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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

A high-throughput screen of the cytotoxic activity of 2000 molecules from a commercial library in three human colon cancer cell lines and two normal cell types identified the acridine acriflavin to be a colorectal cancer (CRC) active drug. Acriflavine was active in cell spheroids, indicating good drug penetration and activity against hypoxic cells. In a validation step based on primary cultures of patient tumor cells, acriflavine was found to be more active against CRC than ovarian cancer and chronic lymphocytic leukemia. This contrasted to the activity pattern of the CRC active standard drugs 5-fluorouracil, irinotecan and oxaliplatin. Mechanistic studies indicated acriflavine to be a dual topoisomerase I and II inhibitor. In conclusion, the strategy used seems promising for identification of new diagnosis-specific cancer drugs. (Cancer Sci 2011; 102: 2206–2213)

Incremental advances in surgery and systemic chemotherapy over many years, including the development of new targeted drugs, have provided quantifiable improvements in the overall survival of patients with metastatic colorectal cancer (CRC).(1) However, despite this progress, most CRC patients do not benefit from drug treatment in the adjuvant setting, and in advanced disease most patients will die from disease progression. Therefore, there is need for new therapeutic options for CRC patients, including more active drugs.

There are three main strategies currently in use for development of new cancer drugs: rational drug design, drug screening and development of analogues.(2) Historically, screening and analogue development have contributed most, but due to the significant progress in basic cancer biology research, rational drug design based on identification of tumor-specific targets followed by development of target inhibitors has come into focus. Although theoretically promising, the rational drug design approach has so far provided only limited progress for the major cancer types.(3,4) Thus, a revival of the other approaches using improved algorithms and techniques might be worthwhile.

Based on these considerations and our interest in the treatment of CRC, we hypothesized that it would be possible to identify molecules with promising activity against CRC tumor cells using our previous experience from small molecule cancer drug screening and characterization using human tumor cell lines and primary cultures of tumor cells from patients.(5–7) In this attempt, the Spectrum Collection compound library, which contains 2000 substances, synthetic as well as natural, provides a wide range of biological activities and structural diversity and was screened in human colon cancer cell lines and normal cell types followed by further characterization of a candidate drug (CD) in patient tumor samples and by gene expression-based mechanistic evaluation. Using this approach, we successfully identified acriflavine as a promising drug for treatment of CRC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell lines.  The colorectal cell lines HCT116, HT29 and CC20 were cultured in McCoy’s and Dulbecco’s Modified Eagle’s Medium (DMEM). HCT116 and HT29 were obtained from ATCC (Manassas, VA, USA) and the CC20 cell line was a gift from Dr Christian Sundberg (Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden).(8) The breast cancer epithelial cell line MCF7 was obtained from ATCC, was cultured in DMEM with 1 mM sodium pyruvate, and was used for the genomics-based mechanistic analysis. The untransformed retinal epithelial cell line hTERT-RPE1 (Clontech Laboratories, Palo Alto, CA, USA), cultured in DMEM nutrient mixture F-12 Ham, was included as a reference cell type. A panel of 10 cell lines of different histological origins and containing pairs of parental drug-sensitive and drug-resistant sublines was used for phenotypic characterization of the hit molecule pattern of drug resistance, as described previously.(5) These cell lines were grown in RPMI 1640 medium. All cell lines were grown in media supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 μg/mL streptomycin and 100 IU/mL penicillin (all from Sigma Aldrich, St Louis, MO, USA). The resistant cell lines were tested regularly for maintained resistance to the selected drugs.

Tumor cells from patients and peripheral blood mononuclear cells.  The tumor sampling for in vitro drug sensitivity testing was approved by the Regional Ethical Review Board in Uppsala (nos. 2007/37 and 21/93). All patients provided written informed consent, except for a few patients sampled before 2007 for which the consent was verbal and documented in the patient file. This was in accordance with the ethical standards of the Review Board. All tumor sampling was performed at the University hospital in Uppsala.

Tumor samples for this investigation were obtained from 51 patients with CRC who were scheduled for cytoreductive surgery and intraperitoneal chemotherapy for peritoneal carcinomatosis, from 15 patients with advanced ovarian carcinoma (OC) at second-look laparotomy or sampling from malignant ascites, and from peripheral blood samples from 12 patients newly diagnosed with chronic lymphocytic leukemia (CLL). For CRC, 36 samples were from patients previously treated with chemotherapy and 15 samples were from previously untreated patients. All but one of the OC samples were from previously treated patients and all CLL samples were from treatment naive patients. As a screening reference cell type, drug sensitivity was also investigated in 27 preparations of mononuclear cells (MNC) isolated from peripheral blood from healthy blood donors.

