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

Treatment regimens for head and neck squamous cell carcinoma (HNSCC) typically include cisplatin and radiotherapy and are limited by toxicities. We have identified naturally derived withalongolide A triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting HNSCC. We hypothesized that combining WGA-TA with cisplatin may allow for lower, less toxic cisplatin doses. HNSCC cell lines were treated with WGA-TA and cisplatin. After treatment with the drugs, the cell viability was determined by MTS assay. The combination index was calculated using CompuSyn. The expression of proteins involved in the targeting of translational initiation complex, epithelial to mesenchymal transition (EMT), and apoptosis were measured by western blot. Invasion and migration were measured using the Boyden-chamber assay. Treatment of MDA-1986 and UMSCC-22B cell lines with either WGA-TA or cisplatin alone for 72 h resulted in a dose dependent decrease in cell viability. Cisplatin in combination with WGA-TA resulted in significant synergistic cell death starting from 1.25 μM cisplatin. Combination treatment with WGA-TA resulted in lower cisplatin dosing while maintaining the downregulation of translational initiation complex proteins, the induction of apoptosis, and the blockade of migration, invasion, and EMT transition. These results suggest that combining a low concentration of cisplatin with WGA-TA may provide a safer, more effective therapeutic option for HNSCC that warrants translational validation

Stress responsive pathways in <i>clk-1</i> worms are upregulated during larval development and decline with age.

<p><i>Pgst-4</i>, <i>Phsp-6</i> and <i>Pnhr-57</i> reporter constructs were used to monitor the upregulation of oxidative stress response, mitochondrial unfolded protein response (mitoUPR) and hypoxia response during development and aging in <i>clk-1</i> worms. All three pathways were increased on day 1 of adulthood. <b>A</b>. The upregulation of the oxidative stress response decreases with age. <b>B</b>. The increase in mitoUPR in <i>clk-1</i> worms continues to increase until day 3 of adulthood and then decreases with age. <b>C</b>. In contrast, the mild upregulation of the hypoxia response is relatively constant with increasing age. <b>D</b>. On day 1 after hatching the mitoUPR and hypoxia response are already activated in <i>clk-1</i> worm. <b>E</b>. On day 2 after hatching, all three stress response pathways are upregulated in <i>clk-1</i> worms. Error bars indicate SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

<div><p>Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the <i>clk-1</i> mitochondrial mutant, but not other long-lived mitochondrial mutants such as <i>isp-1</i> or <i>nuo-6</i>. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (<i>sod</i>) genes in <i>clk-1</i> mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in <i>clk-1</i> worms. In contrast, deletion of either of the two cytoplasmic <i>sod</i> genes, <i>sod-1</i> or <i>sod-5</i>, significantly decreases the lifespan of <i>clk-1</i> worms. Further, we show that increasing mitochondrial superoxide levels through deletion of <i>sod-2</i> or treatment with paraquat can still increase lifespan in <i>clk-1;sod-1</i> double mutants, which live shorter than <i>clk-1</i> worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of <i>clk-1</i> worms.</p></div

Mitochondrial superoxide increases the lifespan of worms with a detrimental level of cytoplasmic superoxide.

<p>To further test the extent to which superoxide has different effects on lifespan depending on which subcellular compartment it is in, mitochondrial superoxide levels were increased through treatment with 0.1 mM paraquat. <b>A,B</b>. Despite the fact that <i>clk-1;sod-1</i> and <i>clk-1;sod-5</i> worms have decreased lifespan compared to <i>clk-1</i> worms resulting from elevated cytoplasmic superoxde levels, both double mutants showed increased lifespan when treated with paraquat. <b>C</b>. This is in contrast to <i>clk-1;sod-2</i> worms that exhibit a decrease in lifespan when mitochondrial superoxide levels are further increased. <b>D,E</b>. <i>clk-1;sod-3</i> worms exhibited a small but significant increase in lifespan when treated with paraquat, while <i>clk-1;sod-4</i> worms exhibit a robust increase. <b>F</b>. <i>clk-1;sod-1;sod-2</i> worms show increased lifespan compared to <i>clk-1;sod-1</i> worms, but decreased lifespan compared to <i>clk-1;sod-2</i> worms. This indicates that increasing mitochondrial superoxide through the deletion of <i>sod-2</i> can increase the lifespan of <i>clk-1;sod-1</i> worms, while increasing cytoplasmic superoxide through the deletion of <i>sod-1</i> can decrease the lifespan of <i>clk-1;sod-2</i> worms. This indicates that mitochondrial and cytoplasmic superoxide have opposing effects on lifespan.</p

<i>clk-1</i> worms are sensitive to acute exposure to oxidative stress but resistant to chronic exposure.

