Formononetin Defeats Multidrug-Resistant Cancers by Induction of Oxidative Stress and Suppression of P-Glycoprotein
<p>Inhibitory effect of formononetin on P-gp efflux of <span class="html-italic">ABCB1</span>/Flp-In<sup>TM</sup>-293 cells. (<b>A</b>) Chemical structure of formononetin. (<b>B</b>) P-gp overexpressing <span class="html-italic">ABCB1</span>/Flp-In<sup>TM</sup>-293 cells were incubated with serial-dose formononetin or verapamil, a positive control, for 30 min. The intracellular calcein fluorescence was determined to indicate the inhibition of P-gp. Each data are expressed as the mean ± standard error of at least two experiments, each performed in triplicate. Verapamil has used as positive control of P-gp inhibitor. The abbreviations of verapamil and formononetin are VER and FMN, respectively. * denotes <span class="html-italic">p</span> < 0.05 as compared to control group.</p> "> Figure 2
<p>(<b>A</b>,<b>B</b>) The effects of FMN on P-gp ATPase activity was detected and data were analyzed as RLUs. After P-gp membranes were incubated with or without FMN, the unmetabolized ATP transformed into a luminescence. FMN could significantly stimulate basal and verapamil-stimulated P-gp ATPase activity. Verapamil (200 µM) was used as positive control. (<b>A</b>,<b>B</b>) Data were presented as the difference between Na3VO<sub>4</sub>-treated samples. Each data are expressed as the mean ± standard error of at least two experiments, each performed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control group. (<b>C</b>) P-gp substrates were identified by MDR1 shift assay, UIC2 fluorescence was increased during the binding of substrate on P-gp. Vinblastine (22.5 µM), a P-gp standard substrate, was used as positive control. The abbreviations of verapamil and formononetin are VER and FMN, respectively.</p> "> Figure 3
<p>The kinetic interactions of formononetin on rhodamine 123 or doxorubicin. Michaelis–Menten kinetics of P-gp efflux were determined by the extracellular fluorescence of rhodamine 123 (<b>A</b>) or doxorubicin (<b>C</b>). Lineweaver–Burk plot analysis of formononetin inhibitory mechanism on rhodamine 123 and doxorubicin efflux is shown in (<b>B</b>,<b>D</b>), respectively. Each datum is expressed as the mean ± standard error of at least two experiments, each performed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to nontreatment control.</p> "> Figure 3 Cont.
<p>The kinetic interactions of formononetin on rhodamine 123 or doxorubicin. Michaelis–Menten kinetics of P-gp efflux were determined by the extracellular fluorescence of rhodamine 123 (<b>A</b>) or doxorubicin (<b>C</b>). Lineweaver–Burk plot analysis of formononetin inhibitory mechanism on rhodamine 123 and doxorubicin efflux is shown in (<b>B</b>,<b>D</b>), respectively. Each datum is expressed as the mean ± standard error of at least two experiments, each performed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to nontreatment control.</p> "> Figure 4
<p>Effects of formononetin combined with chemotherapeutic agents on cell viabilities of drug-sensitive HeLaS3 cells and MDR KBvin cells. (<b>A</b>,<b>B</b>) The combination effects of formononetin and vincristine or doxorubicin on cell viability by SRB assay in cervical cancer HeLaS3 cells and MDR KBvin cells. Cells were pretreated with drugs alone or compound drug for 72 h. (<b>C</b>) Normalized isobologram for non-constant ratio combination of formononetin and chemotherapeutic drugs. The co-treatment of vincristine or doxorubicin with formononetin for 72 h at different combined concentrations. The actual drug dose was normalized with its corresponding IC<sub>50</sub> and used to determine the synergetic effect of the co-treatments in KBvin cells. The line on the isobologram denotes the half effect from each drug. Antagonism, additive, or synergism effects were indicated above, on, or below the line, respectively. ⊡ formononetin 25 µg/mL + vincristine 1000 nM/doxorubicin 10,000 nM ⊙ formononetin 25 µg/mL + vincristine 100 nM/doxorubicin 1000 nM; ▽ formononetin 20 µg/mL + vincristine 1000 nM; △ formononetin 20 µg/mL + vincristine 100 nM; ⟐ formononetin 20 µg/mL + vincristine 1000 nM; ╳ formononetin 20 µg/mL + vincristine 1000 nM. Data presented as mean ± SE of at least two experiments, each in duplicate. * indicates <span class="html-italic">p</span> value < 0.05 compared with doxorubicin only or vincristine-only group. The abbreviations of vincristine, doxorubicin, and formononetin are VIN, DOX, and FMN, respectively.</p> "> Figure 4 Cont.
