The Potential Targets and Mechanisms of a Carbazole and Pyrazole Containing Anticancer Compound

Abstract

Aims:

Characterization of a small anticancer compound.

Background:

The development of small molecules as new anti-cancer therapeutics is necessary to improve anti-tumor efficacy and reduce toxicities, especially for the treatment of brain tumors, where only small molecules can effectively cross the brain-blood barrier. Several novel hits were previously selected by concurrently screening colon and glioma cancer cell lines with a sensor-conjugated reporter system. Here, we focused on one of them.

Objective:

Elucidating the potential target(s) of a novel anticancer compound.

Methods:

Computer-assisted structural and motif analysis (least absolute shrinkage and selection operator or LASSO score) was used to assess compound’s targets, then direct kinase activity assays were used for the confirmation; Western blot of phosphorylated kinases, as well as FACS and caspase 3/7 activity assays, were used to decipher the action mechanisms. Finally, the expression profiling of proteins involved in various G-protein pathways by real-time PCR was performed.

Result:

The small chemical, (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl-4,5-dihydro-1H-pyrazol-5-one, with a formula C18H17N5O and MW of 319.36, designated as VUGX01, was predicted to be a ligand/inhibitor to receptor tyrosine kinases (RTKs) by computer analysis (least absolute shrinkage and selection operator or LASSO score). However, direct analysis with recombinant kinases showed that it is not an effective inhibitor to the popular receptor kinases at 1μM concentration. This compound can activate caspases in some tumor cell lines but has minimal effects on the cell cycle. Drug treatments lead to the changes in phosphorylation of AKT and c-RAF, as well as the expression level of MAP2K, suggesting this compound may interact with G-protein coupled receptors (GPCRs). The expression profiling of 82 proteins involved in various G-protein pathways by real-time PCR showed that the treatment up-regulates the expression of several proteins, including angiotensinogen, angiotensin II receptor, and IP3-kinase catalytic subunit gamma.

Conclusion:

VUGX01 can effectively block proliferation and induce apoptosis of certain types of cancer cells, even it is predicted by high LASSO score, but it is not an effective RTKs inhibitor, it may inhibit cell growth through acting as a novel ligand to one or several GPCRs.

1. INTRODUCTION

Two major approaches for drug development begin with either target-based or phenotypic-based screening of a library of compounds. In the past 25 years, molecular target-based screening has become the most common approach to early drug discovery. G protein-coupled receptors (GPCRs), ion channels and enzymes have been the most common and successful molecular targets for drug discovery [1]. However, most lead compounds discovered by a target-based approach have shown poor drug efficacy in the later stages of development. As a result, there has been renewed interest in the use of phenotypic screening for drug discovery [2]. Phenotypic screening, also known as ‘forward pharmacology’ or ‘forward chemical biology’ [3], is usually more physiologically relevant because intact cells and native cellular environments are used [2a, 4]. In a previous study, a phenotypic screening reporter system based on controlled and self-amplified protein degradation was developed [5]. Using that approach, we concurrently screened approximately 4000 compounds against both colon and glioma cancer cell lines [6]. Several cardiac glycosides used as cardiotonic drugs were found to be specific and effective for killing colon cancer cells. By contrast, statins (hypolipidemic agents) used as cholesterol-lowering drugs are relatively more effective for killing glioma cells. In addition, several novel hits of unknown properties and targets were also identified. To facilitate future studies of structure–activity relationships (SAR) and the optimization of lead candidates, we partially characterized one compound, (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl-4,5-dihydro-1H-pyrazol-5-one, with a formula C18H17N5O and MW of 319.36, which consist with a carbazole and pyrazole linked by hydrazine, and was predicted by computer analysis (LASSO score) to be a ligand/inhibitor to receptor associated kinases, and was designated as VUGX01. However, further experimental studies did not fully support this prediction, and suggest that it is more likely to inhibit cell growth by acting as a novel ligand to one or several G-protein coupled receptors (GPCRs) or through other mechanisms.

