Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives

Background A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. Methodology/Principal Findings IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 μM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). Conclusions Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer.


Introduction
The KRAS gene encodes a G-protein which serves as a molecular switch between the endothelial growth factor receptor and the nucleus, controlling several signaling pathways important for cell growth and survival. KRAS GTPase exists in two states, a GTP-bound active state and a GDP-bound inactive state. Further, KRAS mutations increase KRAS affinity for GTP leading to the constitutive activation of the protein. Importantly, deletion of mutant KRAS allele in colon cancer cell lines dramatically reduces cellular proliferation [1], highlighting the fact that many tumors harboring mutant KRAS are KRAS-dependent. KRAS mutations are mostly prevalent in pancreatic (90-60%), colorectal (30-50%) and lung (20-30%) carcinomas [2]. Due to the high incidence of these cancers worldwide [3] and the increased resistance to conventional chemotherapy [4], the search for new targets has been intensified in past recent years.
The importance of therapeutic modulation of KRAS signaling have been widely recognized and several approaches have been reported in the past, but none has provided an approved new anticancer drug to date [5]. An innovative therapeutic approach being studied is the use of miRNAs, since they play an important role in chemo-sensitization [6]. We have previously demonstrated that miRNA-143, which also reduces KRAS expression, chemosensitizes colon cancer cells to 5-fluorouracil [7], and reduces tumor growth in vivo, with increased apoptosis and reduced proliferation [8].
Continuing our studies that aim to discover novel and selective anticancer drugs, pursued through the development of several chemical systems to be used in diverse therapeutic strategies [9], [10], [11], we report here on an approach to targeting KRAS signaling in cancer cells by directly modulating KRAS expression at the gene level. It has been recently demonstrated that a guanine-rich strand within the promoter of KRAS can fold into an intra-molecular Gquadruplex structure (G4), which has an important role in the regulation of KRAS transcription [12], [13]. G4 arrangements are nucleic acid higher-order structures, formed by sequences containing repetitive guanine(G)-rich tracts [14]. Several studies have provided evidence supporting the existence of G4s in eukaryotic telomeres and oncogene promoters, including those of KRAS, HRAS, HSP90, c-MYC, c-KIT, BCL-2 and VEGF genes, and that small molecules stabilizing G4 structures are able to down-regulate oncogene transcription in tumor cell lines, inhibit telomerase activity and induce cancer cell growth arrest [15], [16], [17]. G4 structures have also been found in RNA sequences, including in the 5 0 untranslated region (UTR) of KRAS mRNA, and shown to have translation regulatory functions [18], [19], [20].
Indoloquinolines are natural alkaloids able to target DNA structures, some of which have potential for development into anticancer drugs [21], [22]. Indolo [3,2-b]quinoline derivatives have been shown to be potent G4 ligands, and to inhibit cell proliferation and oncogene (c-MYC) transcription [21], [23], [24]. Moreover, we have recently discovered that indolo [3,2-b] quinolines with a 7-carboxylate group and three alkylamine side chains (1a and 2a in Fig 1) are promising selective anticancer leads [25]. These compounds selectively inhibited (100-fold) the growth of KRAS mutant HCT116 colon cancer cells compared to primary rat hepatocytes, while also decreasing KRAS protein levels. In order to exploit this scaffold towards the discovery of novel and improved anticancer drugs, we have extended the chemical diversity of these indoloquinolines and studied their potential anticancer mechanism of action. Previous structure-activity studies with mono-alkylamine indolo [3,2-b]quinolines have shown that optimal G4 stabilization was induced by compounds with propyl side chains and basic amine groups (pKa 8) [25]. Thus, compounds 1a-d and 2a-d (Fig 1) were designed, synthesized and evaluated for selective G4 thermal stabilization comparing to duplex DNA, together with inhibition of cancer cell proliferation, induction of apoptosis and down-regulation of KRAS and HSP90 transcription and protein expression. In order to improve the anticancer activity profile and KRAS oncogene down-regulation capacity of our target indoloquinolines, we have used four cell lines with differing KRAS and TP53 genotypes, as well as two positive controls, the anticancer drug 5-fluorouracil (5-FU) and the G4 ligand TMPyP4 (Fig 1).

