A novel quinoline, MT477: suppresses cell signaling through Ras molecular pathway, inhibits PKC activity, and demonstrates in vivo anti-tumor activity against human carcinoma cell lines
- First Online:
- Cite this article as:
- Jasinski, P., Welsh, B., Galvez, J. et al. Invest New Drugs (2008) 26: 223. doi:10.1007/s10637-007-9096-x
- 152 Views
MT477 is a novel thiopyrano[2,3-c]quinoline that has been identified using molecular topology screening as a potential anticancer drug with a high activity against protein kinase C (PKC) isoforms. The objective of the present study was to determine the mechanism of action of MT477 and its activity against human cancer cell lines. MT477 interfered with PKC activity as well as phosphorylation of Ras and ERK1/2 in H226 human lung carcinoma cells. It also induced poly-caspase-dependent apoptosis. MT477 had a dose-dependent (0.006 to 0.2 mM) inhibitory effect on cellular proliferation of H226, MCF-7, U87, LNCaP, A431 and A549 cancer cell lines as determined by in vitro proliferation assays. Two murine xenograft models of human A431 and H226 lung carcinoma were used to evaluate tumor response to intraperitoneal administration of MT477 (33 μg/kg, 100 μg/kg, and 1 mg/kg). Tumor growth was inhibited by 24.5% in A431 and 43.67% in H226 xenografts following MT477 treatment, compared to vehicle controls (p < 0.05). In conclusion, our empirical findings are consistent with molecular modeling of MT477’s activity against PKC. We also found, however, that its mechanism of action occurs through suppressing Ras signaling, indicating that its effects on apoptosis and tumor growth in vivo may be mediated by Ras as well as PKC. We propose, therefore, that MT477 warrants further development as an anticancer drug.
KeywordsMT477Protein Kinase CRas-MEK-ERK pathway inhibitionCaspase-dependent apoptosisNew drug development
Protein kinase C
Glycogen synthase kinase-3β
PKC has been implicated in many malignancies with possible roles in tumor growth, differentiation, metastasis and apoptosis [3, 4]. Overexpression of several PKC isoforms has been shown to promote the progression of a variety of human malignancies [5–7]. In MCF-7 human breast cancer cells, PKCα regulates the cell-cycle and apoptosis, and overexpression of this isoform has been shown to induce malignant transformation and proliferation in these cells, leading to the development of an aggressive phenotype of breast cancer . In A549 human lung adenocarcinoma cells, it has been shown that overexpression of PKC̣ɩ is required for transformed growth of this malignancy . Both PKCα and PKCδ have also been shown to play important roles in proliferation and apoptosis of U87 human malignant glioma cells .
PKC activation has been linked to different downstream pathways active in tumor cell signaling. It may trigger signaling through direct activation of the Ras kinase pathway, which may be involved in the progression of tumor cell proliferation and inhibition of apoptosis [11–14]. PKC may also have a direct effect on Raf-1, a small GTP protein, and ERK1/2 kinase, suggesting that PKC may be involved in the Ras-Raf-1-MEK-ERK signaling cascade [15–17]. Goode et al. have shown that PKC may also activate glycogen synthase kinase-3β (GSK3β), which is linked to the development of tumor proliferation and progression .
Ras proteins belong to a large superfamily of low-molecular weight GTP-binding proteins. Ras proteins include the isoforms H-RAS, K-RAS and N-RAS. About 30% of all human tumors have mutations of a Ras gene [19, 20]. The activated form of Ras, which is bound to GTP, selectively interacts with several effector proteins, such as Raf, PI3K and Ral, and activates these molecular pathways . The biologic importance of Ras in malignant formation is mainly to deregulate tumor cell proliferation, survival, invasiveness and programmed cell death [21, 22]. Taken together, these reports suggest that PKC and Ras are important therapeutic targets in several types of human cancer.
In the present study, we examined the activity of MT477 in six cancer cell lines, assessed its mechanism of action in the H226 and A549 cell lines and evaluated its in vivo activity against A431 and H226 xenografts in mice.
Materials and methods
The following carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA): NCI-H226 (human squamous cell lung carcinoma) and A549 (human lung adenocarcinoma), MCF-7 (breast carcinoma), A431 (epidermoid carcinoma), LNCaP (prostate carcinoma) and U87 (brain glioblastoma). Cell lines were cultured and expanded in media according to ATCC guidelines.
