|Year : 2019 | Volume
| Issue : 3 | Page : 188-200
Identification of a novel anticancer compound through screening of a drug library on multicellular spheroids
Department of Pharmacognosy, Pharmaceutical and Drug Industries Division, Drug Bioassay-Cell Culture Laboratory, National Research Centre, Dokki, Giza, Egypt
|Date of Submission||18-Mar-2019|
|Date of Acceptance||30-May-2019|
|Date of Web Publication||26-Sep-2019|
Department of Pharmacognosy, Pharmaceutical and Drug Industries Division, National Research Centre, Dokki, Giza 12622
Source of Support: None, Conflict of Interest: None
Background and objectives A multicellular cancer spheroid model has proven to mimic the in-vivo tumors more closely compared with the conventionally used monolayer model. Thus, the spheroid model estimates the in-vivo activity more accurately than its counterpart the monolayer model. Accordingly, a library of 320 chemically diverse compounds was screened for their cytotoxicity against MCF7 human breast carcinoma spheroids, aiming for identification of novel compounds active against this type of solid tumor.
Materials and methods MCF7 spheroids were generated in 96-well plates by a centrifugation method. The spheroids took 5 days to reach ∼500-μm diameter and were ready for treatment. The initial screen was performed at 50 μM in triplicates. A dose–response study followed the initial screen. A counterscreen was carried out using RPE1 normal cell spheroids to identify the selectivity of active compounds. The acid phosphatase method was applied to measure the cytotoxicity of compounds. A clonogenic assay was used to investigate the viability of remaining cells after treatment with test compounds.
Results and conclusion The compound (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate was identified in this study for the first time with reasonable toxicity on MCF7 cancer spheroids. This compound is suggested as a lead compound for the development of more active derivatives against solid tumors. Additionally, the multicellular spheroid model was proved as a useful and applicable platform for identification of novel compounds for the treatment of solid tumors.
Keywords: breast carcinoma, cancer, MCF7, RPE1, screening and anticancer drug discovery, spheroids, therapeutic window
|How to cite this article:|
Fayad W. Identification of a novel anticancer compound through screening of a drug library on multicellular spheroids. Egypt Pharmaceut J 2019;18:188-200
|How to cite this URL:|
Fayad W. Identification of a novel anticancer compound through screening of a drug library on multicellular spheroids. Egypt Pharmaceut J [serial online] 2019 [cited 2019 Oct 20];18:188-200. Available from: http://www.epj.eg.net/text.asp?2019/18/3/188/264088
| Introduction|| |
Despite the enormous development in understanding the molecular basis of malignant tumors, the cure rates of cancers that require systemic treatment are still limited to 4% . Thus, there is a pressing need for the development for more efficient drugs for curing cancer. Cancer chemotherapy development started since the mid-20th century. The most organized large system for drug discovery of anticancer drugs is the Developmental Therapeutic Program run by the National Cancer Institute. Paclitaxel is a prominent example of approved antineoplastic agents that have been discovered by Developmental Therapeutic Program . Most currently used chemotherapeutic agents were identified in cell-based cytotoxicity assays where cancer cells are grown as monolayers . It is thought that this type of screen will continue to play an important role in cancer drug discovery. However, it was found that monolayer screening is not necessarily predictive for in-vivo activity . As an attempt to model solid tumors in vitro, Sutherland et al.  developed the multicellular spheroid model. Spheroids were shown to be superior to monolayers in modeling solid tumors in terms of growth kinetics , gene expression , three-dimensional structure, multicellular resistance , and similarity of extracellular matrix . Thus, spheroids are considered more efficient in predicting in-vivo anticancer activity compared with their monolayers counterpart. Indeed, there is an international increasing interest in cancer spheroid model reflected in the substantial increase in the number of cancer spheroid-related publications per year, as shown by searching PubMed website for the key words ‘cancer’ and ‘spheroids’ ([Figure 1]).
|Figure 1 Number of publications per year according to searching the PubMed website for the keywords: ‘cancer’ and ‘spheroids.’|
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In the current study, 320 chemically diverse compounds were purchased and were screened for their cytotoxic activity on MCF7 human breast carcinoma spheroids. The screen led to the identification of (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate compound. Further dose–response and clonogenic studies on both cancer and normal spheroids showed that this compound was selectively cytotoxic toward breast cancer spheroids at concentration of 6.25 μM.
| Materials and methods|| |
A total of 320 chemically diverse identified compounds were purchased from Specs Company (Bleiswijkseweg 552712 PB Zoetermeer, The Netherlands). The compounds were delivered in a 96-well format as dry films of 2-μmol quantities each and were dissolved in DMSO.
MCF7 (human breast carcinoma) and RPE1 (human normal immortalized retinal epithelial) cell lines were kindly gifted by Prof. Stig Linder, Karolinska Institute, Stockholm, Sweden. Both cell lines were grown in DMEM-F12 medium supplemented with 10% FBS and antibiotic-antimycotic (1%), and were kept at 37°C in 5% CO2 and 95% humidity.