GPCRs regulate many fundamental biological processes, such as growth, metabolism and homeostasis [7]. Tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin-releasing peptide receptor, have been well studied [8]. GPCRs’ potential roles as anti-cancer targets remain under-exploited but have been considered to be among the most useful drug targets against many solid tumors. Thus, the development of selective ligands directly targeting GPCRs, or more selectively targeting specific downstream signaling components, may provide novel and effective treatment strategies against cancers.

2. MATERIALS AND METHOD

2.1. Cell Culture

Rat glioma C6 and human HEK293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA), and human colon cancer cells DLD1, Difi, and HCT116 were provided by one of our collaborators (Dr. Robert J Coffey, Vanderbilt University Medical Center, Nashville, TN). All cells used in the lab were registered and approved by the Vanderbilt University Medical Center Institutional Biosafety Committee (Approval #. VBMR-0150). All cells were cultured in DMEM medium supplemented with 10% FBS and 50 units/ml penicillin and 50μg/ml streptomycin (Invitrogen, CA) under standard culture conditions in a humidified incubator maintained at 5% CO2 and 37°C.

2.2. Compound Library

VUGX01 was purchased from Aldrich Market Select (Sigma-Aldrich, St. Louis, MO), which provides customized syntheses of chemicals at a small scale, and the purity and MW were verified by mass spectroscopy.

2.3. Kinase Analysis

The compound was tested in single-dose, duplicate mode at a concentration of 1μM. Staurosporine was used as a control compound, tested in 10-dose IC50 mode with 4-fold serial dilution starting at 20μM. Reactions were carried out in reaction buffer (20mM Hepes (pH 7.5), 10mM MgCl₂, 1mM EGTA, 0.02% Brij35, 0.02mg/ml BSA, 0.1mM Na3VO4, 2mM DTT, 1% DMSO) plus tested recombinant kinases. ³³P-ATP (specific activity 10μCi/μl) was added into the reaction mixture to initiate the reaction. The kinase reactions lasted 2 hours at room temperature and were terminated by spotting onto P81 ion exchange paper. Protein kinases and their assays were provided and performed by Reaction Biology Corp. (Malvern, PA).

2.4. Caspase Assay

VUGX01 (VU0007775) was prepared at 20mM in DMSO. Glioma cells (C6), colorectal cancer lines (Difi, DLD1, HCT116), and human embryonic kidney cells Hek293, were each plated in multiple wells (1×10⁴ cells per well) of 96-well plates. 24 hours after the seeding, VUGX01 or Cetuximab (purchased from Vanderbilt Hospital Pharmacy) was added into the wells of the plates for a 24 hours treatment, then apoptosis assays based on Caspase 3/7 activity were performed (caspase-Glo kit, Promega, Madison, WI) according to manufacturer-recommended procedures.

2.5. FACS Analysis

1×10⁶ each of control or treated cells at different time point were trypsinized to single-cell suspensions. After washing with PBS, cells were resuspended with 500μl of 1% BSA in PBS, and then fixed with 3x volume of cold (−20 °C) absolute ethanol and kept at −20 °C for at least one hour. The cells were then spun down and washed twice with PBS buffer, and resuspended with propidium iodide (PI) staining solution (3.8mM sodium citrate, 50μg/ml PI, 0.5μg/ml RNase A in PBS) to a final cell concentration approximately 1×10⁶ cell/ml, and incubated 3 or more hours at 4 °C before FACS analysis. Samples were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA) flow cytometer with multiple lasers. Propidium iodide fluorescence was collected using a 617nm emission filter with excitation at 535nm. A minimum of 10,000 cells was analyzed per sample. The data were collected and analyzed with CellQuest software.