Cell proliferation assay
Cell lines were plated at 5 × 103 cells per well in 96-well plates using an appropriate medium containing 10% FBS for each cell line, according to ATCC instructions. Cells were treated with several concentrations of MT477 (0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mM) for 1 h, and then the wells were washed with PBS before addition of fresh medium. After incubation for 24 h, cell viability was determined by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche, Penzberg, Germany). Absorbance was determined at 575 nm. The MT477 concentration required to inhibit growth of cancer cells by 50% was assessed using ADAPT Software . All experiments were performed at least three times with samples run in triplicate or six replicates.
H226 cells (3.0 × 106) were plated on tissue culture dishes (100 × 20 mm) in an RPMI medium containing 10% FBS, 2% HEPES and sodium bicarbonate, 1% sodium pyruvate, and 1% penicillin–streptomycin. Cultures were incubated with 0.05 mM of MT477 for 1, 2, and 4 h continuously. Cells were trypsinized, counted, resuspended in binding buffer (BD Biosciences, San Jose, CA) and incubated with 5μg/ml FITC-conjugated AnnexinV in the presence of 5μg/ml propidium iodide, according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Cells were screened by flow cytometer (Facscalibur, BD Biosciences, San Jose, CA). The FL-1 and FL-2 channels were used simultaneously to gate Annexin V-positive cells and PI-positive cells, respectively.
H226 cells (6.0 × 106) were plated on tissue culture dishes (100 × 20 mm) with regular medium containing 10% FBS. Cell cultures were treated with 0.05 mM of MT477 for 24 h continuously. For comparison, another cell culture was treated for 1 h with the same drug concentration, washed with PBS and incubated in fresh medium for 24 h. Cells were then trypsinized and transferred into a V-bottomed 96-well microplate (1.0 × 106 cells/well). Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at 25°C. Permeabilization of cells was achieved by incubation with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was then carried out according to the manufacturer’s instructions (In situ Cell Death Detection Fluorescein kit, Roche, Penzberg, Germany). Cells were screened by flow cytometer (Facscalibur, BD Biosciences, San Jose, CA).
Quantification of histone-complexed DNA fragments
H226 cells were plated in a 96-well, flat-bottom microplate (5,000 cells/well) and incubated over night in 5% CO2 and 37°C. Cells were then treated for 1 h with MT477 (0.200 mM, 0.100 mM, 0.050 mM, 0.025 mM and 0.012 mM), camptothecin (4,000 ng/ml), 1% DMSO or left untreated as a control. Cells were then washed, and all groups were incubated in fresh medium for 24 h. The Cell Death Detection ELISAPLUS (Roche Applied Science) assay was performed according to the kit instructions for all groups. Absorbance was measured at 405 nm on an ELISA plate reader using softmax pro software.
Apoptosis/caspase detection assay
Activated poly-caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9) in living H226 cells were detected using carboxyfluorescein-labeled fluromethyl ketone (FMK)-peptide-inhibitor substrate of caspases (Immunochemistry Technologies, LLC, Bloomington, MN). The Poly-Caspases (FLICA) activity kit was used as the FAM-VAD-FMK inhibitor. This is a carboxyfluorescein (FAM) derivate of valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK), a potent inhibitor of caspase activity. Cells (1 × 106 per well) were plated in a six-well plate with 2 ml of medium. Cells were treated with 0.05 mM of MT477 for 1, 2 and 4 h. The medium was then removed, and the FAM-VAD-FMK FLICA reagent was added in concentrations indicated in the manufacturer’s instructions. After 1 h of incubation, the suspended solution was removed. Cells were washed once with the wash buffer provided with the kit, tripsinized with 1 ml of 0.25% trypsin and resuspended in 2 ml of fresh medium. Cells were counted and spun. Supernatant was removed, and cells were resuspended in wash buffer at a concentration of 1 × 106 cells/ml. A fixative was added into the cell solution in 1:10 ratio. Cells were analyzed using a FACSCalibur machine, using CellQuest software (BD Biosciences, Franklin Lakes, NJ). H226 cells were irradiated with 10 Gy as a positive control for FLICA test, and camptothecin (4,000 ng/ml) was used as a positive control for AnnexinV-PI and TUNEL staining as well as for the ELISA assay.