<p>Sensitivity to oxidative stress was assessed during development and adulthood using two superoxide-generating compounds: paraquat (PQ) and juglone. Chronic assays of oxidative stress could only be performed with paraquat because the toxicity of juglone is decreased within 8 hours. During development, <i>clk-1</i> L2 larvae have decreased survival compared to wild-type worms under conditions of oxidative stress: <b>A</b>. 200 mM paraquat or <b>B</b>. 180 μM juglone. Similarly, in acute assays of oxidative stress assay on day 1 of adulthood, <i>clk-1</i> worms show decreased survival compared to wild-type worms after exposure to either <b>C</b>. 200 mM paraquat or <b>D</b>. different concentrations of juglone (180–300 μM), indicating increased sensitivity to oxidative stress. <b>E</b>. In a chronic oxidative stress assay where worms are exposed to 4 mM paraquat beginning on day 1 of adulthood after development on NGM plates, <i>clk-1</i> worms survive significantly longer than wild-type worms. <b>F</b>. However, <i>clk-1</i> worms remain sensitive to acute oxidative stress throughout adulthood when exposed to 180 μM juglone. Overall, this shows that <i>clk-1</i> worms are sensitive to acute oxidative stress throughout development and adulthood but are resistant to chronic oxidative stress during adulthood. Error bars indicate SEM. * p<0.05, *** p<0.001.</p

Increased superoxide has a compartment specific effect on <i>clk-1</i> lifespan.

<p>Genetic deletion of individual <i>sod</i> genes allows for a compartment specific increase in the levels of superoxide. <b>A,B</b>. Deletion of either of the cytoplasmic <i>sod</i> genes (<i>sod-1</i>, <i>sod-5</i>) decreases <i>clk-1</i> lifespan. <b>C</b>. In contrast, deletion of the primary mitochondrial <i>sod</i> gene (<i>sod-2</i>) results in a marked increase in longevity. <b>D,E</b>. Loss of the inducible mitochondrial sod gene (<i>sod-3</i>) or the extracellular <i>sod</i> gene (<i>sod-4</i>) has no effect on <i>clk-1</i> lifespan. <b>F</b>. While the loss of both cytoplasmic <i>sod</i> genes decreases <i>clk-1</i> lifespan, <i>clk-1;sod-2;sod-3</i> mutants, which have no mitochondrial matrix SOD, still live longer than <i>clk-1</i> worms. <b>G,H</b>. Mean and maximum lifespan for <i>clk-1</i> double mutants lacking individual <i>sod</i> genes. The p-values shown indicate differences from <i>clk-1</i> worms. All <i>clk-1</i> double mutants had lifespans and maximum lifespans that were significantly different from wild-type. The fact that deletion of <i>sod-1</i> or <i>sod-5</i> decreases <i>clk-1</i> lifespan, while deletion of <i>sod-2</i> increases <i>clk-1</i> lifespan demonstrates that increasing mitochondrial and cytoplasmic superoxide has opposing effects on lifespan. Error bars indicate SEM. *** p < 0.001. NS = not significant.</p

Deletion of individual <i>sod</i> genes results in a compartment specific effect on sensitivity to oxidative stress in <i>clk-1</i> worms.

<p><b>A</b>. Deletion of <i>sod-1</i> or <i>sod-2</i> increases <i>clk-1</i> sensitivity to oxidative stress during development. <b>B</b>. Deletion of <i>sod-1</i>, <i>sod-4</i> or <i>sod-5</i> increases <i>clk-1</i> sensitivity to oxidative stress after acute exposure to juglone on day 1 of adulthood. In contrast, deletion of either mitochondrial <i>sod</i> gene, <i>sod-2</i> or <i>sod-3</i>, reverts stress sensitivity to wild-type. <b>C</b>. Deletion of <i>sod-1</i>, <i>sod-2</i> or <i>sod-5</i> increases <i>clk-1</i> sensitivity to oxidative stress during chronic exposure to 4 mM paraquat beginning on day 1 of adulthood. Note that deletion of <i>sod-1</i> and <i>sod-2</i> both increase sensitivity to paraquat in <i>clk-1</i> worms despite having opposite effects on lifespan. Significance indicates difference from <i>clk-1</i> worms. Error bars indicate SEM. * p<0.05, ** p<0.01, *** p<0.001. FA = fertile adult.</p

<i>clk-1</i> worms have increased antioxidant defenses.

<p>The expression of antioxidant genes was examined in day 1 adult worms by quantitative real-time RT-PCR. Compared to WT worms (white), <i>clk-1</i> worms (blue) showed increased expression of four different types of antioxidant genes including: (<b>A</b>) superoxide dismutases (<i>sod</i>), (<b>C</b>) peroxiredoxins (<i>prdx</i>), (<b>D</b>) catalases (<i>ctl</i>) and (<b>E</b>) thioredoxins (<i>trx</i>). The expression of SOD-3 protein is also significantly increased in <i>clk-1</i> worms as indicated by an increase in GFP intensity in <i>clk-1</i> worms expressing a SOD-3:GFP transgene under the <i>sod-3</i> gene promoter (<b>B</b>). Error bars indicate SEM. * p<0.05, ** p<0.01, *** p<0.001.</p