Generation of spheroids
Round-bottom 96-well plates coated with poly-HEMA (cat. no. P3932; Sigma, Munich, Germany)  were used for production of spheroids as previously described. The spheroid generation was based on the centrifugation method described by Ivascu and Kubbies . Cell suspensions of 10 000 cell/well for MCF7 and 50 000 cell/well for RPE1 were seeded and centrifuged at 1000g for 10 min at 4°C. Plates were kept in an incubator overnight and then shaken with TITRAMAX 1000 shaker (Fisher Scientific Company, Waltham, Massachusetts, USA) at 450 rpm for further 4 days in the CO2 incubator.
Spheroids were treated for 3 h, then media was changed, and then they were incubated for further 5 days till cytotoxicity assessment. Media was changed every other day during the incubation. The initial screen was performed at 50 μM on MCF7 spheroids in triplicates. The dose–response study for the identified compound was performed on MCF7 and RPE1 spheroids at final concentrations of 50, 25, 12.5, 6.25, 3.12, and 1.56 μM in triplicates. For the study, 2-μM staurosporine was used as positive control, and 0.5% DMSO as negative control.
The acid phosphatase assay was used to measure the cytotoxicity according to the method previously described . Spheroids were washed twice with 200 μl PBS, and then 100 μl of para-nitro phenyl phosphate (Santa Cruz, Heidelberg, Germany) dissolved in a buffer solution (0.1 M sodium acetate, 0.1% triton X-100, pH=5) at a concentration of 2 mg/ml was added per well and incubated for 2 h at the CO2 incubator.
Absorbance was measured at 405 nm. Blank was subtracted from the readings, and percent cytotoxicity was calculated by the formula [1−(D/S)] ×100, where D and S denote the optical density of drug-treated and solvent-treated spheroids, respectively.
Both MCF7 and RPE1 spheroids were cultured and treated at the same settings performed at screening and dose–response studies. After the 5 days of incubation, the spheroids were washed with 200 μl PBS once, trypsinized for 15 min, and gently pipetted, and then spheroids were transferred to 6-well plates. The plates were incubated for further 13 days, with media change every third day. Colonies were washed with PBS, fixed with 100% methanol, stained with Geimsa and counted. The 2-μM staurosporine-treated spheroids were used as positive control, whereas 0.5% DMSO-treated spheroids were the negative control. All treatments were performed in triplicates. Clonogenicity percent reduction was calculated compared with the negative control: [1−(ND/NNC)]×100, where ND is the number of colonies in the drug-treated spheroids, and NNC is the number of colonies in the negative control spheroids.
| Results|| |
The drug library was screened at 50-μM final concentration on MCF7 spheroids in triplicates. Staurosporine (2 μM) was used as a positive control and caused 74.9±3.6% cytotoxicity. The % cytotoxicity results are presented in [Table 1].
|Table 1 Percent cytotoxicity results of the 320 compounds on MCF7 spheroids|
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The compound number 237 in [Table 1] was selected for further studies, as it caused the highest cytotoxicity (81%). For simplicity, the compound was given the code SP1 ([Figure 2]).
|Figure 2 Compound SP1 (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate.|
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A parallel dose–response study for SP1 was performed on both MCF7 and RPE1 spheroids ([Figure 3]) at six concentrations: 50, 25, 12.5, 6.25, 3.12, and 1.56 μM. The results are presented in [Figure 4].
|Figure 4 Dose–response results of compound SP1 on MCF7 and RPE1 spheroids.|
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Staurosporine (2 μM) was used as a positive control and caused 76.7±2.9% cytotoxicity in MCF7 spheroids, and 81.2±4.1% cytotoxicity in RPE1 spheroids. The IC50 values of SP1 on MCF7 and RPE1 spheroids were 29 and 18 μM, respectively, computed by GraphPad Prism program.
Both MCF7 and RPE1 spheroids were treated in triplicates at four different concentrations of SP1: 50, 25, 12.5, and 6.25 μM. The percent reduction in clonogenicity was calculated in reference to negative control (0.5% DMSO). The results are presented in [Figure 5]. Staurosporine (2 μM) was used as positive control and caused 100% reduction in clonogenicity in both cell lines.
|Figure 5 Effect of the compound SP1 on clonogenicity of MCF7 and RPE1 spheroids.|
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| Discussion|| |
The multicellular cancer spheroid model is gaining an increasingly interest to be employed in the anticancer drug discovery procedures. The inability of the conventional cancer monolayer model to accurately predict the in-vivo activity suits the three-dimesional models, including the cancer spheroids, in replacing the monolayer model or bridging the gap between the monolayer and animal models . Of particular interest, the hypoxic quiescent subpopulation that has been found both in solid tumors and cancer spheroids represents an obstacle for achieving a successful treatment . The hypoxic cells have been shown to be resistant to chemotherapeutic agent and are able to repopulate the tumor between doses . This subpopulation lies far from blood vessels that carry the chemotherapy. Thus, the drug must possess a penetrating property to access these cells in a therapeutic concentration . The three-dimensional cancer models are able to select for such compounds, whereas monolayers are blind to identify compounds with penetrating property.