Protein kinase C (PKC) inhibition assay
Inhibition of PKC isozyme activity by MT477 was determined using a peptide pseudosubstrate, which can be phosphorylated by PKC. The pseudosubstrate was pre-coated on an ELISA plate. Cancer cells (6.0 × 106) were plated on a 100 × 20 mm tissue culture dish in 10% FBS medium and treated with 0.05 mM of MT477 for 0.5, 1, 2, and 4 h. Cells were collected from the plate and centrifuged. Cell pellets were washed twice in ice-cold PBS and suspended in 1 ml cold sample preparation buffer (50 mM Tris–HCl, 50 mM β-mercaptoethanol, 10 mM EGTA, 10 mM Benzamidine, 5 mM EDTA, 1 mM PMSF, pH 7.5). Cells were then sonicated on ice four times for 10 s each and after that centrifuged at 100,000×g for 60 min at 4°C. Protein concentration was determined by BioRad protein assay (BioRad Laboratories, Hercules, CA). Samples (12 μl) containing equal amounts of protein were used in the PKC activity reaction, according to the manufacturer’s instruction (Protein Kinase Assay Kit, EMD Biosciences, Darmstadt, Germany). Adenosine 5’-Triphosphate, Disodium Salt (EMD Biosciences, Darmstadt, Germany), which was used for the reaction, was dissolved and prepared just prior to the assay. Color intensity was measured on a microplate reader at 492 nm, and the relative kinase activity (compared with untreated, baseline controls) of samples was calculated from absorbance measurements.
Immunoprecipitation assays and immunoblotting analysis
Cclls (3.0 × 106) were seeded in tissue culture dishes (100 × 20 mm) in 10% FBS medium and incubated with MT477 (0.05 mM) for 0.5, 1, 2 or 4 h. Cells were next resuspended in 1 ml of lysis buffer for 2 min, scraped and immediately snap-frozen in liquid nitrogen. For measurement of Ras, lystates were resuspended in Mg2 + /wash buffer (25 mM Hepes at pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA and 2% Glycerol). For assessment of ELK1 and ERK1/2, cell lysates were resuspended in cell lysis buffer (20 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). Protein concentration was determined with the BioRad protein assay (BioRad Laboratories, Hercules, CA).
Ras affinity precipitation was performed with Raf-1 RBD agarose beads according to the manufacturer’s instructions (RAS activation assay kit; Upstate, Temecula, CA). ELK1 affinity precipitation was performed with immobilized phospho-p44/42 MAP Kinase (Thr202/Tyr204) monoclonal antibody (p44/42 MAP Kinase Assay Kit, nonradioactive; Cell Signaling, Boston, MA).
Cell lysates were subjected to SDS-PAGE followed by electroblotting onto nitrocellulose membranes. The nitrocellulose membranes were incubated overnight at 4°C with one of the following primary antibodies: phospho-Elk1, anti-Erk1/2, phospho-Erk1/2, phospho-AKT (Cell Signaling, Boston, MA), anti-RAS (Upstate, Temecula, CA), and actin (Santa Cruz Biotechnology, Santa Cruz, CA). ). Phospho-GSK3β affinity precipitation was performed with immobilized Akt (1G1) monoclonal antibody and GSK-3 fusion protein (Akt Kinase Assay Kit, nonradioactive; Cell Signaling, Boston, MA).
Immunoreactive proteins were detected by incubating blots with alkaline phosphatase-conjugated secondary antibody for 1 h followed by ECF fluorescent substrate for 5 to 7 min. The Storm™ fluorescent scanning system (GE Healthcare, Piscataway, NJ) was used to visualize immunoreactive proteins.
Animal xenograft models
To determine the effect of MT477 on epidermal tumor and squamous cell lung tumor growth in vivo, A431 (5 × 106) and H226 (5 × 106) cells were injected subcutaneously into the right mediodorsal flank of 6-week-old male nude mice. Once the tumors reached a measurable size, animals were randomized into three groups: control group (injection of vehicle; DMSO in PBS), MT477 group (injection of 1 mg/kg, 100 μg/kg or 33 μg/kg of MT477 in A431 cells and one dose of MT477 1 mg/kg in H226 cells) and positive control group (injection of cisplatin 2 mg/kg). In A431 tumor xenografs, MT477 was injected intraperitoneally once on days 1, 4, 8, 16, 18 and 20. H226 xenografts were given one intraperitoneal injection of MT477 on days 1, 4, 8, 20, 24, and 28. Cisplatin (2 mg/kg) was used as a positive control at the same time-points as MT477 treatment in both xenograft models. Each group consisted of six to eight mice. Tumor size was determined with a caliper, and tumor volume was calculated by using the formula (a × b × c)/2, where a and b are the shorter and longer diameter of the tumors and c is the depth. All experiments with animals were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.