Results and Discussion
Compounds 1a-d and 2a-d were synthesized in four steps following the procedure previously described [25] with some modifications (S1 Text). Structures of 1a-d and 2a-d were completely elucidated by bidimensional 1 H (COSY and NOESY) and 13 C heterocorrelation NMR experiments (HMQC and HMBC) and purity (> 95%) confirmed by HPLC-ELSD-MS (S1 Fig). The capacity of compounds 1a-d, 2a-d and the standard G4 ligand TMPyP4 (Fig 1) [16] to bind and stabilize KRAS [26] and HSP90A [27] G4 DNA structures as well as duplex DNA (Tloop) was evaluated by a Fluorescence Resonance Energy Transfer (FRET) melting assay. The increase in the melting temperatures induced by different concentrations of compounds is presented in Table 1 and S2 Fig. Our results show that tri-alkylamine indolo [3,2-b]quinolines (IQ3A) are potent and selective ligands for the KRAS and HSP90A G4 structures. As previously observed for mono-and di-alkylamine analogues [25] and other polyaromatic-fused G4 ligands [28], [29], compounds with propylamine side chains (1d and 2d) are superior G4 stabilizers (ΔT m between 18 and 23°C at 2 μM of ligand) than compounds with shorter alkylamine side chains (1a-b and 2a-b; ΔT m values between 7 and 17°C at 2 μM ligand concentration). It was observed that the basicity of side chains correlates positively with thermal G4 stabilization of all DNA sequences up to an optimal pKa~8.0-9.0 (Fig 2). Heterocyclic amines at the end of alkyl side chain (1b and 2b) appear to improve binding to G4s and complex stabilization compared to the di-ethylamine group (1a and 2a), which cannot be explained by differences in basicity between terminal groups. Additionally, and in line with what was previously observed for di-alkylamine indoloquinolines [25], this study suggests that N5,N10,COO tri-substitution with heterocyclic amine groups at alkyl side chain termini increase ligand affinity to G4, as 2b, d-G4 DNA complexes have higher melting temperatures than complexes of the correspondent isomers 1b,d. However, this was not observed for the isomeric pair 1a / 2a. Despite the increased G4 stabilization capacity, ligands 2b and 2d were not able to significantly discriminate between the two DNA G4 structures (Table 1).
To study the effect of IQ3A compounds on cancer and non-cancer cells, we selected compound 2d showing the best G4 stabilization capacity in vitro and the pair 1a and 2a, which despite showing lower ΔT m values for the respective complexes with G4 DNA, are able to reduce KRAS expression when incubated in HCT116 colorectal cancer cells, as we have previously shown [25].
The short-term effect of compounds at 72 h on cell growth was studied using colorectal carcinoma cell lines with different KRAS and TP53 genotypes: human colorectal carcinoma HCT116 (mut Kras, wild-type (wt) p53), and human metastatic colorectal adenocarcinoma SW620 (mut Kras, mut p53). In parallel, we also used the immortalized human embryonic kidney cell line HEK293T (wt Kras, wt p53) and normal human colon fibroblast cells CCD18co (wt Kras, wt p53). The IC 50 and IC 65 values in Table 2 show that compounds 2a and 2d display superior anti-proliferative activity compared to 1a and 5-FU, particularly in metastatic SW620 cells, where 2d gave an IC 50 value (0.28 μM), almost 20-fold lower than that of the standard anticancer drug 5-FU (IC 50 = 5.39 μM). Interestingly, colorectal cancer cells HCT116 and SW620, which express mutant KRAS, were particularly insensitive to the porphyrin derivative TMPyP4, in contrast to non-malignant HEK293T cells expressing wild type KRAS. In addition, IQ3A compounds and 5-FU, were not selective for cancer cells expressing mutant KRAS, since they were equally active against HCT116, SW620 and HEK293T cells. Nevertheless, we observed some selectivity (S.I. > 2.4; Table 2) toward the colon cancer cell line HCT116 compared to normal colon fibroblasts (CCD18co).
