Prediction of Drug Synergism between Peptides and Antineoplastic Drugs Paclitaxel, 5-Fluorouracil, and Doxorubicin Using In Silico Approaches

Abstract

Chemotherapy is the main treatment for most early-stage cancers; nevertheless, its efficacy is usually limited by drug resistance, toxicity, and tumor heterogeneity. Cell-penetrating peptides (CPPs) are small peptide sequences that can be used to increase the delivery rate of chemotherapeutic drugs to the tumor site, therefore contributing to overcoming these problems and enhancing the efficacy of chemotherapy. The drug combination is another promising strategy to overcome the aforementioned problems since the combined drugs can synergize through interconnected biological processes and target different pathways simultaneously. Here, we hypothesized that different peptides (P1-P4) could be used to enhance the delivery of chemotherapeutic agents into three different cancer cells (HT-29, MCF-7, and PC-3). In silico studies were performed to simulate the pharmacokinetic (PK) parameters of each peptide and antineoplastic agent to help predict synergistic interactions in vitro. These simulations predicted peptides P2-P4 to have higher bioavailability and lower T_max, as well as the chemotherapeutic agent 5-fluorouracil (5-FU) to have enhanced permeability properties over other antineoplastic agents, with P3 having prominent accumulation in the colon. In vitro studies were then performed to evaluate the combination of each peptide with the chemotherapeutic agents as well as to assess the nature of drug interactions through the quantification of the Combination Index (CI). Our findings in MCF-7 and PC-3 cancer cells demonstrated that the combination of these peptides with paclitaxel (PTX) and doxorubicin (DOXO), respectively, is not advantageous over a single treatment with the chemotherapeutic agent. In the case of HT-29 colorectal cancer cells, the combination of P2-P4 with 5-FU resulted in synergistic cytotoxic effects, as predicted by the in silico simulations. Taken together, these findings demonstrate that these CPP6-conjugates can be used as adjuvant agents to increase the delivery of 5-FU into HT-29 colorectal cancer cells. Moreover, these results support the use of in silico approaches for the prediction of the interaction between drugs in combination therapy for cancer.

Keywords: cancer therapy; cell-penetrating peptides; drug combination; drug synergism; in silico.

Scheme 1

Chemical structure of the peptides evaluated in silico and in vitro in this manuscript. (A) P1, (B) P2, (C) P3, and (D) P4.

Figure 1

Plasma concentration of 5-FU generated using the GastroPlus^® software.

Figure 2

Plasma concentration of DOXO generated using the GastroPlus^® software.

Figure 3

Plasma concentration of PTX generated using the GastroPlus^® software.

Figure 4

Plasma concentration of P1 generated using the GastroPlus^® software.

Figure 5

Plasma concentration of P2 generated using the GastroPlus^® software.

Figure 6

Plasma concentration of P4 generated using the GastroPlus^® software.

Figure 7

Plasma concentration of P3 generated using the GastroPlus^® software.

Figure 8

Representation of Blood/Plasma concentration ratio versus P_eff for peptides P1–P4.

Figure 9

Cytotoxic results of MCF-7 after exposure to increasing concentrations of peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3). * Statistically significant vs. control (vehicle) at p < 0.05, ** statistically significant vs. control (vehicle) at p < 0.01.

Figure 10

Morphological evaluation of MCF-7 after exposure to increasing concentrations of P1–P4 (0.01–100 μM) for 48 h. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 11

Cytotoxic results of HT-29 after exposure to increasing concentrations of peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay and the results are given as the mean ± SEM (n = 3). * Statistically significant vs. control (vehicle) at p < 0.05, ** statistically significant vs. drug alone at p < 0.01.

Figure 12

Morphological evaluation of HT-29 after exposure to increasing concentrations of P1–P4 (0.01–50 μM) for 48 h. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 13

Cytotoxic results of PC-3 after exposure to increasing concentrations of peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3). * Statistically significant vs. control (vehicle) at p < 0.05.

Figure 14

Morphological evaluation of PC-3 after exposure to increasing concentrations of P1–P4 (0.01–50 μM) for 48 h. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 15

Comparison of cell viability results of (A) MCF-7, (B) HT-29, and (C) PC-3 cells after exposure to increasing concentrations of the peptides P1–P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3).

Figure 16

Cytotoxic results of MCF-7 after exposure to combinations of 3 nM of paclitaxel with increasing concentrations of the peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3). * Statistically significant vs. drug alone at p < 0.05, ** statistically significant vs. drug alone at p < 0.01, *** statistically significant vs. drug alone at p < 0.001, **** statistically significant vs. drug alone at p < 0.0001.

Figure 17

Morphological evaluation of MCF-7 after exposure to a combination of IC₅₀ of PTX (3 nM) with increasing concentrations of P1–P4 (0.01–50 μM) for 48 h. Both substances were added at the same time. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 18

Cytotoxic results of PC-3 after exposure to combinations of 8 μM of doxorubicin with increasing concentrations of the peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3). **** Statistically significant vs. drug alone at p < 0.0001.

Figure 19

Morphological evaluation of PC-3 after exposure to a combination of 8 μM of DOXO with increasing concentrations of P1–P4 (0.01–50 μM) for 48 h. Both substances were added at the same time. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 20

Cytotoxic results of HT-29 after exposure to combinations of 3 μM of 5-FU with increasing concentrations of the peptides (A) P1, (B) P2, (C) P3, and (D) P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3). * Statistically significant vs. drug alone at p < 0.05, ** statistically significant vs. drug alone at p < 0.01, *** statistically significant vs. drug alone at p < 0.001, **** statistically significant vs. drug alone at p < 0.0001.

Figure 21

Morphological evaluation of HT-29 after exposure to a combination of the IC₅₀ of 5-FU (3 μM) with increasing concentrations of P1–P4 (0.01–50 μM) for 48 h. Both substances were added at the same time. Control cells were treated with the vehicle (0.01% DMSO). These images are representative of three independent experiments. Scale bar is 200 μM.

Figure 22

Comparison of the cell viability results of (A) MCF-7, (B) HT-29, and (C) PC-3 cells after exposure to combinations of chemotherapeutic drugs with increasing concentrations of the peptides P1–P4 (0.01–50 μM) for 48 h. Control cells were treated with 0.01% DMSO (vehicle). Cell viability was obtained using the MTT assay, and the results are given as the mean ± SEM (n = 3).

Figure 23

Effect level–combination index (Fa-CI) plot obtained for HT-29 cells after exposure to the combination of 5-FU with increasing concentrations of the peptides P1–P4, obtained using the Chou–Talalay method. CI < 1—synergism; CI = 1—additivity CI > 1—antagonism.