<p>Effects of formononetin combined with chemotherapeutic agents on cell viabilities of drug-sensitive HeLaS3 cells and MDR KBvin cells. (<b>A</b>,<b>B</b>) The combination effects of formononetin and vincristine or doxorubicin on cell viability by SRB assay in cervical cancer HeLaS3 cells and MDR KBvin cells. Cells were pretreated with drugs alone or compound drug for 72 h. (<b>C</b>) Normalized isobologram for non-constant ratio combination of formononetin and chemotherapeutic drugs. The co-treatment of vincristine or doxorubicin with formononetin for 72 h at different combined concentrations. The actual drug dose was normalized with its corresponding IC<sub>50</sub> and used to determine the synergetic effect of the co-treatments in KBvin cells. The line on the isobologram denotes the half effect from each drug. Antagonism, additive, or synergism effects were indicated above, on, or below the line, respectively. ⊡ formononetin 25 µg/mL + vincristine 1000 nM/doxorubicin 10,000 nM ⊙ formononetin 25 µg/mL + vincristine 100 nM/doxorubicin 1000 nM; ▽ formononetin 20 µg/mL + vincristine 1000 nM; △ formononetin 20 µg/mL + vincristine 100 nM; ⟐ formononetin 20 µg/mL + vincristine 1000 nM; ╳ formononetin 20 µg/mL + vincristine 1000 nM. Data presented as mean ± SE of at least two experiments, each in duplicate. * indicates <span class="html-italic">p</span> value < 0.05 compared with doxorubicin only or vincristine-only group. The abbreviations of vincristine, doxorubicin, and formononetin are VIN, DOX, and FMN, respectively.</p> "> Figure 5
<p>Mechanism of formononetin MDR reversal ability on cancer cells. (<b>A</b>) The effects of formononetin on vincristine-induced cytotoxicity was assessed by apoptosis assay. Apoptotic cells were stained with 5 µL of Annexin V–FITC and propidium iodide (PI) and analyzed by flow cytometry. (<b>B</b>) <span class="html-italic">ABCB1</span> mRNA expression was determined by real-time RT PCR. Cells were pretreated with formononetin 10 µg/mL or 25 µg/mL in HeLaS3 and KBvin for 72 h. Each datum is expressed as the mean ± standard error of at least two experiments, each performed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control group. The abbreviations of vincristine and formononetin are VIN and FMN, respectively.</p> "> Figure 5 Cont.
<p>Mechanism of formononetin MDR reversal ability on cancer cells. (<b>A</b>) The effects of formononetin on vincristine-induced cytotoxicity was assessed by apoptosis assay. Apoptotic cells were stained with 5 µL of Annexin V–FITC and propidium iodide (PI) and analyzed by flow cytometry. (<b>B</b>) <span class="html-italic">ABCB1</span> mRNA expression was determined by real-time RT PCR. Cells were pretreated with formononetin 10 µg/mL or 25 µg/mL in HeLaS3 and KBvin for 72 h. Each datum is expressed as the mean ± standard error of at least two experiments, each performed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control group. The abbreviations of vincristine and formononetin are VIN and FMN, respectively.</p> "> Figure 6
<p>The docking results showed the superimposition of docked poses of compounds in the P-gp-binding pocket of the 3D structure of formononetin, rhodamine 123, and doxorubicin on P-gp: (<b>A</b>) formononetin, (<b>B</b>) rhodamine 123, (<b>C</b>) doxorubicin.</p> "> Figure 6 Cont.