Subsequently, the Guava ViaCount assay was used to evaluate the effects of IQ3A compounds on cell death induction in cancer (HCT116 and SW620) and non-malignant (HEK293T and CCD18co) cells, compared to 5-FU and TMPyP4 at equitoxic concentrations (IC 50 and IC 65 ). Down-regulation of mutant KRAS expression by antisense oligonucleotides in colorectal cancer cells [7], [8], [30] and by a MAZ-binding oligonucleotide decoy in pancreatic cancer cells [31] has been associated with increased apoptosis and cell growth arrest. Also, anticancer drugs, such as 5-FU and G4 stabilizers of telomeric and oncogene promoter sequences such as TMPyP4, are known to induce a generalized cell response leading to cell growth arrest and cell death by apoptosis (DNA damage response) [15], [32], which involves activation of the pro-apoptotic transcription factor p53 among other pathways in the case of 5-FU [34].   Conversely, all compounds were unable to induce significant cell death in HEK293T and CCD18co cells ( Fig 3A and 3B, lower panels), whereas in SW620 cells only 5-FU was able to induce apoptosis in a dose-dependent manner ( Fig 3A and 3B, upper right panels). The effect of TMPyP4 on SW620 was not studied, as this cell line was not sensitive to this compound up to a 20 μM concentration.
To further validate the effect of the IQ3A compounds on the induction of apoptosis in HCT116 cells, apoptosis was ascertained by evaluating changes in nuclear morphology by Hoechst staining, and also by the Nexin assay, following 72 h of incubation with IQ3A compounds at the IC 50 concentrations. CCD18co cells were used in parallel to confirm that IQ3A do not elicit cell death in normal cells. These results showed that IQ3A significantly induce apoptosis in HCT116 cells, with compound 1a producing a marked increase in apoptosis compared to vehicle (Fig 4A and 4B). In addition, we also confirmed that IQ3A compounds do not induce apoptosis in normal colon fibroblasts CCD18co. The p53 protein plays a central and pivotal role in human cancers [33], having a potent tumor suppressive activity via pleiotropic mechanisms. Therefore, the steady-state levels of p53 protein were evaluated by immunoblotting, following 72 h of incubation with IQ3A compounds at the IC 50 concentrations, to ascertain their involvement on the mechanism of apoptosis elicited by IQ3A. Our data ( Fig 5A) clearly demonstrate that IQ3A increased p53 protein steady state levels in HCT116 by 4-7 fold (p < 0.01) (Fig 5A, left panel), which may be correlated with the higher cell death verified by Guava ViaCount assay. In SW620 cells, no significant increase in p53 steady-state expression was detected, possibly due to the mutant status of p53 ( Fig 5A, middle panel). Finally, in HEK293T cells, IQ3A showed no influence on p53 protein expression, in accord with the absence of cell death in the viability assay ( Fig 5A, right panel). In addition, we further explored the relevance of p53 in the mechanism of apoptosis elicited by IQ3A, by silencing KRAS in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines, and evaluating its impact on cell death, and on IQ3A-induced cell death ( Fig 5B). Our results clearly show that KRAS silencing induced a higher level of cell death compared to siRNA control (p < 0.05), and a higher level of IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-) (p < 0.05 for 2a and 1a). Further, in siRNA control transfected cells, all IQ3A significantly induced a higher level of cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-)  Targeting KRAS Oncogene with Indolo[3,2-b]quinolines (p < 0.05). However, in p53 mutant SW620 cells, IQ3A compounds did not significantly elicit cell death (Fig 3), nor did they increase p53 protein steady-state