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Concurrent versus sequential sorafenib therapy in combination with radiation for hepatocellular carcinoma.

Wild AT, Gandhi N, Chettiar ST, Aziz K, Gajula RP, Williams RD, Kumar R, Taparra K, Zeng J, Cades JA, Velarde E, Menon S, Geschwind JF, Cosgrove D, Pawlik TM, Maitra A, Wong J, Hales RK, Torbenson MS, Herman JM, Tran PT - PLoS ONE (2013)

Bottom Line: Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not.Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone.In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology & Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G1-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.

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Mechanism of sorafenib-mediated radioprotection.Immunostaining for γ-H2AX foci, and then staining for DAPI were performed as detailed in Materials and Methods (note: the t = 0 time point actually represents cells that were fixed at 30 minutes post-irradiation). Fluorescent images were captured at 40× using a confocal microscope with uniform exposures of 50 ms for DAPI and 900 ms for Alexa Fluor 488 used for all images. (A) Representative images are shown for the HepG2 cell line at 0 and 24 h for each of the treatment arms. Sample images for the other 3 cell lines are shown in Figure S3. The percent of nuclei demonstrating high (>25), moderate (10–25), low (<10), or no γ-H2AX foci was quantitated for each cell line at each time point by counting at least 5 representative high-power fields (HPF). The results of this quantitation are shown in graphical form with SEM for each treatment arm of each cell line (B–E). For all cell lines, radiation (RT) with or without SOR resulted in a significantly greater percent of nuclei with a high number of foci at t = 0 compared to the non-irradiated SOR and control arms. At t = 24 h, 3 of the 4 cell lines (HepG2, HCC-4-4, Hep3b) demonstrated a significantly greater percent of nuclei with a high number of persistent foci in the RT arm compared to the concurrent RT-SOR (CONC) arm (B–D). The fourth cell line (HuH7) showed no difference in number of nuclei manifesting any level of γ-H2AX foci between the RT and CONC treatment arms (E). Asterisks represent significant differences between treatment arms by Fisher's exact test as indicated by accompanying brackets. Dotted lines are merely included in cases of overlapping brackets to avoid confusion. Cover slips with treated/fixed cells were prepared in triplicate.
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pone-0065726-g004: Mechanism of sorafenib-mediated radioprotection.Immunostaining for γ-H2AX foci, and then staining for DAPI were performed as detailed in Materials and Methods (note: the t = 0 time point actually represents cells that were fixed at 30 minutes post-irradiation). Fluorescent images were captured at 40× using a confocal microscope with uniform exposures of 50 ms for DAPI and 900 ms for Alexa Fluor 488 used for all images. (A) Representative images are shown for the HepG2 cell line at 0 and 24 h for each of the treatment arms. Sample images for the other 3 cell lines are shown in Figure S3. The percent of nuclei demonstrating high (>25), moderate (10–25), low (<10), or no γ-H2AX foci was quantitated for each cell line at each time point by counting at least 5 representative high-power fields (HPF). The results of this quantitation are shown in graphical form with SEM for each treatment arm of each cell line (B–E). For all cell lines, radiation (RT) with or without SOR resulted in a significantly greater percent of nuclei with a high number of foci at t = 0 compared to the non-irradiated SOR and control arms. At t = 24 h, 3 of the 4 cell lines (HepG2, HCC-4-4, Hep3b) demonstrated a significantly greater percent of nuclei with a high number of persistent foci in the RT arm compared to the concurrent RT-SOR (CONC) arm (B–D). The fourth cell line (HuH7) showed no difference in number of nuclei manifesting any level of γ-H2AX foci between the RT and CONC treatment arms (E). Asterisks represent significant differences between treatment arms by Fisher's exact test as indicated by accompanying brackets. Dotted lines are merely included in cases of overlapping brackets to avoid confusion. Cover slips with treated/fixed cells were prepared in triplicate.

Mentions: Based on the observed sorafenib-mediated effects on the cell cycle in three of the HCC cell lines we examined, we hypothesized that concurrent treatment with sorafenib may cause radioprotection by promoting reassortment into less radiosensitive phases of the cell cycle. To investigate this possibility, cells were incubated with sorafenib for 24 h, then irradiated with 6 Gy, fixed at time points of 30 minutes and 24 h, probed for γ-H2AX foci indicative of double-strand breaks (DSBs), and quantitated with a confocal microscope (Fig. 4A–E). Among all four HCC cell lines, irradiated cells exhibited a much greater proportion of nuclei with a high number (>25) of foci at 30 m following irradiation than cells treated with sorafenib or vehicle control alone, as expected (all p<0.001, Fisher's exact test). Treatment with radiation resulted in similar amounts of foci per nucleus at 30 m post-irradiation regardless of treatment with sorafenib, with almost all nuclei displaying a high number of foci after concurrent sorafenib-radiation or radiation alone (p>0.05 for all cell lines, Fisher's exact test). At 24 h post-irradiation, HuH7 cells did not exhibit significantly different proportions of nuclei with a high number of foci after concurrent sorafenib-radiation compared to radiation alone (p = 0.73). However, the remaining three HCC cell lines did demonstrate a greater proportion of nuclei with a high number of persistent foci after radiation alone versus concurrent sorafenib-radiation (p<0.001 for HepG2 and HCC-4-4; p = 0.02 for Hep3b, Fisher's exact test). Similar to the short term in vitro apoptosis assays conducted above we could not model sequential radiation-sorafenib effects on γ-H2AX foci given the rapid repair of DSBs. Despite this limitation, our data show that concurrent sorafenib-radiation treated HCC cells demonstrated reduced persistence of DSBs compared to HCC cells treated with radiation alone.


