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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 in vitro.HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G1-S versus G2-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay in both cell lines and reduced the number of cells in G2-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's t-test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in Figure S2. Data for unsynchronized HepG2 cells are also shown in Figure S2. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in Figure S2C.
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pone-0065726-g003: Mechanism of sorafenib-mediated radioprotection in vitro.HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G1-S versus G2-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay in both cell lines and reduced the number of cells in G2-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's t-test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in Figure S2. Data for unsynchronized HepG2 cells are also shown in Figure S2. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in Figure S2C.

Mentions: Sorafenib treatment effects on the cell cycle of the four HCC lines were examined (Fig. 3). HuH7 exhibited polyploidy and therefore was not analyzed further (Fig. S2). Unsynchronized Hep3b and HCC-4-4 cells following incubation with sorafenib for 24 h demonstrated a G1-S delay in response to sorafenib (Fig. 3B,C). For Hep3b, 73% (SD 2%) of sorafenib-treated cells were in G1 phase after 24 h as compared to only 48% (SD 1%) of untreated cells (p<0.00001 by Student's t-test) (Fig. 3B). Likewise, for HCC-4-4 at 24 h, 46% (SD 1%) of cells treated with sorafenib were in G1 versus only 35% (SD 1%) of untreated cells (p<0.00001, Student's t-test) (Fig. 3C). Unsynchronized HepG2 cells demonstrated a less pronounced effect on the cell cycle perhaps consistent with generalized cell cycle slowing (Fig. S2). To assess the effects of sorafenib on HepG2 cells further, we synchronized HepG2 cells and then analyzed after incubation with sorafenib for 0, 6, 12, and 24 h (Fig. 3A). Sorafenib treatment caused an overt G1-S delay, with the majority of cells (67%, SD 2%) remaining in G1 phase at 12 h after treatment with sorafenib versus only 17% (SD 1%) of untreated cells (p<0.00001, Student's t-test). As suspected with unsynchronized HepG2 cells, sorafenib treatment resulted in generalized cell cycle slowing that could easily be observed following synchronization with 79% (SD 4%) of cells treated with sorafenib remaining in G1 or S phase at 24 h versus 41% (SD 2%) of untreated cells (p<0.00001, Student's t-test). Altogether, sorafenib treatment resulted in cell cycle reassortment and cell cycle delay of HCC cells.


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 in vitro.HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G1-S versus G2-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay in both cell lines and reduced the number of cells in G2-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's t-test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in Figure S2. Data for unsynchronized HepG2 cells are also shown in Figure S2. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in Figure S2C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675179&req=5

pone-0065726-g003: Mechanism of sorafenib-mediated radioprotection in vitro.HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G1-S versus G2-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G1, S, and G2 phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G1-S delay in both cell lines and reduced the number of cells in G2-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's t-test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in Figure S2. Data for unsynchronized HepG2 cells are also shown in Figure S2. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in Figure S2C.
Mentions: Sorafenib treatment effects on the cell cycle of the four HCC lines were examined (Fig. 3). HuH7 exhibited polyploidy and therefore was not analyzed further (Fig. S2). Unsynchronized Hep3b and HCC-4-4 cells following incubation with sorafenib for 24 h demonstrated a G1-S delay in response to sorafenib (Fig. 3B,C). For Hep3b, 73% (SD 2%) of sorafenib-treated cells were in G1 phase after 24 h as compared to only 48% (SD 1%) of untreated cells (p<0.00001 by Student's t-test) (Fig. 3B). Likewise, for HCC-4-4 at 24 h, 46% (SD 1%) of cells treated with sorafenib were in G1 versus only 35% (SD 1%) of untreated cells (p<0.00001, Student's t-test) (Fig. 3C). Unsynchronized HepG2 cells demonstrated a less pronounced effect on the cell cycle perhaps consistent with generalized cell cycle slowing (Fig. S2). To assess the effects of sorafenib on HepG2 cells further, we synchronized HepG2 cells and then analyzed after incubation with sorafenib for 0, 6, 12, and 24 h (Fig. 3A). Sorafenib treatment caused an overt G1-S delay, with the majority of cells (67%, SD 2%) remaining in G1 phase at 12 h after treatment with sorafenib versus only 17% (SD 1%) of untreated cells (p<0.00001, Student's t-test). As suspected with unsynchronized HepG2 cells, sorafenib treatment resulted in generalized cell cycle slowing that could easily be observed following synchronization with 79% (SD 4%) of cells treated with sorafenib remaining in G1 or S phase at 24 h versus 41% (SD 2%) of untreated cells (p<0.00001, Student's t-test). Altogether, sorafenib treatment resulted in cell cycle reassortment and cell cycle delay of HCC cells.

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