expression (Fig 5). In agreement, KRAS and/or HSP90 silencing were unable to induce cell death in this cell line, whereas KRAS silencing induced a dose-dependent effect in the reduction of SW620 cell proliferation (S3 Fig). Collectively, these data further confirm the importance of p53 for IQ3A induction of apoptosis. Finally, the capacity of IQ3A compounds to repress KRAS expression was evaluated by quantifying KRAS protein in cancer cell lines. Since IQ3As have also shown to be effective stabilizers of G4 sequences in the HSP90 oncogene promoter region (Table 1), the expression of this protein was also evaluated. Fig 6A shows that G4 ligands 1a, 2a, 2d and TMPyP4 downregulated the expression of mutant KRAS by 35-60% in HCT116 and SW620 cells, with exception of 2a (p < 0.01). In addition, IQ3A also reduced HSP90 protein steady levels, but to a lesser extent compared to KRAS. Therefore, we next investigated the ability of IQ3A compounds to down-regulate KRAS transcription in colon cancer cells. KRAS mRNA steady-state levels were evaluated by RT-PCR after 72 h incubation of cells with the compounds at their IC 50 concentrations and compared with the effect of TMPyP4 at equitoxic concentrations. G4 ligands such as TMPyP4 have been shown to bind to G4 structures from the KRAS promoter region and from the 5'-UTR of KRAS mRNA, and also repress both gene transcription and translation [13,19]. Fig 6B shows that 1a, 2a and 2d were able to down-regulate KRAS transcription by ca 40% in HCT116 cells, but not significantly so in SW620 cells. A possible explanation is the time point at which we evaluated KRAS mRNA and protein steady-state levels. After 72 h of IQ3A exposure, there may no longer be significant repression of KRAS promoter activity in the SW620 cell line, whereas protein levels remain reduced as a result of accumulated IQ3A effects. Also, TMPyP4 was unable to reduce KRAS mRNA steady-state levels in the HCT116 cell line, in contrast to a decrease of ca 80% of the protein steady-state levels (Fig 6A and 6B). These results are in agreement with the reported ability of TMPyP4 to preferentially accumulate in the cytoplasm of cells [19], where it can inhibit KRAS mRNA translation.
To validate that the mechanism of anti-proliferative activity and apoptotic induction by IQ3A compounds involves repression of KRAS gene expression as a result of the stabilization of G4-forming sequences present in the promoter, the effects of compounds on the KRAS gene promoter were directly evaluated by a luciferase reporter assay. For this purpose, we used two different size promoter constructs containing the G4 region of the KRAS gene promoter, cloned into the pGL3 Basic backbone: pGL-Ras0.5, pGL-Ras2.0, and pGL3 Basic empty (Firefly Luciferase negative control/no promoter), co-transfected together with pRL-TK (transfection efficiency normalization) into HEK293T cells, as a G4 negative control. This construct does not harbor G4 sequences and is insensitive to G4-related effects/regulation. Our data clearly demonstrate that IQ3A compounds, similarly to TMPyP4, were able to significantly reduce KRAS transcription, we suggest by interacting with the G4 region of the KRAS gene promoter, suppressing downstream coding-region expression from 40 to 60% versus DMSO control (p < 0.01) (Fig 6C). Using both plasmids, with 500 and 2000 bp upstream to the transcription start site, we also show that the target region of the IQ3A compounds is within this region, thus coinciding with the polypurine G-rich strand responsible for G-quadruplex structure assembly [12]. Importantly, we were also able to show that IQ3A compounds, similarly to TMPyP4, were able to significantly reduce KRAS promoter activity in HCT116 and SW620 cells (Fig 6D).