In the present study, the breast carcinoma spheroids have been the primary platform for screening 320 chemically diverse compounds for anticancer activity. In the design of the experiment, the cancer spheroids were treated for 3 h and then media was changed aiming to mimic the physiologic conditions of clearance of the drugs from the body in comparable periods. In addition, such setting allows selection of penetrating compounds and not for cytotoxic compound that kill the spheroids layer by layer on incubating for long nonphysiological periods (>24 h). Moreover, 5 days of incubation was selected not to miss relatively late-acting drugs.
This screen identified only one compound that caused more than 80% cytotoxicity. This compound was (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate, and was termed SP1 ([Figure 2]). On reviewing literature, no reported biological activity was found for this compound, and to the author’s knowledge, this is the first report indicating its anticancer activity on cancer spheroids. On reviewing the literature, no biological activity at all was reported for this compound.
The spheroids of RPE1 normal human cell line have been previously shown to be completely nondividing , a state similar to most of adult normal tissues. This model was used to test the therapeutic window of SP1. The compound caused significant cytotoxicity (>59%) on RPE1 spheroids at doses from 12.5 to 50 μM ([Figure 4]). However, at the dose 6.25 μM, it was totally safe on RPE1 spheroids and caused 21% cytotoxicity on MCF7 spheroids at the same concentration ([Figure 4]). In the clonogenic study performed on the same two cell lines, a similar phenomenon was observed. SP1 caused high clonogenicity reduction in both cell line spheroids at concentrations from 12.5 to 50 μM, whereas was selectively toxic to cancer spheroids at concentration 6.25 μM ([Figure 5]). Interestingly, the compound induced more than 99% reduction in clonogenicity at 6.25 μM, whereas scored 21% cytotoxicity at the same concentration on MCF7 spheroids. These results suggest that the residual living cells after the drug treatment (∼80%) lost the clonogenic ability, possibly owing to senescence. Another possible explanation is that these residual cells die at longer time than 5 days after treatment, and thus were not clonogenic after seeding.
From these results, it can be concluded that although SP1 has minor activity on tumor tissue at low concentrations, at such concentrations, it is safe to normal cells. Thus, it can be anticipated that at multiple lower concentration treatments, a corresponding loss of regrowth of in-vivo tumors during recovery period can be expected after a chemotherapeutic dose of SP1 compound, which might lead to a successful remedy.
The results obtained both from the cytotoxicity and clonogenicity assays put the clonogenic assay in a significant position to correctly evaluate an anticancer agent and is recommended to be included in antitumor drug discovery procedures.
Screening of a large number of compounds on cancer and normal spheroids is highly encouraged. According to the author’s point of view, this would most likely identify curative compounds that can be tolerated by patients with cancer.
| Conclusion|| |
The cytotoxicity profile of (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate on both cancer and normal cell spheroids presents it as a promising anticancer compound that deserves further pharmacological and chemical studies.
The author is grateful for Professor Stig Linder (Karolinska Institute, Stockholm, Sweden) for kindly providing the cell lines.
Financial support and sponsorship
This work is supported by National Research Centre, project number: 10010114.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Beckwith MC, Tyler LS. Cancer chemotherapy manual. St Louis, MO: Facts and Comparisons; 2001.
Balis FM. Evolution of anticancer drug discovery and the role of cell-based screening. J Natl Cancer Inst 2002; 94:78–79.
Chabner BA, Roberts TGJr. Timeline: chemotherapy and the war on cancer. Nat Rev Cancer 2005; 5:65–72.
Sutherland RM, McCredie JA, Inch WR. Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 1971; 46:113–120.
Sherratt JA, Chaplain MA. A new mathematical model for avascular tumour growth. J Math Biol 2001; 43:291–312.
Chang TT, Hughes-Fulford M. Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes. Tissue Eng Part A 2009; 15:559–567.
Desoize B, Jardillier J. Multicellular resistance: a paradigm for clinical resistance? Crit Rev Oncol Hematol 2000; 36:193–207.
Longati P, Jia X, Eimer J, Wagman A, Witt MR, Rehnmark S et al.
3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 2013; 13:95.
Herrmann R, Fayad W, Schwarz S, Berndtsson M, Linder S. Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids. J Biomol Screen 2008; 13:1–8.
Ivascu A, Kubbies M. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 2006; 11:922–932.
Friedrich J, Eder W, Castaneda J, Doss M, Huber E, Ebner R et al.
A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay. J Biomol Screen 2007; 12:925–937.
LaBarbera DV, Reid BG, Yoo BH. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert Opin Drug Discov 2012; 7:819–830.
Tredan O, Galmarini CM, Patel K, Tannock IF. Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst 2007; 99:1441–1454.
Kennedy KA. Hypoxic cells as specific drug targets for chemotherapy. Anticancer Drug Des 1987; 2:181–194.
Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer 2006; 6:583–592.
Fayad W, Rickardson L, Haglund C, Olofsson MH, D’Arcy P, Larsson R et al.
Identification of agents that induce apoptosis of multicellular tumour spheroids: enrichment for mitotic inhibitors with hydrophobic properties. Chem Biol Drug Des 2011; 78:547–557.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]