2.6. SDS PAGE and Western Blot

Cells were collected by trypsin/EDTA treatment and washed with PBS, and lysed on ice for 15 minutes with MPER Mammalian Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA), supplemented with a cocktail of protease inhibitors (Sigma, St. Louis, MO) at a concentration of 10⁷ cells/ml. Nuclei and insoluble debris were cleared by centrifugation at maximum spin (17000rpm) with a microcentrifuge for 30 minutes. Protein concentration was determined by the Bradford method (Bio-Rad Protein Assay Kit, Bio-Rad). 30μg of total cell lysate proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were then pre-blocked with 5% non-fat milk in TBST and then incubated overnight at 4°C with various primary antibodies as indicated. All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA), and diluted with 1% BSA in TBST. After washing three times with TBST, the corresponding HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) were added and incubated for 1hr at room temperature. The protein bands were visualized by enhanced chemiluminescence (Pierce, Rockford, IL). As a loading control, the same membranes were also blotted with β-tubulin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

2.7. Profiling of GPCR Signaling Pathway

GPCR signal pathway profiling was performed by analyzing the expression level of key genes in the GPCR pathway with or without the treatment of compound. The expression levels were determined by using real-time quantitative PCR. A GPCR Signaling PCR Array (PathwayFinder RT2 Profiler) with 82 selective genes was purchased from Qiagen (Valencia, CA). The total RNA was extracted from C6, DLD1, HCT116 and Difi cells treated with or without 5μM of VUGX01 for 48 hours, and RNAs were reverse transcribed to cDNA. cDNAs from DLD1 cells were analyzed with a 96-well PCR array plate containing primer pairs of 82 human GPCR pathway genes. Comparison (subtraction) analysis of GPCR gene expression between control and treated cells was performed by online software provided by Qiagen. Positive candidates were further confirmed by using cDNAs from rat C6 and three colon cancer cell lines with primer pairs in different coding regions. Real-time PCR was performed with SYBR Green PCR reagent from Bio-Rad by following the recommended procedure.

3. RESULTS

3.1. VUGX01 Structure and LASSO Analysis

Our previous screening led to several novel hits [6], here we focused on one compound designated as VUGX01 which can inhibit the growth of both DLD1 and C6 cells through apoptosis (more effective toward DLD1 cells), a selective example of the compound on DLD1 cell proliferation is provided in the (Fig. 1) of supplemental data. The full molecule name of VUGX01 is (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl-4,5-dihydro-1H-pyrazol-5-one, with a formula C18H17N5O and MW of 319.36. The structure of VUGX01 is illustrated in Fig. (1), which consists of a carbazole and pyrazole linked by hydrazone. Carbazole skeleton has been the key structural motif of many biologically active compounds in natural and synthetic products [9], such as kinase inhibitor [10]. Pyrazole is also a well-known heterocyclic compound with a variety of bioactivities, including anticancer, angiotensin-converting enzyme (ACE) inhibitory, cholecystokinin-1 receptor antagonist, and estrogen receptor (ER) ligand activity [11]. This compound was predicted to be a potential ligand to several receptor tyrosine kinases (RTKs) based on computer-assisted structural analysis. The structural analysis system used was developed by Simulated Biomolecular Systems Inc (Etobicoke, Canada), and it uses a systematic search algorithm to find the minimum energy pose for a ligand in proteins’ binding pockets. By screening active protein motifs from public databases, it generates a score (ranging from 0 to 100, the higher the score, the higher the probability) for a bioactive molecule to match different motifs [12]. As summarized in Table 1, epidermal growth factor receptor (EGFr) and cyclin-dependent kinase 2 (CDK2) are two kinases with top LASSO (least absolute shrinkage and selection operator) scores.

Fig. (1).

Structure of VUGX01 (C18H17N5O MW 319.36), Full name (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl-4,5-dihydro-1H-pyrazol-5-one, which consist with a carbazole in the left end and pyrazole in the right end, linked by hydrazine in the middle.

Table 1.

Predicted potential targets for VUGX01 based on structural analysis.