Tumor tissue from solid samples was minced into small pieces and tumor cells were then isolated by collagenase dispersion followed by Percoll (Pharmacia Biotech, Uppsala, Sweden) density-gradient centrifugation.(9) OC cells from ascites fluid were collected by centrifugation, followed by purification of the cells by Ficoll-Paque (Pharmacia Biotech) and Percoll density-gradient centrifugation. CLL cells and MNC were isolated from peripheral blood by density gradient centrifugation on 1.077 g/mL Ficoll-Paque (Pharmacia Biotech) gradient.(9) Cell viability was routinely >90% for the samples included, as determined by the trypan blue exclusion test, and the proportion of tumor cells in the preparations, as judged by inspection of May-Grünwald–Giemsa-stained cytospin preparations, was >70%. Cell culture medium RPMI 1640 (supplemented as described above) was used throughout.

Screening compounds and standard drugs.  The Spectrum Collection (MicroSource Discovery Systems, Gaylordsville, CT, USA) contains 2000 compounds, both synthetic and natural, covering a wide range of biological activities and structural diversity. The compounds were supplied as 10-mM solutions in DMSO and were further diluted with phosphate-buffered saline (PBS) and transferred to 384-well microplates (NUNC Brand Products, Roskilde, Denmark) using a Biomek 2000 pipetting station (Beckman Coulter, Fullerton, CA, USA). All compounds were screened at a final concentration of 10 μM (0.1% DMSO v/v) in the three colon cancer cell lines as well as in hTERT-RPE1 and MNC for cytotoxic activity by means of the fluorometric microculture cytotoxicity assay (FMCA; see below). Acriflavine (Sigma-Aldrich; CAS 8063-24-9) was stored as a 10-mM stock solution in DMSO, further diluted with PBS and tested in duplicates at five concentrations from a maximum of 20 μM and at fivefold serial dilutions.

The clinically used CRC active cytotoxic drugs 5-fluorouracil (5-FU), irinotecan and oxaliplatin were supplied from the Uppsala University Hospital pharmacy and were diluted with sterile PBS. The clinical preparation of irinotecan was chosen in the present study instead of its active metabolite SN-38 as irinotecan was previously shown to better reflect the clinical efficacy of this drug in vitro.(10) The standard drugs were tested in duplicate at three concentrations at 10-fold serial dilutions from a maximum of 1000 μM for 5-FU and irinotecan and of 100 μM for oxaliplatin.

Drug sensitivity testing.  The semi-automated FMCA, described in detail previously was used to assess drug sensitivity.(11) The method is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate (FDA) to fluorescein by cells with intact plasma membranes. Using the pipetting robot BioMek 2000 (Beckman Coulter), 384-well microplates (NUNC) were prepared with 5 μL drug solution in 10 times the final drug concentration. The plates were then stored at −70°C until further use.

Tumor cells from cell lines (5000 cells/well) or patient samples (5000 cells/well for CRC and OC, 40 000 cells per well for CLL) were seeded in the drug-prepared 384-well plates using the pipetting robot Precision 2000 (Bio-Tek Instruments, Winooski, VT, USA). Three columns without drugs served as controls and one column with medium only served as a blank. The plates were incubated at 37°C for 72 h and were then analyzed using the FMCA.

Cell survival, expressed as survival index (SI), is defined as fluorescence in test wells divided by fluorescence of control wells, with blank values subtracted, ×100. Quality criteria for a successful assay included a mean coefficient of variation of <30% in the control and a fluorescence signal in control wells of more than five times the blank.

Selection of hits from the screen.  The Small Laboratory Information and Management System (SLIMS) was used for screening data management.(12) Raw fluorescence data files were loaded into the SLIMS software, which calculates percent inhibition according to the formula: percent inhibition = [1 − (exp − blank/control − blank)] × 100, where exp denotes fluorescence from experimental wells. Screening data was subsequently exported to Vortex (Dotmatics, Bishop’s Stortford, UK) software for visualization and data analysis. More than or equal to 70% inhibition in all three colon cancer cell lines and <30% in counter-screens with hTERT-RPE1 and MNC cultures were set as the criteria for qualifying as hit compounds using the filtering tools in Vortex. Structural similarity to other compounds in the library was calculated based on a structural fingerprint consisting of chemical fragments located within the compound and which are computed for each compound by Vortex. Clustering of compounds based on these fingerprints was then performed within the vortex program.

