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Figure 6. Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs9 and cell viability were evaluated by MTT and SRB ass ays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus fluphenazine on cell viability and (B) cell protein synthesis. (C) The effect of 5-FU plus fluoxetine on cell viability and (D) cell protein synthesis. (E) The effect of 5-FU plus benztropine on cell viability and (F) cell protein synthesis. (G) The effect of 5-FU plus artesunate on cell viability and (H) cell protein synthesis. Values are expressed in percentage of control and represent means 4 ** statist. t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.

Figure 6 Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs9 and cell viability were evaluated by MTT and SRB ass ays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus fluphenazine on cell viability and (B) cell protein synthesis. (C) The effect of 5-FU plus fluoxetine on cell viability and (D) cell protein synthesis. (E) The effect of 5-FU plus benztropine on cell viability and (F) cell protein synthesis. (G) The effect of 5-FU plus artesunate on cell viability and (H) cell protein synthesis. Values are expressed in percentage of control and represent means 4 ** statist. t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.

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Scheme 1. Chemical structures of the drugs used in combination. (A) CNS drugs: (1) selegiline, (2) safinamide, (3) enta- capone, (4) tolcapone, (5) latrepirdine, (6) fluphenazine, (7) thioridazine, (8) fluoxetine, (9) benztropine, (10) carbidopa, (11) bromocriptine, (12) nepicastat, (13) scopolamine, (14) carbamazepine, (15) sertraline and (16) rivastigmine. (B) Antimalarial drugs: (18) mefloquine, (19) chloroquine and (20) artesunate.
Scheme 1. Chemical structures of the drugs used in combination. (A) CNS drugs: (1) selegiline, (2) safinamide, (3) enta- capone, (4) tolcapone, (5) latrepirdine, (6) fluphenazine, (7) thioridazine, (8) fluoxetine, (9) benztropine, (10) carbidopa, (11) bromocriptine, (12) nepicastat, (13) scopolamine, (14) carbamazepine, (15) sertraline and (16) rivastigmine. (B) Antimalarial drugs: (18) mefloquine, (19) chloroquine and (20) artesunate.
Table 1. CNS drugs used in this work and their mechanism of action. 2. Results
Table 1. CNS drugs used in this work and their mechanism of action. 2. Results
Figure 1. The effect of 5-FU on HT-29 cells. (A) Cell viability and (B) dose-response. Cells were cultured in the presence of increasing concentrations of 5-FU, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3) *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 1. The effect of 5-FU on HT-29 cells. (A) Cell viability and (B) dose-response. Cells were cultured in the presence of increasing concentrations of 5-FU, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3) *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 2. The effects of some CNS drugs on HT-29 cells. (A) The effect of latrepirdine on cell viability and (B) the dose- response curve. (C) The effect of fluphenazine on cell viability and (D) the dose-response curve. (E) The effect of fluoxetine on cell viability and (F) the dose-response curve. (G) The effect of benztropine on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means -- SEM. Each experiment was done three times independently (n = 3); ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 2. The effects of some CNS drugs on HT-29 cells. (A) The effect of latrepirdine on cell viability and (B) the dose- response curve. (C) The effect of fluphenazine on cell viability and (D) the dose-response curve. (E) The effect of fluoxetine on cell viability and (F) the dose-response curve. (G) The effect of benztropine on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means -- SEM. Each experiment was done three times independently (n = 3); ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 3. The effects of some CNS drugs and antimalarial drugs on HT-29 cells. (A) The effect of thioridazine on cell viability and (B) the dose-response curve. (C) The effect of sertraline on cell viability and (D) the dose-response curve. (E) The effect of mefloquine on cell viability and (F) the dose-response curve. (G) The effect of artesunate on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MT] [ assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means a t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 3. The effects of some CNS drugs and antimalarial drugs on HT-29 cells. (A) The effect of thioridazine on cell viability and (B) the dose-response curve. (C) The effect of sertraline on cell viability and (D) the dose-response curve. (E) The effect of mefloquine on cell viability and (F) the dose-response curve. (G) The effect of artesunate on cell viability and (H) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MT] [ assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means a t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
t Drugs selected for combinations with 5-FU. Table 2. Cytotoxicity of 5-FU, several CNS drugs and some antimalarials agents in HT-29 colon cancer cells. [C59 values are given as mean.