An unpaired Student’s t test was performed and two-tailed P values are reported comparing experimental and control groups in in vitro studies. For tumor volume calculations, log volume data were analyzed with one-way ANOVA and assessed by Tukey–Kramer’s method using Instat Software (GraphPad Software, Inc., San Diego, CA).
Antiproliferative effect of MT477
Proapototic activity of MT477
To elucidate the time-dependent effect of MT477 on apoptosis, H226 cells were treated with 0.05 mM of MT477 for 1 and 24 h. Apoptotic cells were quantified by counting TUNEL-labeled cells. A significant percentage of cells underwent apoptosis after 1 h of treatment (18.6 ± 4.9%) and after 24 h of treatment (98 ± 0.2%; p < 0.003 vs. untreated control; Fig. 3c), suggesting that MT477 strongly induces cell apoptosis and cell necrosis in a time-dependent manner.
To evaluate the dose-dependent effects of MT477 on cell death, we measured oligonucleosomal fragmentations using cell death detection ElisaPLUS. H226 cells were treated for 1 h with MT477 concentrations between 0.012 and 0.200 mM and incubated for 24 h with fresh medium. These analyses showed that MT477 also induced cell apoptosis at drug concentrations as low as 0.012 and 0.025 mM (Fig. 3b).
To evaluate how MT477 may influence the main downstream effectors of PKC, we evaluated the phosphorylation status of AKT, GSK3β, Ras, and ERK pathways after incubating H266 cells with 0.05 mM of MT477 for 0.5, 1, 2 or 4 h. Notably, Ras-GTP protein levels were reduced even after 0.5 h of treatment (Fig. 5b), suggesting that MT477 may have a direct effect on the Ras protein because PKC is first inhibited after 1 h of treatment (Fig. 5b). Erk phosphorylation was inhibited beginning at 1 h and persisting for 4 h. Similarly, Elk1 phosphorylation declined during the same time period, which would be expected because Elk1 is a downstream target of the ERK1/2 kinase pathway (Fig. 5c). Western blot analysis of p-AKT and p-GSK3β showed no changes in phosphorylation levels (data not shown).
Because MT477 appears to act through a Ras-mediated pathway, we next evaluated the effect of MT477 in the K-Ras mutated A549 cell line. A549 is a non-small lung carcinoma cell line that has an oncogenic K-Ras mutation [24, 25]. We observed a reduction in the level of Ras-GTP in A549 cells exposed for 60 min to 0.05 mM of MT477 (data not shown).
MT477 inhibits tumor growth in xenografts
MT477 is a novel quinoline (Fig. 1) discovered by computer simulation to be a potentially potent anti-cancer molecule. The results of our study show that MT477 strongly inhibits the growth of A431 squamous cell skin carcinoma and H226 squamous cell lung carcinoma cell lines (Fig. 6a–b). Within an hour, MT477 dramatically induces apoptosis of H226 lung cancer cells involving activation of the caspase cascade (Fig. 4). Annexin V/PI assay also implicated apoptosis as the main mechanism underlying these growth inhibitory effects, although indications of apoptosis were only evident after 2–4 h of MT477 treatment (Fig. 3a). Our dose-dependent data (Fig. 3b) show that histone complexes in MT477-treated groups were present even at low molecular doses, suggesting that MT477 has robust anti-tumor activity in mice, even at doses that reasonably approximate plasma concentrations close to the IC50. Results from our TUNEL assay, which preferentially labels apoptosis in comparison to necrosis [26, 27], confirm the proapoptotic activity of MT477 after 1 h of treatment, as well as near-complete cell cytotoxicity after continuous treatment for 24 h (Fig. 3c). Within 1–2 h of MT477 treatment, we also observed a large number of cells detach from the dish surface in tissue culture experiments (data not shown), which may be caused by the destruction of the cytoskeleton during cell death.
Cell apoptosis is characterized by fragmented nuclei with condensed chromatin and in most cases, in contrast to necrosis, is mediated by caspases that are activated by extrinsic or intrinsic pathways [27–29]. However, cell necrosis causes rapid loss of plasma membrane integrity without severe nuclear damage and initiates, in contrast to apoptosis, inflammation and tissue damage . Our previous study on caspase-3 activation (data not shown) showed no changes after 1, 2 or 4 h of MT477 treatment; however, a FLICA assay (Fig. 4), which detects caspase-1, -3, -4, -5, -6, -7, -8 and -9, clearly suggests the involvement of different proteases in the molecular pathway of MT477. Our findings demonstrate apoptosis-specific caspase activation after 1, 2, or 4 h of treatment, as well as the induction of apoptosis and necrosis after longer, continuous treatments of 2, 4 or 24 h. Further studies are needed to clarify the multiple signaling pathways of apoptosis induction by MT477.