Assessment of acriflavine activity in tumor spheroids and on clonogenic growth.  Five-day-old multicellular spheroids of HCT116 cells, established using the hanging drop technique and with homogeneous diameters, were exposed to different concentrations (0.2, 1 and 5 μM) of acriflavine or various control drugs for 6 h and then washed.(13) The spheroids were then incubated for 5 days followed by measurement of spheroid diameters using a caliper in a microscope for volume calculations. The spheroids were then trypsinized in PBS, seeded in 6-well plates and left in the incubator for 10 days. Clonogenicity was then assessed by staining with Giemsa followed by clone counting.

Mechanistic characterization of acriflavine using Connectivity Map gene expression analysis.  The experiments and analysis were performed as previously described.(14,15) Briefly, MCF7 cells were exposed to 10 μM acriflavine or vehicle (DMSO) for 6 h. RNA was isolated using Qiagen columns (Qiagen GmbH, Hilden, Germany). Total RNA (2 μg) from each sample were used to prepare biotinylated fragmented cRNA, which was hybridized to Human Genome U133 Plus 2.0 Arrays according to the GeneChip Expression Analysis Technical Manual (Rev. 5; Affymetrix, Santa Clara, CA, USA).

The raw data from the microarray expression analysis was normalized using the Mas5 software. Only probes with present call in both treated and vehicle control were used in the analysis. Probes were ranked according to the expression differences compared to the vehicle-treated control, and the 30 most induced and reduced probes (Table S1) were used as up and down tags in the Connectivity Map (cmap) analysis (http://www.broad.mit.edu/cmap), as described in the Results section.

Topoisomerase enzyme assays.  Tests of topoisomerase inhibition were performed using Topoisomerase I and II Drug Screening Kits, Recombinant Hu Topoisomerase I and Human Topoisomerase II enzymes (TopoGen Aces Alley, Port Orange, FL, USA) according to the instructions of the manufacturer. All incubations were performed in the presence of solvent (deionized water) for 30 min at 37°C followed by agarose gel electrophoresis in the absence of ethidium bromide. Gels were then stained with ethidium bromide, washed and photographed.

Data presentation and statistical methods.  Dose-response SI data were used to calculate the 50% inhibitory concentrations, the drug concentration producing an SI of 50% (IC50). This was performed in GraphPadPrism (GraphPad Software Inc., San Diego, CA, USA) using nonlinear regression and a standard sigmoidal dose-response model. Resistance factors were defined as IC50 in the resistant subline divided by IC50 in the parental cell line.

Data are presented as mean values ± standard error for the number of experiments/samples indicated. Statistical inferences between several means were performed by one-way analysis of variance using Tukey’s multiple comparison post test of group means or, for comparison of two means, using Student’s t-test, in GraphPadPrism.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Drug screen identifies acriflavine as a potential colorectal cancer active drug.  Screening results for HCT116, MNC and hTERT-RPE1 are shown in the 3-D plot in Figure 1(A). Based on the criteria for hit qualification, six compounds were identified and are highlighted in blue in the radar plot encompassing all cell line models subjected to screening (Fig. 1B). Clustering of the compounds according to chemical structure using Vortex revealed that three of the hits are of similar chemical structure and related to the natural product gambogic acid (i.e. gambogic acid, gambogic acid amide and tetragambogic acid).(16) Two other hit compounds, obutsaquinone(17) and pristimerin,(18) are also natural products. Only one of the hits, acriflavine, is a synthetic compound. As judged by the radar plot (Fig. 1C), the hit group of compounds was within two standard deviations of the library with respect to molecular weight, hydrogen donors and hydrogen acceptor, whereas logP was generally higher. Four of the hits (pristimerin and gambogic acid with derivatives) had logP above 5, thus violating Lipinski’s rule of five criteria for druggable compounds.(19) Based on the above results and considerations, the synthetic compound acriflavine with a logP of 4.35 was selected for further evaluation. The chemical structure of acriflavine is shown in Figure 1(D).

