Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma
<p>MDA-1986 and UMSCC-22B cells were treated with varying concentrations of either cisplatin alone or WGA-TA alone and the cell viability after 72 h was measured via MTS assay. The IC50 values (μM) shown below were calculated using GraphPad Prism.</p> "> Figure 2
<p>(<b>A</b>,<b>B</b>). Western blot showing treatment effect on the mTOR pathway and downstream substrates. MDA1986 cells were treated with varying concentrations of WGA-TA alone or in combination with 1.25 (<b>A</b>) or 2.5 (<b>B</b>) μM cisplatin as well as cisplatin alone for 24 h. Solvent treated cells were used as control. Cells were collected, lysed, and immunoblotted for mTOR pathway proteins.</p> "> Figure 3
<p>(<b>A</b>,<b>B</b>). Western blot showing the treatment effect on translation initiation. MDA-1986 cells were treated with either 1.25 (<b>A</b>) or 2.5 (<b>B</b>) µM cisplatin either alone or in combination with either 0.125 or 0.25 or 0.50 µM TA-WGA for 24 h. Solvent treated samples served as control. Post-treatment cells were collected, and equal amounts of proteins (20 µg) were loaded on SDS-PAGE gel and transferred onto a nitro cellulose membrane. The membranes were incubated with primary antibodies of translation complex proteins. The membranes were then treated with appropriate secondary antibodies and the blots were then developed using ECL.</p> "> Figure 4
<p><b>A</b> (<b>Top</b>)<b>.</b> MDA1986 cells were treated either alone or in combination for 24 h. The induction of apoptosis and necrosis was detected using a RealTime-Glo annexin V apoptosis necrosis assay. The increase in luminescence induction of apoptosis and changes in fluorescence indicates the induction of secondary necrosis. <b>B</b> (<b>Bottom</b>). Western blot showing treatment effect on protein markers for the induction of apoptosis. MDA-1986 cells were treated with either cisplatin or WGA-TA alone or in combination for 24 h. Post-treatment cells were immunoblotted for PARP. Actin was used as a loading control.</p> "> Figure 4 Cont.
<p><b>A</b> (<b>Top</b>)<b>.</b> MDA1986 cells were treated either alone or in combination for 24 h. The induction of apoptosis and necrosis was detected using a RealTime-Glo annexin V apoptosis necrosis assay. The increase in luminescence induction of apoptosis and changes in fluorescence indicates the induction of secondary necrosis. <b>B</b> (<b>Bottom</b>). Western blot showing treatment effect on protein markers for the induction of apoptosis. MDA-1986 cells were treated with either cisplatin or WGA-TA alone or in combination for 24 h. Post-treatment cells were immunoblotted for PARP. Actin was used as a loading control.</p> "> Figure 5
<p>Immunoblot analysis of MDA-1986 cells treated with either 1.25 or 2.5 μM cisplatin alone or in combination with 0.25 or 0.50 μM WGA-TA for 24 h.</p> "> Figure 6
<p>(<b>A</b>,<b>B</b>) The Boyden chamber assay utilized approximately 100,000 MDA-1986 cells treated with either 1.25 or 2.5 μM cisplatin alone or in combination with 0.25 or 0.50 μM WGA-TA for 24 h. Control inserts were used for migration (<b>A Left</b>) and Matrigel coated inserts were used for invasion (<b>B Right</b>). Post-treatment, the cells were fixed with 2% paraformaldehyde, stained with 1% crystal violet in 20% methanol, washed and imaged using a light microscope. Quantification for the migration and invasion are given below (<b>C</b>).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Viability Assay
2.2. Western Blot Analysis
2.3. Boyden Chamber Assay for Migration and Invasion
2.4. Statistical Analysis
3. Results
3.1. Determination of IC50 Values for Single Drug Treatment
3.2. Synergistic Effect of Combination Treatment
3.3. Effect of WGA-TA and Combination Treatment on the mTOR Pathway and Its Substrates
3.4. Effect of WGA-TA and Combination Treatment on Translation Initiation
3.5. Effect of WGA-TA and Combination Treatment on the EMT, Apoptosis, Migration and Invasion
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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A | ||||
Cisplatin Dose (μM) | WGA-TA Dose (μM) | Combination Effect | CI Value | Cell Lines |
1.25 | 2.50 | 0.017 | 0.04 | UMSCC-22B |
1.25 | 1.25 | 0.078 | 0.07 | |
1.25 | 0.63 | 0.178 | 0.07 | |
1.25 | 0.31 | 0.473 | 0.12 | |
1.25 | 0.16 | 0.864 | 0.31 | |
1.25 | 0.08 | 0.972 | 0.69 | |
2.50 | 2.50 | 0.017 | 0.04 | |
2.50 | 1.25 | 0.064 | 0.06 | |
2.50 | 0.63 | 0.146 | 0.06 | |
2.50 | 0.31 | 0.490 | 0.13 | |
2.50 | 0.16 | 0.795 | 0.21 | |
1.25 | 2.50 | 0.020 | 0.42 | MDA-1986 |
1.25 | 1.25 | 0.130 | 0.72 | |
1.25 | 0.63 | 0.140 | 0.39 | |
1.25 | 0.31 | 0.410 | 0.44 | |
1.25 | 0.16 | 0.760 | 0.54 | |
1.25 | 0.08 | 0.920 | 0.66 | |
2.50 | 2.50 | 0.010 | 0.30 | |
2.50 | 1.25 | 0.080 | 0.55 | |
2.50 | 0.63 | 0.140 | 0.38 | |
2.50 | 0.31 | 0.600 | 0.36 | |
2.50 | 0.16 | 0.740 | 0.28 | |
B | ||||
Cisplatin Dose (μM) | WGA-TA Dose (μM) | Combination Effect | CI Value | Cell Lines |
1.25 | 5.000 | 0.016 | 0.002 | |
1.25 | 2.500 | 0.212 | 0.010 | UMSCC-22B |
2.50 | 5.000 | 0.021 | 0.003 | |
2.50 | 2.500 | 0.239 | 0.012 | |
2.50 | 1.250 | 0.479 | 0.014 | |
1.25 | 5.000 | 0.008 | 0.556 | |
1.25 | 0.625 | 0.520 | 0.710 | MDA-1986 |
2.50 | 5.000 | 0.003 | 0.375 | |
2.50 | 1.250 | 0.310 | 0.953 | |
2.50 | 0.313 | 0.710 | 0.523 |
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Subramanian, C.; Spielbauer, K.K.; Pearce, R.; Kovatch, K.J.; Prince, M.E.; Timmermann, B.N.; Cohen, M.S. Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma. Nutrients 2022, 14, 5398. https://doi.org/10.3390/nu14245398
Subramanian C, Spielbauer KK, Pearce R, Kovatch KJ, Prince ME, Timmermann BN, Cohen MS. Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma. Nutrients. 2022; 14(24):5398. https://doi.org/10.3390/nu14245398
Chicago/Turabian StyleSubramanian, Chitra, Katie K. Spielbauer, Robin Pearce, Kevin J. Kovatch, Mark E. Prince, Barbara N. Timmermann, and Mark S. Cohen. 2022. "Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma" Nutrients 14, no. 24: 5398. https://doi.org/10.3390/nu14245398