To confirm the mechanism of action of MT477 suggested by in silico analyses, we first explored how MT477 may interfere with PKC activity. Our in vitro kinase assay demonstrates that MT477 significantly reduces PKC activity (Fig. 5a), which led us to evaluate the downstream targets of PKC. We first determined the phosphorylation status of phospho-AKT and p-GSK3β, both downstream molecules of PKC. However, treatment of H226 cells with 0.05 mM of MT477 resulted in no changes in the phosphorylation status of p-AKT or p-GSK3β after 1–4 h of treatment (data not shown), which suggested MT477 may be involved in a different branch of the PKC pathway. Ras-GTP protein is another possible direct downstream protein of PKC [13, 30, 31]. We found that MT477 had an inhibitory effect on Ras-GTP, even 0.5 h after treatment. We also established a correlation between phosphorylated ERK1/2 and ELK1, confirming the possible inhibition of small active molecule Ras (Fig. 5b–c). However, the initiation of Ras inhibition (0.5 h) occurred before the onset of PKC inhibition (1 h), suggesting a direct inhibitory effect of MT477 on the small molecule Ras. The ERK1/2 pathway, a major downstream pathway of Ras, may also be directly affected by PKC [15, 32, 33]. This raises the possibility that MT477 may inhibit the ERK1/2 pathway through direct inhibition of both PKC and Ras. Erk1/2 inhibition resulted in a lower phosphorylation status of the transcription factor Elk1 (Fig. 5c), which is specifically activated through the ERK1/2 molecular pathway and is strongly implicated in regulating c-fos gene expression. Targeting c-fos expression may be a promising strategy for treating many different human cancers, such as sarcomas [34, 35], breast cancer  and epitheloid adenocarcinoma ; therefore, indirect Elk1 inhibition by MT477 may represent a critical finding of our study.
We were surprised to find evidence indicating MT477 directly inhibited Ras-GTP phosphorylation in the H226 cell line. Tumor resistance to antineoplastics is often associated with mutations of Ras. We therefore decided to study the effect of MT477 inhibition of Ras activity in the K-Ras mutated A549 cell line. We found that Ras activity was inhibited within 60 min of exposure to MT477 (0.05 mM). This finding is consistent with the dramatic drop in A549 proliferation seen at this same dose and time of exposure. These findings support the notion that MT477 blocks overactive Ras signaling and suggest that MT477 may be able to overcome Ras-mediated tumor survival by inhibiting Ras. MT477’s activity in a K-Ras mutated carcinoma is clearly relevant to clinical applications. Activating mutations in codon 12 of the K-Ras gene are the most common genetic alterations in many malignancies, though it is possible that MT477 is active against H-Ras and N-Ras isoforms as well.
Molecular topology studies using QSAR analysis predicted that MT477 would target PKC. MT477 was only one of many molecules defined by Dr. Galvez and Medisyn Technologies as having specific anti-cancer activity. Data gathered in our experiments enrich the predictions regarding the mechanism of action of topologically similar chemical structures and can be used in refining the QSAR computer model. The unexpected finding that MT477 may target Ras also raises the possibility of drug specificity, and we are currently assessing other possible molecular targets of MT477 in cancer cell lines and normal tissue.
In conclusion, we show that MT477 exhibits a direct effect on human tumor cells in vitro and in vivo, inducing apoptosis and suppressing proliferation of a wide array of cultured human tumor cells. MT477 targets Ras/Raf/MEK/ERK/ELK and PKC pathways, which are strongly involved in growth, differentiation, proliferation, transformation and cell cycle control in mammalian cells [38–42]. In addition, this study confirms the predictions of an in silico model based upon the approach of Galvez et al. . We believe that further development of MT477 as an anti-cancer drug is warranted.
This study was partially supported by Experimental Therapeutics Fund from University of Minnesota (we are grateful to Audrey and Denis Anderson for ongoing support for this Fund) and a grant from Medisyn Technologies. We would like to thank Michael Franklin for editorial support.