control), 25 or 50 µM NC381, harvested 16 h after treatment, washed with cold PBS twice, and fixed in 70% ethanol at 4°C at least 4 h. The fixed cells were centrifuged at 1500 rpm for 4 min at 4°C, washed with cold PBS containing 2% FBS twice, treated with 3 mg/ml ribonuclease (Sigma Chemical, Oakville, ON, Canada) and 50 µg/ml propidium iodide (Sigma Chemical) for 30 min at 37°C. The fluorescence of stained cells was measured using a FACScan flow cytometer and the Cell Quest program (BD Biosciences, San Jose, CA). Data were evaluated using Modfit software (Verity software House, Topsham, ME).
Western Blot Analysis. NCI-H460 cells were synchronized as described above and then released into complete medium containing 0.1% DMSO (vehicle control), 25 or 50 µM NC381 for the duration indicated in the text. After the treatments, the cells were harvested and the total extracts were prepared in denaturing lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 7.2 mM 
-mercaptoethanol). Protein concentration in the lysates was quantified by trichloroacetic acid method or with a Bio-Rad protein assay kit using bovine serum albumin as the standard. Total protein lysates (40 µg/lane) were resolved on 10% SDS-polyacrylamide gels and protein transferred to polyvinylidene difluoride membranes. Blots were treated with blocking agent, 5% nonfat milk in Tris-buffered saline, for 1 h at room temperature. Protein expression was subsequently detected with primary antibodies (described above) against the different antigens at a dilution described in the text. After washing with Tris-buffered saline/Tween 20, three times, a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology and Amersham Biosciences Inc., Piscataway, NJ) at a dilution of 1:10,000 was added and incubated at room temperature for 1 h. The blots were washed and the immune complexes detected using an enhanced chemiluminescence detection reagent kit (Amersham Biosciences Inc.) and exposed to Kodak X-OMAT AR film for autoradiography.
Immunoprecipitation. Immunoprecipitations were performed as follows. NCI-H460 cells were treated as described above, washed with cold PBS once, and lysed for 10 min on ice in 1x cell lysis buffer (Cell Signaling Technology; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4, 20 mM NaF, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 13,000 rpm for 5 to 10 min at 4°C, and a total of 500 µg of cleared lysates was incubated with anti-cyclin D1 or control (VEGF-R1) antibodies for 1 h at 4°C. The immune complexes were collected on protein A-Sepharose TM 4 Fast Flow beads for 1 h at 4°C (Amersham Pharmacia AB, Uppsala, Sweden) and washed for four times in cold cell lysis buffer. The beads were then resuspended in denaturing lysis buffer, incubated for 15 min at room temperature, heat denatured, and resolved on 10 to 12% SDS-polyacrylamide gels. Protein expression was detected by immunoblot analyses using anti-cyclin D1 and anti-CDK4 antibodies. As an internal control for each sample used in the immunoprecipitation experiment, GAPDH expression was quantified using anti-GAPDH antibody.
In Vivo Tumorigenicity Assay. Five- to six-week-old female CD-1 athymic nude mice (Charles River Canada, Montreal, QC, Canada) were acclimatized in a pathogen-free facility at Sunnybrook and Women's Health Science Center for at least 1 week. Animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals in Canada and approved by the Animal Care Committees of the Sunnybrook and Women's College Health Science Center and the University of Toronto. Approximately 5 x 106 NCI-H460 cells in 100 µl of PBS were subcutaneously injected into the right flank of each mouse. Once tumors reached an approximate volume of 100 mm3, 4 days post-tumor cell injection, mice were randomized by tumor size into three groups (n = 20). NC381 was administrated orally at a dose of 25 or 75 mg/kg everyday 5 days a week for 2 weeks followed by every other day for 4 days. Control animals received vehicle alone [50 µl of soybean oil/Capmul/Tween 80 (45:50:5)] for the same period. The tumor dimensions (length, width, and height) were measured using calipers twice a week over the treatment period. The mice were sacrificed when the tumor burden reached approximately 10% of total body weight and excised tumors were weighed. A standard bar graph was used to demonstrate the differences in tumor weights with each bar representing mean tumor weight.