Category	Target Code	Protein Name	PDB Code	LASSO Score
Kinases	EGFr	Epidermal growth factor receptor	1m17	0.97
Kinases	CDK2	Cyclin dependent kinase 2	1ckp	0.90
Kinases	VEGFr2	Vascular endothelial growth factor receptor	1vr2	0.85
Kinases	SRC	Tyrosine kinase SRC	2src	0.81
Kinases	PDGFrb	Platelet derived growth factor receptor kinase	N/A	0.70
Kinases	FGFrl	Fibroblast growth factor receptor kinase	1agw	0.39

3.2. VUGX01 is not an Effective Inhibitor of Predicted or other Popular Protein Kinases

LASSO structural analysis predicted VUGX01 to be a potential ligand to several receptor tyrosine kinases, so we directly tested if VUGX01 can act as an inhibitor to these kinases. Since most commercialized or FDA approved EGFR kinase inhibitors have an IC-50 in sub μM range, therefore VUGX01 was tested in single doses at a concentration of 1μM for its effects on ten well-known kinases. The assay was performed as duplicates and repeated once a day. Staurosporine was used as a positive control in all the assays to assure the tested kinase was active and the assay condition was optimized. The data are shown in Table 2, VUGX01 shows minimal effects on EGFr (highest LASSO scorer) but did show some (5 to 6%) inhibition on CDK2, as well as to fibroblast growth factor receptor 2 (FGFR2) and platelet-derived growth factor receptor alpha (PDGFRα) to a smaller degree. By comparison, staurosporine exhibits more than 50% inhibition on these kinases at lower concentrations.

Table 2.

Effects of 1μM VUGX01 on the Activities of Selected Kinases.

-	% Enzyme Activity (Relative to DMSO Controls – 100%)		IC50 (μM) Staurosporine
Kinase:	Data 1	Data 2
c-Src	99.17	98.97	3.15E-03
CDK2/cyclin A	94.36	94.21	1.09E-03
EGFR	99.09	97.84	1.09E-01
ERBB2/HER2	99.34	98.15	8.83E-02
FGFR1	106.68	104.35	6.50E-03
FGFR2	96.10	94.89	2.50E-03
FLT1/VEGFR1	100.42	99.75	1.21E-02
KDR/VEGFR2	98.30	97.95	1.28E-02
PDGFRa	96.32	96.21	1.10E-03
PDGFRb	100.49	99.88	3.95E-03

3.3. VUGX01 Causes Tumor Cell Death Partly through Activation of Caspase

Next, the effect of this compound on cellular caspase activities on selected cell lines has been analysed. As shown in Fig. (2A), VUGX01 can greatly activate caspase 3/7 in colorectal DLD1 cancer cells, but with minimal effect on another colon cancer line HCT116 up to 5μM concentration, and with only slight activation of caspase in HEK293 cells. Cetuximab has been well studied and acts as a ligand to EGFR in colon cancer cells and can effectively induce apoptosis, so we compared the effect of VUGX01 with Cetuximab on Difi cells, as shown on Fig. (2B), VUGX01 is less potent but is effective in the activation of caspase in Difi cells. By contrast, VUGX01 can also effectively activate caspases in rat brain glioma C6 cell lines, where Cetuximab shows no effects at any of the tested concentrations (Fig. 2C).

Fig. (2).

VUGX01 induced caspase activities on cancer cell lines. All cells (1×10⁴) were plated in multiple wells of 96-well plate. 24 hours after seeding, DMSO or indicated concentration of VUGX01 or Cetuximab were added to multiple wells of these cells. 24 hours later, the cell caspase activities were quantified by using Promega’s caspase-Glo kit. No drug treatment (DMSO alone) group was normalized as 1, data from HCT116, DLD1 and HEK293 treated with three concentrations (0.2, 1 and 5μM) were plotted in Panel A. In Panel B and C, VUGX01’s effect on Difi (B) or C6 (C) cells were compared to those from Cetuximab. All the data from the treatment group were normalized against their individual cell line’s non-treatment controls. Each data point was the average from 6 sample wells; standard deviations were shown as error bars. p-value of each data set is less than 0.05.