t Drugs selected for combinations with 5-FU. Table 2. Cytotoxicity of 5-FU, several CNS drugs and some antimalarials agents in HT-29 colon cancer cells. [C59 values are given as mean.
administered together, mainly in the MTT assays. Additionally, for the higher concentra- tions (4 x ICs9), the results obtained for the combined drugs did not show improvements concerning the repurposed drugs alone (Figure 6). Microscopically, at concentrations of 4 x ICs, differences between cells were only found between 5-FU, control cells and treated cells; differences between single drugs and drug combinations were very subtle and both treatments resulted in the decreasing of cell number, less aggregate formation and rounded cells (Figure 7).
administered together, mainly in the MTT assays. Additionally, for the higher concentra- tions (4 x ICs9), the results obtained for the combined drugs did not show improvements concerning the repurposed drugs alone (Figure 6). Microscopically, at concentrations of 4 x ICs, differences between cells were only found between 5-FU, control cells and treated cells; differences between single drugs and drug combinations were very subtle and both treatments resulted in the decreasing of cell number, less aggregate formation and rounded cells (Figure 7).
Figure 4. Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs59 and the cell viability was evaluated by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus latrepirdine on cell viability and (B) cell protein synthesis. (C) The effect of 5-FU plus thioridazine on cell viability and (D) cell protein synthesis. (E) The effect of 5-FU plus sertraline on cell viability and (F) cell protein synthesis. (G) The effect of 5-FU plus mefloquine on cell viability and (H) cell protein synthesis. Values are expressed in percentage of control and represent means a t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 4. Growth inhibition of HT-29 after 48 h of combination therapy, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs59 and the cell viability was evaluated by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of 5-FU plus latrepirdine on cell viability and (B) cell protein synthesis. (C) The effect of 5-FU plus thioridazine on cell viability and (D) cell protein synthesis. (E) The effect of 5-FU plus sertraline on cell viability and (F) cell protein synthesis. (G) The effect of 5-FU plus mefloquine on cell viability and (H) cell protein synthesis. Values are expressed in percentage of control and represent means a t SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 5. Microscopic cellular visualisation of HT-29 cells after 48 h of incubation with vehicle (A), 5-FU (B), latrepirdine (C), latrepirdine + 5-FU (D), thioridazine (E), thioridazine + 5-FU (F), sertraline (G), sertraline + 5-FU (H), mefloquine (I) and mefloquine + 5-FU (J) at concentrations of 4 x ICs9 of each drug.
Figure 5. Microscopic cellular visualisation of HT-29 cells after 48 h of incubation with vehicle (A), 5-FU (B), latrepirdine (C), latrepirdine + 5-FU (D), thioridazine (E), thioridazine + 5-FU (F), sertraline (G), sertraline + 5-FU (H), mefloquine (I) and mefloquine + 5-FU (J) at concentrations of 4 x ICs9 of each drug.
Figure 8. Chou-Talalay method Fa-CI plot of 5-FU plus latrepirdine (A), fluoxetine (B), benztropine (C), thioridazine (D), sertraline (E), mefloquine (F), artesunate (G) and fluphenazine (H). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI < 1, CI= 1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Figure 8. Chou-Talalay method Fa-CI plot of 5-FU plus latrepirdine (A), fluoxetine (B), benztropine (C), thioridazine (D), sertraline (E), mefloquine (F), artesunate (G) and fluphenazine (H). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI < 1, CI= 1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Table 3. CI values and the respective fractional effect of different combinations of 5-FU plus CNS agents and antimalarial drugs. CI in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the ICs9 of each drug (total dose).
Table 3. CI values and the respective fractional effect of different combinations of 5-FU plus CNS agents and antimalarial drugs. CI in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the ICs9 of each drug (total dose).
Table 3. Cont.
Table 3. Cont.
Figure 9. Bliss (left) and HAS (right) synergy plots of 5-FU plus latrepirdine (A,B), thioridazine (C,D), sertraline (E,F) and mefloquine (G,H).
