Radiosensitization by statins in aggressive head and neck cancers
SCC61 (radiosensitive) and SQ20B (radioresistant) were treated with pitavastatin in presence or absence of IR and a colony formation assay was performed. SCC61 cell proliferation decrease as long as the irradiation dose is increased, however the result remains the same in presence of pitavastatin (Fig 1-A) indicating that SCC61 cell proliferation is not sensitive to this statin.
On the other hand, increasing amount of irradiation reduces the number of colonies in SQ20B with a stronger result when irradiation is combined with pitavastatin (Fig 1-B). Notice that pitavastatin concentration for SQ20B cells is four times less than for SCC61, demonstrating the strength of SQ20B sensitivity, compared to SCC61.
The combination of different pitavastatin concentration with 4 Gy of irradiation, demonstrates that effective radiosensitizing dose of pitavastatin for cell proliferation on SQ20B may be 2.5 μM (Fig 1-C), at least to detect proliferation differences in HNSCC.
Other statins are also affecting radioresistant HNSCC. SCC61 and SQ20B were treated with different concentration of several statins alone or in combination with irradiation and then stained with crystal violet to assess cell growing (Fig 1-D). As expected, pitavastatin reduces SQ20B growth at 2.5 μM, and other lipophilic statins have the same effect but at higher concentrations (simvastatin, atorvastatin and lovastatin). Hydrophilic statins like pravastatin or rosuvastatin have a weaker effect. The effect of all the statins on SQ20B cells is increased with irradiation. In SCC61, upon irradiation there is a decrease in growing, but is not influenced by the presence of statins. Some effect with 10 μM of atorvastatin is observed, but doesn’t seem to be relevant.
Senescence of cells treated with pitavastatin with or without irradiation was measured by flow cytometry 6 days after treatment (Fig 1-E). Pitavastatin alone slightly increased senescence in both cell lines but the major increase is observed in SQ20B cells when the statin is combined with irradiation. As expected, irradiation significantly increases senescence in SCC61 and not SQ20B.
To know if the effect of pitavastatin on radioresistant HNSCC is due to DNA DSB, a neutral comet assay was performed 20 hours after pitavastatin treatment alone or in combination with IR. As seen in figure 2-A, while irradiation is the responsible of increase DNA damage on SCC61, amount of DSB in SQ20B cells is enhanced upon statin treatment without irradiation. The result is significantly enhanced when pitavastatin is administered with irradiation. Amount of DSB in irradiated SQ20B was considerably high for a radioresistant cell line.
To test if DNA damage is different in SCC61 and SQ20B in vivo upon statin treatment, we examined by IHC the amount of γH2AX after pitavastatin with or without irradiation on xenograft tumors in athymic nude mice (Fig. 2-B). Treating mice with SCC61 xenografts only with pitavastatin increases the amount of γH2AX but the highest effect is observed with irradiation, being the effect similar when pitavastatin and irradiation are combined. A comparable effect was obtained in SQ20B xenografts after pitavastatin treatment. The presence of irradiation and pitavastatin increases the amount of γH2AX in SQ20B cells with not too much significance compared to only pitavastatin treated animals. The increase of γH2AX only with irradiation in SQ20B xenografts is not as significant as those treated with pitavastatin.
- Data contributed by Dr. Natalia Ricco, Post-Doctoral Scholar, Kron Lab. 2016.
Repurposing cephalosporin antibiotics as pro-senescent radiosensitizers
A. Tumor bearing mice were treated for 12 days with cephalexin (30 mg/kg twice daily by gavage) alone or in combination with two doses of 20 Gy. Groups were control (n = 10), 20 + 20 Gy (n = 10), cephalexin (n = 10), and cephalexin + 20 Gy + 20 Gy (n = 6). Combined treatment suppressed tumor growth compared to either treatment alone.
B. Scatter plot of individual tumors in corresponding treatment groups at day 12 showing the distribution of tumor volumes and the presence of experimental outliers. Means ± SEM are shown.
C. Cephalexin combined with IR produced marked tissue destruction and loss of cellularity. Combined treatment increases DNA damage, suppresses proliferation and induces senescence in B16.SIY tumors. H&E staining revealed extensive tissue destruction 7 days post combined treatment. Treatment with cephalexin + IR further increased the number of γ-H2AX positive cells, decreased the number of Ki-67 positive cells and enhanced SA-β-Gal staining compared to either treatment alone. Representative images are shown. Scale bar 100 μm.
D. Cephalexin plus IR induce oxidative damage in vivo. Tissue sections were examined by immunohistochemistry for oxidative damage to proteins (protein damage marker nitrotyrosine, nY), DNA (DNA damage marker 8-hydroxy-2’-deoxyguanosine, 8-OHdG), or lipids (lipid damage marker 4-hydroxy-2-nonenal, 4-HNE). Compared to control, both cephalexin and radiation increased staining for each oxidative stress marker while the combination produced an increase in each marker suggesting interactive effects. Representative images are shown. Scale bar 100 μm.
DNA-damage foci persistence and accelerated senescence as determinants of radiosensitization by PARP inhibitors
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- Poster contributed by Dr. Elena Efimova, Kron Lab. 2015.