3.4. The Effect of VUGX01 on the Cell Cycle of Cancer Cells

To explore the molecular mechanism and pathway of VUGX01’s action, the effect of the compound on the cell cycle distribution of several cancer cell lines was then assessed, as summarized in Fig. (3). VUGX01 had minimal effects on G1 or G2/M phases for all the cell lines at low concentrations. At 5μM concentration, VUGX01 shifts the cell population from G1 to G2 phase of HEK293 cells. In addition, VUGX01 caused a noticeable amount of cell death in HEK293 cells as demonstrated by the cells in sub G0 phase (see quantification of cell cycles in Table 1 of supplemental data).

Fig. (3).

FACS analysis of cell cycle distribution treated with different concentrations of VUGX01 for 48 hours. 1×10⁶ each of control or treated cells at different time point were collected, fixed and stained with propidium iodide (PI). Samples were analyzed on a flow cytometer. A minimum of 10,000 cells was analyzed per sample.

3.5. VUGX01 Affects the Phosphorylation AKT and c-raf

Cell proliferation and apoptosis are highly regulated processes, and kinase phosphorylation plays an essential role in each pathway. Next, we tested if VUGX01 treatment can lead to a change of phosphorylation status. Western blots were used to examine the expression and phosphorylation level of several key proteins involved in cell signaling pathways. As shown in Fig. (4A), lysate from untreated and treated C6 cells was analyzed for S6, phosphorylated S6 protein, poly (ADP-ribose) polymerase (PARP), total AKT and phosphorylated AKT at serine 473, and LC3B (autophagy-related). For all the tested proteins, the only noticeable change in the treatment group was a minor increase in the phosphorylation of AKT at serine 473. Guided by this clue, several other protein kinases were further tested in other cell lines after treatments at different concentrations. As shown in Fig. (4B), VUGX01 had no effect in HEK293 cells on tested proteins. However, in colon HCT116 and DLD1 cells, p-c-raf (Ser259) and map2k increased at low concentrations and decreased at high concentrations, and similar results were observed in C6 cells. For phosphoAKT (ser473), there were very weak bands in colon cells, VUGX01 treatment may slightly reduce Akt phosphorylation, had no effects on the expression level of total AKT. Surprisingly, in C6 cells, when active (phosphorylated) Akt is constitutively displayed at a high level, VUGX01 furth0er slightly increased the level of phosphorylation of AKT at serine 473. Attempts to detect the phosphorylation status of AKT at threonine 308 were not successful.

Fig. (4).

Western blots analysis. Whole-cell lysates were extracted from various cell lines (C6 in Panel A, HCT116, HEK293, DLD1, C6 in Panel B) treated with the indicated concentration of VUGX01 for 48 hours. 30μg of protein were separated by SDS-PAGE and blotted with antibodies against different antigens as labeled on the right. Tubulin was used as loading controls.

3.6. GPCR Signaling Pathway Profiling

Western data suggest the potential involvement of Akt/mTOR, which acts as the central node of many receptor signaling pathways, including RTKs and GPCRs. After we ruled out that RTKs are not likely a direct target of this compound, we investigated if another major membrane-associated receptor - GPCRs may be the targets of VUGX01; therefore the expression levels of 82 key genes (listed in supplemental Table 2) in various GPCR pathways with or without compound treatment were analyzed by real-time quantitative PCR. The relative expression profiles of those 82 genes in human DLD1 cells are plotted in Fig. (5A). As expected, a majority of genes show no or minimal changes, which are represented as dark black spots. VUGX01 treatment causes mostly overexpression of some tested genes. 15 of them show statistically significant differences, they fall outside the two boundaries of lines and are labeled as gray spots (Fig. 5A); their gene names and the expression changes are listed in Fig. (5B). Two PCR primer pairs (each pair for rat and human respectively) in different coding regions were re-synthesized for these preliminarily selected genes (info of primer pairs is provided in Table 3 of supplementary data), and real-time PCRs were performed for further confirmation of these genes by using cDNAs from four cell lines (C6, HCT116, DLD1, and Difi). 9 of them (AGT, AGTR1, AGTR2, GCGR, MMP9, PIK3CG, PTGS2, SCTR, TSHR) were found be elevated by VUGX01 treatment in all 4 cell lines (Table 4 of supplementary data). These results suggest that VUGX01 may directly or indirectly interfere with GPCR pathways.