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Results |
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; Aktas et al., 1998). Furthermore, NC381 exhibited a unique activity profile as demonstrated by mid-log differential activity in the standard 48/72 h assay and as further determined by the COMPARE algorithm (Developmental Therapeutics Program of the U.S. National Cancer Institute, unpublished data). In vitro time-course assays confirmed the cytostatic nature of NC381, particularly against NCI-H460 NSCLC cells (Fig. 3). Exposure durations, as short as 0.75 h, were highly effective in achieving growth inhibition with a GI50 value of approximately 60 µM. In addition, it was apparent that progressively longer exposure of NCI-H460 NSCLC cells to NC381 allowed incrementally lower drug concentrations necessary to achieve GI50. The lowest GI50 value was reached at approximately 11 µM after a 6-day exposure to NC381. These data support the exposure durations chosen for subsequent mechanism of action experiments (see below).
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NC381 Arrests Cell Cycle in G0-G1 Phase. Flow cytometric analysis demonstrated inhibitory effects of NC381 on cell cycle progression (Fig. 4). Cycling NCI-H460 cells were treated with increasing concentration of NC381 in complete medium and the effective dose was determined to be within the 25 to 50 µM range to obtain significant cell cycle arrest in G0-G1 phase (data not shown). Cell cycle inhibitory effects were augmented when cells were treated with NC381 after the synchronization by serum starvation. When cells were released into the complete medium and treated with vehicle alone (DMSO) for 16 h, cell cycle progression was evident with significant increase in number of cells progressing into S and G2-M phases (Fig. 4A, serum-starved versus DMSO). However, the cell cycle progression was dramatically blocked in G0-G1 when cells were treated with NC381. In the presence of 25 µM NC381, fewer cells exited out of G0-G1 phase (55.02% versus 32.17%), and consequently fewer cells progressed to S phase (54.28% versus 35.38%). Higher dose of NC381 (50 µM) induced a 2-fold increase in the number of cells still remaining in G0-G1 phase at 60.09% compared with cells in vehicle-treated group (Fig. 4A). Progressive decrease in the number of cells exiting G0-G1 with increasing drug concentrations was accompanied by concomitant decrease in number of cells in S and somewhat in G2-M phases. These results are clearly illustrated in Fig. 4B when the data from three independent experiments were pooled and the percentage of cells in each phase plotted. There was no indication of NC381-induced apoptosis because there was no evidence showing any increase in the sub-G1 population of cells (Fig. 4A). At present, the data do not differentiate between a block in progression from G0 or arrest in G1. Studies are ongoing to assess the relative contribution of NC381 to each of these possibilities.
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Effects on the Expression of Cell Cycle-Related Proteins in Cells Treated with NC381. D-type cyclins are induced in resting cells after growth factor stimulation (Matsushime et al., 1991) and are expressed throughout G1 phase and contribute to G1-S transition (Sherr, 1996). Conversely, cyclin B accumulation peaks at G2-M phase and modulates the catalytic activity of CDC2, which is restricted to mitosis (Morgan, 1995). We investigated whether the effects induced by NC381 were at the level of G1-S transition-related protein (i.e., cyclin D1) and/or at the level of G2-M transition-related protein (i.e., cyclin B). As expected for cells released into complete medium after synchronization by serum starvation, the expression of cyclin D1 increased dramatically in NCI-H460 cells at 4 h and progressively decreased with time (Fig. 5A, DMSO). However, the expression pattern of cyclin D1 was significantly altered in cells treated with NC381. By 4 h, cyclin D1 was decreased relative to DMSO-treated cells at the same time point, and by 24 h cyclin D1 was barely detectable, indicating almost complete down-regulation (Fig. 5A, NC381). As expected, there was a substantial and steady increase in cyclin B expression after serum stimulation up to 24 h (Fig. 5A, DMSO). The expression pattern of cyclin B in cells treated with NC381 remained basically unaltered during the same period. Immunoprecipitation experiments further demonstrated that significant decrease in cyclin D1 expression observed in NC381-treated cells was accompanied by substantial reduction in the amount of CDK4 that associates with cyclin D1 (Fig. 5B).