Figure 9. Bliss (left) and HAS (right) synergy plots of 5-FU plus latrepirdine (A,B), thioridazine (C,D), sertraline (E,F) and mefloquine (G,H).
Figure 10. Bliss (left) and HAS (right) synergy plots of 5-FU plus fluphenazine (A,B), fluoxetine (C,D), benztropine (E,F) and artesunate (G,H).
Figure 10. Bliss (left) and HAS (right) synergy plots of 5-FU plus fluphenazine (A,B), fluoxetine (C,D), benztropine (E,F) and artesunate (G,H).
Figure 11. The three combination schedules designed for evaluating 5-FU combination with fluphenazine, fluoxetine, benztropine and artesunate. Schedule A represents cells treated con- comitantly with 5-FU and each repurposed drug for 48 h. Schedule B represents cells pre-treated with 5-FU for 24 h followed by each repurposed drug for another 24 h. For schedule C, cells were pre-treated with each repurposed drug for 24h, followed by 5-FU for another 24 h.
Figure 11. The three combination schedules designed for evaluating 5-FU combination with fluphenazine, fluoxetine, benztropine and artesunate. Schedule A represents cells treated con- comitantly with 5-FU and each repurposed drug for 48 h. Schedule B represents cells pre-treated with 5-FU for 24 h followed by each repurposed drug for another 24 h. For schedule C, cells were pre-treated with each repurposed drug for 24h, followed by 5-FU for another 24 h.
Figure 12. Cont.
Figure 12. Cont.
Figure 12. HT-29 cells were exposed to sequential 5-FU and (A,B) fluphenazine (C,D) fluoxetine (E,F) benztropine and (G,H) artesunate using constant ratios of the ICs5q dose. Values are expressed in percentage of control and represent means + SEM Each experiment was done three times independently (n = 3).
Figure 12. HT-29 cells were exposed to sequential 5-FU and (A,B) fluphenazine (C,D) fluoxetine (E,F) benztropine and (G,H) artesunate using constant ratios of the ICs5q dose. Values are expressed in percentage of control and represent means + SEM Each experiment was done three times independently (n = 3).
Table 4. CI values and respective fractional effect for three different combination schedules of 5-FU plus fluphenazine, fluoxetine, benztropine and artesunate. CI in red indicates concentrations of drug pairs that are synergic.
Table 4. CI values and respective fractional effect for three different combination schedules of 5-FU plus fluphenazine, fluoxetine, benztropine and artesunate. CI in red indicates concentrations of drug pairs that are synergic.
Figure 13. Chou-Talalay method Fa-CI plot of three schedule-dependent combinations of 5-FU plus fluphenazine (A), fluoxetine (B), benztropine (C) and artesunate (D). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI < 1, CI = 1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Figure 13. Chou-Talalay method Fa-CI plot of three schedule-dependent combinations of 5-FU plus fluphenazine (A), fluoxetine (B), benztropine (C) and artesunate (D). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI < 1, CI = 1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Figure 14. The effects of some CNS drugs on MCF-7 cells. (A) The effect of fluoxetine on cell viability and (B) the dose- response curve. (C) The effect of sertraline on cell viability and (D) dose-response curve. (E) The effect of thioridazine on cell viability and (F) the dose-response curve. (G) The effect of fluphenazine on cell viability and (H) the dose-response curve. (I) The effect of benztropine on cell viability and (J) the dose-response curve. (K) The effect of latrepirdine on cell viability and (L) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. **** statistically significant vs. control at p < 0.0001.
Figure 14. The effects of some CNS drugs on MCF-7 cells. (A) The effect of fluoxetine on cell viability and (B) the dose- response curve. (C) The effect of sertraline on cell viability and (D) dose-response curve. (E) The effect of thioridazine on cell viability and (F) the dose-response curve. (G) The effect of fluphenazine on cell viability and (H) the dose-response curve. (I) The effect of benztropine on cell viability and (J) the dose-response curve. (K) The effect of latrepirdine on cell viability and (L) the dose-response curve. Cells were cultured in the presence of increasing concentrations of each drug, and after 48 h, the MTT assay was performed to measure the cellular viability. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. **** statistically significant vs. control at p < 0.0001.