Fig. (5).

GPCR Signaling Pathway Profiling. 82 selective genes in PathwayFinder RT2 Profiler PCR Array was screened by cDNA from DLD1 (control group) and VUGX01 treated group), their relative expression level was plotted against each other, genes corresponding to the spots outside triple lines (represented by gray light-colored spots) was considered to be significant in their expression difference (Panel A). Positive candidates were listed Panel B, they were further confirmed by another primer pairs by using cDNAs from rat C6 and three colon cancer cell lines. 9 genes marked with bold fonts were confirmed and showed significant change induced by VUGX01 treatment in all four cell lines.

4. DISCUSSION

Our previous screening of 3920 compounds from the Spectrum and unbiased libraries led to several new hits [6]. Here we focused on one chemical which mainly causes apoptosis in DLD1 cells. The computer-based structural analysis suggested that it can act as a potential ligand to several receptor types of the kinase (Table 1). However, direct kinase assays do not support this possibility. VUGX01 showed only minor inhibition to CDK2 and PDGFRa (Table 2) at 1μM concentration (for comparison, staurosporine has an IC50 in nM range for most of the tested kinases, see Table 2), and suggested that it is not an effective inhibitor. Most kinase inhibitors act by binding kinase domains of enzymes and blocking the transfer of a phosphate group to their substrates (target proteins). Computer analysis can predict the association of a small molecule to known kinase domains based on fitness, but the affinity or the capability to block activity is mainly determined by the molecule’s subtle structural features. VUGX01 seems not only to bind to these popular kinase domains with high affinity, but it may still represent the backbone of a new class of kinase inhibitors. It is possible that the inhibition of these kinases may increase with increased compound concentrations, CDK2 and other kinases may, therefore still be potential targets of VUGX01 at the current form. It has been believed that additional optimization or modification is needed to make this molecule as an effective kinase inhibitor at sub-uM concentrations.

To explore the molecular mechanism by which VUGX01 inhibits the growth of tumor cells, we analyzed the effect of VUGX01 on cellular caspase activities in several cell lines. VUGX01 can greatly activate caspase 3/7 in colorectal cancer DLD1 cells, has less impact on Difi cells, and minimal effects on HCT116 cells. EGFR is overexpressed on Difi cells, and Cetuximab acts as a strong apoptosis inducer for Difi cells [13], but it has almost no effects on C6 glioma cells, so quite likely, VUGX01 has different targets than those of Cetuximab. Along with the negative results from direct kinase assay, these data suggest that EGFR is not likely the target of VUGX01. However, computer analysis (with second-highest LASSO score of 70) and direct kinase assay (highest kinase inhibition of 6% at 1μM) indicate that CDK2 is a potential target, therefore the effects of VUGX01 on the cell cycle distributions of several cancer cell lines were examined. The data show VUGX01 had minimal effects on the cell cycles for both colon and glioma cells. It increases the cell population only in the G2 phase and causes a noticeable amount of cell death for HEK293 cells. This cell death is unlikely the result of apoptosis by an external pathway since caspase 3/7 activation in HEK293 is lower than in DLD1 cells.

