3.1. NCI 60 Cell Line Panel In Vitro ScreeningThe NCI in vitro screen comprises a panel of sixty distinct human tumor cell lines (from nine different organ sites), against which compounds are tested over a specific concentration range (10 nM–100 µM) to evaluate the relative degree of growth inhibition or cytotoxicity against each cell line. For each studied drug, a characteristic profile or “fingerprint” of cellular response is established [35]. The “dose–response curves” and “mean graphs” are the components of the NCI screening data report package [36] that most investigators are interested in. The dose–response curve provides three response parameters: the 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% lethal concentration (LC50) “net cell killing” or “cytotoxicity parameter”, which are the concentrations of the test drug at which the percentages of the cell growth (PG) are +50%, 0%, and −50%, respectively [37]. The mean graph is a pattern made by plotting positive and negative values, referred to as “deltas”, obtained from PG, GI50, TGI or LC50 data for a given chemical evaluated against each cell line in the NCI in vitro screen. Deltas are represented as horizontal bars in relation to a vertical line representing the mean panel [35]. Each bar represents the divergence of a single cancer cell line from the overall mean value of all the tested cells. The mean graph displays the relative resistance and sensitivity of all the cell lines at three levels of effect: GI50, TGI and LC50. Relatively sensitive cell lines have negative deltas and right-extending bars. While relatively resistant cell lines have positive deltas and bars that extend to the left [38].The methodology of NCI 60 panel in vitro screening consists of two consecutive stages: the NCI 60 cell one-dose screen and the NCI 60 cell five-dose screen [35]. In the first phase, all the submitted compounds are initially evaluated at a single high concentration (10−5 M) against a panel of sixty distinct human cancer cell lines. The data from a single dose are presented as a mean graph of the percent growth (PG) of the treated cells, allowing for the detection of both growth inhibition (values > 0) and fatality (values 39]. Only compounds that meet the pre-determined inhibitory threshold requirements in a minimum number of cell lines will advance to the second phase. Compounds that display significant growth inhibition in the one-dose screen are routinely tested against the panel of sixty cells at five 10-fold dilutions (10 nM, 100 nM, 1 µM, 10 µM and 100 µM) in the five-dose screen. Each test compound in the comprehensive screen produces sixty dose-response curves and three mean graphs of the three response parameters GI50, TGI and LC50 [35,38,40].Bis-triazole MS47 was selected by the National Cancer Institute for testing against the NCI 60 panel of human tumor cell lines. The mean graph of the initial single-dose (10 μM) screen of MS47 (named as NSC778438) is presented in Figure 2. The percentage growth (PG) that is altered due to treatment represents the anticancer activity. The majority of melanoma and CNS cancer cell lines are relatively very susceptible to MS47. The highest cytotoxic activity for MS47 (yellow highlight) is 96.85% against the melanoma cell line SK-MEL-5, followed by 94.25% against the breast cancer BT-549 cell line, followed by 92.76% against the CNS cancer SNB-75 cell line and 92.63% against the prostate cancer DU-145 cell line. Although leukemic cell lines showed resistance to the lethality of MS47 (LC50 > 100 µM) (Table 1), MS47 demonstrates the highest growth inhibition of 97.98% against the leukemia K-562 cell line of all the tested cells, followed by the breast cancer HS 578T cell line, and the renal cancer A498 and RXF 393 cell lines with growth inhibitions of 85.84%, 85.43% and 80.06%, respectively.

Renal cancer cell lines demonstrate an exceptional resistance to MS47. The renal cancer TK-10 and UO-31 cell lines, ovarian cancer NCI/ADR-RES cell line, and colon cancer HCT-15 cell line have the lowest growth inhibition and highest relative resistance.

Figure 2. The mean graph of the one-dose (10 μM) screen of MS47 (NSC778438) illustrates the sensitivity of the sixty human tumor cell lines to the cytotoxic activity of MS47. Yellow highlights represent the most sensitive cell lines to MS47 cytotoxic (lethal) activity and their PG values in the range of 90%.

Figure 2. The mean graph of the one-dose (10 μM) screen of MS47 (NSC778438) illustrates the sensitivity of the sixty human tumor cell lines to the cytotoxic activity of MS47. Yellow highlights represent the most sensitive cell lines to MS47 cytotoxic (lethal) activity and their PG values in the range of 90%.