Conclusions
We have investigated in this study the ability of a group of 7-carboxylate indolo[3,2-b]quinoline tri-alkylamine derivatives (IQ3A), which are potent stabilizers of DNA G4 structures KRAS mRNA steady-state expression was evaluated by Taqman Real-time RT-PCR using specific Taqman Assays for KRAS and β-Actin for normalization. KRAS mRNA steady-state expression levels were calculated by the ΔΔCt method, using DMSO (vehicle control) for calibration; and C. HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5.000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO); D. HCT116, SW620 and HEK293T cells were cotransfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, together with pRL-TK. Twentyfour hours later, cells were replated in 96-well plates, at 5,000 cells per well and exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h (C.) or 24 h (D.) after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. Results are expressed as mean ± SEM of at least three independent experiments; *p < 0.05 and §p < 0.01 from DMSO (vehicle control); and †p <0.05 and ‡p < 0.01 from 5-FU. doi:10.1371/journal.pone.0126891.g006 Targeting KRAS Oncogene with Indolo[3,2-b]quinolines present in the KRAS promoter, to down-regulate KRAS expression and induce cell death by apoptosis, particularly in KRAS-dependent colon cancer cell lines. The IQ3A compounds markedly showed anti-proliferative activity in an IC 50 range lower than 2.69 μM in HCT116 and SW620 cells. Particularly, in HCT116 cells, IQ3A compounds at the IC 65 concentration increased cell death up to 4.7 fold (p < 0.01) and 2.4 fold (p < 0.01) in comparison to DMSO or 5-FU, respectively. In addition, in non-malignant cell lines the IQ3A compounds did not cause significant cell death. Further, they markedly reduced KRAS mRNA expression and protein levels in colon cancer cells possibly through direct transcriptional repression of the KRAS promoter. Our data to date shows a correlative relationship between transcriptional repression and binding to the G4 region within this promoter, although further studies are needed in order to demonstrate a direct link between the two. The repression effect was accompanied by increased p53 protein steady-state levels in HCT116, and only a slight reduction of HSP90 levels in the three cell lines tested, indicating that these compounds are not also having an effect on some other possible G4 targets such as in the HSP90 promoter [27]. Rather the IQ3A compounds are selectively targeting the KRAS driver genes in the colon cancer cell lines. Collectively these results show that G4-binding ligands, such as these indoloquinoline derivatives, display potential to be employed as novel anticancer therapeutic agents, especially for the treatment of human cancers driven by KRAS mutations, which are still in large part cancers of high unmet clinical need.

Evaluation of cell viability and general cell death
Cell viability was evaluated by the tetrazolium dye (MTS) Short-Term Cytotoxicity Assay. In brief, cells were seeded in 96 well plates at 5,000 cells/well. Twenty-four hours after cell plating, media was removed and replaced with fresh media containing test compounds IQ3A, TMPyP4 and 5-FU, or vehicle control (DMSO). Following 72 h of compound exposure, cell viability was evaluated using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described [34] [35]. Cell viability data were expressed as mean ±SD from at least three independent experiments. IC 50 and IC 65 values were determined using GaphPad Prism v.5.00 (GraphPad Software). In selected experiments, cell viability was also assessed by trypan blue exclusion assay [34], and general cell death by LDH activity from cell culture supernatants [7].

Guava ViaCount assay
ViaCount assay was used with Guava easyCyte 5HT Flow cytometer (Guava Technologies, Inc., Hayward, CA, USA), as previously described [36] to evaluate viable, apoptotic and dead cell populations, on HCT116, SW620, HEK293T and CCD18co cells exposed to test compounds IQ3A, TMPyP4 and 5-FU at IC 50 and IC 65 concentrations, and a vehicle control (DMSO). The ViaCount Assay distinguishes viable mid apoptotic and dead cells based on differential permeability of two DNA-binding dyes in the GuavaVia Count Reagent. The nuclear dye stains only nucleated cells, while the viability dye brightly stains dying cells. HCT116, SW620, HEK293 T and CCD18co cells were seeded in 24-well plates 50,000 cells/well. Twentyfour hours later, cells were exposed to compounds for 72 h. After treatment, cell culture supernatants were collected and adherent cells were detached with TrypLE (Invitrogen). Next, detached cells were pooled with cell culture supernatants and centrifuged for 5 min (650 g). Supernatants were discarded and the cells were resuspended in 50-500μl phosphate buffered saline (PBS) with 2% FBS. Subsequently, 15 μl of cell suspension were mixed with 135 μl of Guava ViaCount reagent, and incubated for 5 min at room temperature. Sample acquisition and data analysis were performed using the ViaCount software module.

