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Biomimetic Magnetic Nanocarriers Drive Choline Kinase Alpha Inhibitor inside Cancer Cells for Combined Chemo-Hyperthermia Therapy

Jabalera, Ylenia, Sola-Leyva, Alberto, Peigneux, Ana, Vurro, Federica, Iglesias, Guillermo R., Vilchez-Garcia, Jesus, Pérez-Prieto, Inmaculada, Aguilar-Troyano, Francisco J., López-Cara, Luisa C., Carrasco-Jiménez, María P., Jimenez-Lopez, Concepcion·2019·Pharmaceutics
DOI 10.3390/pharmaceutics11080408PMC PMC6722936
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Abstract

Choline kinase α1 (ChoKα1) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoKα1 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoKα1. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia.

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Figure 1 Chemical structure of choline kinase α 1 (ChoKα1) inhibitor Ff35.
Figure 1 Chemical structure of choline kinase α 1 (ChoKα1) inhibitor Ff35.CC BY · PubMed Central
Figure 2 Biomimetic magnetic nanoparticles (BMNPs) characterization. (A) TEM image of BMNPs and (B) crystal size distribution. (C) Hysteresis cycle of BMNPs at 300 K and 5 K. (D) Detail of the hysteresis cycle in the absence of external magnetic field at 300 K and 5 K. (E) Zero field cooling-field cooling (ZFC-FC) curves of BMNPs.
Figure 2 Biomimetic magnetic nanoparticles (BMNPs) characterization. (A) TEM image of BMNPs and (B) crystal size distribution. (C) Hysteresis cycle of BMNPs at 300 K and 5 K. (D) Detail of the hysteresis cycle in the absence of external magnetic field at 300 K and 5 K. (E) Zero field cooling-field cooling (ZFC-FC) curves of BMNPs.CC BY · PubMed Central
Figure 3 Adsorption kinetics (A) and adsorption isotherm (B) of Ff35 on BMNPs. The line represents the nonlineal weighted least-squares (NWLS) fitting of the experimental data according to LF model. In the case of adsorption kinetics, the vertical error bars are smaller than the symbol.
Figure 3 Adsorption kinetics (A) and adsorption isotherm (B) of Ff35 on BMNPs. The line represents the nonlineal weighted least-squares (NWLS) fitting of the experimental data according to LF model. In the case of adsorption kinetics, the vertical error bars are smaller than the symbol.CC BY · PubMed Central
Figure 4 Effects of Ff35, BMNPs, and Ff35-BMNPs on HepG2 cell proliferation. HepG2 cells growing in the log phase were incubated with Minimum Essential Medium containing 10% heat-inactivated fetal bovine serum (MEM/10% FBS) in the presence or absence of Ff35 up to 10 μM (A) or Ff35 1 μM, BMNPs 300 µg mL−1 or Ff35-BMNPs (concentration of Ff35 was 1 μM and that of BMNPs was 300 µg mL−1) (B). Cell viability was determined by MTT assay and normalized to that of the respective control cells. These experiments were performed twice in triplicate. * p < 0.05, ** p < 0.001 compared to their respective controls.
Figure 4 Effects of Ff35, BMNPs, and Ff35-BMNPs on HepG2 cell proliferation. HepG2 cells growing in the log phase were incubated with Minimum Essential Medium containing 10% heat-inactivated fetal bovine serum (MEM/10% FBS) in the presence or absence of Ff35 up to 10 μM (A) or Ff35 1 μM, BMNPs 300 µg mL−1 or Ff35-BMNPs (concentration of Ff35 was 1 μM and that of BMNPs was 300 µg mL−1) (B). Cell viability was determined by MTT assay and normalized to that of the respective control cells. These experiments were performed twice in triplicate. * p < 0.05, ** p < 0.001 compared to their respective controls.CC BY · PubMed Central
Figure 5 (A) Effects of Ff35 on ChoKα1 activity assayed in purified ChoKα1 exposed to different Ff35 concentrations. The results are expressed as percentage of enzymatic inhibition. (B) Choline uptake assay in HepG2 cells. Choline uptake was determined in cells treated for 10 min, 24 h, and 48 h soluble Ff35 or Ff35-BMNPs. Results are normalized to their respective control. These experiments were performed three times in triplicates. * p < 0.05, ** p < 0.001 compared to their respective controls.