Ligand MS47 (NSC778438) satisfied the pre-determined criteria for the threshold inhibition in a minimum number of cells in the NCI 60 cell one-dose screening, so it was tested against the panel of sixty tumor cell lines of NCI at five small doses. Table 1 and Figure 3 illustrate the GI50, TGI and LC50 values (µM) and dose–response curves of the MS47 (NSC778438) against the panel of NCI 60 human cancer cell lines that resulted from the NCI 60 cell five-dose screening. While the mean graphs of the log10 values (Molar) of GI50, TGI and LC50 of MS47 (NSC778438) obtained from the NCI 60 cell line experiments can be found in the Supplementary Materials (Figure S1).The results in Table 1 and dose–response curves in Figure 3 reflect the potent anticancer activity (cellular growth inhibition, total growth inhibition and lethality) of MS47 (NSC778438) against most of the NCI 60 human cancer cell lines. The NCI 60 GI50 values range from 0.111 µM to 24.1 µM with the exception of the ovarian cancer NCI/ADR-RES and renal cancer CAKI-1 cell lines, which showed significant resistance to MS47 with GI50 values > 100 µM. The most sensitive cell line to MS47 was the melanoma MALME-3M cell line, with the lowest sub-micromolar GI50 value being 0.111 µM, followed by the ovarian cancer OVCAR-4, breast cancer MCF7 and MDA-MB-468, colon cancer COLO 205, CNS cancer U251, SNB-75 and SNB-19, melanoma MDA-MB-435 and LOX IMVI, renal cancer SN12C, and non-small cell lung cancer NCI-H522 cell lines, showing sub-micromolar GI50 values MS47 was for the renal cancer 786-0 cell line (GI50 = 24.1 µM).

The total growth inhibition activity of MS47 (NCS778438) against the NCI 60 human cancer cell lines can be described by the NCI 60 TGI values, which range from 0.26 µM to 38.1 µM. Resistance to MS47 (TGI values > 100 µM) can be found in the leukemia CCRF-CEM, colon cancer HCT-15, ovarian cancer NCI/ADR-RES, and renal cancer 786-0 and CAKI-1 cell lines. MS47 showed the highest potency in total growth inhibition against the ovarian cancer OVCAR-4 cell line (TGI = 0.26 µM), followed by the melanoma MALME-3M cell line (TGI = 0.265 µM), and other cell lines showing sub-micromolar TGI values less than 0.4 µM. While the lowest potency in the total growth inhibitory activity was against the renal cancer UO-31 cell line.

The NCI 60 LC50 values show the lethality and cytotoxicity of MS47 (NCS778438) against the NCI 60 human cancer cell lines. The values range from 0.515 µM to 86.1 µM. The ovarian cancer OVCAR-4 cell line showed the highest sensitivity to MS47 (LC50 = 0.515 µM), followed by the melanoma MDA-MB-435 and MALME-3M, colon cancer COLO 205, and CNS cancer U251 cell lines, with sub-micromolar LC50 values of 0.602, 0.636, 0.644 and 0.694 µM, respectively, and other cell lines with LC50 values of <1 µM. The least lethal activity shown by MS47 was for the renal cancer UO-31 cell line (LC50 = 86.1 µM). All of the leukemia cell lines showed interesting resistance to MS47 cytotoxic activity, in addition to the colon cancer HCT-15, ovarian cancer NCI/ADR-RES, renal cancer 786-0 and CAKI-1, prostate cancer PC-3 and breast cancer HS 578T cell lines, with LC50 values of >100 µM. The resistance and sensitivity shown by the panel of sixty human cancer cell lines reflected a significant selective cytotoxic (lethal) activity for MS47.

Table 1. The anticancer activity of MS47 (NSC778438) against the NCI 60 human cancer cell lines illustrated by its GI50, TGI and LC50 values (µM). Yellow highlights represent the highest potency of MS47, while green highlights represent its lowest potency against NCI 60 panel.

Table 1. The anticancer activity of MS47 (NSC778438) against the NCI 60 human cancer cell lines illustrated by its GI50, TGI and LC50 values (µM). Yellow highlights represent the highest potency of MS47, while green highlights represent its lowest potency against NCI 60 panel.