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The effects elicited by NC381 treatment on the level of other cell cycle-related proteins such as p16Ink4 and p27Kip1 were also shown in Fig. 6. Both p16Ink4 and p27Kip1 can directly block CDK4 activity and cause G1 phase arrest (Serrano et al., 1993; Kamb et al., 1994; Polyak et al., 1994; Toyoshima and Hunter, 1994). There clearly was significant decrease in p16Ink4 expression as the cells progressed further into the cell cycle after serum stimulation (Fig. 6, DMSO). In the presence of NC381, however, the level of p16Ink4 remained unchanged (Fig. 6, NC381). Although less obvious, p27Kip1 levels fell in response to serum stimulation during 24-h incubation, whereas the kinetics of reduction was somewhat impeded over the same duration in cells treated with NC381 (Fig. 6). The p27Kip1 levels were the same at 4 h and dropped to 62% of the 4 h level at 8h in both vehicle (DMSO) and NC381 treated cells. However, by 24 h, the p27Kip1level dropped further to 40% of the 4 h level in vehicle treated cells, whereas the p27Kip1 level remained unaltered at about 60% in NC381-treated cells.
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NC381 Inhibits Phosphorylation of pRb. Phosphorylation of pRb by the cyclin D/CDK4 complex leads to the dissociation of E2F transcription factor from pRb and the transcriptional activation of a variety of cell cycle-related genes (Sherr, 1996). Thus, pRb inactivation by phosphorylation and concomitant release of E2F late in G1 is a key molecular event leading to the S-phase commitment of cells at the G1 restriction point. To examine the effects of NC381 on the phosphorylation status of pRb, serum-starved NCI-H460 tumor cells were treated with 50 µM NC381 as described above, and resolved proteins were immunoblotted using an antibody that can detect total pRb, as well as antibodies that are specific to pRB phosphorylated at three phosphorylation sites (serine 780, serine 795, and serine 807/811). Although not clearly separated, pRb antibody against total pRb detected a doublet that represented both nonphosphorylated and phosphorylated forms of pRb (pRb versus ppRb; Fig. 7). At 4 h after serum stimulation, there was a significant increase in the phosphorylaton of pRb and the increase persisted up to 24 h (Fig. 7, DMSO). In contrast, NC381 treatment resulted in significant reduction in the total pRb (both pRb and ppRb). The complete absence of ppRb band at 24 h indicated that molecular events that culminate in the phosphorylation of pRb, and the release of E2F seems to be severely compromised in NC381-treated cells (Fig. 7, NC381).
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The blot shown in Fig. 7 further demonstrated that the phosphorylation of serines at three positions, 780 (S780), 795 (S795), and 807/811(S807/S811), was dramatically inhibited in NCI-H460 cells treated for 24 h with NC381. Notably, the inhibition of phosphorylation of pRb at S780 and S795 results in the suppression of transactivation activity of E2F, an activity that is critical to G1-S progression (Kitagawa et al., 1996; Grana et al., 1998). In addition, NC381 inhibited the phosphorylation of S807/811 that can abolish the interaction between c-Abl and pRb (Knudsen and Wang, 1996). The phosphorylation results in activation of c-Abl, a proto-oncogene overexpressed or mutated in a number of malignant tumors (Chung et al., 1996).
Inhibition of Tumor Growth in Vivo. Antitumor efficacy of NC381 was evaluated in a mouse model of NCI-H460 human NSCLC xenografts. As illustrated in Fig. 8, oral administration of NC381 at 25 and 75 mg/kg results in inhibition of tumor growth compared with that observed in the vehicle-treated group. Individual tumors excised from the vehicle or two groups of NC381-treated animals were weighed and the average tumor weights were determined to be 1125.0 ± 195.9 versus 574.1 ± 150.2 versus 345.0 ± 157.8 mg, respectively (Fig. 8). The differences in tumor weights were statistically significant with P value less than 0.025 and 0.004. Although vehicle-related toxicity caused death of several mice from each group, including the vehicle-treated group, no obvious drug-related systemic toxicity was observed in the NC381-treated animals. NC381 did not cause significant body weight losses in treated animals and no obvious abnormalities were detected upon gross observation of the brain, heart, liver, spleen, kidney, and gastrointestinal tract (data not shown). Preliminary toxicology studies have been completed and pending a detailed analysis, the results will be published elsewhere. Briefly, there were no gross pathological findings and no systemic toxicity from histology studies of NC381 administered at 80 mg/kg/day (data not shown).