<p>The docking results showed the superimposition of docked poses of compounds in the P-gp-binding pocket of the 3D structure of formononetin, rhodamine 123, and doxorubicin on P-gp: (<b>A</b>) formononetin, (<b>B</b>) rhodamine 123, (<b>C</b>) doxorubicin.</p> "> Figure 7
<p>Intracellular ROS production and mitochondria membrane potential changes in the MDR KBvin cells. The intracellular ROS production was detected in the HeLaS3 (<b>A</b>) and KBvin cells (<b>B</b>). Formononetin with or without chemotherapeutic drugs were treated for 1 h. Menadione was used as a positive control. The mitochondria membrane potential changes were measured in the HeLaS3 (<b>C</b>) and KBvin cells (<b>D</b>). The cells were treated with formononetin or chemotherapeutic drugs only or formononetin combined with chemotherapeutic drug for 6 h. Menadione was used as a positive control. Each datum is expressed as the mean ± standard error of at least two experiments, each per-formed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control. N-acetylcysteine was applied as the antioxidant, and the cell viabilities were further evaluated by SRB assay in HeLaS3 (<b>E</b>) and KBvin cells (<b>F</b>). * denotes <span class="html-italic">p</span> < 0.05 as compared to with doxorubicin only or vincristine only group. # indicates <span class="html-italic">p</span> < 0.05 compared with formononetin combined doxorubicin or vincristine.</p> "> Figure 7 Cont.
<p>Intracellular ROS production and mitochondria membrane potential changes in the MDR KBvin cells. The intracellular ROS production was detected in the HeLaS3 (<b>A</b>) and KBvin cells (<b>B</b>). Formononetin with or without chemotherapeutic drugs were treated for 1 h. Menadione was used as a positive control. The mitochondria membrane potential changes were measured in the HeLaS3 (<b>C</b>) and KBvin cells (<b>D</b>). The cells were treated with formononetin or chemotherapeutic drugs only or formononetin combined with chemotherapeutic drug for 6 h. Menadione was used as a positive control. Each datum is expressed as the mean ± standard error of at least two experiments, each per-formed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control. N-acetylcysteine was applied as the antioxidant, and the cell viabilities were further evaluated by SRB assay in HeLaS3 (<b>E</b>) and KBvin cells (<b>F</b>). * denotes <span class="html-italic">p</span> < 0.05 as compared to with doxorubicin only or vincristine only group. # indicates <span class="html-italic">p</span> < 0.05 compared with formononetin combined doxorubicin or vincristine.</p> "> Figure 7 Cont.
<p>Intracellular ROS production and mitochondria membrane potential changes in the MDR KBvin cells. The intracellular ROS production was detected in the HeLaS3 (<b>A</b>) and KBvin cells (<b>B</b>). Formononetin with or without chemotherapeutic drugs were treated for 1 h. Menadione was used as a positive control. The mitochondria membrane potential changes were measured in the HeLaS3 (<b>C</b>) and KBvin cells (<b>D</b>). The cells were treated with formononetin or chemotherapeutic drugs only or formononetin combined with chemotherapeutic drug for 6 h. Menadione was used as a positive control. Each datum is expressed as the mean ± standard error of at least two experiments, each per-formed in triplicate. * denotes <span class="html-italic">p</span> < 0.05 as compared to control. N-acetylcysteine was applied as the antioxidant, and the cell viabilities were further evaluated by SRB assay in HeLaS3 (<b>E</b>) and KBvin cells (<b>F</b>). * denotes <span class="html-italic">p</span> < 0.05 as compared to with doxorubicin only or vincristine only group. # indicates <span class="html-italic">p</span> < 0.05 compared with formononetin combined doxorubicin or vincristine.</p> "> Figure 8
<p>Formononetin combined with paclitaxel suppressed MDR KBvin cell growth in a xenotransplantation model. (<b>A</b>) Formononetin did not express significant toxicity at 48 hpi. (<b>B</b>) In an MDR KBvin xenograft model, we demonstrated that formononetin synergistically suppressed tumor size in combination with paclitaxel. The intensity of red fluorescence is proportional to the tumor size. Scale bar represents 1 mm. * <span class="html-italic">p</span> < 0.05 compared with the control; @ <span class="html-italic">p</span> < 0.05 compared to paclitaxel only group. hpf: hours post-fertilization; hpi: hours post-treatment or post-injection. The abbreviations of paclitaxel and formononetin are PXL and FMN, respectively.</p> "> Figure 8 Cont.