Panel/Cell LineResponse Parameters of Ligand MS47 (µM)GI50TGILC50Leukaemia CCRF-CEM0.251>100>100HL-60(TB)0.2260.643>100K-5620.59714.4>100MOLT-40.2260.600>100RPMI-82260.6293.86>100SR0.4575.35>100Non-Small Cell Lung Cancer A549/ATCC1.433.177.04EKVX1.543.056.04HOP-621.542.915.52HOP-920.4231.405.88NCI-H2261.563.126.24NCI-H230.2670.79424.8NCI-H322M0.2891.043.45NCI-H4600.3441.386.24NCI-H5220.1940.4814.76Colon Cancer COLO 2050.1740.3350.644HCC-29980.3171.124.49HCT-1160.2831.143.57HCT-1522.9>100>100HT290.3210.90033.8KM120.5442.176.39SW-6200.4071.737.40CNS Cancer SF-2680.3261.456.88SF-2951.362.725.46SF-5390.2020.4200.875SNB-190.1820.3710.755SNB-750.1760.4211.01U2510.1750.3480.694Melanoma LOX IMVI0.1870.4892.62MALME-3M0.1110.2650.636M141.583.387.24MDA-MB-4350.1760.3260.602SK-MEL-20.2190.5538.81SK-MEL-280.9132.466.24SK-MEL-50.2270.7052.70UACC-2570.7112.427.07UACC-621.052.566.26Ovarian Cancer IGROV10.2330.6873.79OVCAR-30.2610.7617.37OVCAR-40.1310.2600.515OVCAR-50.5922.529.07OVCAR-81.563.66-NCI/ADR-RES>100>100>100SK-OV-31.995.3236.9Renal Cancer 786-024.1>100>100A4981.616.0226.0ACHN9.5623.254.6CAKI-1>100>100>100RXF 3933.6015.254.4SN12C0.1850.3710.745TK-1011.130.181.3UO-3116.938.186.1Prostate Cancer PC-32.407.52>100DU-1451.032.234.83Breast Cancer MCF70.1440.3580.894MDA-MB-231/ATCC0.25310.261.4HS 578T2.889.17>100BT-5491.733.165.78MDA-MB-4680.1730.3820.843

Figure 3. Dose–response curves of the cytotoxic activity of MS47 (NSC778438) against the panel of sixty human cancer cell lines. Value of 100 for the growth percentage indicates the growth of untreated cells, while a growth percentage value of 0 indicates no net growth throughout the period of the experiment, and a growth percentage value of −100 represents that all of the cells were killed by MS47.

Figure 3. Dose–response curves of the cytotoxic activity of MS47 (NSC778438) against the panel of sixty human cancer cell lines. Value of 100 for the growth percentage indicates the growth of untreated cells, while a growth percentage value of 0 indicates no net growth throughout the period of the experiment, and a growth percentage value of −100 represents that all of the cells were killed by MS47.

The NCI 60 cell five-dose screening results indicated that MS47 (NSC778438) has potent and selective cell growth inhibition and cytotoxic activities against tumor cell lines isolated from distinct organs, making it a promising anticancer drug candidate for further development for the treatment of multiple carcinomas, such as renal, melanoma, ovarian, colon, breast, and CNS cancers.

The cytotoxicity of MS47 indicated by the NCI 60 panel in vitro screen results and the higher selective potent growth inhibitory shown by MS49 indicate that further preclinical evaluations of both ligands are warranted. Specifically notable is the sensitivity of the melanoma MDA-MB-435 cell line (LC50 = 0.602 µM); thus, this cell line was selected for more thorough interrogation of anticancer activity.

3.2. MTT AssayThe growth inhibitory activities of MS47 and MS49 were tested further in vitro using an MTT assay [20,41,42] against the human melanoma MDA-MB-435 cell line, which was chosen in particular for further in vitro anticancer evaluation of both ligands as it is one of the most sensitive cell lines to the lethal effect of MS47, as shown by the NCI 60 panel data.The estimated concentrations at 50% cell growth inhibition (GI50) were determined from the dose–response curves (Figure 4) after 72 h exposure of melanoma MDA-MB-435 cells to MS47 and MS49, and are provided in Table 2. The GI50 values resulting from examination of the growth inhibition by both ligands in human non-tumorigenic MRC-5 (embryonic lung fibroblasts) to evaluate their putative selective cytotoxic activities are also presented in Table 2. In the concentration range (0.01–100 µM), both MS47 and MS49 inhibit the growth of the human melanoma MDA-MB-435 cell line potently. However, ligand MS49 (GI50 value of 75 nM) shows more potent growth inhibitory effect than MS47 (GI50 value of 226 nM). Comparing the GI50 values with those of human normal lung MRC-5 fibroblasts, both ligands demonstrate greater potency in the melanoma cell line than in non-tumorigenic lung fibroblasts, with indicated cancer selectivity indices (SI) of 9.8 for MS47 and 17.7 for MS49, revealing their good selective growth inhibitory effects for cancer cells over normal cells.