Concurrent versus sequential sorafenib therapy in combination with radiation for hepatocellular carcinoma.

Wild AT, Gandhi N, Chettiar ST, Aziz K, Gajula RP, Williams RD, Kumar R, Taparra K, Zeng J, Cades JA, Velarde E, Menon S, Geschwind JF, Cosgrove D, Pawlik TM, Maitra A, Wong J, Hales RK, Torbenson MS, Herman JM, Tran PT - PLoS ONE (2013)

Mechanism of sorafenib-mediated radioprotection.Immunostaining for γ-H2AX foci, and then staining for DAPI were performed as detailed in Materials and Methods (note: the t = 0 time point actually represents cells that were fixed at 30 minutes post-irradiation). Fluorescent images were captured at 40× using a confocal microscope with uniform exposures of 50 ms for DAPI and 900 ms for Alexa Fluor 488 used for all images. (A) Representative images are shown for the HepG2 cell line at 0 and 24 h for each of the treatment arms. Sample images for the other 3 cell lines are shown in Figure S3. The percent of nuclei demonstrating high (>25), moderate (10–25), low (<10), or no γ-H2AX foci was quantitated for each cell line at each time point by counting at least 5 representative high-power fields (HPF). The results of this quantitation are shown in graphical form with SEM for each treatment arm of each cell line (B–E). For all cell lines, radiation (RT) with or without SOR resulted in a significantly greater percent of nuclei with a high number of foci at t = 0 compared to the non-irradiated SOR and control arms. At t = 24 h, 3 of the 4 cell lines (HepG2, HCC-4-4, Hep3b) demonstrated a significantly greater percent of nuclei with a high number of persistent foci in the RT arm compared to the concurrent RT-SOR (CONC) arm (B–D). The fourth cell line (HuH7) showed no difference in number of nuclei manifesting any level of γ-H2AX foci between the RT and CONC treatment arms (E). Asterisks represent significant differences between treatment arms by Fisher's exact test as indicated by accompanying brackets. Dotted lines are merely included in cases of overlapping brackets to avoid confusion. Cover slips with treated/fixed cells were prepared in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675179&req=5

pone-0065726-g004: Mechanism of sorafenib-mediated radioprotection.Immunostaining for γ-H2AX foci, and then staining for DAPI were performed as detailed in Materials and Methods (note: the t = 0 time point actually represents cells that were fixed at 30 minutes post-irradiation). Fluorescent images were captured at 40× using a confocal microscope with uniform exposures of 50 ms for DAPI and 900 ms for Alexa Fluor 488 used for all images. (A) Representative images are shown for the HepG2 cell line at 0 and 24 h for each of the treatment arms. Sample images for the other 3 cell lines are shown in Figure S3. The percent of nuclei demonstrating high (>25), moderate (10–25), low (<10), or no γ-H2AX foci was quantitated for each cell line at each time point by counting at least 5 representative high-power fields (HPF). The results of this quantitation are shown in graphical form with SEM for each treatment arm of each cell line (B–E). For all cell lines, radiation (RT) with or without SOR resulted in a significantly greater percent of nuclei with a high number of foci at t = 0 compared to the non-irradiated SOR and control arms. At t = 24 h, 3 of the 4 cell lines (HepG2, HCC-4-4, Hep3b) demonstrated a significantly greater percent of nuclei with a high number of persistent foci in the RT arm compared to the concurrent RT-SOR (CONC) arm (B–D). The fourth cell line (HuH7) showed no difference in number of nuclei manifesting any level of γ-H2AX foci between the RT and CONC treatment arms (E). Asterisks represent significant differences between treatment arms by Fisher's exact test as indicated by accompanying brackets. Dotted lines are merely included in cases of overlapping brackets to avoid confusion. Cover slips with treated/fixed cells were prepared in triplicate.
Mentions: Based on the observed sorafenib-mediated effects on the cell cycle in three of the HCC cell lines we examined, we hypothesized that concurrent treatment with sorafenib may cause radioprotection by promoting reassortment into less radiosensitive phases of the cell cycle. To investigate this possibility, cells were incubated with sorafenib for 24 h, then irradiated with 6 Gy, fixed at time points of 30 minutes and 24 h, probed for γ-H2AX foci indicative of double-strand breaks (DSBs), and quantitated with a confocal microscope (Fig. 4A–E). Among all four HCC cell lines, irradiated cells exhibited a much greater proportion of nuclei with a high number (>25) of foci at 30 m following irradiation than cells treated with sorafenib or vehicle control alone, as expected (all p<0.001, Fisher's exact test). Treatment with radiation resulted in similar amounts of foci per nucleus at 30 m post-irradiation regardless of treatment with sorafenib, with almost all nuclei displaying a high number of foci after concurrent sorafenib-radiation or radiation alone (p>0.05 for all cell lines, Fisher's exact test). At 24 h post-irradiation, HuH7 cells did not exhibit significantly different proportions of nuclei with a high number of foci after concurrent sorafenib-radiation compared to radiation alone (p = 0.73). However, the remaining three HCC cell lines did demonstrate a greater proportion of nuclei with a high number of persistent foci after radiation alone versus concurrent sorafenib-radiation (p<0.001 for HepG2 and HCC-4-4; p = 0.02 for Hep3b, Fisher's exact test). Similar to the short term in vitro apoptosis assays conducted above we could not model sequential radiation-sorafenib effects on γ-H2AX foci given the rapid repair of DSBs. Despite this limitation, our data show that concurrent sorafenib-radiation treated HCC cells demonstrated reduced persistence of DSBs compared to HCC cells treated with radiation alone.

Bottom Line: Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not.Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone.In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology & Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G1-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.

Show MeSH
Related in: MedlinePlus