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Discussion |
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). As such, the cell cycle regulatory pathway may represent a useful target for drug and gene therapeutic approaches (Hunter and Pines, 1994). CLT blocks the cell cycle specifically in G1 by reducing expression of G1 cyclins such as cyclin D1, thereby inhibiting associated CDK activity (Aktas et al., 1998). However, the hepatotoxicity associated with CLT precludes its application as an anticancer therapeutic drug. To eliminate the toxicity associated with the imidazole ring of CLT, we set out to synthesize and identify structural analogs that retained antiproliferative efficacy of CLT (Al-Qawasmeh et al., 2004). The current study demonstrates that NC381, one of these CLT analogs, inhibits NCI-H460 NSCLC cell growth in a time-dependent manner. The results obtained from flow cytometric analysis and from evaluating the effects of NC381 on the expression of cell cycle-related proteins suggest that the growth inhibition is associated with the arrest of cells in G0-G1 phase and with significant deregulation of several key proteins involved in the cell cycle progression. Feasibility of NC381 as a therapeutic agent was further evaluated in a mouse model of human NCI-H460 NSCLC xenograft. The results suggest that NC381may become an effective antiproliferative agent that can be orally administered and acts through unique mechanism of action.
CLT inhibited in vitro proliferation of human cancer cells by a novel mechanism involving Ca2+ store-mediated inhibition of translation initiation (Benzaquen et al., 1995). NC381, an analog of CLT, also inhibits translation initiation by phosphorylation of eIF2 (manuscript in preparation), which leads to the inhibition of cyclin D1 expression and the concomitant reduction of cyclin D1/CDK4 complex formation as shown in the current study. Marked decrease in cyclin D1 level was accompanied by strong inhibition of pRb phosphorylation. In contrast, NC381 had little effects on cyclin B expression, suggesting that the blockage of NCI-H460 cell growth is mainly induced by the down-regulation of G1 cyclin, specifically, cyclin D1 rather than by modulating the level of G2-M phase cyclin. Given that expression of cyclin B was induced equally 4 h after serum-stimulation in both vehicle- and NC381-treated cells suggests that the expression of cyclin B is not directly affected by NC381. The level of cyclin B, however, remained unaltered in the presence of NC381, whereas the level increased in the control cells. This may be a secondary effect of cyclin D1 deregulation, which results in fewer cells progressing into the later part of cell cycle in which cyclin B is normally up-regulated.
Progression through the G1 phase of the cell cycle requires pRb phosphorylation, a major cellular target of the cyclin D1-dependent CDK4/6 kinases. The importance of pRb is exemplified by its frequent mutation in a number of human tumors (Maelandsmo et al., 1996; Dublin et al., 1998; Gorgoulis et al., 1998). The current study indicates that phosphorylation of serines at different positions [780 (S780), 795 (S795), and 807/811(S807/S811)] is dramatically inhibited by NC381. Inhibition of phosphorylation at S780 and S795 affects G1-S progression, whereas inhibition at S807/811 may affect the activity of the c-Abl proto-oncogene. Together, these results suggest that NC381 inhibits pRb phosphorylation at multiple sites and leads to the inhibition of cell cycle progression at the G1-S phase transition and/or the suppression of c-Abl function as a consequence of the perturbation of cyclin D1 expression. These results may impose a limitation on the clinical application of NC381 to tumors where pRb is functional. This caveat is further supported by studies using antisense or antibody inhibition of cyclin D1 expression in which it was demonstrated that effectiveness was limited to cells with active pRb (Tam et al., 1994; Lukas et al., 1995). However, in the NCI 60 cell line screen (our unpublished data), it was found out that NC381 possesses antiproliferative activity in tumor cells with pRb deficiency. This observation suggests that the effects of NC381 are not limited to the cyclin D1/CDK4/pRb pathway and that it may be a useful therapeutic agent in the treatment of a variety of tumors, with or without functional pRb. In this respect, there is evidence that CDK4 may play a role in G2-M transition (Gabrielli et al., 1999). The inhibition of cyclin D3 expression may cause alterations in cyclin D-dependent kinase (CDK4), via a pRb-independent mechanism, leading to a rate-limiting situation in the G2-M transition. The effects of NC381 on cyclin D3 expression are currently being explored in cell lines that do not possess functional pRb.