<p>Formononetin combined with paclitaxel suppressed MDR KBvin cell growth in a xenotransplantation model. (<b>A</b>) Formononetin did not express significant toxicity at 48 hpi. (<b>B</b>) In an MDR KBvin xenograft model, we demonstrated that formononetin synergistically suppressed tumor size in combination with paclitaxel. The intensity of red fluorescence is proportional to the tumor size. Scale bar represents 1 mm. * <span class="html-italic">p</span> < 0.05 compared with the control; @ <span class="html-italic">p</span> < 0.05 compared to paclitaxel only group. hpf: hours post-fertilization; hpi: hours post-treatment or post-injection. The abbreviations of paclitaxel and formononetin are PXL and FMN, respectively.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Modulation of P-gp Functions by Formononetin in ABCB1 Overexpressing Cells
2.2. Formononetin Inhibits P-gp Efflux Function by Inducing ATPase Activity
2.3. Inhibition Kinetics of the P-gp Substrate Rhodamine 123 and Doxorubicin
2.4. The Synergetic Effects of Formononetin with Chemotherapeutic Agents on MDR Cancer Cell Line
2.5. The Docking Model of Formononetin on P-gp
2.6. Formononetin Induced ROS Production and Mitochondria Membrane Potential Changes
2.7. Formononetin Resensitized MDR Cancer In Vivo
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Cell Lines and Cell Culture
4.3. Rhodamine 123 and Doxorubicin Efflux Assay
4.4. Determination of Cytotoxicity
4.5. Calcein–AM Uptake Assay
4.6. P-gp ATPase Assay
4.7. MDR1 Shift Assay
4.8. Quantitative Real-Time RT-PCR
4.9. Apoptosis Assay
4.10. Molecular Docking Simulations
4.11. Measurement of Intracellular Total ROS Activity and Mitochondrion Membrane Potential Changes
4.12. Zebrafish Xenograft Assay
4.13. Data and Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Nonlinear Kinetic Parameters | |||
---|---|---|---|
Vm (pmol/mg protein/10 min) | Km (μM) | ||
Nonlinear regression | |||
Rhodamine123 only | 12.68 ± 0.67 | 19.28 ± 1.93 | |
+ formononetin 10 μM | 6.11 ± 0.52 * | 9.4 ± 1.11 * | |
+ formononetin 20 μM | 3.45 ± 0.1 * | 4.81 ± 0.91 * | |
efflux IC50 (μM) | 9.04 ± 0.38 | ||
Vm (pmol/mg protein/120 min) | Km (μM) | ||
Nonlinear regression | |||
Doxorubicin only | 55.99 ± 1.89 | 20.15 ± 2.83 | |
+ formononetin 10 μM | 33.46 ± 0.95 * | 11.9 ± 0.65 * | |
+ formononetin 20 μM | 23.6 ± 1.26 * | 8.71 ± 2.51 * | |
efflux IC50 (μM) | 9.30 ± 0.14 |
Chemotherapeutics Agent (nM) | Formononetin (µg/mL) | Fa a | CI b | Pharmacological Effect |
---|---|---|---|---|
Vincristine | ||||
100 | 10 | 0.63 | 0.3 | Strong Synergism |
1000 | 10 | 0.22 | 0.1 | Strong Synergism |
100 | 20 | 0.60 | 0.5 | Synergism |
1000 | 20 | 0.25 | 0.2 | Strong Synergism |
100 | 25 | 0.51 | 0.5 | Synergism |
1000 | 25 | 0.20 | 0.2 | Strong Synergism |
Doxorubicin | ||||
1000 | 25 | 0.65 | 0.7 | Synergism |
10,000 | 25 | 0.55 | 0.5 | Synergism |
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Chang, Y.-T.; Wu, I.-T.; Sheu, M.-J.; Lan, Y.-H.; Hung, C.-C. Formononetin Defeats Multidrug-Resistant Cancers by Induction of Oxidative Stress and Suppression of P-Glycoprotein. Int. J. Mol. Sci. 2024, 25, 8471. https://doi.org/10.3390/ijms25158471
Chang Y-T, Wu I-T, Sheu M-J, Lan Y-H, Hung C-C. Formononetin Defeats Multidrug-Resistant Cancers by Induction of Oxidative Stress and Suppression of P-Glycoprotein. International Journal of Molecular Sciences. 2024; 25(15):8471. https://doi.org/10.3390/ijms25158471
Chicago/Turabian StyleChang, Ying-Tzu, I-Ting Wu, Ming-Jyh Sheu, Yu-Hsuan Lan, and Chin-Chuan Hung. 2024. "Formononetin Defeats Multidrug-Resistant Cancers by Induction of Oxidative Stress and Suppression of P-Glycoprotein" International Journal of Molecular Sciences 25, no. 15: 8471. https://doi.org/10.3390/ijms25158471