image

Figure 1.  In (A) screening for colon cancer drug activity results are displayed and expressed as percent inhibition (see Materials and Methods for definition) for HCT116, hTERT-RPE1 and peripheral blood mononuclear cells (MNC) with the hit compounds marked with blue circles. In (B) a radar plot displaying the activity of the hit compounds in all five cell line models is indicated with blue lines. The brown area indicates the area between two to three standard deviations (SD) of the library. In (C) a radar plot shows the position of the hit compounds with respect to log P, molecular weight, hydrogen donors and hydrogen acceptors. (i.e. compliance with Lipinski’s rule of five). The brown area indicates the area between two to three SDs of the library. (D) shows the structural formulae of acriflavine. Acriflavine is a mixture of trypaflavine (3,6-diamino-10-methylacridinium chloride; above) and proflavine (3,6-diaminoacridine; below).

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Acriflavine shows novel activity against tumor cells from colorectal cancer.  Acriflavine was tested in parallel with the CRC standard drugs 5-FU, irinotecan and oxaliplatin in a panel of patient tumor samples. The CRC standard drugs 5-FU, irinotecan and oxaliplatin were all significantly (P < 0.001) more active in OC and CLL compared with CRC. In contrast, the IC50 for acriflavine was lower for CRC (1.38 μM) than for the comparator diagnoses (4.23 and 2.58 μM for OC and CLL, respectively), although the difference did not reach statistical significance (Table 1). At 4 μM acriflavine, the concentration producing the greatest scatter of effects among the tumor samples included, the SI value for CRC (20%) was statistically significantly lower than that for OC (53%; P < 0.001) and tended to be lower than for CLL (34%; not significant, data not shown). This finding of an “inverse” sensitivity compared with the standard drugs is a novel finding that we have not previously observed for any standard or experimental drug tested in patient tumor cells in vitro (Fig. 2).

Table 1.   Mean drug concentrations (μM) corresponding to a survival index of 50% (IC50 concentrations) for the indicated drugs and tumor types. The number of samples investigated is given in parentheses and 95% confidence intervals are indicated in the second row for each condition
DrugColorectal cancerOvarian cancerChronic lymphocytic leukemiaMononuclear cells
  1. *P < 0.001 versus ovarian cancer, chronic lymphocytic leukemia and mononuclear cells for the same drug.

Acriflavine1.4 (22)4.2 (10)2.6 (8)1.4 (23)
1.1–1.72.4–7.61.8–3.61.1–1.7
5-FU755.2 (51)*562.8 (15)658.2 (12)429.8 (25)
587.5–970.5338.1–937.5449.7–963.8324.3–568.8
Irinotecan89.6 (50)*75.3 (15)29.3 (12)25.4 (18)
73.8–113.843.7–106.920.9–41.122.1–29.3
Oxaliplatin26.1 (51)*10.9 (15)7.6 (12)2.9 (27)
20.2–33.76.8–17.45.5–10.52.6–3.2
image

Figure 2.  Drug concentration: Response relationships for acriflavine (A), 5-FU (B), irinotecan (C) and oxaliplatin (D) in tumor samples from patients with colorectal cancer (––––), chronic lymphocytic leukemia (- - - - -) and ovarian cancer (······). The number of samples for each condition is indicated in Table 1.

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This finding was also illustrated by calculation (based on the data presented in Table 1) of the ratio of IC50 values in MNC, the most sensitive cell type among those investigated for all drugs, to that in tumor cells. A ratio below 1 indicates higher activity in MNC compared with the patient tumor cells. Whereas this ratio was 0.57 or lower in CRC and lower for CRC than in OC and CLL for the standard drugs, it was 1.0 for acriflavine compared with 0.32 and 0.53 for ovarian cancer and CLL, respectively (data not shown). Prior treatment status did not affect the acriflavine sensitivity of the CRC samples. The mean IC50 values for samples from previously untreated (n = 7) and treated (n = 15) patients were 1.29 and 1.42 μM, respectively (P = 0.9439).