+ Drugs selected for combinations with PTX. Table 5. Comparison between the ICs9 of several CNS drugs in HT-29 and MCF-7 cancer cells. ICs 9 values are given as mean.
+ Drugs selected for combinations with PTX. Table 5. Comparison between the ICs9 of several CNS drugs in HT-29 and MCF-7 cancer cells. ICs 9 values are given as mean.
Figure 15. Growth inhibition of MCF-7 after 48 h of combination therapy with PTX, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs9 and the cell viability was evaluated by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of PTX plus fluoxetine on cell viability and (B) cell protein synthesis. (C) The effect of PTX plus sertraline on cell viability and (D) cell protein synthesis. (E) The effect of PTX plus thioridazine on cell viability and (F) cell protein synthesis. (G) The effect of PTX plus fluphenazine on cell viability and (H) cell protein synthesis. (I) The effect of PTX plus benztropine on cell viability and (J) cell protein synthesis. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 15. Growth inhibition of MCF-7 after 48 h of combination therapy with PTX, by MTT (left) and SRB assays (right). Cells were exposed to concentrations of each drug of 0.25, 0.5, 1, 2 and 4 times their ICs9 and the cell viability was evaluated by MTT and SRB assays. The drugs in combination were co-administered at the same time. (A) The effect of PTX plus fluoxetine on cell viability and (B) cell protein synthesis. (C) The effect of PTX plus sertraline on cell viability and (D) cell protein synthesis. (E) The effect of PTX plus thioridazine on cell viability and (F) cell protein synthesis. (G) The effect of PTX plus fluphenazine on cell viability and (H) cell protein synthesis. (I) The effect of PTX plus benztropine on cell viability and (J) cell protein synthesis. Values are expressed in percentage of control and represent means + SEM. Each experiment was done three times independently (n = 3); * statistically significant vs. control at p < 0.05. ** statistically significant vs. control at p < 0.01. *** statistically significant vs. control at p < 0.001. **** statistically significant vs. control at p < 0.0001.
Figure 16. Microscopic cellular visualisation of MCF-7 cells after 48 h of incubation with vehicle (A), PTX (B), fluoxetine (C), fluoxetine + PTX (D), sertraline (E), sertraline + PTX (F), thioridazine (G), thioridazine + PTX (H), fluphenazine (I) fluphenazine + PTX (J), benztropine (K) and benztropine + PTX (L) at concentrations of 4 x ICsq of each drug. Scale bar: 50 um.
Figure 16. Microscopic cellular visualisation of MCF-7 cells after 48 h of incubation with vehicle (A), PTX (B), fluoxetine (C), fluoxetine + PTX (D), sertraline (E), sertraline + PTX (F), thioridazine (G), thioridazine + PTX (H), fluphenazine (I) fluphenazine + PTX (J), benztropine (K) and benztropine + PTX (L) at concentrations of 4 x ICsq of each drug. Scale bar: 50 um.
Figure 17. Chou-Talalay method Fa-CI plot of PTX plus fluoxetine (A), sertraline (B), thioridazine (C), fluphenazine (D) and benztropine (E). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI <1,CI=1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Figure 17. Chou-Talalay method Fa-CI plot of PTX plus fluoxetine (A), sertraline (B), thioridazine (C), fluphenazine (D) and benztropine (E). CI was plotted on the y-axis as a function of effect level (Fa) on the x-axis to evaluate drug synergism. CI <1,CI=1 and CI > 1 refers to synergism, additivity and antagonism, respectively.
Table 6. CI values and respective fractional effect of different combinations of PTX plus CNS agents. Cl in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the ICs9 of each drug (total dose).
Table 6. CI values and respective fractional effect of different combinations of PTX plus CNS agents. Cl in red indicates concentrations of drug pairs that are synergic. Cells were treated with 0.25, 0.5, 1, 2 and 4 times the ICs9 of each drug (total dose).
Figure 18. Bliss synergy plots of PTX plus fluoxetine (A), sertraline (B), thioridazine (C), fluphenazine (D) and benztropine (E).
Figure 18. Bliss synergy plots of PTX plus fluoxetine (A), sertraline (B), thioridazine (C), fluphenazine (D) and benztropine (E).
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