Guava Nexin assay
Nexin assay was used with Guava easyCyte 5HT Flow cytometer (Guava Technologies, Inc., Hayward, CA, USA) to evaluate viable, early and late apoptotic cell populations, on HCT116 and CCD18co cells exposed to either test compounds IQ3A, TMPyP4 and 5-FU at IC 50 concentration, or vehicle control (DMSO). The Nexin Assay distinguishes viable, early and late apoptotic cells based on the externalization of phosphatidylserine to the cell surface, where Annexin V can readily bind them. The membrane dye stains with higher intensity early and late apoptotic cells. HCT116 and CCD18co cells were seeded in 24-well plates 50,000 cells/well. Twenty-four hours later, cells were exposed to compounds for 72 h. After treatment, cell culture supernatants were collected and adherent cells were detached with TrypLE (Invitrogen). Next, detached cells were pooled with cell culture supernatants and centrifuged for 5 min (650 g). Supernatants were discarded and the cells were resuspended in 50-500 μl phosphate buffered saline (PBS) with 2% FBS. Subsequently, 50 μl of cell suspension were mixed with 50 μl of Guava Nexin reagent, and incubated for 20 min at room temperature. Sample acquisition and data analysis were performed using the Nexin software module.

Total protein extraction and immunoblotting
HCT116, SW620 and HEK293 T cells were seeded in 35 mm plates at 300,000 cells per well. Test compounds IQ3A, TMPyP4, 5-FU and a vehicle (DMSO) were added to the cells 24 h after plating, at IC 50 equitoxic concentration. After 72 h of compound exposure, cells were collected and processed for total protein extraction, as previously described [8]. Briefly, samples were homogenized in ice-

KRAS promoter activity Luciferase Reporter Assay
HEK293T cells were seeded in 35 mm plates at 300,000 cells per well. Twenty-four h later, cells were transiently co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK (Promega, Madison, WI, USA). KRAS promotor luciferase reporters respectively harbor 500 bp and 2000 bp of the human KRAS promotor region, kindly provided by Prof. Kim Nam-Soon. pGL3 Basic empty was used as negative control, and pRL-TK simultaneously for transfection efficiency normalization and as a G4 negative control. This construct does not harbor G4 sequences, therefore being insensitive to G-quadruplex-related effects/regulation. Transfections were performed using Lipofectamine 3000 (Invitrogen), according to the manufacturer's instructions. Twenty-four h after transfection, cells were replated in 96-well plates, at 5,000 cells per well. Subsequently, 24 h after replating, test compounds IQ3A, TMPyP4, and vehicle (DMSO) were added to the cells at IC 50 equitoxic concentration. Finally, 72 h after compound incubation, cells were lysed and firefly and renilla luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega). KRAS promoter activity levels were expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. In parallel, HCT116, SW620 and HEK293T cells were transfected with PGL-Ras0.5 together with pRL-TK (Promega, Madison, WI, USA), replated in 96-well plates and simultaneously exposed to test compounds IQ3A, TMPyP4, and vehicle (DMSO) at IC 50 equitoxic concentration. Twenty-four h later, cells were lysed and renilla luciferase activities were measured and expressed as above. The results are expressed as the mean ± SEM fold-change compared to DMSO exposure, from three independent experiments.
Supporting Information