Acriflavine is insensitive to common mechanisms of drug resistance.  By using pairs of parental and drug-resistant cell lines, it is possible to assess the mechanisms of drug resistance for new drugs. Acriflavine showed resistance factors mostly close to l. The greatest difference in sensitivity between a parental line and its subline was observed for NCI-H69 and its subline H69AR with a ratio <3 (Table 2), indicating that the activity of acriflavine is not severely affected by the major mechanisms described for cytotoxic drug resistance.(20)

Table 2.   Resistance factors for acriflavine and the three standard cytotoxic agents in the cell line panel with cell line pairs representing the resistance types indicated. Previously published data from our database for one typical drug for each type of resistance was also included as a reference.(20)
Parental cell lineCCRF-CEMNCI-H69RPMI 8226RPMI 8226U937-GTB
Resistant sublineCEM/VM-1H69AR8226/Dox408226/LR-5U937/Vcr
DrugResistance factor
  1. Resistance factor was defined as the IC50 value in the resistant subline divided by that in its parental cell line. Resistance mechanisms for the cell line pairs: CCRF-CEM/ CEM/VM-1, Topoisomerase II-associated resistance; NCI-H69/H69AR, Multidrug-resistance MRP associated protein; RPMI 8226/8226-Dox40, P-glycoprotein 170 associated resistance; RPMI 8226/8226-LR5, Glutathione-associated resistance; U937-GTB/ U937Vcr, Tubulin-associated multidrug resistance.

Acriflavine1.12.71.30.92.2
5-FU6.70.1150.22.3
Oxaliplatin1.30.81.70.61.6
Irinotecan1.83.03.70.71.0
Vincristine#0.68.93602.243
Doxorubicin#1.242240.91.4
Etoposide#122.0291.22.5
Melphalan#1.00.91.43.01.0

The notion of low cross-resistance between acriflavine and established cancer drugs was supported by statistically non-significant correlation coefficients and comparatively flat linear regression lines between acriflavine and the standard CRC active drugs 5-FU, oxaliplatin and irinotecan in the patient samples from CRC (Fig. 3). In comparison, these standard drugs correlated significantly to each other.

image

Figure 3.  Linear regression with Pearson’s correlation coefficients (r) for the activity, expressed as survival index (SI%), for the indicated drugs in 51 (panels A–C) or 22 (panels D–F) patient samples of colorectal cancer. The drug concentrations (μM) used were: oxaliplatin 10, 5-FU 1000, irinotecan 100 and acriflavine 0.8. ***P < 0.0001; **P < 0.01; ns, not statistically significant.

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Acriflavine is active on 3-D tumor spheroids.  We next assessed the activity of acriflavine in the multicellular spheroid model. HCT116 colon cancer spheroids (approximately 500 μM diameter) were exposed to acriflavine for 6 h and then incubated in drug-free medium for 5 days. Treatment with 5 μM significantly (P < 0.001) reduced the spheroid volume to approximately one-third of unexposed control spheroids, whereas lower concentrations were ineffective (Fig. 4A,B). Staurosporine (1 μM), a potent apoptosis inducing agent, reduced the spheroid volume to approximately half (P < 0.01) (Fig. 4B).

image

Figure 4.  Effect of 6-h exposure to the indicated concentrations of acriflavine or staurosporine on HCT116 cells grown as multicellular spheroids for 5 days (A and B). (A) shows a typical experiment comparing unexposed control spheroids with those exposed to 5 μM acriflavine, which resulted in decreased spheroid volume and outer layer loosening up. (B) shows results from three independent experiments presenting mean spheroid volumes ± standard error for the drugs and concentrations indicated. *P < 0.01, **P < 0.001 versus control. (C and D) Clonogenic outgrowth of HCT116 cells dispersed from spheroids exposed to the drugs and concentrations indicated. (C) shows a typical clonogenic assay in a 6-well plate with three unexposed controls with hundreds of colonies and three wells with cells exposed to 5 μM acriflavine for 6 h prior to seeding. Outgrowth time was 10 days. (D) shows number of clones in percent of controls for the indicated drugs and concentrations using the same set-up. Results are presented as mean values ± standard error for three independent experiments.

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Clonogenic outgrowth of cells dispersed from drug-exposed spheroids was strongly affected (Fig. 4C,D). Thus, acriflavine at 5 μM reduced the number of clones to <5% of the control. In comparison, the established topoisomerase I inhibitor irinotecan was less active (Fig. 4D).