Figure 5 (A) Effects of Ff35 on ChoKα1 activity assayed in purified ChoKα1 exposed to different Ff35 concentrations. The results are expressed as percentage of enzymatic inhibition. (B) Choline uptake assay in HepG2 cells. Choline uptake was determined in cells treated for 10 min, 24 h, and 48 h soluble Ff35 or Ff35-BMNPs. Results are normalized to their respective control. These experiments were performed three times in triplicates. * p < 0.05, ** p < 0.001 compared to their respective controls.CC BY · PubMed Central
Figure 6 Internalization of BMNPs and Ff35-BMNPs. Ultrastructural alterations produced by Ff35-BMNPs. (A,B): Control HepG2 cells. (C,D): HepG2 cell exposed to BMNPs has ultrastructure similar to control cells. (E,F): Treatment with Ff35-BMNPs (concentration of BMNPs was 300 µg mL−1 and Ff35 was 1 μM). Perinuclear space (Ps) is visible. The two nuclear membranes were separated showing a dilatation of the Ps. It is notably mitochondrial (M) rarefaction, disorganization, and dilatation. Both BMNPs and Ff35-BMNPs were internalized by HepG2 cells via endocytosis and compartmentalized in endosomes (Ed). Scale bar corresponds to 2 µm (A,C,E) and 1 µm (B,D,F).
Figure 6 Internalization of BMNPs and Ff35-BMNPs. Ultrastructural alterations produced by Ff35-BMNPs. (A,B): Control HepG2 cells. (C,D): HepG2 cell exposed to BMNPs has ultrastructure similar to control cells. (E,F): Treatment with Ff35-BMNPs (concentration of BMNPs was 300 µg mL−1 and Ff35 was 1 μM). Perinuclear space (Ps) is visible. The two nuclear membranes were separated showing a dilatation of the Ps. It is notably mitochondrial (M) rarefaction, disorganization, and dilatation. Both BMNPs and Ff35-BMNPs were internalized by HepG2 cells via endocytosis and compartmentalized in endosomes (Ed). Scale bar corresponds to 2 µm (A,C,E) and 1 µm (B,D,F).CC BY · PubMed Central
Figure 7 Effects of Ff35-BMNPs and hyperthermia on cell proliferation in HepG2 cells. HepG2 cells growing in log-phase were incubated with MEM/10% FBS in the absence or presence Ff35-BMNPs (concentration of Ff35 was 1 μM and BMNPs was 300 µg mL−1) for 24 h, and exposed to an alternative magnetic field for 1, 2, or 3 h. Cell number was determined by the MTT assay and expressed as percentage of control cells. Percentage of viability is normalized to that for the control cells. Results represent the mean ± SEM of three independent experiments conducted in triplicate. ** p < 0.001, when compared with their respective control values.
Figure 7 Effects of Ff35-BMNPs and hyperthermia on cell proliferation in HepG2 cells. HepG2 cells growing in log-phase were incubated with MEM/10% FBS in the absence or presence Ff35-BMNPs (concentration of Ff35 was 1 μM and BMNPs was 300 µg mL−1) for 24 h, and exposed to an alternative magnetic field for 1, 2, or 3 h. Cell number was determined by the MTT assay and expressed as percentage of control cells. Percentage of viability is normalized to that for the control cells. Results represent the mean ± SEM of three independent experiments conducted in triplicate. ** p < 0.001, when compared with their respective control values.CC BY · PubMed Central

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Research works printed but not linked: 28

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Metadata

Authors
Jabalera, Y., Sola-Leyva, A., Peigneux, A., Vurro, F., Iglesias, G. R., Vilchez-Garcia, J., Pérez-Prieto, I., Aguilar-Troyano, F. J., López-Cara, L. C., Carrasco-Jiménez, M. P., & Jimenez-Lopez, C.
Venue
Pharmaceutics
Publisher
MDPI AG
Published
2019-08-12
Volume
11
Issue
8
Pages
408
ISSN
1999-4923
DOI
10.3390/pharmaceutics11080408
PMC
PMC6722936
Cited by
27
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Ministerio de Economía y Competitividad (CGL2013-46612, CGL2016-76723)
  • Ramón y Cajal Programme (RYC-2014-16901)
  • Andalusian regional government (CTS-236)

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