In addition to CDK other components in cell cycle regulation, i.e., the tumor suppressors p16Ink4 and p27Kip1 may also be modulated by NC381. In control cells, the levels of p16Ink4 and p27Kip1 were initially unaltered postserum stimulation but were down-regulated as a function of time in response to serum stimulation. This was as expected for cells progressing normally through the cell cycle. In contrast, the protein levels did not fall in NC381-treated cells upon serum stimulation. This finding suggests that NC381 modulates the expression of p16Ink4 and p27Kip1 and as a result acts as an inducer of tumor suppression. Although attractive, this possibility may be unlikely considering that the protein levels did not increase beyond basal levels at any evaluated time point. Alternatively, the observed effects may be due to stabilization or continued basal expression of these proteins, as a secondary consequence of most of the cells being arrested in the G0-G1 phase in the presence of NC381. Nevertheless, the stabilization of p16Ink4 level can contribute to the inhibition of CDK4 activity by sustained binding of p16Ink4 to CDK4 and by preventing the formation of CDK4-cyclin D1 complexes. In addition, CDK4/6-cyclin D complexes bind a family of cell cycle inhibitors, the Cip/Kip family, including p27Kip1, and the formation of resulting heterotrimeric CDK4/6-cyclin D-Cip/Kip complexes contribute to G1 progression by removal of these inhibitors from binding and inactivating CDK2-cyclin E complexes (Sherr and Roberts, 1999). The sustained levels of p16Ink4 may promote the dissociation of Cip/Kip inhibitors from CDK4/6-cyclin D complexes and force the redistribution of Cip/Kip inhibitors to the CDK2-cyclin E complexes, causing down-regulation of this kinase and contributing further to the cell cycle inhibitory effects of NC381 (Reynisdottir et al., 1995; Jiang et al., 1998). Studies are underway to elucidate the molecular mechanisms of NC381 action on the pathways involving these two tumor suppressor proteins.
A variety of tumors, including NSCLC, have disproportionate levels of genetic alterations in cyclin D1, CDK4/6, pRb, and p16Ink4 that are involved in regulating G1 restriction point traversal (Malumbres and Barbacid, 2001), whereas genes required late in G1 for controlling proper G1-S transition and initiation of S phase such as p27Kip1, cyclin E, and CDK2 are rarely altered during tumorigenesis (Ho and Dowdy, 2002; Ortega et al., 2002). Recent studies have demonstrated that strategies to target proteins associated with early cell cycle progression and the G1 restriction point may be useful in anticancer therapy (Hunter and Pines, 1994; Ortega et al., 2002). Overexpression of cyclin D1 has been found in a variety of human cancers, including breast tumors, esophageal cancer, colorectal carcinoma, head and neck carcinomas, and lung cell tumors (Weinberg, 1995; Sherr, 1996; Donnellan and Chetty, 1998). Furthermore, cyclin D1 has been shown to be a downstream target of several signal transduction pathways mediated by such oncogenes as Neu (c-erbB-2) (Lee et al., 2000), Ras (Robles et al., 1998), and -catenin (Tetsu and McCormick, 1999). Given that NC381 is capable of inducing cell cycle arrest through the inhibition of cyclin D1 expression and pRb phosphorylation, the observed antiproliferative activity of NC381 against a large panel of tumor types is not unexpected and further supports the development of NC381 as an alternative or complementary approach in the treatment of NSCLC. With this in mind, we evaluated the therapeutic effects of NC381 in mice harboring NCI-H460 human NSCLC tumor xenografts. These studies demonstrated adequate therapeutic efficacy without obvious signs of systemic toxicity as the gross examination of selected organs including the size of the liver and spleen did not show any obvious changes. Although hepatotoxicity may have been reduced by the removal of imidazole moiety, definitive demonstration for the lack of hepatotoxicity of NC381 requires systematic in vitro and histological studies, which are currently underway. In addition, possible adverse effects of NC381 on cardiac function as suggested for CLT (Thomas et al., 1999) need to be fully explored for NC381 to be a viable therapeutic agent. Nonetheless, the present study highlights the potential of NC381 and its analogs as effective therapeutic agents that act through a novel mechanism of action in the treatment of human nonsmall cell lung carcinoma.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: NSCLC, nonsmall cell lung cancer; CDK, cyclin dependent kinase; CLT, clotrimazole; FBS, fetal bovine serum; pRb, product of the retinoblastoma susceptibility gene; GI50, 50% of growth inhibition; Ink4, inhibitor of CDK4; Kip1, kinase inhibitory protein 1; Cip, CDK interacting protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.
Address correspondence to: Dr. Ming-Yu Cao and Dr. Yoon Lee, Lorus Therapeutics Inc., 2 Meridian Rd., Toronto, ON, Canada, M9W 4Z7. E-mail: mcao{at}lorusthera.com or ylee{at}lorusthera.com
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