Acriflavine is identified as a topoisomerase inhibitor.  To characterize the mechanism of action of acriflavine, we used the cmap approach to connect the activity of acriflavine to drugs with similar biological activity at the gene expression level.(14,15) The current version of cmap contains genome-wide expression data for 6100 perturbations (instances), representing 1309 distinct small molecules. We exposed MCF7 cells, chosen because they are the most frequently used cell line in cmap, for 6 h with acriflavine and compared the changes in gene expression pattern to the untreated control. Strikingly, the six most enriched instances (highest enrichment score) were topoisomerase II inhibitors (Fig. 5A).

image

Figure 5.  (A) Gene expression-based analysis after treatment with 10 μM acriflavine. The 30 most up and down regulated genes were used as query signature in the Connectivity Map (cmap) database. Out of the 1309 substances in cmap, the topoisomerase II inhibitors hycanthone, mitoxantrone, ellipticine and daunorubicin showed the greatest similarity to the query signature. Score according to the cmap database. (B) Inhibition of topoisomerase I and II activity. Plasmid DNA was separated by agarose gel electrophoresis followed by staining with ethidium bromide. Mobility depends on plasmid conformation. Left panel: Lane 1: supercoiled (sc) plasmid + topo I (leading to generation of relaxed form); Lane 2: sc plasmid control; Lane 3: marker for relaxed forms of plasmid; Lane 4: plasmid + topo I + 5 μM acriflavine (inhibition of relaxation, compare lane 1). Right panel: Lane 1: sc plasmid + topo II (leading to generation of relaxed forms); Lane 2: sc plasmid control; Lane 3: marker for linear form of the plasmid; Lane 4: plasmid + topo II + 5 μM acriflavine (inhibition of relaxation, compare lane 1).

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Based on this cmap-based gene-expressing analysis, pointing towards topoisomerase inhibition as a mechanism of action of acriflavine, its inhibitory activity against topoisomerase I and II was tested directly in a cell-free system. Acriflavine clearly inhibited the activity of both topoisomerase I and II at concentrations considered to be low for this type of assay (Fig. 5B).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Drug screening in vitro, mostly using tumor cell lines and cell death as the endpoint, has been used extensively in cancer drug development and has identified several useful drugs now in clinical practice.(2) Screening in cell lines clearly reports cytotoxicity and provides mechanistic information, but the tumor type specific activity to be expected in the clinic of screening hits is questionable.(21) In the present work, we challenged the latter notion by searching for drugs with activity against CRC by selecting hit molecules from a drug screen in human colon cancer cell lines and counter screen in normal cell types using stringent criteria for the definition of hit molecules expected to be specifically CRC active. In this way, acriflavine was identified as a potentially CRC active molecule.

This finding required confirmation in a model system more closely reflecting clinical drug activity to be considered interesting. Primary cultures of tumor cells from patients as an in vitro model adequately reflects the clinical activity of directly acting small molecule cancer drugs, both on the diagnosis and the individual patient level.(7,9) This model was, therefore, chosen for the confirmatory in vitro step. Patient samples from OC and CLL were chosen as comparators because they represent a drug-sensitive solid tumor type and a generally very drug-sensitive malignancy, respectively.

The finding that acriflavine was more active in CRC samples than in OC and CLL points to the relevance of drug screening in human tumor cell lines to identify diagnosis-specific activity if a rigid qualifying algorithm for drug selection is used. The activity pattern of acriflavine in the patient samples becomes even more conspicuous in comparison with the pattern for the CRC-active drugs 5-FU, irinotecan and oxaliplatin that were all less active in the CRC samples compared with those from OC and CLL. A lack of cytotoxic selectivity between malignant and normal mononuclear cells is a feature shared by many antineoplastic agents, including those in clinical use and was confirmed here for 5-FU, irinotecan and oxaliplatin against colorectal cancer.(22) In contrast, the activity of acriflavine was similar in patient CRC and normal mononuclear cells, indicating a more advantageous therapeutic ratio for acriflavine compared with these standard drugs. Thus, we believe that the activity pattern observed for acriflavine is promising from a clinical point of view and that it is worthwhile further investigating acriflavine as a lead molecule for development of an active CRC drug.

Acriflavine might, of course, be active against other tumor types and further investigations in vitro in patient tumor samples from a broader spectrum of cancer diagnoses could point to promising tumor types for further investigation. Whether drug screening in cell lines along the strategy used here really identified a drug with novel activity against CRC can ultimately only be confirmed by clinical testing of acriflavine in patients with CRC. Furthermore, the generalizability of the screening strategy used here needs to be assessed by testing a similar strategy for other relatively drug-resistant tumor types.