To decipher the molecular mechanisms of VUGX01’s action, the expression level and phosphorylation status of several key proteins were examined. These proteins (S6, p-S6, parp, AKT, p-AKT, LC3B) play essential roles in cell proliferation, signaling pathways, DNA repair and cell death. The results show that VUGX01 had no direct effect on cell proliferation and cell death by the autophagy pathway (no effect on S6, Parp and LC3B) in C6 cells. The only noticeable difference was p-Akt. Akt, also known as protein kinase B (PKB), sits in a central node of a signaling pathway consisting of many components that play critical roles in cell growth and tumorigenesis, whose upstream components include GPCRs and phosphatidylinositol 3-kinase (PI3K). Akt activation has been observed in many cancers [14]. When correctly positioned at the membrane via binding of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), Akt can be phosphorylated by its activating kinases, phosphoinositide-dependent kinase 1 (PDPK1 at threonine 308) and the mammalian target of rapamycin complex 2 (mTORC2 at serine 473) [15]. Subsequently, AKT activates mTOR kinase, which phosphorylates p70 S6 kinase (S6K), and in turn, S6K phosphorylates the ribosomal protein S6 to increase translation of mRNAs and protein synthesis. However, in C6 cells, the increased p-AKT (which generally is associated with promoting proliferation or anti-apoptosis) after VUGX01 treatment did not lead to increases of either S6 or p-S6, so there must be an alternate pathway or VUGX01’s action may be neutralized by a different mechanism.

Next, along with AKT, two protein kinases upstream of AKT were further tested in four cell lines after being treated by VUGX01 at different concentrations. The results show VUGX01 had no effect in HEK293 cells for these tested proteins but causes p-c-raf (Ser259) and map2k to go up in the colon (HCT116 and DLD1) and glioma C6 cells. These results are consistent and indicate that VUGX01 targets some upstream molecules in the signaling pathway of c-raf and map2k. By observing that most of the VUGX01 (based on its brownish color) is outside cell membranes, we speculated VUGX01 likely interacts with some cell surface receptors. G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular effector pathways and are major membrane receptors (with about 900 members). By using Qiagen’s GPCR pathways finder array (with 82 key genes in various pathways), we found that 9 of them (AGT, AGTR1, AGTR2, GCGR, MMP9, PIK3CG, PTGS2, SCTR, TSHR) were elevated by the treatment of VUGX01 in all 4 cell lines. These results suggest that Rhodopsin-Like receptors may be the target of VUGX01, and in turn, it interferes with the protein serine/threonine kinase signaling pathway. GPCRs regulate a wide variety of normal biological processes and play a role in the pathophysiology of many diseases upon dysregulation of their downstream signaling activities. GPCR ligands include neurotransmitters, hormones, cytokines, and lipid signaling molecules. However, the unknown ligands for many so-called “orphan” GPCRs are still actively being sought. GPCRs with known ligands represent the most prominent family of validated pharmacological targets in medicine [7]. Only a small number of these GPCRs are targeted by current drugs, so efforts are currently being made to exploit the remaining receptors, including approximately 120 members for which no existing ligands have been identified [16].

There is multilayered crosstalk between GPCRs and growth factor receptors, which regulate cancer growth, angiogenesis and metastasis. Molecules such as estrogen can cause trans-activation of EGFR through the autocrine and paracrine release of EGF-like ligands tethered at the cell surface, and the subsequent generation of intracellular signaling promotes cancer progression [17]. Indeed, the functional crosstalk between GPCRs and EGFR contributes to the progression of breast and other cancers [18]. Different effects of VUGX01 on various cell lines may be caused by its non-specific interaction with more than one target, so future efforts should be made to characterize its target, modify or optimize the structure to increase the specificity to a single target, reduce or mitigate side effects and improve their anticancer ability.

CONCLUSION

A novel anticancer chemical compound ((4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl-4,5-dihydro-1H-pyrazol-5-one, VUGX01) has been discussed here. It can effectively block proliferation and induce apoptosis of certain types of cancer cells. Structural analysis suggests that this compound can be a ligand/inhibitor to several receptor tyrosine kinases, but direct kinase assays showed that it is not an effective RTKs inhibitor. It may inhibit cell growth by acting as a novel ligand to one or several GPCRs.