Table 2. Growth inhibition effects of MS47 and MS49 on human melanoma MDA-MB-435 and human normal lung MRC-5 cell lines. Values of GI50 are presented as mean ± standard deviation of at least three separate experiments (n = 8 per trial). “SI: Selectivity index (GI50 MRC-5/GI50 melanoma cell line)”.

Table 2. Growth inhibition effects of MS47 and MS49 on human melanoma MDA-MB-435 and human normal lung MRC-5 cell lines. Values of GI50 are presented as mean ± standard deviation of at least three separate experiments (n = 8 per trial). “SI: Selectivity index (GI50 MRC-5/GI50 melanoma cell line)”.

LigandGI50 (μM) ± S.D.SIMDA-MB-435 Cell LineMRC-5 Cell Line470.226 ± 0.0572.219 ± 0.0769.8490.075 ± 0.0101.325 ± 0.13717.7

Figure 4. Dose–response curves that show the growth inhibiting effects of MS47 and MS49 against melanoma MDA-MB-435 cell line. Values are mean ± SD, n = 8, graphs are representative of experiments performed on at least three separate occasions.

Figure 4. Dose–response curves that show the growth inhibiting effects of MS47 and MS49 against melanoma MDA-MB-435 cell line. Values are mean ± SD, n = 8, graphs are representative of experiments performed on at least three separate occasions.

3.4. Cell Cycle AnalysisTo determine whether the MDA-MB-435 cell cycle was perturbed by treatment with MS47 and MS49 and help establish the cell death modality, both a cell cycle analysis and an apoptosis assay were performed [43,44].The effect of treating the human melanoma MDA-MB-435 cells with MS47 and MS49 on the cell cycle was investigated by flow cytometry. Cells were exposed to MS47 and MS49 (0.5 × GI50, 1 × GI50 and 2 × GI50) for 24, 48 and 72 h (Figure 6). Two-way ANOVA indicated that both MS47 and MS49 caused no significant change in the numbers of events in specific cell cycle phases among all treated groups compared to the untreated control group after 24 and 48 h of treatment (Figure 6A,B). In contrast, after 72 h of exposure, Tukey’s multiple comparison test found that MS47 (2 × GI50) caused significant arrest in the G0/G1 and G2/M phases of the cell cycle in the human melanoma MDA-MB-435 cells (** ppFigure 6C,D). While MS49 (2 × GI50) did not show any significant difference compared to the control untreated group and other treatments after 72 h of exposure. Thus, in terms of cell cycle arrest, MS47 (2 × GI50) exerts a more significant effect on treated cells in comparison to MS49 (2 × GI50) (* pMS47 may trigger cell death following the arrest of cells at G0/G1.For both MS47 (2 × GI50) and MS49 (2 × GI50), we observed the presence of sub-G0/G1 events after 72 h, indicating the apoptotic death of the cancer cells. Additionally, the G2/M phase was depleted in both ligands in comparison to other treated groups in an observable manner. However, this depletion was significant only after treating the cells with MS47 (2 × GI50) (* pSupplementary Materials (Figure S2).

Figure 6. Statistical analyses of cell cycle phases: G0/G1, S and G2/M in human melanoma MDA-MB-435 cells, treated with 0.5 × GI50, 1 × GI50 and 2 × GI50 of MS47 and MS49 compared to the untreated cells (control group) for (A) 24 h, (B) 48 h and (C) 72 h. MS47 (2 × GI50) evoked significant arrest in the G0/G1 and G2/M phases (* p < 0.05 and ** p < 0.005; experiments were repeated ≥ three times, n = 2). (D) Flow cytometric cell cycle analysis (histograms) of human melanoma MDA-MB-435 cells, treated with MS47 and MS49 of 2 × GI50 concentrations for 72 h compared to the control cells and stained with propidium iodide (PI).