Acriflavine, a mixture of two very closely related acridine molecules (Fig. 1D) was discovered almost 100 years ago and has been used as an antimicrobial agent, mostly topically but sometimes also systemically administered.(23) It has also been found to be active in sensitive tumor xenografts models in rodents when administered locally or systemically.(24–27) In these models, acriflavine has a short pharmacokinetic half-life, reaches peak plasma concentrations well above the IC50 values observed in the present investigation and is generally well tolerated.(24–28) With this background, further clinical development of acriflavine as a cancer drug seems considerably easier than the development of a new chemical entity.

Given the described good local tolerance to acriflavine when administered intraperitoneally combined with its short half-life in plasma, acriflavine speculatively could have a future in intraperitoneal chemotherapy following cytoreductive surgery for peritoneal carcinomatosis of CRC origin.(29) Other features making acriflavine an interesting candidate for cancer drug development are low cross-resistance and preserved activity also under hypoxic conditions, as illustrated by its effects in tumor spheroids. Acriflavine also fulfils features for a druggable molecule according to Lipinski’s rule of five.(19)

A key question is what makes the CRC tumor cells extraordinarily sensitive towards acriflavine. A common property of acridine derivatives, such as acriflavine, is their ability to intercalate nucleic acids and inhibit topoisomerase II.(30) Furthermore, acriflavine binds to and disrupts the cell surface membrane,(31) also leading to inhibition of protein kinase C, considered to account for some of its anti-tumor effects.(32) Acriflavine also effectively inhibits NF-κB activation in vivo and in vitro, which is associated with its anti-inflammatory effect.(33) Acriflavine was recently found to inhibit hypoxia inducible factor 1α (HIF-1α), which was associated with anti-angiogenesis effects and inhibition of tumor growth in vivo.(27) However, the cytotoxic effect of acriflavine against CRC is hard to explain from the above pharmacodynamic properties. Alkylating agents, as well as and inhibitors of topoisomerase II, protein kinase-C, NF-κB and angiogenesis, have been found to be poorly active or modestly active, at best, in CRC compared with other tumor types. The novel CRC activity of acriflavine as observed here thus remains to be elucidated.

In an effort to further clarify the mechanism of action of acriflavine we took advantage of a recently described genomics-based approach, the cmap, that connects short-term induced gene-expression perturbations from a specific drug with that of a large number of other drugs, allowing for establishment of drug activity relationships giving clues to testable hypotheses on mechanisms of action. Applying this cmap approach to acriflavine showed strong connection to topoisomerase II inhibitors. This mechanism of action was then confirmed in experiments showing acriflavine inhibition of topoisomerase II but also of topoisomerase I.

The finding that acriflavine is a topoisomerase I and II inhibitor is in line with the dual topoisomerase I/II inhibitory effect of other acridine derivatives, such as DACA,(34) a derivative that interestingly showed antitumor activity in a colon adenocarcinoma xenograft model in mice.(35) In addition, acriflavine inhibition of topoisomerase I and II is also compatible with the recent findings of its HIF-1α interaction, because topoisomerase I inhibitors like camptotecin have a strong HIF-1α suppressive effect,(36) with the ability of the acridine derivatives to intercalate nucleic acids and inhibit topoisomerase II.(30) However, acriflavine may have additional properties that remain to be elucidated and that can explain the novel activity against CRC.

The utility of genomics-based analysis using the cmap approach could now be regarded as established. We and others have repeatedly found it to correctly connect patterns of gene expression changes with druggable molecules that provide relevant clues to mechanisms of action as well as a basis for development of mechanistically new drugs for clinical use.(14,15)

In conclusion, using a stringent algorithm for cell line-based screening for identification of tumor type selective drug activity, the acridine derivative acriflavine was identified to have novel activity against CRC tumor cells in a clinically relevant in vitro model and to be a combined topoisomerase I and II inhibitor. The clinical relevance of these findings remains to be elucidated but points towards the possibility of fast-track development of a new active drug against CRC as well as a more general strategy for expedient identification and characterization of cancer drugs.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

The technical support of Wassim Moued, Lena Lenhammar, Christina Leek and Maria Rydåker is gratefully acknowledged. This study was supported by grants from the Swedish Cancer Society, the Swedish Foundation for Strategic Research and the Lions Cancer Research Fund.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Table S1. Expression values for the 30 most up and down regulated probes after acriflavine treatment (MCF7, 10 μM, 6 h).

FilenameFormatSizeDescription
CAS_2097_sm_tS1.xls46KSupporting info item

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