Figure 6. Statistical analyses of cell cycle phases: G0/G1, S and G2/M in human melanoma MDA-MB-435 cells, treated with 0.5 × GI50, 1 × GI50 and 2 × GI50 of MS47 and MS49 compared to the untreated cells (control group) for (A) 24 h, (B) 48 h and (C) 72 h. MS47 (2 × GI50) evoked significant arrest in the G0/G1 and G2/M phases (* p < 0.05 and ** p < 0.005; experiments were repeated ≥ three times, n = 2). (D) Flow cytometric cell cycle analysis (histograms) of human melanoma MDA-MB-435 cells, treated with MS47 and MS49 of 2 × GI50 concentrations for 72 h compared to the control cells and stained with propidium iodide (PI).

3.5. Annexin V-FITC and Propidium Iodide Apoptosis AssayTo investigate the cell death mechanism and test the hypothesis that MS47 and MS49 may initiate apoptosis, human melanoma MDA-MB-435 cells were treated with both ligands (0.5 × GI50, 1 × GI50 and 2 × GI50) for 24, 48 and 72 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry (Figure 7). Corresponding scatter plots are shown in the Supplementary Materials (Figure S3).After 24 h of the treatment, the two-way ANOVA test found that MS47 (0.5 × GI50) and (2 × GI50) have a direct effect on the cell viability as the number of healthy cells decreased significantly compared to the control untreated cells (* pMS47 (0.5 × GI50) and (2 × GI50) decreased the percentage of healthy cells compared to that recorded in the control untreated cells (* pMS47 (2 × GI50), as the number of apoptotic cells was ten-fold higher in the MS47-treated (2 × GI50) cells compared to the control cells (** pMS49, the number of viable cells decreased significantly when the cells were treated with (1 × GI50) and (2 × GI50) MS49 compared to the control untreated group at (* ppMS47 (2 × GI50), MS49 (2 × GI50) showed a 10-fold significant increase in the percentage of late apoptotic cells compared to that of the control untreated group (** pFigure 7A.The same cytotoxic (apoptotic) effect was observed after 48 h of treatment. The two-way ANOVA found that MS47 caused significant decrease in the percentage of viable cells (* pppMS47 (2 × GI50). Similarly, MS49 caused significant programmed cell death; the late apoptotic population increased, while the viable cell population decreased in a significant manner when the cells were treated with MS49 (2 × GI50) for 48 h compared to the other treatments and the control untreated cells at (*** ppFigure 7B).After 72 h exposure to MS47 and MS49, the same cytotoxic (apoptotic) effect persisted. Tukey’s multiple comparison test found that for the MS47-treated cells (2 × GI50), the percentage of viable cells decreased while the percentage of late apoptotic cells increased significantly compared to that of the control group (*** pMS49, 1 × GI50 exposure caused a significant decrease in the viable cell population (* ppMS49 at 2 × GI50 showed a strong significant decrease in the number of viable cells compared to all the other treated groups (**** ppFigure 7C,D).

Figure 7. Statistical analysis of the percentages of healthy, apoptotic (early and late) and necrotic human melanoma MDA-MB435 cells, treated with MS47 and MS49 (0.5 × GI50, 1 × GI50 and 2 × GI50) compared to the untreated cells (control group) for (A) 24 h, (B) 48 h and (C) 72 h. Both ligands caused significant increase in the percentage and number of the late apoptotic cells (* p < 0.05, ** p < 0.005, *** p < 0.0005; experiments were repeated ≥ three times, n = 2). (D) Flow cytometric analysis (histograms) of cell apoptosis mechanism (apoptosis/necrosis) in human melanoma MDA-MB435 cells, treated with MS47 and MS49 of 2 × GI50 concentrations compared to the untreated cells (control group) and stained with FITC-conjugated annexin V and propidium iodide (PI) for 72 h.

Figure 7. Statistical analysis of the percentages of healthy, apoptotic (early and late) and necrotic human melanoma MDA-MB435 cells, treated with MS47 and MS49 (0.5 × GI50, 1 × GI50 and 2 × GI50) compared to the untreated cells (control group) for (A) 24 h, (B) 48 h and (C) 72 h. Both ligands caused significant increase in the percentage and number of the late apoptotic cells (* p < 0.05, ** p < 0.005, *** p < 0.0005; experiments were repeated ≥ three times, n = 2). (D) Flow cytometric analysis (histograms) of cell apoptosis mechanism (apoptosis/necrosis) in human melanoma MDA-MB435 cells, treated with MS47 and MS49 of 2 × GI50 concentrations compared to the untreated cells (control group) and stained with FITC-conjugated annexin V and propidium iodide (PI) for 72 h.